EP4426136B1 - Verfahren zur behandlung von tabakmaterial und behandeltes tabakmaterial - Google Patents

Verfahren zur behandlung von tabakmaterial und behandeltes tabakmaterial

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Publication number
EP4426136B1
EP4426136B1 EP22813527.3A EP22813527A EP4426136B1 EP 4426136 B1 EP4426136 B1 EP 4426136B1 EP 22813527 A EP22813527 A EP 22813527A EP 4426136 B1 EP4426136 B1 EP 4426136B1
Authority
EP
European Patent Office
Prior art keywords
tobacco material
tobacco
fermentation
content
lactic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
EP22813527.3A
Other languages
English (en)
French (fr)
Other versions
EP4426136C0 (de
EP4426136A1 (de
Inventor
Lucien Bovet
Simon HAENNI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Philip Morris Products SA
Original Assignee
Philip Morris Products SA
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Publication date
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Publication of EP4426136A1 publication Critical patent/EP4426136A1/de
Application granted granted Critical
Publication of EP4426136C0 publication Critical patent/EP4426136C0/de
Publication of EP4426136B1 publication Critical patent/EP4426136B1/de
Active legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
    • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
    • A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
    • A24B15/10—Chemical features of tobacco products or tobacco substitutes
    • A—HUMAN NECESSITIES
    • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
    • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
    • A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
    • A24B15/18—Treatment of tobacco products or tobacco substitutes
    • A24B15/20—Biochemical treatment
    • A—HUMAN NECESSITIES
    • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
    • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
    • A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
    • A24B15/18—Treatment of tobacco products or tobacco substitutes
    • A24B15/28—Treatment of tobacco products or tobacco substitutes by chemical substances
    • A24B15/30—Treatment of tobacco products or tobacco substitutes by chemical substances by organic substances
    • A24B15/305—Treatment of tobacco products or tobacco substitutes by chemical substances by organic substances of undetermined constitution characterised by their preparation
    • A24B15/307—Treatment of tobacco products or tobacco substitutes by chemical substances by organic substances of undetermined constitution characterised by their preparation using microorganisms or enzymes as catalysts
    • A—HUMAN NECESSITIES
    • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
    • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
    • A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
    • A24B15/18—Treatment of tobacco products or tobacco substitutes
    • A24B15/28—Treatment of tobacco products or tobacco substitutes by chemical substances
    • A24B15/30—Treatment of tobacco products or tobacco substitutes by chemical substances by organic substances
    • A24B15/32—Treatment of tobacco products or tobacco substitutes by chemical substances by organic substances by acyclic compounds
    • A—HUMAN NECESSITIES
    • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
    • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
    • A24B3/00—Preparing tobacco in the factory
    • A24B3/12—Steaming, curing, or flavouring tobacco

Definitions

  • the present invention relates to a method for treating tobacco and a tobacco material treated by fermentation.
  • the invention also relates to an aerosol generating article containing tobacco material treated by fermentation.
  • the fermentation is an anaerobic fermentation.
  • Treatments to enhance or add flavours and aromas to the tobacco material at a later stage of tobacco processing often involve the addition of one or more additive(s) to the tobacco and can require additional processing steps and equipment, which can be costly and time-consuming. Furthermore, the addition of additives to the tobacco may be not well perceived by some consumers.
  • GB 2 548 543 A discloses a method of treating a slurry of tobacco material comprising the steps of: (a) providing a slurry of tobacco material; (b) in the presence of homolactic acid bacteria and limited oxygen, fermenting the slurry of tobacco material; and (c) obtaining a slurry of fermented tobacco material in which the lactic acid content therein is increased and the malic acid content therein is decreased as compared to the slurry of tobacco material prepared in step (a).
  • GB 2 542 623 A discloses a tobacco product comprising tobacco stem material produced by a method comprising, in any order, the steps of (a) processing the tobacco stem material; and (b) fermenting the tobacco stem material in the presence of homolactic acid bacteria and limited oxygen for a time from about 4 days to about 21 days at a temperature of from about 15°C to about 45°C.
  • CN 108 541 999 A discloses a method for treating anaerobic solid-state fermented stems.
  • the invention relates to a method for treating tobacco material, the method comprising providing a tobacco material and fermenting the tobacco material to obtain a fermented tobacco material.
  • the fermenting step includes: incubating the tobacco material under anaerobic conditions.
  • the fermented tobacco material at the sensory level, consistently delivers higher smoothness characters compared to the corresponding unfermented cured tobacco material.
  • fermented tobacco material delivers in use floral notes or scent notes.
  • the tobacco fermented material has a smoother character reducing harshness sensation. Therefore, the organoleptic properties of the tobacco material are increased and the experience for the user is improved.
  • tobacco material may ferment.
  • Tobacco plants may host microorganisms which in turn may include bacteria, mould and actinomyces. Studies have shown that bacteria occupy most of the present microorganisms in the tobacco, while mould and actinomyces are minorities. Yeast has a low concentration or cannot be detected at all.
  • Fermented tobacco can be made by various suitable techniques known in the art, for example as described in " Research Progress in Tobacco Fermentation" published by Yang Yang et al, Journal of Biosciences and Medicines 2018, 6, 105-114 available on line at: http://www.scirp.org/journal/jbm ; or in US 5372149 , or in US 4528993 , and others.
  • tobacco fermentation includes adjusting the moisture content of cured, aged tobacco to a moisture content of from about 20 percent to about 60 percent, and allowing the moistened tobacco to ferment in piles. Fermentation can be terminated for example by drying or cold storage. Tobacco fermentation does not need the addition of microorganisms because, as mentioned above, microorganisms are generally naturally present in the tobacco plants.
  • the fermentation takes place under anaerobic condition.
  • Anaerobic fermentation is defined as the conversion of complex organic compounds into smaller molecules in the absence of oxygen.
  • the term can be also defined as the conditions in which, as a result of both chemical equilibria and biochemical activities, oxygen is not available for redox reactions. Instead, other oxidized compounds may be present which can be used by micro-organisms for specific types of energy metabolism.
  • Anaerobic conditions may coexist with aerobic ones: oxygen in gaseous form may be unavailable to microorganisms in micro-environments (such as aggregates of detritus suspended in water) while at the same time it may be present in the macro-environment (water).
  • micro-environments such as aggregates of detritus suspended in water
  • the main energy extraction pathway may be coming from glycolysis, some amino acids being also used as carbon/nitrogen sources.
  • the preferred nitrogenous compounds usually include glutamine, alanine, serine, threonine, aspartate, asparagine, urea, and arginine.
  • fertilization conditions it is meant that the tobacco is subjected to anaerobic conditions.
  • a certain amount of oxygen may be present in the fermentation environment but Oxygen is not available for redox reactions.
  • the anaerobic fermentation conditions do not impact the content of alkaloids and in particular of nicotine of the tobacco material. At the same time, the organoleptic properties of the tobacco material are increased
  • the method provides for stopping the fermenting step when at least one desired condition is satisfied.
  • the method comprises stopping the fermenting step when the content of Lactic Acid is more than 10 times, preferably more than 20 times, more preferably more than 50 times an initial amount of Lactic Acid in the tobacco material.
  • the method comprises stopping the fermentation when the content of Lactic Acid is more than 70 times, preferably more than 80 times, an initial amount of Lactic Acid in the tobacco material.
  • the increase of Lactic Acid is a reliable indicator of the fermentation of the tobacco material.
  • the amount of the Lactic Acid in the tobacco material after fermentation may depend on the type of the tobacco material, or also on the initial amount of Lactic Acid in the tobacco material. Additionally the amount of Lactic Acid in the tobacco material after fermentation may depend on the content of other compounds in the tobacco material before fermentation. On the contrary, the increase of the Lactic Acid in the tobacco material is a reliable indicator of the degree of fermentation of the tobacco material. This parameter is not dependent on the type of the tobacco material or on the composition of the same, and it is not influenced by further external parameters.
  • Lactic Acid has two enantiomers L-Lactic Acid D-Lactic Acid.
  • Lactic Acid it is intended the sum of two enantiomers L-Lactic Acid D-Lactic Acid.
  • L-Lactic Acid in fermented tobacco mostly the enantiomer L-Lactic Acid is present, however some D-Lactic Acid is also produced.
  • 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).
  • D-Lactic Acid and L-Lactic Acid are non-volatile, and therefore not transfer into the aerosol produced with the tobacco material.
  • the content of Lactic Acid in the tobacco material is determined by spectrophotometry, according to MP 0309 rev 5 2012, Chelab S.r.l.
  • the method comprises stopping the fermenting step when 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.
  • Reducing Sugars are converted by anaerobic fermentation to pyruvate, and pyruvic acid is a precursor of many other flavour compounds and provides caramel brown sugar and sour notes. Therefore, the decrease of the content of the Reducing Sugars in the tobacco material that is subjected to anaerobic fermentation is a good indicator of the degree of fermentation of the tobacco material.
  • the amount of the Reducing Sugars in the tobacco material may depend on different factors, like for example the type of tobacco, the harvesting area, etc., but the reduction rate of the Reducing Sugars indicates the degree of the fermentation process. Most abundant naturally present sugars in tobacco leaves are glucose, fructose and sucrose. Differences in sugar content may be present among tobacco varieties.
  • Virginia has high level of sugar (generally in a range from 8 percent to 30 percent) while Burley is characterized with low content of sugars (generally in a range of 1 percent to 2 percent).
  • Burley is characterized with low content of sugars (generally in a range of 1 percent to 2 percent).
  • Changes in the amount of Reducing Sugars may change the organoleptic properties of the tobacco material and of the smoke or aerosol produced with it.
  • the content of Reducing Sugars in the tobacco material is determined by continuous flow analyzer, according to CORESTA recommended method N° 38.
  • the method comprises stopping the fermenting step when 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 absolute content of indole-3 Lactic Acid was determined by UPLC-MS, calculated based on extracted standard curves.
  • the Indole-3 Lactic Acid is present only in traces in tobacco leaf but it is produced during anaerobic fermentation in cured leaf material and indicates that the tobacco material has been subjected to an anaerobic fermentation.
  • the method comprises stopping the fermenting step when the content of caffeic acid is more than 4 time, preferably more 10 times, preferably more than 20 times, an initial amount of caffeic acid in the tobacco material.
  • the presence of the caffeic acid increases the organoleptical properties of the tobacco material improving the experience for the user.
  • the content of caffeic acid in the tobacco material is estimated by metabolomic analyses using UPLC-MS, comparison of caffeic acid in non-fermented vs treated tobacco material, i.e. fermented or partly fermented tobacco material.
  • the method comprises stopping the fermenting step when 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 quinic acid in the tobacco material is estimated by metabolomic analyses using UPLC-MS, comparison of caffeic acid in non-fermented vs treated tobacco material, i.e. fermented or partly fermented tobacco material.
  • the method comprises stopping the fermenting step when 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 asparagine is determined by ion-exchange chromatography, according to MP 2442 rev 0 2021, Chelab S.r.l.
  • the reduction of asparagine implies a reduced conversion to acrylamide.
  • Tobacco material contains a certain quantity of amino acids.
  • the amino acids may contribute substantially to the level of certain components in the smoke or aerosol produced by the final product where the fermented tobacco material is contained, and to the sensory properties of the smoke or aerosol.
  • Different type of tobaccos may contain different quantities of amino acids.
  • the growing location for the tobacco may alter the ratios of the levels of different amino acids, but rather similar profiles for the same tobacco amino acid are generally maintained. Regardless of the tobacco type and origin, during fermentation under the fermentation conditions of the invention, it has been observed that the asparagine content in the tobacco material decreases.
  • the method comprises stopping the fermenting step when 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 Glutamine is determined by ion-exchange chromatography, according to MP 2442 rev 0 2021, Chelab S.r.l.
  • the reduction of Glutamine allows the flavourings of the tobacco material to be increased since the reduction of Glutamine implies that an umami savory note is released form the tobacco material in use.
  • the method comprises stopping the fermenting step when 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-Ornithine is determined by ion-exchange chromatography, according to MP 2442 rev 0 2021, Chelab S.r.l.
  • the increase of L-Ornithine in the fermented tobacco material is a good indicator of the degree of fermentation of the tobacco material independently on the type of tobacco material subjected to fermentation and to the initial composition thereof. It has been found that independently on the type of tobacco material, the fermentation causes an increase of L-Ornithine in the tobacco material.
  • the method comprises stopping the fermenting step when 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-Leucine is determined by ion-exchange chromatography, according to MP 2442 rev 0 2021, Chelab S.r.l.
  • the method comprises stopping the fermenting step when 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 content of L-Lysine is determined by ion-exchange chromatography, according to MP 2442 rev 0 2021, Chelab S.r.l.
  • the increase of the L-Leucine and L-Lysine is a good indicator of the degree of fermentation of the tobacco material independently on the type of tobacco material subjected to fermentation and to the initial composition thereof.
  • the method comprises stopping the fermenting step when the 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 an initial amount of 3-sec-Butylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione in the tobacco material.
  • BHHPPD 3-sec-Butylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione
  • the method comprises stopping the fermenting step when the content of Secoisolariciresinol (SECO) that is more than 10 times, preferably more than 20 times, more preferably more than 40 times, more preferably more than 50 times an initial amount of Secoisolariciresinol (SECO) in the tobacco material.
  • SECO Secoisolariciresinol
  • the method comprises stopping the fermenting step when the fermentation index is more than 50, preferably more than 100, more preferably more than 250, more preferably more 400.
  • 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.
  • the Fermentation index (Fr) allows also the little change in the composition of the tobacco material under fermentation to be detected. Moreover this parameter changes during the fermentation independently from the type of tobacco material under fermentation, therefore the parameter is highly reliable.
  • Stopping the fermenting step when at least one of the above conditions is satisfied allows obtaining a treated tobacco material having desired organoleptic properties.
  • the tobacco material treated with the method of the invention could be fully fermented or partly fermented. Independently of the degree of fermentation of the tobacco material, by stopping the fermenting step when one of the above conditions is satisfied allows for obtain a tobacco material having certain features.
  • Treated tobacco material in the aim of the invention indicates fermented or partly fermented tobacco material.
  • the method further comprises an initial measuring step for measuring the 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, or fermentation index in the tobacco material before the fermenting step so as to obtain an initial amount respectively 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, or
  • Initial measuring step provides for measuring the features of the tobacco material before fermentation, in other words the features of the non-fermented tobacco material are measured in the initial measuring step.
  • the initial amount of the measured features corresponds to the value of the feature in the non-fermented tobacco material.
  • the method further comprises an 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 fermentation index in the tobacco material during fermentation.
  • an 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,
  • the method provides for stopping the fermenting step when 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.
  • the presence of 2,3 butanediol confers a natural odor of cocoa butter.
  • the presence of 2,3 butanediol contributes to a nice flavoured volatiles present in anaerobic fermented tobacco material.
  • the method provides for stopping the fermenting step when the content of diacetyl is more than 5 times, preferably more than 10 times, an initial amount of diacetyl in the tobacco material.
  • the presence of diacetyl confers some organoleptic properties to the tobacco material.
  • the presence of diacetyl contributes to the presence of nice flavoured volatiles in anaerobic fermented tobacco material.
  • the method comprises 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.
  • the method further comprises a measuring step for measuring the content of 2,3 butanediol or diacetyl in the tobacco material during fermentation.
  • the method comprises providing a database containing 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 fermentation index, or butanediol, or diacetyl in the non-fermented tobacco material.
  • a database containing 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]pyrazin
  • the method comprises providing a database containing 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 fermentation index, or butanediol, or diacetyl in the non-fermented tobacco material for a plurality of different tobacco materials.
  • a database containing 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 Butylhexahydropyrrol
  • Each database may contain the usual 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 fermentation index, or butanediol, or diacetyl for each type of tobacco material in the non-fermented conditions.
  • Lactic Acid or Reducing Sugars
  • Indole-3 Lactic Acid or quinic acid, or caffeic acid
  • L-Ornithine or Asparagine
  • Glutamine or L-Leucine
  • L-Lysine or Butylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione, or Secoisolariciresinol
  • the method provides for retrieving 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 fermentation index in the tobacco material before the fermentation from at least one database.
  • Lactic Acid or Reducing Sugars
  • Indole-3 Lactic Acid or quinic acid, or caffeic acid
  • L-Ornithine or Asparagine
  • Glutamine or L-Leucine
  • L-Lysine or Butylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione
  • SECO Secois
  • the method comprises providing a database containing 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 fermentation index, or butanediol, or diacetyl in the fermented and/or partly fermented tobacco material.
  • a database containing 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,
  • the method comprises providing a database containing 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 fermentation index, or butanediol, or diacetyl in the fermented and/or partly fermented tobacco material for a plurality of different tobacco materials.
  • a database containing 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 Butylhe
  • the method provides for retrieving from at least one database 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 fermentation index, or butanediol, or diacetyl and comparing the retrieved value with the corresponding measured value in the tobacco material under fermentation.
  • Lactic Acid or Reducing Sugars
  • Indole-3 Lactic Acid or quinic acid, or caffeic acid
  • L-Ornithine or Asparagine
  • Glutamine or L-Leucine
  • L-Lysine or Butylhexahydropyrrolo[1,2-a]pyrazine-1,4
  • the method provides for stopping or continuing the fermenting step on the basis of the result of the comparing step.
  • the methods provides for continuing the fermenting step if the measured value 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 is lower than the corresponding retrieved value and/or the value of Reducing Sugars, or Asparagine, or Glutamine, or fermentation index is higher than the corresponding retrieved value.
  • the invention relates to a method for treating tobacco material, the method comprising providing a tobacco material and fermenting the tobacco material to obtain a fermented tobacco material.
  • the fermenting step includes: incubating the tobacco material under anaerobic conditions and stopping the fermenting step when at least one of the following conditions is satisfied:
  • the anaerobic conditions are achieved by placing the tobacco material in a container and closing the container.
  • the anaerobic conditions are achieved placing the tobacco material in a container, removing air from the container and closing the container.
  • the container is not closed in a complete air-tight fashion, so that it is allowed for the CO 2 or other gases produced during fermentation escaping from the container. In this way, the accumulation of these substances in the container is avoided.
  • the container is so isolated from the external environment to allow anaerobic conditions to be maintained inside the container.
  • the container is closed with a closure avoiding an overpressure to be realised in the container.
  • pressure is applied in order to remove the air from the tobacco material.
  • the applied pressure forces the air out from the tobacco material, so that, after the container is closed, oxygen is not present anymore, or it is present only in minimal quantity, in the container.
  • the fermenting step includes: applying a pressure to the tobacco material comprised between 1000 kilograms per square meter (Kilograms/m 2 ) and 15000 kilograms per square meter (Kilograms/m 2 ), 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.
  • a pressure to the tobacco material comprised between 1000 kilograms per square meter (Kilograms/m 2 ) and 15000 kilograms per square meter (Kilograms/m 2 ), 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.
  • the pressure applied to the tobacco material is maintained in the above range during the fermenting step.
  • the pressure may be applied to the tobacco material by any means.
  • the pressure may be applied pumping an inert gas in the container.
  • the pressure may be applied putting a weight on the tobacco material causing the desired pressure range to be applied to the tobacco material.
  • the container may be filled with wet tobacco material and, as a "lid" of the container, a weight is placed in contact to the tobacco material till water seeps out of the container.
  • the pressure is applied by means of a hydraulic actuator pressing on the closure of the container and/or on the tobacco material contained therein so as so establish the desired pressure in the container.
  • a hydraulic actuator is used for applying the desired pressure on the tobacco material.
  • the hydraulic actuator may be chosen in dependence of the level of pressure to be realised in the container. Hydraulic actuators are known in the art for applying pressure to containers, such as for example wine presses.
  • the tobacco material is inserted in a container and a weight is located on top or above the tobacco material to exert the desired pressure.
  • the container is closed, leaving the weight inside the container, so that the weight may keep applying pressure to the tobacco material.
  • the fermenting step includes keeping the moisture content of the tobacco material during fermentation comprised between 10 and 50 percent in weight of the total weight of the tobacco material (weight by weight percentage). More preferably, the fermenting step includes keeping the moisture content of the tobacco material during fermentation comprised between 35 percent in weight and 45 percent in weight of the total weight of the tobacco material (weight by weight percentage). More preferably, the fermenting step includes keeping the moisture content of the tobacco material during fermentation at about 40 percent in weight (weight by weight percentage). After curing, the moisture of the tobacco material is generally low. Therefore, preferably water is added to the tobacco material to reach a desired moisture level.
  • water is added also during the fermentation process in order to keep the moisture of the tobacco material comprised between 10 and 50 percent in weight, preferably 35 percent in weight and 45, more preferably about 40 percent in weight of the total weight of the tobacco material for at least one month, more preferably for at least 2 months, preferably for at least 6 months, even more preferably at least 8 months, preferably for at least 12 months, preferably for at least 24 months.
  • the tobacco material is wetted with water. Water is added to the tobacco material.
  • the tobacco material is wetted with water before being introduced in the container where the anaerobic conditions are created and kept.
  • the moisture content of the tobacco material is monitored.
  • the container may be opened, and the moisture of the tobacco material may be measured when the container is opened.
  • the container is opened at regular intervals in order to perform the measurement of the tobacco moisture.
  • the moisture may be measured by a moisture sensor provided inside the container. In this way, the moisture may be measured also when the tobacco material is in the closed container.
  • the fermenting step 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.
  • the fermenting step lasts at least 12 months.
  • fermenting step lasts at least 24 months.
  • tobacco material is subjected to the fermentation conditions for a fermenting time less than 36 months, more preferably for a fermenting time less than 24 months.
  • the fermenting step comprises a plurality of fermenting phases each fermenting phase lasting for a fermenting period.
  • the overall fermenting step 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.
  • the fermentation time may depend on the type of the tobacco material, or by the features of the tobacco material, or on at least one of the parameters of the fermenting step like for example Temperature, Pressure, Relative Humidity, etc..
  • the application of the fermentation conditions may be continuous for all the claimed time (for example, longer than one month, or longer than two months, or longer than 6 months, or longer than 12 months, or longer than 24 months).
  • the fermenting step comprises a plurality of fermenting phases lasting a certain fermenting period.
  • the fermentation conditions may be applied during a plurality of time intervals forming a sequence of time intervals. The different fermenting phases are separated one from the other by interruptions in which the fermenting conditions are not applied.
  • an interruption a period of time in which one or more of the following conditions is interrupted: presence of anaerobic conditions, amount of moisture comprised between 25 percent in weight and 40 percent in 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.
  • An interruption may take place in order check the tobacco material. For example, the moisture of the tobacco material may be measured during an interruption. An interruption may take place to turn or mix the tobacco material, so that a uniform treated tobacco material may be obtained. An interruption may last up to 6 hours.
  • the fermentation time is thus the total period during which the tobacco is subjected to the above fermentation conditions and it is to be calculated adding the duration of all the fermenting periods of each fermenting phases during which the fermentation conditions are indeed applied.
  • the fermentation time can be calculated starting from the moment in which the fermentation conditions are applied for the first time and terminating when the fermentation conditions are applied for the last time, and then subtracting the duration of the interruptions.
  • a fermentation time T is selected, where with “fermentation time” the total period during which the tobacco material is subjected to the fermentation conditions is meant, the following cases are possible.
  • the fermentation conditions are applied continuously for a total duration equal to T, in this case there is only one fermentation phase.
  • the time gap between a fermentation period t j and the subsequent time interval t j+1 is the interruption.
  • This total fermentation time T is of at least one month, or of at least two months, or of at least 6 months, or of at least 12 months, or of at least 24 months).
  • each fermenting phase lasts about 15 days, more preferably about 30 days.
  • each fermenting phase lasts between about 30 days and about 60 days.
  • the method provides for 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.
  • the drying step is preferably carried out after the fermenting step under fermentation conditions has been terminated.
  • the treated tobacco material is preferably removed from the container where it was placed, and the pressure applied to the tobacco is decreased.
  • the treated tobacco material is then dried to a water content of between 1 percent and 15 percent in weight of the total weight of the tobacco material, more preferably between 5 percent and 10 percent. The drying is performed so that the treated tobacco material may be easily processed is subsequent steps
  • the method comprises the step of curing the tobacco material before the fermenting step.
  • the tobacco material processed according to the method of the invention may comprise post-curing tobacco.
  • post-curing tobacco refers to tobacco that has been cured.
  • the curing of the tobacco is preferably realized according to standard procedures and may depend on the type of tobacco which is included in the tobacco material.
  • the tobacco material may include tobacco of different types and having had different curing.
  • the tobacco of different types may be blended and then treated according to the invention.
  • the tobacco material is placed in a container and air is removed and replaced with water.
  • the container in which the tobacco material is placed is for example a barrel.
  • the barrel is made of wood, or concrete, or metal or a combination of any of these three materials.
  • the anaerobic conditions are kept for the desired duration of the fermenting step.
  • the method provides for evaluating the colour of the tobacco material.
  • the evaluation of the colour of the tobacco material may be performed also in addition to the measurement of a desired chemical substance.
  • the colour of the tobacco material changes during the fermenting step.
  • the evaluation of the colour of the tobacco material allows obtaining in an easy way an indication about the degree of fermentation.
  • the method provides for stopping the fermenting step when the colour of the tobacco material reaches a desired colour.
  • the fermentation condition can be applied till the desired colour of the tobacco material is obtained. In this way, it is possible to improve the efficacy of the method of the invention.
  • the method provides for keeping the temperature of the tobacco material during the fermenting step comprised between 21 degrees Celsius and 35 degrees Celsius, preferably between 25 degrees Celsius and 31 degrees Celsius.
  • the temperature of the tobacco material during the fermenting step remains comprised in a range of between 21 degrees Celsius and 35 degrees Celsius, more preferably between 25 degrees Celsius and 31 degrees Celsius.
  • the temperature of the tobacco material is substantially maintained within this range during the whole fermenting step.
  • the temperature is maintained by the fermentation itself, there is no need of providing or subtracting heat to the tobacco material.
  • This temperature of the tobacco material during fermentation is obtained when the ambient temperature of the ambient where the tobacco material is located is preferably comprised between 15 degrees Celsius and 25 degrees Celsius.
  • the method includes the step of turning the tobacco material, during the fermenting step.
  • the turning of the tobacco material may provide an improved homogenization.
  • Turning the tobacco material may means turning the tobacco material upside down.
  • Turning the tobacco material may means overturning the tobacco material.
  • the interruption of the fermentation conditions caused by the turning may be used also to measure certain parameters of the tobacco material, for example the moisture content.
  • the fermentation conditions may be not applied any more.
  • the fermentation process may be interrupted, in the sense that the tobacco material may not be subjected to anaerobic condition during turning.
  • the fermentation conditions are re-applied to the tobacco material.
  • the method includes the step of turning the tobacco material at a time interval of about 30 days.
  • the method includes the step of turning the tobacco material at a time interval of about 15 days.
  • the different fermenting phases of the fermenting step are preferably interrupted by a turning step.
  • the method comprises: securing the tobacco material within a moisture retaining material.
  • This step of securing the tobacco material preferably takes place before the tobacco material is subjected to the fermentation conditions. It is desirable for the moisture-retaining material to be resistant to degradation during the tobacco treatment process (the 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 plastic material. Alternatively, or in addition, the moisture-retaining material may comprise a rigid material.
  • the container in which the tobacco material is introduced may function as a moisture retaining material. In this case, the material of the container may include for example metal, wood, plastic, or concrete.
  • the method further comprises mixing a certain quantity of non-fermented tobacco material with a desired quantity of fermented or partly fermented tobacco material to obtain a tobacco material in which the amount of the fermented tobacco material in the tobacco material is comprised between 5 percent in weight and 10 percent in weight of the total tobacco material.
  • the mixing step is preferably provided before the fermenting step. Preferably, it is provided for subjecting the tobacco material to the fermenting step after the mixing step.
  • the method comprises a cutting step in which the tobacco material is cut in pieces having dimensions comprised between about 0.3 millimeters and about 1.4 millimeters.
  • the cutting step is performed before the fermenting step. This would improve the efficiency of the fermenting step. This would also reduce the duration of the fermenting step, i.e. the time required for obtaining the desired changes of composition of the tobacco material.
  • the method further provided for maintaining the pH of the tobacco material during fermentation comprised between 4.5 and 5.5, preferably about 4.8 and 5.4.
  • the treated tobacco material is 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 of at least 2 pH units. In different tobacco material, the pH may remain substantially unchanged.
  • the tobacco material treated according to the method of the invention may comprise tobacco that has been re-graded, green-leaf blended, conditioned, de-stemmed or threshed (or not in the case of whole leaf), dried or packed.
  • the invention relates to a tobacco material obtained by a process comprising fermenting the tobacco material to obtain treated tobacco material, the method including incubating the tobacco material under anaerobic conditions.
  • the treated tobacco material comprises Lactic Acid.
  • the treated tobacco material has a content of Lactic Acid 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.
  • Lactic Acid is known to be a related catabolic product.
  • the Lactic Acid may have a "smoothing effect" regarding nicotine harshness.
  • the Lactic Acid may be responsible in the lowering of the pH of the treated tobacco material.
  • the treated tobacco material has a content of Reducing Sugars 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.
  • the treated tobacco material comprises an amount of total Reducing Sugars at least 50 percent, more preferably 60 percent, even more preferably 85 percent lower than the amount of total Reducing Sugars contained in the same tobacco material before the treatment according to the method of the previous aspect.
  • the amount of Reducing Sugars is at least 50 percent, more preferably 60 percent, even more preferably 85 percent lower than the amount of Reducing Sugars contained in the same tobacco material before the treatment.
  • Reducing Sugars are the sum of the following: glucose, fructose, sucrose, maltose. The majority of the Reducing Sugars in the treated tobacco material may be converted. Reducing Sugars resources such as glucose and fructose present in the starting tobacco material may be used as a source of energy by the anaerobic bacteria. In the absence of oxygen, the glycolysis pathway transforms glucose (or fructose) into pyruvate. The altered levels of these compounds may contribute to the desirable taste and aroma of the treated tobacco material.
  • the treated tobacco material has a content of Indole-3 Lactic Acid 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.
  • the treated tobacco material has a content of caffeic acid that is more than 4 times, preferably more than 10 times, an initial amount of caffeic acid in the tobacco material.
  • the treated tobacco material has a content of quinic acid that is more than 2 times, preferably more than 4 times, an initial amount of quinic acid in the tobacco material.
  • the treated tobacco material has a content of asparagine 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.
  • the treated tobacco material has a content of Glutamine that is lower than 0.5, preferably lower than 0.4 an initial amount or Glutamine in the tobacco material.
  • the treated tobacco material has a content of L-Leucine that is more than 2 times, preferably more than 6 times, an initial amount of L-Leucine in the tobacco material in the tobacco material.
  • the treated tobacco material has a content of L-Lysine that is more than 2 times, preferably more than 6 times, an initial amount of L-Lysine in the tobacco material.
  • the invention relates to a tobacco treated material containing at least one of the following features:
  • the treated tobacco material having one or more of the previous indicated parameters has improved organoleptic properties.
  • the above indicated parameters allow evaluating the quality and the possible appreciation of the tobacco material form a user. Additionally the above indicated parameters affect the organoleptic properties and thus the final taste of the tobacco material. Therefore it is possible, on the basis of the values of the above indicated parameters, to foreseen the group of users 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 foresee which users will appreciate more the tobacco material.
  • 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 an initial amount of 3-sec-Butylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione in the tobacco material.
  • the treated tobacco material has a content of Secoisolariciresinol (SECO) that is more than 10 times, preferably more than 20 times, more preferably more than 40 times, more preferably more than 50 times an initial amount of Secoisolariciresinol (SECO) in the tobacco material.
  • SECO Secoisolariciresinol
  • the invention relates to a tobacco material containing at least 20 milligrams per gram, preferably at least 50 milligrams per gram of Lactic Acid in total dry weight basis.
  • the invention relates to a tobacco material comprising less than 3 percent of total Reducing Sugars in total dry weight basis.
  • the invention relates to a tobacco material comprising less than 300 milligrams per kilogram of asparagine in total dry weight basis.
  • the invention relates to a tobacco material comprising less than 70 milligrams per kilogram of glutamine in total dry weight basis.
  • the invention relates to a tobacco material comprising more than 10000 milligrams per kilogram of asparagine in total dry weight of total free amino acids.
  • the invention relates to a tobacco material comprising more than 10 times, preferably more than 20 times, an initial amount of L-Ornithine in the tobacco material.
  • the invention relates to a tobacco material containing more than 50 milligrams per kilograms, preferably more than 80 milligrams per kilograms, of L-Ornithine in total dry weight basis.
  • the tobacco material of any one of the previous aspects may be obtained by a process comprising fermenting the tobacco material, the method including incubating the tobacco material under anaerobic conditions.
  • the tobacco material is preferably cured.
  • the tobacco material is preferably cured before being subjected to the fermenting step.
  • Dark tobacco material having a content of 2,3 butanediol that is more than 5 times, preferably more than 10 times an initial amount of 2,3 butanediol in the tobacco material.
  • an 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.
  • the percentages are indicated by weight in total dry weight basis of a tobacco fermented material according to any one of the previous aspects.
  • an aerosol generating article containing at least 5 milligrams per gram, preferably 10 milligrams per gram in total dry weight basis of Lactic Acid.
  • an aerosol generating article containing less than 300 milligrams per kilogram of asparagine in total dry weight basis.
  • an aerosol generating article containing less than 70 milligrams per kilogram of glutamine in total dry weight basis.
  • an aerosol generating article containing more than 10000 milligrams per kilogram of asparagine in total dry weight of total free amino acids.
  • the tobacco material includes hand - stripped leaves from which ribs have been removed.
  • the tobacco material comprises Kasturi tobacco.
  • the tobacco material comprises Virginia tobacco.
  • the tobacco material comprises Dark tobacco.
  • the treated tobacco according to the method of the invention may change its chemical composition with respect to the untreated tobacco.
  • "Treated tobacco material” in the present context means tobacco material that underwent the treatment as described in the previous process, that is, tobacco material that has been subjected for at least one month to the fermentation conditions.
  • "Untreated tobacco material” in the present context means tobacco material that did not undergo the treatment as described in the previous method, that is, tobacco material that has not been subjected to the fermenting step. Untreated tobacco material is for example the tobacco material which is inserted in the container before the treatment of the invention starts.
  • the treated tobacco material is compared with the same tobacco material which did not undergo the treatment according to the invention (the untreated tobacco material).
  • the decrease in asparagine may be associated with an increase in aspartate. This suggests that fermenting bacteria produce specific asparaginase(s) to assimilate C and N from amino acid resources. This reaction may produce ammonia.
  • Non fermented tobacco material in the present context means tobacco material that did not undergo the treatment as described in the previous method, that is, tobacco material that has not been subjected to the fermenting step.
  • Fermented or partly fermented tobacco material in the present context means tobacco material that underwent the treatment as described in the previous method that is, tobacco material that has been subjected to the fermenting step for a certain period of time.
  • the terms "change” or “changed” are used in the context of the flavour or organoleptic properties to mean that there is a modification from one overall taste or sensory character to another, as identified by expert smokers. This may include an improvement.
  • initial it is considered the amount of a chemical substance or in general the value of a parameter of the tobacco material before subjecting the tobacco material to the fermenting step.
  • initial may be considered synonymous of non-fermented.
  • fermenting step or “fermentation” or “fermentation conditions” all indicate that the material is subjected for a desired extent of time to conditions suitable for causing the fermentation of the material.
  • tobacco material refers to any part of a tobacco plant or a mixture of different tobacco plants and includes without limitation tobacco leaf scraps, tobacco green leaf scraps, tobacco stems, tobacco dust created during tobacco processing, and tobacco leaf prime lamina strip and a combination thereof.
  • the tobacco material can have the form of processed tobacco parts or pieces, cured and aged tobacco in essentially natural lamina or stem form, a tobacco extract or a mixture of the foregoing, for example, a mixture that combines extracted tobacco pulp with granulated cured and aged natural tobacco lamina.
  • the tobacco material can be in solid form, in liquid form, in semi-solid form, or the like.
  • the term "tobacco material” includes any part and any related by-product, such as for example the leaves or stems, of any member of the genus Nicotiana.
  • the tobacco material for use in the present invention is preferably from the species Nicotiana tabacum. Any type, style or variety of tobacco may be treated. Examples of tobacco which may be used include but are not limited to Virginia, Burley, and Oriental tobaccos, and blends of any of these types.
  • the tobacco material comprises Kasturi tobacco.
  • the tobacco material to be treated may comprise or consist of post-curing tobacco.
  • post-curing tobacco refers to tobacco that has been cured but has not undergone any further treatment process to alter the taste or aroma of the tobacco material.
  • the post-curing tobacco may have been blended with other styles, varieties or types of tobacco.
  • the tobacco material to be treated may comprise or consist of tobacco that has been re-graded, green-leaf blended, conditioned, de-stemmed or threshed (or not in the case of whole leaf), dried or packed.
  • the tobacco material comprises lamina tobacco material.
  • the tobacco may comprise between about 70percent and 100percent lamina material.
  • the lamina may be in whole leaf form.
  • the tobacco material comprises cured whole leaf tobacco.
  • the tobacco material substantially comprises cured whole leaf tobacco.
  • the tobacco material consists essentially of cured whole leaf tobacco.
  • the tobacco material comprises stem tobacco material.
  • the tobacco may comprise up to a 30 percent of stem material.
  • the process of "curing" green tobacco depends on the type of tobacco harvested. For example, Virginia flue (bright) tobacco is typically flue-cured, whereas Burley and certain dark strains are usually air-cured.
  • the flue-curing of tobacco typically takes place over a period of five to seven days compared to one to two months for air-curing. Many major chemical and biochemical changes begin during the curing process and continue through the early phases of leaf drying.
  • the conversion of the tobacco from its yellow to brown colour generally results in formation and substantial accumulation of nitrosamines, and an increased microbial content.
  • Virginia tobacco is generally 'flue-cured.' The tobacco leaves are hung in curing barns, where heated air is generated to dry the leaves. As the leaves lose moisture, they develop their distinct aroma, texture, and colour. The farmer must carefully guide this process, which takes up to a week, during which time the temperature of the heated air must be constantly monitored and gradually increased. Too much or too little heat at any stage of the process will have a negative impact on the quality of the tobacco.
  • Burley and oriental tobaccos are cured differently. Burley is 'air-cured' in barns where the heat and humidity come from natural ventilation. The curing process takes up to two months. Oriental tobacco is 'sun-cured' by hanging the leaves outdoors in the sun for about two weeks.
  • the invention could be inter alia defined by the following Examples.
  • 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.
  • 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").
  • 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.
  • Pressure is applied to each barrel.
  • the pressure is comprised between between 1000 kilograms per square meter and 4000 kilograms per square meter.
  • example called 1T 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.
  • 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").
  • 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.
  • Pressure is applied to each barrel.
  • the pressure is comprised between between 1000 kilograms over square meter and 4000 kilograms over square meter.
  • example called 1T 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.
  • the given value represents an average of several values obtained for each sample of the same type.
  • the pH of the samples of tobacco material 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.
  • Figs. 1 and 2 show the presence of Lactic Acid in the tobacco material.
  • figures 1 represent Lactic Acid content in HS leaves and figure 2 in CC leaves
  • figures 1 represent Lactic Acid content in HS leaves and figure 2 in CC leaves
  • 3T three samples for tobacco material
  • 3T three samples for tobacco material
  • all samples, both CC and HS leaves show the presence of Lactic Acid, albeit in variable amount.
  • 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 Figure 3 (HS leaves) and Figure 4 (Chopped leaves, 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.
  • 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.
  • TSNA tobacco specific nitrosamines
  • NNN 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.
  • 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.
  • Figures 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.
  • Figures 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.
  • 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).
  • 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 60percent 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.
  • tryptophan degradation the pathway has been described by Ummadi and Weimer (2001, J. Dairy Sci. 84:1773-1782) for cheese bacteria and adapted accordingly.
  • more than 78percent of the tryptophan present in the starting tobacco material is catabolized after 6 months of fermentation (samples 3T) in both HS and CC leaves.
  • CGA Chlorogenic acid
  • Example 1 and 2 have also been tested for some additional chemical compounds which could be connected with the fermentation.
  • Fig. 17 shows the change of Indole-3-Lactic Acid in the tobacco material of example 1 (RAJ) and of example 2 (HS) between the non-fermented material (control) and the fermented material (HF). These data show an increase of the content of Indole-3-Lactic Acid in the fermented material.
  • Fig. 18 shows the change of L-Ornithine in the tobacco material of example 1 (RAJ) and of example 2 (HS) between the non-fermented material (control) and the fermented material (HF), indicated as quantity of L-Ornithine, and also as absolute change.
  • Fig. 19 shows the change of BHHPPD in the tobacco material of example 1 (RAJ) and of example 2 (HS) between the non-fermented material (control) and the fermented material (HF).
  • Fig. 20 shows the change of Secoisolariciresinol in the tobacco material of example 1 (RAJ) and of example 2 (HS) between the non-fermented material (control) and the fermented material (HF). These data show an increase of the content of Secoisolariciresinol in the fermented material.
  • 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.
  • the tobacco material in the two barrels has been turned monthly during the 7 months of the experiment.
  • the tobacco material has been turned and the moisture content of the tobacco material has been readjusted to approximatively 30 percent ⁇ 5 percent.
  • the pH of the tobacco material did not change significantly during the fermentation run (T1 to AF) staying at 5.1 ⁇ 0.3.
  • VG-AF the color of the tobacco material at the end of the fermentation process
  • VG-SM the starting material
  • VG-T4 4 month of anaerobic fermentation
  • 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.
  • VG-AF anaerobic fermentation
  • the Virginia tobacco material has a darker color than after 4 months, thus indicating that a full fermentation of Virginia tobacco material took place.
  • nice scented and floral odors were perceived after 8 months.
  • the given value represents an average of several values obtained for each sample of the same type, replicate.
  • Figure 11 shows the evolution of total alkaloids (TA) during the fermentation process, Reducing Sugars (RS) and ammonia (NH3) 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. No increase of ammonia were observed when using Virginia as fermented material, and the nitrate which was not present in this Virginia material did not shown any increase during and after the fermentation process.
  • Fig. 11 shows the TA (Total Alkaloids), RS (Reducing Sugars) and ammonia (NH3) in non-fermented Virginia (VG) material (SM) during the fermentation process (T1 toT7) and after fermentation (AF). The data are expressed in percent DW.
  • TA Total Alkaloids
  • RS Reducing Sugars
  • NH3 ammonia
  • Reducing Sugars were used as substrate by fermenting bacteria. Therefore about 60percent 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.
  • RS Reducing Sugars
  • the analyses show a strong significant increase ( ⁇ 10x) 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).
  • Fig. 12 shows catabolism of tobacco Reducing Sugars, citrate and malate followed by an accumulation of pyruvate and Lactic Acid (Lactic Acid fermentation) during the anaerobic fermentation of Virginia tobacco.
  • Fig. 14 shows the change in the content of quinic acid and Caffeic acid.
  • quinic and caffeic acid largely increased after the fermentation when the content of these compounds in the fermented tobacco material is compared with the content in the non-fermented tobacco material. This is probably as the result of cinnamoyl esterase catabolizing chlorogenic acid.
  • caffeic acid has demonstrated antimicrobial activity and may be promising in the treatment of dermal diseases.
  • Quinic acid is also a potent drug candidate to combat prostate cancer.
  • Indole-3-Lactic Acid ( Figure 15 ), largely increased (>10x) during the tobacco fermentation process and originates from the catabolism of tryptophan. Therefore, as Lactic Acid, the organic acid indole-3-Lactic Acid is also a good marker for Lactic Acid fermentation.
  • 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.
  • 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.
  • FC tobacco strips from Brazil 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 kilogramsof 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.
  • 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.
  • the tobacco material in the two barrels has been turned every two months during the 6 months of the experiment.
  • the given value represents an average of several values obtained for each sample of the same type, replicate.
  • Figure 16 shows the difference between the content of total alkaloids (TA) during the fermentation process, Reducing Sugars (RS), Nitrate (NO 3 ) and ammonia (NH 3 ) before fermentation and after three months of fermentation and at the end of the fermentation process.
  • Total alkaloids (TA) are not impacted by the anaerobic fermentation process.
  • Reducing Sugars are consumed by the anaerobic bacteria and their content in the tobacco material decrease with fermentation. At the end of the fermentation the level of Reducing Sugars in the tobacco material is very low. In this case Reducing Sugars were almost completely consumed after 6 month fermentation and one turning only.
  • 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.
  • Fig. 17A shows the change of Indole-3-Lactic Acid in the tobacco material of Example 4 (VG-CH).
  • the content of the Indole-3-Lactic is indicated in micrograms per gram.
  • the graph shows a considerable increase in the content of Indole-3-Lactic Acid in the fermented tobacco material.
  • the above indicated data for Indole-3-Lactic Acid have been obtained using Ultra Performance Liquid Chromatography polar and lipid positive and GC-MS; 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 a measurement in polar negative ionization mode.
  • RP-UPLC Waters ACQUITY Reversed Phase Ultra Performance Liquid Chromatography
  • ESI ElectroSpray Ionization source
  • Orbitrap Orbitrap
  • TOF Time of Flight
  • UPLC-MS measurements of the aqueous phase enabled the detection of the polar and semi-polar primary and secondary metabolites, and the organic phase the detection of the lipid and lipophilic content.
  • GC-MS measurements allow the analyses of the primary metabolites.
  • 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.
  • 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.
  • RP-UPLC Waters ACQUITY Reversed Phase Ultra Performance Liquid Chromatography
  • 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 datatbase and confirmed by measured standards.
  • 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.
  • 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.
  • the samples were measured on an Agilent Technologies GC coupled to a Leco Pegasus HT mass spectrometer which consists of an El ionization source and a TOF mass analyzer.
  • 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.
  • 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
  • HS CH VG-19-20 indicates the tobacco material of Example 4.
  • Measurements of D-Lactic Acid and L-Lactic Acid 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
  • 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.
  • Fr (F LA/ NF LA ): (F RS /NF RS ) wherein:
  • 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.
  • 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.
  • 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.
  • the anaerobic process does not negatively impact the content of alkaloids of the tobacco material.

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Claims (13)

  1. Verfahren für eine Behandlung von Tabakmaterial, das Verfahren umfassend:
    Vorsehen eines Tabakmaterials;
    Fermentieren des Tabakmaterials, um fermentiertes Tabakmaterial zu erhalten, der Fermentierungsschritt umfassend:
    Inkubieren des Tabakmaterials unter anaeroben Bedingungen;
    Beenden der Fermentation, wenn wenigstens eine der folgenden Bedingungen erfüllt ist:
    ✔ der Gehalt an Milchsäure beträgt mehr als das 10-Fache, bevorzugt mehr als das 20-Fache, noch bevorzugter mehr als das 50-Fache, noch bevorzugter mehr als das 70-Fache, bevorzugt mehr als das 80-Fache einer Ausgangsmenge an Milchsäure in dem Tabakmaterial,
    ✔ der Gehalt an reduzierenden Zuckern ist niedriger als 0,5, bevorzugt niedriger als 0,4, noch bevorzugter unter 0,2, noch bevorzugter unter 0,1 einer Anfangsmenge oder reduzierenden Zuckern in dem Tabakmaterial,
    ✔ der Gehalt an Indol-3-Milchsäure beträgt mehr als das 5-Fache, bevorzugt mehr als das 10-Fache, bevorzugt mehr als das 20-Fache, einer Anfangsmenge an Indol-3-Milchsäure in dem Tabakmaterial,
    ✔ der Gehalt an Kaffeesäure beträgt mehr als das 4-Fache, bevorzugt mehr als das 10-Fache, bevorzugt mehr als das 20-Fache einer Anfangsmenge an Kaffeesäure in dem Tabakmaterial,
    ✔ der Gehalt an Chinasäure beträgt mehr als das 2-Fache, bevorzugt mehr als das 4-Fache einer Anfangsmenge an Chinasäure in dem Tabakmaterial,
    ✔ der Gehalt an Asparagin ist niedriger als 0,5, bevorzugt niedriger als 0,4, bevorzugt niedriger als 0,3 einer Anfangsmenge oder Asparagin in dem Tabakmaterial,
    ✔ der Gehalt an Glutamin ist niedriger als 0,5, bevorzugt niedriger als 0,4 einer Anfangsmenge oder Glutamin in dem Tabakmaterial,
    ✔ der Gehalt an L-Ornithin beträgt mehr als das 10-Fache, bevorzugt mehr als das 20-Fache, bevorzugt mehr als das 50-Fache, bevorzugt mehr als das 100-Fache einer Ausgangsmenge an L-Ornithin in dem Tabakmaterial,
    ✔ der Gehalt an L-Leucin beträgt mehr als das 2-Fache, bevorzugt mehr als das 4-Fache, einer Anfangsmenge an L-Leucin in dem Tabakmaterial,
    ✔ der Gehalt an L-Lysin beträgt mehr als das 2-Fache, bevorzugt mehr als das 6-Fache, einer Anfangsmenge an L-Lysin in dem Tabakmaterial,
    ✔ der Fermentationsindex ist größer als 50, bevorzugt größer als 100, noch bevorzugter größer als 250, noch bevorzugter größer als 400, wobei der Fermentationsindex durch Dividieren des Verhältnisses zwischen dem Gehalt an Milchsäure in dem behandelten Tabakmaterial und dem Gehalt an Milchsäure in dem nicht fermentierten Tabakmaterial durch das Verhältnis zwischen dem Gehalt an reduzierenden Zuckern in dem behandelten Tabakmaterial und dem Gehalt an reduzierenden Zuckern in dem nicht fermentierten Tabakmaterial erhalten wird.
  2. Verfahren nach Anspruch 1 und ferner umfassend einen Anfangsmessschritt für ein Messen des Anfangsgehalts an wenigstens einer Milchsäure oder reduzierenden Zuckern oder Indol-3-Milchsäure oder Chinasäure oder Kaffeesäure oder L-Ornithin oder Asparagin oder Glutamin oder L-Leucin oder L-Lysin oder Fermentationsindex in dem Tabakmaterial.
  3. Verfahren nach Anspruch 1 oder 2 und ferner umfassend einen Messschritt für ein Messen des Gehalts an wenigstens einer Milchsäure oder reduzierenden Zuckern oder Indol-3-Milchsäure oder Chinasäure oder Kaffeesäure oder L-Ornithin oder Asparagin oder Glutamin oder L-Leucin oder L-Lysin oder eines Fermentationsindexes in dem Tabakmaterial während des Fermentierungsschritts.
  4. Verfahren nach einem der Ansprüche 1 bis 3, wobei, wenn das Tabakmaterial dunklen Tabak enthält, vorgesehen ist, die Fermentation zu beenden, wenn wenigstens eine der folgenden Bedingungen erfüllt ist: der Gehalt an 2,3-Butandiol beträgt mehr als das 5-Fache, bevorzugt mehr als das 10-Fache einer Ausgangsmenge an 2,3-Butandiol in dem Tabakmaterial, oder der Gehalt an Diacetyl beträgt mehr als das 5-Fache, bevorzugt mehr als das 10-Fache einer Ausgangsmenge an Diacetyl in. dem Tabakmaterial.
  5. Verfahren nach Anspruch 4 und ferner umfassend einen Anfangsmessschritt für ein Messen des Anfangsgehalts an 2,3-Butandiol oder Diacetyl in dem Tabakmaterial vor der Fermentation, um jeweils eine Anfangsmenge an 2,3-Butandiol oder Diacetyl in dem Tabakmaterial zu erhalten.
  6. Verfahren nach Anspruch 1, wobei während des Fermentierungsschritts vorgesehen ist, auf das Tabakmaterial einen Druck zwischen 1000 Kilogramm pro Quadratmeter und 15000 Kilogramm pro Quadratmeter, bevorzugt zwischen 3000 Kilogramm pro Quadratmeter und 12000 Kilogramm pro Quadratmeter, noch bevorzugter zwischen 5000 Kilogramm pro Quadratmeter und 10000 Kilogramm pro Quadratmeter aufzubringen.
  7. Verfahren nach Anspruch 1, wobei vorgesehen ist, den Fermentierungsschritt für eine Fermentationszeit von wenigstens 1 Monat, bevorzugt wenigstens 2 Monaten, noch bevorzugter wenigstens 4 Monaten, noch bevorzugter wenigstens 6 Monaten, noch bevorzugter wenigstens 8 Monaten, bevorzugt wenigstens 10 Monaten, noch bevorzugter wenigstens 12 Monaten fortzusetzen.
  8. Verfahren nach Anspruch 1, wobei während des Fermentierungsschritts vorgesehen ist, die Temperatur des Tabakmaterials zwischen 21 Grad Celsius und 35 Grad Celsius, bevorzugt zwischen 25 Grad Celsius und 31 Grad Celsius, zu halten.
  9. Tabakmaterial, umfassend:
    a) wenigstens eine der folgenden Verbindungen:
    - Milchsäure in einer Menge, die mehr als das 10-Fache, bevorzugt mehr als das 20-Fache, noch bevorzugter mehr als das 50-Fache, noch bevorzugter mehr als das 70-Fache, noch bevorzugter mehr als das 80-Fache einer Anfangsmenge an Milchsäure in dem Tabakmaterial beträgt.
    - Reduzierende Zucker in einer Menge, die niedriger ist als 0,5, bevorzugt niedriger als 0,4, noch bevorzugter unter 0,2, noch bevorzugter unter 0,1 einer Ausgangsmenge oder reduzierenden Zuckern in dem Tabakmaterial,
    - Indol-3-Milchsäure in einer Menge, die mehr als das 5-Fache, bevorzugt mehr als das 10-Fache, bevorzugt mehr als das 20-Fache einer Anfangsmenge an Indol-3-Milchsäure in dem Tabakmaterial beträgt.
    - Kaffeesäure in einer Menge, die mehr als das 4-Fache, bevorzugt mehr als das 10-Fache einer Ausgangsmenge an Kaffeesäure in dem Tabakmaterial beträgt,
    - Chininsäure in einer Menge, die mehr als das 2-Fache, bevorzugt mehr als das 4-Fache einer Ausgangsmenge an Chininsäure in dem Tabakmaterial beträgt,
    - Asparagin in einer Menge, die niedriger ist als 0,5, bevorzugt niedriger als 0,4, bevorzugt niedriger als 0,3 einer Ausgangsmenge oder Asparagin in dem Tabakmaterial,
    - Glutamin in einer Menge, die niedriger ist als 0,5, bevorzugt niederer als 0,4 einer Ausgangsmenge oder Glutamin in dem Tabakmaterial,
    - L-Ornithin beträgt mehr als das 10-Fache, bevorzugt mehr als das 50-Fache, bevorzugt mehr als das 100-Fache einer Ausgangsmenge an L-Ornithin in dem Tabakmaterial,
    - L-Leucin in einer Menge, die mehr als das 2-Fache, bevorzugt mehr als das 4-Fache einer Ausgangsmenge an L-Leucin beträgt,
    - L-Lysin in einer Menge, die mehr als das 2-Fache, bevorzugt mehr als das 6-Fache einer Anfangsmenge an L-Lysin beträgt,
    - ein Fermentationsindex ist größer als 50, bevorzugt größer als 100, noch bevorzugter größer als 250, noch bevorzugter größer als 400, wobei der Fermentationsindex durch Dividieren des Verhältnisses zwischen dem Gehalt an Milchsäure in dem Tabakmaterial und dem Gehalt an Milchsäure in dem nicht fermentierten Tabakmaterial durch das Verhältnis zwischen dem Gehalt an reduzierenden Zuckern in dem Tabakmaterial und dem Gehalt an reduzierenden Zuckern in dem nicht fermentierten Tabakmaterial erhalten wird ; oder
    b) weniger als 300 Milligramm pro Kilogramm Asparagin auf Gesamttrockengewichtsbasis; oder
    c) weniger als 70 Milligramm pro Kilogramm Glutamin auf Gesamttrockengewichtsbasis; oder
    d) mehr als 10000 Milligramm pro Kilogramm auf Gesamttrockengewichtsbasis an gesamten freien Aminosäuren; oder
    e) wenigstens 1 Mikrogramm pro Gramm, bevorzugt wenigstens 2 Mikrogramm pro Gramm, noch bevorzugter wenigstens 2,5 Mikrogramm pro Gramm Indol-3-Milchsäure auf Gesamttrockengewichtsbasis.
  10. Tabakmaterial nach dem vorhergehenden Anspruch, wobei das Tabakmaterial durch einen Prozess erhalten wird, umfassend Fermentieren des Tabakmaterials, um behandeltes Tabakmaterial zu erhalten, einschließlich: Inkubieren des Tabakmaterials unter anaeroben Bedingungen.
  11. Tabakmaterial nach Anspruch 9 oder 10, wobei das Tabakmaterial getrocknet ist.
  12. Tabakmaterial nach einem der Ansprüche 9 bis 11, wobei das Tabakmaterial gemahlen ist.
  13. Aerosolerzeugender Artikel, umfassend ein Tabakmaterial, das zwischen etwa 2,5 Gewichtsprozent auf Gesamttrockengewichtbasis und 100 Gewichtsprozent auf Gesamttrockengewichtsbasis, bevorzugt wenigstens etwa 4 Gewichtsprozent auf Gesamttrockengewichtsbasis, bevorzugt wenigstens etwa 10 Gewichtsprozent auf Gesamttrockengewichtsbasis, bevorzugt wenigstens etwa 20 Gewichtsprozent auf Gesamttrockengewichtsbasis des Tabakmaterials nach einem der Ansprüche 9 bis 12 enthält.
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US3070098A (en) * 1960-02-29 1962-12-25 American Sumatra Tobacco Corp Process for curing and preserving plants
US4528993A (en) 1982-08-20 1985-07-16 R. J. Reynolds Tobacco Company Process for producing moist snuff
US5372149A (en) 1992-03-25 1994-12-13 Roth; David S. Sterilization process in the manufacturing of snuff
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