A METHOD FOR TREATING TOBACCO MATERIAL AND TREATED TOBACCO MATERIAL
20260047595 ยท 2026-02-19
Inventors
Cpc classification
A24D1/20
HUMAN NECESSITIES
International classification
Abstract
A method for treating tobacco material, the method comprising: providing a tobacco material, fermenting the tobacco material to obtain fermented tobacco material, the fermenting step including: # incubating the tobacco material under anaerobic conditions; # stopping the fermentation when at least one of the following conditions is satisfied: # 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 an initial amount of Lactic Acid in the tobacco material, # the content of Reducing Sugars is lower than 0.5, preferably lower than 0.4, more preferably below 0.2, more preferably below 0.1 an initial amount or 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, 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, 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, an initial amount of quinic acid in the tobacco material, # the content of asparagine is lower than 0.5, preferably lower than 0.4, preferably lower than 0.3 an initial amount or asparagine in the tobacco material, # the content of Glutamine is lower than 0.5, preferably lower than 0.4 an initial amount or 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 an initial amount of L-Ornithine in the tobacco material, # the content of L-Leucine is more than 2 times, preferably more than 4 times, an initial amount of L-Leucine in the tobacco material, # the content of L-Lysine is more than 2 times, preferably more than 6 times, an initial amount of L-Lysine in the tobacco material, # the fermentation index is more than 50, preferably more than 100, more preferably more than 250, more preferably more 400, wherein the fermentation index is obtained dividing the ratio between the content of Lactic Acid in the treated tobacco material and the content of Lactic Acid in the non-fermented tobacco material by the ratio between the content of Reducing Sugars in the treated tobacco material and the content of Reducing Sugars in the non-fermented tobacco material.
Claims
1. Method for treating tobacco material, the method comprising: providing a tobacco material; fermenting the tobacco material to obtain fermented tobacco material, the fermenting step including: incubating the tobacco material under anaerobic conditions; stopping the fermentation when at least one of the following conditions is satisfied: 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 an initial amount of Lactic Acid in the tobacco material, the content of Reducing Sugars is lower than 0.5, preferably lower than 0.4, more preferably below 0.2, more preferably below 0.1 an initial amount or 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, 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, 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, an initial amount of quinic acid in the tobacco material, the content of asparagine is lower than 0.5, preferably lower than 0.4, preferably lower than 0.3 an initial amount or asparagine in the tobacco material, the content of Glutamine is lower than 0.5, preferably lower than 0.4 an initial amount or 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 an initial amount of L-Ornithine in the tobacco material, the content of L-Leucine is more than 2 times, preferably more than 4 times, an initial amount of L-Leucine in the tobacco material, the content of L-Lysine is more than 2 times, preferably more than 6 times, an initial amount of L-Lysine in the tobacco material, the fermentation index is more than 50, preferably more than 100, more preferably more than 250, more preferably more 400, wherein the fermentation index is obtained dividing the ratio between the content of Lactic Acid in the treated tobacco material and the content of Lactic Acid in the non-fermented tobacco material by the ratio between the content of Reducing Sugars in the treated tobacco material and the content of Reducing Sugars in the non-fermented tobacco material.
2. Method according to claim 1 and further comprising an initial measuring step for measuring the initial content of at least one 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 fermentation index in the tobacco material.
3. Method according to claim 1 and further comprising a measuring step for measuring the content of at least one 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 fermentation index in the tobacco material during the fermenting step.
4. Method according to claim 1, wherein if the tobacco material contains dark tobacco, it is provided for stopping the fermentation when at least one of the following conditions is satisfied: the content of 2,3 butanediol is more than 5 times, preferably more than 10 times, an 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, an initial amount of diacetyl in the tobacco material.
5. Method according to claim 4 and further comprising an initial measurement step for measuring the initial content of 2,3 butanediol or diacetyl in the tobacco material before the fermentation so as to obtain an initial amount respectively of 2,3 butanediol or diacetyl in the tobacco material.
6. Method according to claim 1, wherein during the fermenting step it is provided for applying a pressure to the tobacco material comprised between 1000 kilograms per square meter and 15000 kilograms per square meter, preferably between 3000 kilograms per square meter and 12000 kilograms per square meter, more preferably between 5000 kilograms per square meter and 10000 kilograms per square meter.
7. Method according to claim 1, wherein it is provided for continuing the fermenting step 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. Method according to claim 1, wherein during the fermenting step it is provided for keeping the temperature of the tobacco material comprised between 21 degrees Celsius and 35 degrees Celsius, preferably between 25 degrees Celsius and 31 degrees Celsius.
9. Tobacco material comprising: a) at least one of the following compounds: Lactic Acid in an amount that 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, an initial amount of Lactic Acid in the tobacco material. Reducing Sugars in an amount that is lower than 0.5, preferably lower than 0.4, more preferably below 0.2, more preferably below 0.1 an initial amount or Reducing Sugars in the tobacco material, Indole-3 Lactic Acid in an amount that is more than 5 times, preferably more than 10 times, preferably more than 20 times, an initial amount of Indole-3 Lactic Acid in the tobacco material. caffeic acid in an amount that is more than 4 times, preferably more than 10 times, an initial amount of caffeic acid in the tobacco material, quinic acid in an amount that is more than 2 times, preferably more than 4 times, an initial amount of quinic acid in the tobacco material, asparagine in an amount that is lower than 0.5, preferably lower than 0.4, preferably lower than 0.3 an initial amount or asparagine in the tobacco material, Glutamine in an amount that is lower than 0.5, preferably lower than 0.4 an initial amount or Glutamine in the tobacco material, L-Ornithine is more than 10 times, preferably more than 50 times, preferably more than 100 times an initial amount of L-Ornithine in the tobacco material, L-Leucine in an amount that is more than 2 times, preferably more than 4 times, an initial amount of L-Leucine, L-Lysine in an amount that is more than 2 times, preferably more than 6 times, an initial amount of L-Lysine, a fermentation index is more than 50, preferably more than 100, more preferably more than 250, more preferably more 400, wherein the fermentation index is obtained dividing the ratio between the content of Lactic Acid in the tobacco material and the content of Lactic Acid in the non-fermented tobacco material by the ratio between the content of Reducing Sugars in the tobacco material and the content of Reducing Sugars in the non-fermented tobacco material; or b) less than 300 milligrams per kilogram of asparagine in total dry weight basis; or c) less than 70 milligrams per kilogram of glutamine in total dry weight basis; or d) more than 10000 milligrams per kilogram in total dry weight basis of total free amino acids; or e) at least 1 microgram per gram, preferably at least 2 micrograms per gram, more preferably at least 2.5 micrograms per gram of Indole-3 Lactic Acid in total dry weight basis.
10. Tobacco material according to claim 9, wherein said tobacco material is obtained by a process comprising fermenting the tobacco material to obtain treated tobacco material, including: incubating the tobacco material under anaerobic conditions.
11. Tobacco material according to claim 9, wherein the tobacco material is cured.
12. Tobacco material according to claim 9, wherein the tobacco material is grinded.
13. Aerosol generating article comprising a tobacco material containing between about 2.5 percent by weight in total dry weight basis and 100 percent by weight in total dry weight basis, preferably at least about 4 percent by weight in total dry weight basis, preferably at least about 10 percent by weight in total dry weight basis, preferably at least about 20 percent by weight in total dry weight basis of a the tobacco material according to claim 9.
Description
[0264] Examples will now be further described with reference to the figures in which:
[0265]
[0266]
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[0280]
EXAMPLE 1
[0281] Dark tobacco leaf material has been fully sun-cured for about 10 days. The sun cured leaves have been stripped to keep only the lamina (hand stripped leaves). This tobacco material is referred to as HS.
[0282] The tobacco material was conditioned to obtain a moisture of circa 30 percent. Samples of this tobacco material conditioned but not fermented yet are called 0T (starting material).
[0283] The conditioned tobacco material is then introduced in three barrels, in each barrel circa 100 kilograms of tobacco material is present. Before the introduction, the tobacco material is wrapped in a material maintaining the acquired moisture.
[0284] Pressure is applied to each barrel. The pressure is comprised between 1000 kilograms per square meter and 4000 kilograms per square meter.
[0285] After 1 month (sample called 1T), 2.5 months (sample called 2T), 6 months (sample called 3T) and 8.5 months (sample called 4T), the barrels were opened, and the samples collected at least in triplicate in each barrel before tobacco turning and readjustment of the moisture content to approximatively 30 percent5 percent.
[0286] During the heavy fermentation process under fully anaerobic conditions, the temperature inside the barrels did not particularly increase (it remained within the following temperature range: between 27 degrees Celsius and 31 degrees Celsius). The fermentation has been stopped after 8.5 months.
EXAMPLE 2
[0287] Dark tobacco leaf material has been yellowed for two days and rapidly chopped in cut-filler. This tobacco material contains both lamina and ribs. The chopped leaves containing both lamina and mid-ribs were sun-dried for two days. Samples of this tobacco material are named in the following CC.
[0288] The tobacco material was conditioned to obtain a moisture content of circa 30 percent. Samples of this tobacco material conditioned but not fermented yet are called 0T (starting material).
[0289] The conditioned tobacco material is then introduced in three barrels, in each barrel circa 100 kilograms of tobacco material is present. Before the introduction, the tobacco material is wrapped in a material maintaining the acquired moisture.
[0290] Pressure is applied to each barrel. The pressure is comprised between 1000 kilograms over square meter and 4000 kilograms over square meter.
[0291] After 1 month (sample called 1T), 2.5 months (sample called 2T), 6 months (sample called 3T) and 8.5 months (sample called 4T), the barrels were opened, and the samples collected at least in triplicate in each barrel before tobacco turning and readjustment of the moisture content to approximatively 30 percent5 percent.
[0292] During the heavy fermentation process under fully anaerobic conditions, the temperature inside the barrels did not particularly increase (it remained within the following temperature range: between 27 degrees Celsius and 31 degrees Celsius). The fermentation has been stopped after 8.5 months.
Visual Observations
[0293] The initial tobacco material was changing already after 2.5 month (samples 2T) of fermentation, the color of both HS and CC leaves became darker, the tobacco smell expressing nice caramel-buttery and fermented complex notes. The dark color was more marked in the fermented HS leaves compared to the CC leaves at the end of the process (8.5 month, 4T), likely due to the presence of leaf mid-rib in the CC leaves.
Chemical Analysis
[0294] In the following, when a value relative to a sample is mentioned, the given value represents an average of several values obtained for each sample of the same type.
[0295] The pH of the samples of tobacco material, both CC and HS, became acidic reaching 3.2, after fermentation conditions have been applied for 2.5 months (as found in sample 2T). This reflects the process of anaerobic fermentation involving sugars degradation, which usually produces organic acids like (acetic and/or) Lactic Acids. The starting pH of the tobacco material is generally comprised between 5 pH and 6 pH.
[0296]
[0297] Alkaloids were not or only slightly degraded during the fermentation. The total alkaloids (TA) content in percent in total dry weight basis (indicated as percent DW in the figures) is shown in
[0301] Sample 4T of HS leaves and sample 3T of CC leaves have a p-value<0.01 and Samples 1T and 4T of CC leaves have a p-value<0.001. This indicates a statistical significant difference between the fermented tobacco material and the non-fermented one.
[0302] The nitrate content was not affected by the heavy fermentation process. However, some impact was observed on tobacco specific nitrosamines (TSNA): NNN (N-nitrosonornicotine), NNK (nicotine-derived nitrosamine ketone) and NAT (N-nitrosoanatabine). No changes were measured on NNK and NAT after 8.5 months fermentation. However, an increase of NNN was observed in both HS (3 increase) and CC (5-6 increase). As nornicotine, the precursor of NNN before nitrosation did not increase correspondingly, therefore NAT and NNK, but not NNN, may be partially degraded by bacteria during the fermentation run, since NNK and NAT first increased by a factor 2 till 2.5 months fermentation and then decreased to reach the initial value of non-fermented tobacco. This observation may mean that nitrosation of alkaloids occurs during heavy fermentation.
[0303] The evolution of sugars and free amino acids during the heavy fermentation according to the invention has been analyzed. The measurements performed in the 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 the untreated tobacco material sample (samples 0T) to 8.5 months of fermentation process (samples 4T) under fermentation conditions in barrels containing either hand-stripped (HS) or Chopped (CC) leaves, as in Example 1 and Example 2. All values in the table are in total dry weight basis. The units of Reducing Sugars are in percent in total dry weight basis, while the free amino acids are in milligram per kilogram of total dry tobacco material. A drop of Reducing Sugars appeared after 2.5 month (2T, see Table 1) in phase with the color change and the slurry acidification. Glucose and fructose are two tobacco leaf substrates that anaerobic bacteria may metabolize in the fermentation barrels. Conversely, most of the amino acids increased during the process. Both asparagine and glutamine strongly decreased. Altogether, these observations may indicate that the main fermentation activities occurred between the first and the third month. Proline was not degraded under anaerobic fermentation (see Table 1). Ornithine strongly increased during fermentation (>100 times) in both HS and CC, as well as citrulline (data obtained from metabolomic analyses between 0T and 3T) increasing by a factor 16 in HS and 2 in CC. This may indicate that (plant-derived) Lactic Acid bacteria are active in the tobacco fermenting barrels, since such bacteria are described to produce ornithine and citrulline at high levels (Rakhimuzzaman et al., Biol Pharm Bull. 2019; 42(9):1581-1589).
TABLE-US-00001 TABLE 1 HS-0T HS-1T HS-2T HS-3T HS-4T CC-0T CC-1T CC-2T CC-3T CC-4T Sugars GLUCOSE 3.0 3.2 0.4 0.3 0.5 4.0 4.6 0.7 0.9 1.0 (%/DW) FRUCTOSE 4.3 4.1 0.3 0.2 0.2 4.9 5.1 0.4 0.3 0.3 SUCROSE 0.1 0.0 0.0 0.0 0.0 0.9 0.0 0.0 0.0 0.0 MALTOSE 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 SUM OF SUGARS 7.4 7.3 0.7 0.5 0.7 9.8 9.7 1.1 1.2 1.3 Free ASPARTIC ACID 252.0 951.3 2459.0 2702.3 2933.2 160.7 1492.0 1742.7 2184.7 2074.0 Amino GLUTAMIC ACID 518.7 813.3 1298.7 1185.0 1219.8 787.0 949.7 1201.7 986.0 1002.0 Acids ALANINE 601.3 762.3 1231.3 1374.7 1524.3 293.0 624.7 1084.0 1403.7 1652.2 (mg/ ARGININE 41.3 123.7 147.0 180.0 210.3 17.0 237.0 140.3 267.3 226.8 kg DW) ASPARAGINE 1604.3 1920.7 212.0 42.7 22.3 1747.0 640.0 287.7 124.3 139.0 PROLINE 4165.3 5108.7 4885.0 4838.3 4920.8 2962.3 3502.0 3017.7 3415.3 3417.0 PHENYLALANINE 234.0 400.7 336.0 402.0 414.5 236.8 386.7 243.0 312.7 327.8 GLYCINE 68.0 111.0 251.7 342.3 376.3 48.0 155.7 254.0 344.0 363.8 GLUTAMINE 1456.0 692.0 56.7 25.3 0.0 1921.0 314.3 57.7 17.0 52.5 ISOLEUCINE 31.7 99.9 165.3 217.3 182.0 43.0 197.7 188.0 258.7 215.3 HYSTIDINE 122.7 185.0 142.0 136.0 115.5 160.3 145.7 199.7 109.7 91.0 LEUCINE 113.3 238.3 372.7 464.7 621.5 143.7 473.7 334.3 419.7 585.8 LYSINE 54.0 131.7 183.0 241.0 256.2 40.0 242.3 249.0 323.0 340.5 METHIONINE 30.0 34.0 16.7 11.0 26.3 16.0 38.7 12.0 11.0 28.8 ORNITHINE 0.0 0.0 92.7 113.0 87.8 18.0 0.0 125.0 97.0 89.2 SERINE 297.3 393.3 453.7 495.7 535.3 277.3 444.7 500.7 583.7 609.7 TYROSIN 81.3 135.3 221.9 274.3 247.3 111.0 233.3 246.0 318.7 277.8 THREONINE 156.7 272.0 346.7 433.3 449.5 184.0 350.0 392.7 470.3 473.3 VALINE 279.0 398.3 468.0 523.3 496.0 278.3 593.0 627.3 784.0 626.5 GABA 326.0 465.7 507.0 468.3 438.0 358.7 980.7 842.3 869.7 845.7 TOTAL FREE 10459.7 13270.7 13886.3 14494.0 15225.2 9813.0 12027.7 11658.7 13293.0 13520.2 AMINO ACIDS
[0304] In
[0305] A metabolomic study was performed 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 material (control) of both HS and CC leaves may be used as a source of energy by the anaerobic bacteria (see Table 1). In the absence of oxygen, the glycolysis pathway transforms glucose (or fructose) into pyruvate producing 2 ATP and 2 NADH+H+. Other organic and rich carbon compounds that may be rapidly used by anaerobic bacteria are citrate and malate (Bintsis, T, 2018, AIMS Microbiology, 4(4): 665-684), both being the most abundant organic acids in plants. Citrate and malate, like Reducing Sugars, are also metabolized during the tobacco heavy fermentation: it is shown from chemical analysis of the samples that more than 60 percent of the glucose and fructose, citrate and malate present in the starting tobacco material (samples 0T), hand-stripped and chopped leaves are catabolized after 6 months of heavy fermentation (samples 3T). Another observation that can be coupled to the consumption of such organic molecules is the increase of pyruvate (13-14 times) in both HS and CC fermented tobacco material. Pyruvate is the substrate of several reactions that may occur under anaerobic conditions: (1) the production of D-lactate, mostly to regenerate NAD+ for the glycolytic reaction; (2) the production of acetate, diacetyl and 2,3 butanediol that may contribute to the delivery of aromatic compounds and flavours in heavy fermented tobacco. Pyruvate may lead to the generation of aromatic compounds, like 2,3-butanediol or lactate as a product of Lactic Acid bacteria.
[0306] Two other pathways emerged from the metabolomic analyses of heavy fermented tobacco: (1) the degradation of tryptophan and (2) the catabolism of chlorogenic acid.
[0307] Regarding tryptophan degradation, the pathway has been described by Ummadi and Weimer (2001, J. Dairy Sci. 84:1773-1782) for cheese bacteria and adapted accordingly. In this case, more than 78 percent of the tryptophan present in the starting tobacco material (samples 0T) is catabolized after 6 months of fermentation (samples 3T) in both HS and CC leaves. The pathway indicated that the product resulting from such a catabolic reaction is principally indole-3-Lactic Acid. This is illustrated by an increase of 14 times and 28 times in HS and CC leaves, respectively. No other compound belonging to this pathway showed such an increase. No specific aromatic properties had been reported for this compound.
[0308] Chlorogenic acid (CGA), an important biologically active dietary polyphenol, is produced by certain plant species, like tobacco, and is a major component of coffee. In heavy fermented tobacco leaf, CGA is completely degraded after the anaerobic fermentation process. On the other side, products resulting from the catabolism of CGA, namely quinic and caffeic acids, increased in both HS and CC leaves after 6 months fermentation. This likely results from bacterial cinnamoyl esterase activities as documented by Guglielmetti et al. (2008, Applied and Environmental Microbiology, 74, 4: 1284-1288). Therefore, part of the quinic and caffeic acid pools likely result from the hydrolysis of CGA, whereas none of them was reported to have flavor properties.
[0309] The presence of elevated pyruvate, indole-3-Lactic Acid and the lack of chlorogenic acid in heavy fermented tobacco compared to cured tobacco can make them useful as chemical markers.
[0310] For the purpose of the present description and of the appended claims, except where otherwise indicated, all numbers expressing amounts, quantities, percentages, and so forth, are to be understood as being modified in all instances 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 enumerated herein. In this context, therefore, a number A is understood as A10 percent of A. Within this context, a number A may be considered to include numerical values that are within general standard error for the measurement of the property that the number A represents. The number A, in some instances as used in the appended claims, may deviate by the percentages enumerated above provided that the amount by which A deviates does not materially affect the basic and novel characteristic(s) 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 enumerated herein.
[0311] The tobacco material of Example 1 and 2 have also been tested for some additional chemical compounds which could be connected with the fermentation.
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EXAMPLE 3
[0316] A further trial has been conducted in Asia on sample of Virginia tobacco. Tobacco leaf material has been flue-cured as a standard procedure.
[0317] The tobacco cured material, in form of strips, was then conditioned to obtain moisture of about 30 percent.
[0318] Samples of this tobacco material conditioned but not fermented yet are called SM (starting material before fermentation).
[0319] The conditioned tobacco material is then introduced in two barrels, in each barrel circa 100 kilograms of tobacco material is present. Before the introduction, the tobacco material is wrapped in a material maintaining the acquired moisture. The two barrels containing Virginia tobacco material were subjected to anaerobic fermentation as described in Example 1. The temperature inside the fermentation barrel and the pH of the tobacco material under fermentation were monitored during the whole experiment.
[0320] After 1 month (sample called 1T), 2 months (sample called 2T), 3 months (sample called 3T), 4 months (sampled called 4T), 5 months (sampled called 5T), 6 months (sample called 6T), 7 months (sample called 7T) and 8 months (sample called AF, after fermentation), the barrels were opened.
[0321] The tobacco material in the two barrels has been turned monthly during the 7 months of the experiment.
[0322] Samples have been collected before fermentation (VG-SM: starting material, 6 replicates), during the fermentation process (in all months from VG-T1 to VG-T7, 3 replicates per barrel) and after fermentation (VG-AF: after fermentation, 6 replicates), as indicated in Table 2 reported below. The features of the sample have been analyzed.
TABLE-US-00002 TABLE 2 Tobacco type SM T1 T2 T3 T4 T5 T6 T7 AF VG-01 Jul. 11, 2019 Oct. 12, 2019 Aug. 1, 2020 Jul. 2, 2020 Oct. 3, 2020 Jun. 4, 2020 Jun. 5, 2020 Oct. 6, 2020 Oct. 7, 2020 VG-02 Jul. 11, 2019 Nov. 12, 2019 Aug. 1, 2020 Oct. 2, 2020 Oct. 3, 2020 Jun. 4, 2020 Jun. 5, 2020 Oct. 6, 2020 Oct. 7, 2020
[0323] During the sample collection, the tobacco material has been turned and the moisture content of the tobacco material has been readjusted to approximatively 30 percent5 percent.
[0324] During the heavy fermentation process under fully anaerobic conditions, no major changes of temperature were observed during the fermentation process moving linearly from 30 degrees Celsius at the beginning of the fermentation (VG-T1) to 26 degrees Celsius at the end of the fermentation (VG-AF). The temperature has been measured inside the barrels using captors.
[0325] The pH of the tobacco material did not change significantly during the fermentation run (T1 to AF) staying at 5.10.3.
Visual Observations
[0326] As seen for the Kasturi tobacco material, the color of the tobacco material at the end of the fermentation process (VG-AF) became remarkably darker compared to the starting material (VG-SM). However, after 4 month of anaerobic fermentation (VG-T4), and thus after 4 turnings, the Virginia tobacco material did not show a so dark coloration indicating possibly that 4 months are not sufficient to get full fermentation of Virginia tobacco material at the experimental condition. After 8 months of anaerobic fermentation (VG-AF), and thus after 8 turnings, the Virginia tobacco material has a darker color than after 4 months, thus indicating that a full fermentation of Virginia tobacco material took place. Moreover, nice scented and floral odors were perceived after 8 months.
Chemical Analysis
[0327] In the following, when a value relative to a sample is mentioned, the given value represents an average of several values obtained for each sample of the same type, replicate.
[0328]
[0329] On the other hand, as already observed in Example 1 and Example 2, Reducing Sugars were used as substrate by fermenting bacteria. Therefore about 60 percent of the Reducing Sugars (RS) were oxidized during the 8 month of fermentation, moving from 18.3 percent (VG-SM) to 7.4 (VG-AF) percent in dry weight (DW). It is possible that a longer period of fermentation would lead to a higher percentage of degradation of Reducing Sugars.
[0330] Further chemical analyses have been performed to compare the compounds in the starting material (VG-SM) and in the fermented material (VG-AF).
[0331] The analyses show a strong significant increase (.sup.10) of lactate (HS ID VG-19-20-NF, HS ID VG-19-20-F in Table 3) in the fermented material (VG-AF) compared to non-fermented material (VG-SM). Moreover the amount of glucose and fructose in the fermented material (VG-AF) is considerably lower than the content of glucose and fructose in the starting material (VG-BF). Glucose in fermented material (VG-AF) is lower than 0.6 the glucose in non-fermented material (VG-SM), fructose in fermented material (VG-AF) is lower than 0.4 the fructose in non-fermented material (VG-SM). Thus about 60 percent reduction after fermentation of Reducing Sugars can be observed. In addition, the organic acids, citrate and malate, are also impacted by the fermentation, as also shown in
[0332] Chemical analyses also confirmed that asparagine and glutamine, are on the side to Reducing Sugars also consumed by anaerobic bacteria, that other free amino acids, like glutamate, histidine, proline and tryptophan are also significantly degraded after fermentation of Virginia tobacco and on the contrary other aminoacids increased after fermentation of Virginia tobacco, particularly L-Leucine and L-lysine (see
[0333]
[0334] Indole-3-Lactic Acid (
EXAMPLE 4
[0335] This experiment has been conducted in Switzerland, in the same experimental condition used in Example 3.
[0336] For this experiment have been used metal wine barrels able to press a wet tobacco substrate for a duration between two to ten months. A pressing equipment used for grapes and customized for the needs of the project has been used. The barrel has a flat bottom, custom disc diameters and custom pressure gauge. It can press between 0 and 8 kilograms per square centimeter. The pressing can be either manual or triggered by an electric motor. As an optional part, a wooden disc is attached to the stainless-steel pressing disc. The fermentation tank has been modified for the purpose of the test, having a height of 70 centimeters, a diameter of 57 centimeters, a capacity of 100 kilograms and an opening at the back of the metal tank (barrel) to help unpacking the substrate when turning the material in the middle or at the end of the fermentation run.
[0337] FC tobacco strips from Brazil (CX B) was used as fermenting substrate preconditioned with water to reach a final humidity of between about 30 percent by weight to about 50 percent by weight of water. About 100 kilograms of tobacco is material is loaded in the fermentation tank and pressed. The pressure is kept between 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 percent by weight and at a temperature of about 22 Celsius degrees during the whole fermentation process (6 months). After three months, the tobacco material is unloaded from the barrel, separated, mixed, turned. The tobacco material is then added with between about 30 percent by weight of water to about 50 percent by weight of water and then reloaded in the barrel.
[0338] Pressure and water levels were regularly monitored and corrected to prevent deviation from the targets and any aerobic fermentation. Temperature inside the tank did not change during the whole run, as observed previously.
[0339] After 6 months, the tobacco material was pre-dried on a belt-oven for a total drying time of about 8 minutes at different temperatures ranges. The tobacco material is firstly subjected to a temperature of about 40 Celsius degrees, then to a temperature of about (70 Celsius degrees and then to a temperature of about 60 Celsius degrees. The pre-drying process allows to obtain a tobacco material with about 25 percent by weight of Relative humidity OV [percent] to allow the cutting of the strips into finer particles. The tobacco material is then cut in particles of about 1 millimeters of cut-width. The tobacco material is then dried to its final moisture content of 10 percent by weight to about 15 percent by weight OV [percent]. The drying step took place in a rotary dryer at a temperature comprised between about 90 Celsius degrees to about 100 Celsius degrees and a pressure of about 0.6 bar for a period of about during 10 minutes. The tobacco material is then finally grinded with a specification of 70 micrometers for short storage.
[0340] The tobacco material in the two barrels has been turned every two months during the 6 months of the experiment.
[0341] Samples have been collected before fermentation (VG-SM: starting material, 6 replicates), during the fermentation process after 3 months from the beginning of the fermentation process and after fermentation (VG-AF: after fermentation, 6 replicates).
Visual Observations
[0342] The color of the tobacco material at the end of the fermentation process (VG-AF) became remarkably darker compared to the starting material (VG-SM). After 6 months of anaerobic fermentation the Virginia tobacco material shows a dark coloration indicating that a full fermentation of Virginia tobacco material has occurred.
Chemical Analysis
[0343] In the following, when a value relative to a sample is mentioned, the given value represents an average of several values obtained for each sample of the same type, replicate.
[0344]
[0345] Chemical analyses show that Lactic Acid is produced during heavy fermentation. According to the metabolic pathways present in Lactic Acid bacteria, Lactic Acid originates from the catabolism of Reducing Sugars (glucose and fructose), pyruvic acid, malic acid and citric acid, as already discussed for Example 3.
[0346]
Sample Preparation
[0347] The sample preparation was performed according to metaSysX standard procedure, a modified protocol from Salem et al. (Salem et al., Plant.Methods. 2016 45(12)). 20 (+ 2) milligrams of ground material was used for metabolite extraction. Samples are extracted with MTBE:MetOH:H2O two-phase extraction method. Total (650 l) organic phase is collected and dried down for LC-MS lipids measurement. 450 l of polar phase is collected and dried down for LC-MS polar metabolites measurements and 150 l polar phase is dried down and derivatized for GC-MS measurement.
Standard Curve Preparation
[0348] Stocks of 2 milligrams/ml of indole-3-lactic were solved in water. Standard mixes were prepared in a following concentrations: 10, 5, 2, 1, 0.5, 0.25, 0.125, 0.062 g/ml. Further, 200 l of standard mixes were dried down and subjected to the same extraction procedure as samples (ex) or directly subjected to LC-MS analysis or dried down, derivatized and analysed by GC-MS (no_ex).The extraction procedure for standard and samples was identical with all identical volumes
Calculation of the Absolute Content
[0349] Compounds concentrations were calculated based on extracted standard curves where all points (average of technical replicates) were taken for calculation and are presented as microgram per milligrams of the sample weight.
[0350] These data are reported in the table 3 reported below. This table shows the content of Indole-3-Lactic Acid of the diluted samples (standard), of the samples of the treated tobacco material of Example 4, (VG_IDC_AF), of the non-fermented tobacco material of Example 4 (VG_IDC_BF) and from different tobacco material non subjected to a fermentation process (K326_75, TN90_110, K326_G, K326_110, TN90_G, K326_cured, TN90_75)
TABLE-US-00003 Concen- Peak.ID Mode Analite mz_mean Adduct Intensity tration Units Sample PN_9494 PN Indole-3-Lactic Acid 204.0656671 [M H] 481151684 10 g/ml Standard PN_9494 PN Indole-3-Lactic Acid 204.0656671 [M H] 273727852 5 g/ml Standard PN_9494 PN Indole-3-Lactic Acid 204.0656671 [M H] 100375241.5 2 g/ml Standard PN_9494 PN Indole-3-Lactic Acid 204.0656671 [M H] 46290925 1 g/ml Standard PN_9494 PN Indole-3-Lactic Acid 204.0656671 [M H] 23189084.5 0.5 g/ml Standard PN_9494 PN Indole-3-Lactic Acid 204.0656671 [M H] 11308841.5 0.25 g/ml Standard PN_9494 PN Indole-3-Lactic Acid 204.0656671 [M H] 5366675 0.125 g/ml Standard PN_9494 PN Indole-3-Lactic Acid 204.0656671 [M H] 2644875.5 0.0625 g/ml Standard PN_9494 PN Indole-3-Lactic Acid 204.0656671 [M H] 304903692.5 10 g/ml Standard extracted PN_9494 PN Indole-3-Lactic Acid 204.0656671 [M H] 137499497 5 g/ml Standard extracted PN_9494 PN Indole-3-Lactic Acid 204.0656671 [M H] 40069896 2 g/ml Standard extracted PN_9494 PN Indole-3-Lactic Acid 204.0656671 [M H] 23274504.5 1 g/ml Standard extracted PN_9494 PN Indole-3-Lactic Acid 204.0656671 [M H] 11163531 0.5 g/ml Standard extracted PN_9494 PN Indole-3-Lactic Acid 204.0656671 [M H] 5869694.5 0.25 g/ml Standard extracted PN_9494 PN Indole-3-Lactic Acid 204.0656671 [M H] 2629402 0.125 g/ml Standard extracted PN_9494 PN Indole-3-Lactic Acid 204.0656671 [M H] 1247621.5 0.0625 g/ml Standard extracted PN_9494 PN Indole-3-Lactic Acid 204.0656671 [M H] 488683 0.000153746 g/ml TN90_cured PN_9494 PN Indole-3-Lactic Acid 204.0656671 [M H] NA NA g/mg K326_75 PN_9494 PN Indole-3-Lactic Acid 204.0656671 [M H] NA NA g/mg TN90_110 PN_9494 PN Indole-3-Lactic Acid 204.0656671 [M H] NA NA g/mg K326_G PN_9494 PN Indole-3-Lactic Acid 204.0656671 [M H] NA NA g/mg K326_110 PN_9494 PN Indole-3-Lactic Acid 204.0656671 [M H] NA NA g/mg TN90_G PN_9494 PN Indole-3-Lactic Acid 204.0656671 [M H] 174702 5.83E05 g/mg K326_cured PN_9494 PN Indole-3-Lactic Acid 204.0656671 [M H] NA NA g/mg TN90_75 PN_9494 PN Indole-3-Lactic Acid 204.0656671 [M H] 8009875 0.002520006 g/mg VG_IDC_AF PN_9494 PN Indole-3-Lactic Acid 204.0656671 [M H] 339777 0.000113988 g/mg VG_IDC_BF
C-MS Measurements (Hydrophillic and Lipophilic Analytes
[0351] The samples were measured with 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 the lipophilic and the hydrophilic 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.
LC-MS Data Processing (Hydrophilic and Lipophilic Analytes)
[0352] Extraction of the LC-MS data was accomplished with the software PeakShaper (metaSysX GmbH). Alignment and filtration of the LC-MS data were completed using in-house software. After extraction from the chromatograms, the data is processed, aligned and filtered for redundant peaks. The alignment of the extracted data from each chromatogram was performed according to the criteria that a feature had to be present in all replicates of at least one of the groups. At this stage, the average RT and m/z values are given to the features. The alignment was performed for each type of measurements independently.
LC-MS Data Annotation (Hydrophilic Lipophilic and Analytes)
[0353] The in-house metaSysX database of chemical compounds was used to match features detected in the LC-MS lipophilic platform. The annotation of the compounds of interest was performed by matching with the MSX database and confirmed by measured standards.
The metaSysX (MSX) Database
[0354] The metaSysX in-house database contains mass-to-charge ratio and retention time information of 7500 reference compounds available as pure compounds and measured in the same chromatographic and spectrometric conditions as the measured samples. In addition, 1500 lipids and sugar esters putatively annotated based on the precursor m/z, fragmentation spectrum and elution patterns. The matching criteria for the DGDGs annotations were 5 parts per million and 0.085 minutes deviation from the reference compounds mass-to-charge ratio and retention time, respectively.
GC-MS Measurements
[0355] The samples were measured on an Agilent Technologies GC coupled to a Leco Pegasus HT mass spectrometer which consists of an EI ionization source and a TOF mass analyzer.
[0356] Column: 30 meters DB35; Starting temp: 85 degree Celsius for 2 minute; Gradient: 15 degree Celsius per min up to 360 degree Celsius.
GC-MS Data Processing and Annotation
[0357] NetCDF files that were exported from the Leco Pegasus software were imported to R. The Bioconductor package TargetSearch[3] was used to transform retention time to retention index (RI), to align the chromatograms, to extract the peaks, and to annotate them.
[0358] The analysis performed on the samples indicates that the content of Indole-3-lactic in the fermented tobacco material (VG_IDC_AF) is 0.002520006 micrograms per milligrams; and the content of Indole-3-lactic in the fermented tobacco material (VG_IDC_BF) is 0.000113988 micrograms per milligrams. These data are reported in
[0359] Comparing the results obtained in the above discussed experiments, it is evident that the chemical analyses confirm that Lactic Acid massively increased after anaerobic fermentation in all the experiments conducted. Fully fermented tobacco material contained between about 50 milligrams per gram and about 100 milligrams per gram of Lactic Acid, whereas less than 4 milligrams per gram can be found in non-fermented tobacco. Data of Lactic Acid for different types of tobacco are reported in Table 2 in which 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.
[0360] Rajangan (RAJ) tobacco ID KS-18-19 indicates the tobacco material of Example 2, HS ID KS-18-19 indicates the tobacco material of Example 1, HS ID VG-19-20 indicates the tobacco material of Example 3 and HS CH VG-19-20 indicates the tobacco material of Example 4.
TABLE-US-00004 Lactic Data Samples Acid forms set sd Replicate sd RAJ ID D-LACTIC ACID 378 64 276 47 KS-18-19-NF TOTAL LACTIC ACID 1578 210 1346 186 L-LACTIC ACID 1200 200 1070 180 RAJ ID D-LACTIC ACID 14700 2500 14800 2500 KS-18-19-F TOTAL LACTIC ACID 86700 12300 87800 12300 L-LACTIC ACID 72000 12000 73000 12000 HS ID D-LACTIC ACID 292 50 276 47 KS-18-19-NF TOTAL LACTIC ACID 1202 168 1246 176 L-LACTIC ACID 910 160 970 170 HS ID D-LACTIC ACID 15200 2600 16000 2700 KS-18-19-F TOTAL LACTIC ACID 79200 11300 80000 11300 L-LACTIC ACID 64000 11000 64000 11000 HS ID D-LACTIC ACID 630 110 700 120 VG-19-20-NF TOTAL LACTIC ACID 2260 290 2320 300 L-LACTIC ACID 1630 270 1620 280 HS ID D-LACTIC ACID 5940 1000 5820 980 VG-19-20-F TOTAL LACTIC ACID 22400 3000 22120 2870 L-LACTIC ACID 16500 2800 16300 2700 HS CH D-LACTIC ACID 1110 190 950 160 VG-19-20-NF TOTAL LACTIC ACID 3190 400 2690 340 L-LACTIC ACID 2080 350 1740 300 HS CH D-LACTIC ACID 10800 1800 12700 2200 VG-19-20-F TOTAL LACTIC ACID 68700 10000 68600 9700 L-LACTIC ACID 57900 9800 55900 9400
[0361] Measurements of D-Lactic Acid and L-Lactic Acid (milligrams/kilograms DW) in the non-fermented compared to fermented samples, RAJ ID KS-18-19-NF compared to RAJ ID KS-18-19-F, HS ID KS-18-19-NF compared to HS ID KS-18-19-F; HS ID VG-19-20-NF compared to HS ID VG-19-20-F and HS CH VG-19-20-NF compared to HS CH VG-19-20-F
[0362] In Table 4 it is indicated the quantity of Lactic Acid in the fermented (F) and non-fermented (NF) tobacco material. The quantity of Lactic Acid is indicated as milligram per Kilogram with reference to the dry weight of the tobacco material. The data show that all the tobacco materials have an increase of the content of Lactic Acid after fermentation. All the tobacco samples have similar amount of Lactic Acid, thereby indicating that both fast-cured cut-filler with mid-rib tobacco and the corresponding hand-stripped cured material reach similar levels of Lactic Acid. Virginia tobacco reach similar level 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]).
[0363] This confirms that tobacco fermentation can be performed with different tobacco types, i.e. Dark and Virginia tobacco, sugars and amino acids being a source of carbon and nitrogen for the Lactic Acid bacteria. Even if tests have not been performed with Burley and Oriental tobacco, nonetheless Oriental tobacco has a composition that is often close to that Dark tobacco with usually less alkaloids which are nonetheless not impacted by the anaerobic fermentation process.
[0364] Most of the Lactic Acid measured in fermented tobacco is the enantiomer L-Lactic Acid, which is in line with the biochemical pathway for anaerobic bacteria. However, some D-Lactic Acid is also produced. Compared to L-lactic-acid, D-Lactic Acid can be toxic to human, i.e. the LD50 value level per orally poisoned rats is around 4.5 g/kilograms (Pohanka, 2020). However, D- and L-Lactic Acid are non-volatile, and therefore not transfer into aerosol.
[0365] Moreover tobacco it has been measured the fermentation Ratio for the tobacco material of examples 1-2 and 4-5 and the data are collected in Table 4 and 5 reported below
[0366] The tobacco fermentation ratio is given by the following formula: Fr=(F.sub.LA/NF.sub.LA): (F.sub.RS/NF.sub.RS) wherein:
[0367] Fr=Fermentation Ration; F.sub.LA=content of Lactic Acid in the fermented tobacco material; NF.sub.LA=initial content of Lactic Acid in the tobacco material; F.sub.RS=content of Reducing Sugars in the fermented tobacco material; NF.sub.RS=initial content of Reducing Sugars in the tobacco material.
TABLE-US-00005 TABLE 5 Fermentation ratio for Reducing Sugars (RS) and Lactic Acid, in which VG-ID indicates the tobacco material of Experiment 3, HS-ID indicates the tobacco material of Experiment 1, RJ-ID indicates the tobacco material of Experiment 2, VG-CH indicates the tobacco material of Experiment 4. RS LACTIC ACID NF F F/NF NF F F/NF VG-ID 183.3 74.1 0.4 2.3 22.4 9.7 HS-ID 102.8 18.9 0.2 1.2 79.2 66.0 RJ-ID 106.0 25.0 0.2 1.7 86.7 51.0 VG-CH 18.9 1.0 0.1 3.2 68.7 21.5
TABLE-US-00006 TABLE 6 Fermentation index: ratio between F/NF Lactic Acid (LA) and F/NF RS F/NF(LA):F/NF(RS) VG-ID 24 HS-ID 330 RJ-ID 255 VG-CH 215
[0368] The tobacco fermentation Index gives a further indication about the fermentation of the tobacco material. The tobacco fermentation ration allows monitoring the level of fermentation in a tobacco sample. As shown in Table 5, the fermentation ration increases considerably during the fermentation. Therefore the fermentation ration is a very efficient indicator of the fermentation of the tobacco material.
[0369] In conclusion, metabolomic data suggests that the compounds generated during tobacco fermentation, namely Lactic Acid, indole-Lactic Acid, caffeic acid and quinic acid, are not directly linked to tobacco types, since they are produced with both Dark or Flue-cured tobacco matrices. However, they certainly may quantitatively change regarding the previous abundance of the substrates compounds found in the starting tobacco leaf material. Therefore anaerobic fermentation is a process applicable to different tobacco types and material and causes a change in the content of some substances in the tobacco material independently from the type of tobacco material.
[0370] Some substances may be used as indicator for the degree of fermentation of the tobacco material and these substances may be used as reliable indication of the degree of fermentation.
[0371] The use of many indicators and for example the correlation between the colour changes, the RS consumption and Lactic Acid production by the anaerobic bacteria attest of the degree of fermentation of the tobacco material and give a very reliable indication of the degree of fermentation.
[0372] Moreover the anaerobic process does not negatively impact the content of alkaloids of the tobacco material.
[0373] The experiments conducted lead to define that the following conditions may positively impact the fermentation increasing the efficiency of the process: [0374] Precondition hand-stripped leaf material with water to reach a final humidity of about comprised between about 40 percent by weight and 60 percent by weight indicated as Relative Humidity; [0375] Fill a close barrel, a tank or a container (barrel) with preconditioned strips to reach a capacity of about 100 kilograms; [0376] Maintain a relative humidity comprised between about 30 percent and 50 percent by weight with water in the closed container during the fermentation process; [0377] perform the fermentation at a temperature of about 22 Celsius degree; [0378] maintain almost stable the temperature during the fermentation process; [0379] perform at least one turning, during the fermentation process unloading the tobacco material, mixing the tobacco material and reloading the tobacco material in the fermentation device, in order to homogenize the anaerobic fermentation process within the container [0380] apply a pressure comprised between about 0.5 kilograms per square centimeter and 1 kilogram per square centimeter during the fermentation process, except when turning tobacco. [0381] Dry fermented tobacco material in a two steps drying process [0382] Subjecting the tobacco material to a first drying step in which the tobacco material is subjected to a temperature of about 60 Celsius degree for a period of about 8 min to reach about 25 percent OV [0383] Subjecting the tobacco material to a second drying step at a temperature comprised between about 90 Celsius degree and 100 Celsius degree at a pressure of about 0.6 bar for a period of time of about 10 min to reach 10-15 percent OV.
[0384] On the other hand, as depicted in
[0385] The present invention is also directed to the following embodiments: [0386] 1. Method for treating tobacco material, the method comprising: [0387] providing a tobacco material [0388] fermenting the tobacco material to obtain fermented tobacco material, the fermenting step including: [0389] incubating the tobacco material under anaerobic conditions; [0390] stopping the fermentation when at least one of the following conditions is satisfied: [0391] 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 an initial amount of Lactic Acid in the tobacco material, [0392] the content of Reducing Sugars is lower than 0.5, preferably lower than 0.4, more preferably below 0.2, more preferably below 0.1 an initial amount or Reducing Sugars in the tobacco material, [0393] the content of Indole-3 Lactic Acid is more than 5 times, preferably more than 10 times, preferably more than 20 times, an initial amount of Indole-3 Lactic Acid in the tobacco material, [0394] the content of caffeic acid is more than 4 times, preferably more than 10 times, preferably more than 20 times, an initial amount of caffeic acid in the tobacco material. [0395] the content of quinic acid is more than 2 times, preferably more than 4 times, an initial amount of quinic acid in the tobacco material, [0396] the content of asparagine is lower than 0.5, preferably lower than 0.4, preferably lower than 0.3 an initial amount or asparagine in the tobacco material, [0397] the content of Glutamine is lower than 0.5, preferably lower than 0.4 an initial amount or Glutamine in the tobacco material, [0398] 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 an initial amount of L-Ornithine in the tobacco material, [0399] the content of L-Leucine is more than 2 times, preferably more than 4 times, an initial amount of L-Leucine in the tobacco material, [0400] the content of L-Lysine is more than 2 times, preferably more than 6 times, an initial amount of L-Lysine in the tobacco material, [0401] the fermentation index is more than 50, preferably more than 100, more preferably more than 250, more preferably more 400, wherein the fermentation index is obtained dividing the ratio between the content of Lactic Acid in the treated tobacco material and the content of Lactic Acid in the non-fermented tobacco material by the ratio between the content of Reducing Sugars in the treated tobacco material and the content of Reducing Sugars in the non-fermented tobacco material. [0402] 2. Method according to embodiment 1 and further comprises an initial measuring step for measuring the initial content of at least one 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 fermentation index in the tobacco material. [0403] 3. Method according to embodiment 1 or 2 and further comprising a measuring step for measuring the content of at least one 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 fermentation index in the tobacco material during the fermenting step. [0404] 4. Method according to any one of embodiments 1 to 3, wherein if the tobacco material contains dark tobacco, it is provided for stopping the fermentation when at least one of the following conditions is satisfied: the content of 2,3 butanediol is more than 5 times, preferably more than 10 times, an 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, an initial amount of diacetyl in the tobacco material. [0405] 5. Method according to embodiment 4 and further comprising an initial measurement step for measuring the initial content of 2,3 butanediol or diacetyl in the tobacco material before the fermentation so as to obtain an initial amount respectively of 2,3 butanediol or diacetyl in the tobacco material. [0406] 6. Method for treating tobacco material, the method comprising: [0407] providing a tobacco material [0408] fermenting the tobacco material to obtain fermented tobacco material, the fermenting step including: [0409] incubating the tobacco material under anaerobic conditions, wherein during the fermenting step it is provided for applying a pressure to the tobacco material comprised between 1000 kilograms per square meter and 15000 kilograms per square meter, preferably between 3000 kilograms per square meter and 12000 kilograms per square meter, more preferably between 5000 kilograms per square meter and 10000 kilograms per square meter. [0410] 7. Method for treating tobacco material, the method comprising: [0411] providing a tobacco material [0412] fermenting the tobacco material to obtain fermented tobacco material, the fermenting step including: [0413] incubating the tobacco material under anaerobic conditions, wherein it is provided for continuing the fermenting step 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. [0414] 8. Method for treating tobacco material, the method comprising: [0415] providing a tobacco material [0416] fermenting the tobacco material to obtain fermented tobacco material, the fermenting step including: [0417] incubating the tobacco material under anaerobic conditions, wherein during the fermenting step it is provided for keeping the temperature of the tobacco material comprised between 21 degrees Celsius and 35 degrees Celsius, preferably between 25 degrees Celsius and 31 degrees Celsius. [0418] 9. Tobacco material obtained according to the method of any one of embodiments 1-8. [0419] 10. Tobacco material comprising at least one of the following compounds: [0420] Lactic Acid in an amount that 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, an initial amount of Lactic Acid in the tobacco material. [0421] Reducing Sugars in an amount that is lower than 0.5, preferably lower than 0.4, more preferably below 0.2, more preferably below 0.1 an initial amount or Reducing Sugars in the tobacco material, [0422] Indole-3 Lactic Acid in an amount that is more than 5 times, preferably more than 10 times, preferably more than 20 times, an initial amount of Indole-3 Lactic Acid in the tobacco material. [0423] caffeic acid in an amount that is more than 4 times, preferably more than 10 times, an initial amount of caffeic acid in the tobacco material, [0424] quinic acid in an amount that is more than 2 times, preferably more than 4 times, an initial amount of quinic acid in the tobacco material, [0425] asparagine in an amount that is lower than 0.5, preferably lower than 0.4, preferably lower than 0.3 an initial amount or asparagine in the tobacco material, [0426] Glutamine in an amount that is lower than 0.5, preferably lower than 0.4 an initial amount or Glutamine in the tobacco material, [0427] L-Ornithine is more than 10 times, preferably more than 50 times, preferably more than 100 times an initial amount of L-Ornithine in the tobacco material, [0428] L-Leucine in an amount that is more than 2 times, preferably more than 4 times, an initial amount of L-Leucine, [0429] L-Lysine in an amount that is more than 2 times, preferably more than 6 times, an initial amount of L-Lysine, [0430] a fermentation index is more than 50, preferably more than 100, more preferably more than 250, more preferably more 400, wherein the fermentation index is obtained dividing the ratio between the content of Lactic Acid in the tobacco material and the content of Lactic Acid in the non-fermented tobacco material by the ratio between the content of Reducing Sugars in the tobacco material and the content of Reducing Sugars in the non-fermented tobacco material. [0431] 11. Tobacco material according to the preceding embodiment, wherein said tobacco material is obtained by a process comprising fermenting the tobacco material to obtain treated tobacco material, including: incubating the tobacco material under anaerobic conditions. [0432] 12. Tobacco material comprising at least 20 milligrams per gram, preferably at least 50 milligrams per gram, more preferably at least 60 milligrams per gram of Lactic Acid in total dry weight basis. [0433] 13. Tobacco material comprising: [0434] less than 3 percent of total Reducing Sugars in total dry weight basis, and/or [0435] less than 300 milligrams per kilogram of asparagine in total dry weight basis. [0436] 14. Tobacco material comprising less than 70 milligrams per kilogram of glutamine in total dry weight basis. [0437] 15. Tobacco material comprising more than 10000 milligrams per kilogram in total dry weight basis of total free amino acids. [0438] 16. Tobacco material comprising at least 1 microgram per gram, preferably at least 2 micrograms per gram, more preferably at least 2.5 micrograms per gram of Indole-3 Lactic Acid in total dry weight basis. [0439] 17. Tobacco material according to any one of embodiments from 19 to 27, wherein the tobacco material is cured. [0440] 18. Tobacco material according to any one of embodiments from 19 to 28, wherein the tobacco material is grinded. [0441] 19. Aerosol generating article comprising a tobacco material containing between about 2.5 percent by weight in total dry weight basis and 100 percent by weight in total dry weight basis, preferably at least about 4 percent by weight in total dry weight basis, preferably at least about 10 percent by weight in total dry weight basis, preferably at least about 20 percent by weight in total dry weight basis of a the tobacco material according to any one of embodiments 9 to 18. [0442] 20. Aerosol generating article comprising a tobacco material containing at least 5 milligrams per gram, preferably 10 milligrams per gram in total dry weight basis of Lactic Acid.