A METHOD FOR TREATING TOBACCO MATERIAL AND TREATED TOBACCO MATERIAL

Abstract

The invention relates to a method for treating tobacco material, the method comprising: o fermenting the tobacco material to obtain treated tobacco material, including: #incubating the tobacco material under anaerobic conditions; #applying a pressure to the tobacco material comprised between 1000 kilograms per square meter and 4000 kilograms per square meter; #keeping the moisture content of the tobacco material comprised between 25 percent in weight and 40 percent in weight of the total weight of the tobacco material; o wherein the fermenting lasts at least one month.

Claims

1-15. (canceled)

16. A method for treating tobacco material, the method comprising: fermenting the tobacco material to obtain treated tobacco material, including: incubating the tobacco material under anaerobic conditions; applying a pressure to the tobacco material comprised between 1000 kilograms per square meter and 4000 kilograms per square meter; keeping the moisture content of the tobacco material comprised between 25 percent in weight and 40 percent in weight of the total weight of the tobacco material; wherein the fermenting lasts at least two months.

17. The method according to claim 16, including the step of: drying the tobacco material to obtain a dried tobacco material having a moisture content comprised between 5 percent and 10 percent in weight of the total weight of the tobacco material.

18. The method according to claim 16, comprising the step of: curing the tobacco material before fermenting.

19. The method according to claim 16, including keeping the temperature of the tobacco material comprised between 25 degrees Celsius and 35 degrees Celsius.

20. The method according to claim 16, comprising the step of turning the tobacco material.

21. The method according to claim 16, comprising: securing the tobacco material within a moisture retaining material.

22. The method according to claim 16, comprising: wetting the tobacco material in water before fermenting, so that a moisture content of the tobacco material comprised between 25 percent in weight and 40 percent in weight of the total weight of the tobacco material is achieved.

23. The method according to claim 16, wherein the amount of asparagine in the treated tobacco material becomes at least 50 percent lower than the amount of asparagine contained in the same tobacco material before treatment.

24. The method according to claim 16, wherein the amount of asparagine in the treated tobacco material becomes at least 50 percent lower than the amount of asparagine contained in the same tobacco material before treatment.

25. The method according to claim 16, wherein the amount of reducing sugars in the treated tobacco material becomes at least 50 percent lower than the amount of reducing sugars contained in the same tobacco material before treatment.

26. Tobacco material comprising: less than 3 percent of total reducing sugars in total dry weight basis; less than 300 milligrams per kilogram of asparagine in total dry weight basis.

27. The tobacco material according to claim 26, comprising: less than 70 milligrams per kilogram of glutamine in total dry weight basis.

28. The tobacco material according to claim 26, comprising: more than 10000 milligrams per kilogram in total dry weight basis of total free amino acids.

29. The tobacco material according to claim 26, wherein the tobacco material is cured.

30. An aerosol generating article comprising the tobacco material according to claim 26.

Description

[0120] FIG. 1 and FIG. 2 are histograms representing the quantity of lactic acid in the tobacco material of Example 1 and Example 2, respectively, measured before (0T) and after 6 months (3T) of fermentation according to the invention;

[0121] FIG. 3 and FIG. 4 are histograms representing the quantity of Total alkaloid (TA) levels (percent in total dry weight basis DW) in the tobacco material of Example 1 and Example 2, respectively, measured before (0T) and during fermentation according to the invention;

[0122] FIG. 5 and FIG. 6 are histograms representing the quantity of glutamine and glutamic acid in the tobacco material (in total dry weight basis, DW) of Example 1 and Example 2, respectively, measured before (0T) and during fermentation according to the invention;

[0123] FIG. 7 and FIG. 8 are histograms representing the quantity of asparagine and aspartic acid in the tobacco material (in total dry weight basis, DW) of Example 1 and Example 2, respectively, measured before (0T) and during fermentation according to the invention;

[0124] FIG. 9 and FIG. 10 are histograms representing the quantity of total alkaloids and reducing sugars in the tobacco material (in total dry weight basis, DW) of Example 3, respectively, measured before (VG-BF), during fermentation and after fermentation (VG-AF) according to the invention.

[0125] A first and a second tobacco material of the same tobacco type but having a different processing before fermentation have been prepared. The tobacco material is Kasturi tobacco.

EXAMPLE 1

[0126] 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”.

[0127] 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”).

[0128] 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.

[0129] Pressure is applied to each barrel. The pressure is comprised between 1000 kilograms per square meter and 4000 kilograms per square meter.

[0130] 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 percent±5 percent.

[0131] 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

[0132] 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”.

[0133] 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”).

[0134] The conditioned tobacco material is then introduced in three barrels, in each barrel circa 100 kg of tobacco material is present. Before the introduction, the tobacco material is wrapped in a material maintaining the acquired moisture.

[0135] Pressure is applied to each barrel. The pressure is comprised between between 1000 kilograms over square meter and 4000 kilograms over square meter.

[0136] 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 percent±5 percent.

[0137] 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

[0138] 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

[0139] 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.

[0140] 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.

[0141] FIGS. 1 and 2 show the presence of lactic acid in the tobacco material. As shown by the figures (FIG. 1 represent lactic acid content in HS leaves and FIG. 2 in CC leaves), before fermentation, there is absence of lactic acid in all samples (three samples 0T per tobacco material— CC or HS— are shown). After fermentation (in this case after 6 months, three samples for tobacco material called 3T, shown for both tobacco materials— CC or HS), all samples, both CC and HS leaves, show the presence of lactic acid, albeit in variable amount.

[0142] Alkaloids were not or only slightly degraded during the fermentation. The total alkaloids (TA) content in percent in total dry weight basis (indicated as % DW in the figures) is shown in FIG. 3 (HS leaves) and FIG. 4 (CC leaves). The content of total alkaloids remained quite stable during the fermentation. After 8.5 month (4T), only 4 percent were degraded in HS and 9 percent in CC leaves. Although statistically relevant, such small variation may just result from sampling. Some limited alkaloid hydrolase activities may not be excluded. Total alkaloids were analyzed in samples collected during the heavy fermentation process at start (0T, n=6 samples have been analyzed), after 1(1T, n=9), 2.5(2T, n=9), 6 (3T, n=9) and 8.5 (4T, n=12) months. T-tests (test statistics) were performed for comparison with the control, unfermented cured tobacco (0T). The results are shown in FIGS. 3 and 4 indicating the p-value, where the p-value is shown as follows:

*, p<0.05;
**, p<0.01 and
***, p<0.001.

[0143] 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.

[0144] 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.

[0145] 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-OT HS-1T HS-2T HS-3T HS-4T CC-OT 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 Amino ASPARTIC ACID 252.0 951.3 2459.0 2702.3 2933.2 160.7 1492.0 1742.7 2184.7 2074.0 Acids GLUTAMIC ACID 518.7 813.3 1293.7 1185.0 1219.8 787.0 949.7 1201.7 986.0 1002.0 (mg/kg DW) ALANINE 601.3 762.3 1231.3 1374.7 1524.3 293.0 624.7 1084.0 1403.7 1652.2 ARGININE 41.3 123.7 147.0 180.0 210.3 17.0 237.0 140.3 267.3 226.8 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 234.0 400.7 336.0 402.0 414.5 236.3 386.7 243.0 312.7 327.8 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 99.0 165.3 217.3 182.0 43.0 197.7 188.0 258.7 215.3 HYSTIDINE 122.7 185.0 142.0 136.0 115.5 160.3 145.7 109.7 109.7 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 40.0 242.3 240.0 232.0 340.5 METHIONINE 30.0 34.0 16.7 11.0 20.3 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 81.3 135.3 221.3 274.3 247.3 111.0 233.3 246.0 318.7 277.8 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 326.0 465.7 507.0 468.3 438.0 358.7 980.7 842.3 860.7 845.7 TOTAL FREE 10459.7 13270.7 13886.3 14494.0 15225.2 9813.0 12027.7 11658.7 13293.0 13520.2 AMINO ACIDS

[0146] In FIGS. 5-8, the amount of glutamine and asparagine in the tobacco material is shown. As shown by FIGS. 5-8 and based on the data presented in Table 1, deamination of glutamine and asparagine occurring during the heavy fermentation process of both HS and CC leaves may be correlated with the concomitant increase of glutamate and aspartate, respectively. This suggests that fermenting bacteria produce specific glutaminase(s) and asparaginase(s) to assimilate C and N from amino acid resources. Both reactions produce ammonia that increased twofold during the anaerobic fermentation process of both HS and CC leaves. FIGS. 5 and 6 show the level of glutamine (white histograms) and glutamic acid (black histograms) in HS leaves and CC leaves, respectively. It is clear from the figures that during fermentation glutamine decreases and glutamic acid increases. FIGS. 7 and 8 show the level of asparagine (striped histograms) and aspartic acid (black histograms) in HS leaves and CC leaves, respectively. It is clear from the figures that during fermentation asparagine decreases and aspartic acid increases.

[0147] 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% 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.

[0148] Two other pathways emerged from the metabolomic analyses of heavy fermented tobacco: (1) the degradation of tryptophan and (2) the catabolism of chlorogenic acid.

[0149] 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% 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.

[0150] 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.

[0151] 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.

[0152] 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 A±10 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.

[0153] 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.

[0154] A third tobacco material of a different tobacco type than in Example 1 and Example 2 has been prepared. The tobacco material is Virginia tobacco.

EXAMPLE 3

[0155] Tobacco leaf material has been fully sun-cured for about 10 days. The sun cured leaves have been treated as standard for Virginia tobacco.

[0156] 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 BF (starting material before fermentation).

[0157] 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.

[0158] Pressure is applied to each barrel. The pressure is comprised between 1000 kilograms per square meter and 4000 kilograms per square meter.

[0159] 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.

[0160] During each month, the tobacco material in the two barrels has been turned at least 6 times.

[0161] Samples have been collected before fermentation (VG-BF: 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).

[0162] During the sample collection, the tobacco material has been turned and the moisture content of the tobacco material has been readjusted to approximatively 30 percent±5 percent.

[0163] 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.

[0164] The pH of the tobacco material did not change significantly during the fermentation run (T1 to AF) staying at 5.1±0.3.

[0165] The fermentation has been stopped after 8 months.

Visual Observations

[0166] 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-BF). However, after 4 month of anaerobic fermentation (VG-T4), the Virginia tobacco material did not show the same “darkness as the Kasturi tobacco after the same amount of fermentation, indicating possibly that 4 months are not sufficient to get full fermentation of Virginia tobacco material.

Chemical Analysis

[0167] 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.

[0168] The behavior of lactic acid over time in the tobacco material is very similar to what is depicted in FIGS. 1 and 2. Qualitatively, before fermentation, there is absence of lactic acid in all samples. After fermentation, lactic acid is present, albeit in variable amount.

[0169] FIG. 9 shows the evolution of total alkaloids (TA) during the fermentation process. These data confirm that alkaloids and in particular nicotine (not shown) are not impacted by anaerobic fermentation. The bacteria did not consume major alkaloids as fermenting substrates.

[0170] On the other hand, as depicted in FIG. 10, and as already observed in Example 1 and Example 2, reducing sugars were used as substrate by fermenting bacteria. Therefore about 60% of the reducing sugars (RS) were oxidized during the 8 month of fermentation, moving from 18.3 percent (VG-BF) 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 RS degradation.

[0171] The average (n=6) and SD are presented in FIGS. 9 and 10, as well as t-tests (paired) performed between BF and AF.

[0172] Further chemical analysis showed that the starting material (VG-BF) had a content of 262 micrograms per gram (μg/g) of asparagine on a dry weight basis. The same tobacco material after fermentation, (8 months, VG-AF) has a content of 19 micrograms per gram (μg/g) of asparagine on a dry weight basis.

[0173] The starting material (VG-BF) had a content of 185 micrograms per gram (μg/g) of glutamine on a dry weight basis. The same tobacco material after fermentation, (8 months, VG-AF) has a content of 12 micrograms per gram (μg/g) of glutamine on a dry weight basis.