GB2542623A

GB2542623A - Treatment of tobacco stems

Info

Publication number: GB2542623A
Application number: GB1517078.0A
Authority: GB
Inventors: Di Giacomo Michele; Gunduz Irfan; Zuchuat Fabien
Original assignee: Philip Morris Products SA
Current assignee: Philip Morris Products SA
Priority date: 2014-10-02
Filing date: 2015-09-28
Publication date: 2017-03-29
Also published as: GB201517078D0

Abstract

A tobacco product comprising tobacco stem material produced by a method comprising, in any order, the steps of: processing by rolling or flaking the tobacco stem material; and fermenting the tobacco stem material in the presence of homolactic acid bacteria and limited oxygen for a time between 4 and 21 days at a temperature between 15 ºC and 45ºC. A further step is drying the processed fermented tobacco stem material where the tobacco stem material has a thickness between 0.1mm and 1.2mm. The homolactic acid bacteria are from the genus Lactobacillus, Pediococcus or both in the fermentation step. One or more enzymes have cellulose or cellobiose hydrolase activity are added prior to the fermenting step. Also provided are tobacco products comprising tobacco stem material produced by the method of the invention.

Description

TREATMENT OF TOBACCO STEMS

The present invention relates, in general, to methods for improving the properties of tobacco stems, and to products incorporating the improved tobacco stems.

It is known that tobacco cut filler for smoking articles may be manufactured predominantly from the lamina portion of the tobacco leaf, which is separated from the stem portion of the leaf during a threshing process. In order to increase the amount of the tobacco material that can be used, it has also been previously proposed to add some tobacco stems back into the cut filler together with the lamina. Several methods are known for treating tobacco stem or stalk material. By way of example, it is known that tobacco stems can be passed, usually following a moistening treatment, between rollers which crush and flatten the stems. Subsequently, the rolled stems can be cut into shreds that are adapted to be mixed with shredded tobacco lamina. In another method, tobacco stems that have been impregnated with liquid carbon dioxide are heated to produce expansion of the stems. This procedure is understood to produce an expanded stem material that has an improved filling power compared with cut rolled stems. Tobacco stems may be rolled, flaked, extruded, or refined, before incorporation into the cut filler.

The smoking quality of tobacco stems is generally considered to be inferior to that of tobacco lamina. For some brands, the impact of stem negative quality characteristics is limited by adding casings of organic acids and other humectants. For example, a typical stem casing may include: glycerin, propylene glycol, lactic acid, inverted sugars, or combinations thereof. In some instances it is desirable to reduce or avoid the use of chemical additives - such as flavours - in tobacco products. In some situations, certain chemical additive are banned by government regulation.

It would be desirable to provide an improved method for making tobacco stems so that they become more suitable for use as a blend component of cut filler for smoking articles. The present invention seeks to address the problem of how to improve the properties, for example, the sensory properties of tobacco stems, whilst reducing or avoiding the use of one or more additives in the tobacco.

This present invention relates to tobacco products (including smoking articles) comprising tobacco stem materials wherein the sensory qualities of the tobacco stem materials when smoked in a smoking article, have been modified by a bacterial fermentation process. In this process, lactic acid is produced in the tobacco stems by the conversion of fermentable sugars, such as glucose, and organic acids, such as malic acid, that are naturally present in tobacco stems into lactic acid by bacterial fermentation.

Specifically, in a first aspect the present invention provides 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 42 days at a temperature of from about 15°C to about 45°C.

The invention also provides methods for producing such fermented processed tobacco stem material comprising, in any order, the steps of: (a) mechanically processing tobacco stem materials; and (b) in the presence of homolactic acid bacteria and limited oxygen, fermenting the tobacco stem material.

The bacterial fermentation is suitably homolactic in character meaning that lactic acid is the main or substantially the only fermentation products. The bacterial fermentation can also be malolactic fermentation meaning that malic acid is converted into lactic acid and CO2. Through this process, the level of organic acids in the tobacco stems can be modulated. Specifically the level of at least lactic acid can be increased. The level of malic acid and reducing sugars can be decreased. In addition, the water retention capability of tobacco stems can be improved by the in-situ formation of lactic acid which is a naturally occurring humectant. In addition, the stability of tobacco stems against spoilage by molds and other undesirable bacteria can be improved through the in situ production of lactic acid. Advantageously, the present disclosure can achieve the specific and in situ production of lactic acid in tobacco stems without the formation of alcohols or acetic acid which would otherwise detract from the sensory properties of the tobacco stems.

The terms “processing” or “processed” are used throughout the specification to mean that the tobacco stems are or have been subjected to a mechanical treatment that modifies the structure and morphology, and reduces at least one of the dimensions so that it is comparable to that of cut tobacco strands. The dimensions that can be reduced are length, width, and thickness. Examples of mechanical processing include but is not limited to rolling, cutting, flaking, refining and extrusion. The tobacco stem material which has been subjected to mechanical processing may be referred to interchangeably as stem particle or processed stem mateiral.

In embodiments, the step of processing tobacco stem material suitably comprises providing moist tobacco stems; and rolling the tobacco stems so as to reduce the thickness of the rolled tobacco stem to about 0.6 millimeter (mm) to about 1.2 mm, preferably about 1.1 mm. In various embodiments, after rolling, the stem is flattend resulting in a cross-sectional width that is about two to three times the thickness of the raw stem. In these embodiments, the step of processing tobacco stem material suitably comprises providing moist tobacco stems, optionally rolled moist tobacco stems; and cutting the tobacco stems. In a specifc embodiment, the rolled moist tobacco stems are cut transversely into strips of about 0.15 mm.

In embodiments, the step of processing tobacco stem material suitably comprises providing moist tobacco stems; and flaking the tobacco stems so as to reduce the thickness of the flaked tobacco stem to about 0.1 to about 0.2 mm, preferably about 0.13 mm. In various embodiments, the cross-sectional width of flaked stem is about four to five times the thickness of the raw stem. In embodiments, the step of processing tobacco stem material suitably comprises providing moist tobacco stems, optionally flaked moist tobacco stems; and cutting the tobacco stems. In a specifc embodiment, the flaked moist tobacco stems are cut transversely into strips of about 0.9 mm.

In embodiments, the step of processing tobacco stem material suitably comprises providing moist tobacco stems; and extruding the tobacco stems so as to obtain tobacco stem particles. The extruding step may be carried out for example by a twin screw extruder. In embodiments, the step of processing tobacco stem material suitably comprises providing moist tobacco stems; and refining the tobacco stems to obtain a tobacco stem particles. The refining step may be carried out for example by means of a disc refiner or a cone refiner.

In various embodiments, the tobacco stems may be processed mechanically as described above before or after bacterial fermentation.

Suitably, the tobacco stem material is not subjected to expansion or puffing by steam, before or after fermentation. Suitably, the tobacco stem material may be subjected to expansion or puffing by fluids other than steam, such as carbon dioxide, before or after fermentation.

Suitably, the method of the invention further comprises measuring the levels of lactic acid, malic acid or both in the tobacco stem material, wherein one or more of said steps of measuring may be performed before, during or after the step of fermenting. In embodiments, the fermentation is continued until the level of lactic acid and/or malic acid and/or the ratio of malic acid to lactic acid reaches a predetermined value indicative of the desired degree of fermentation.

Suitably, the method of the invention further comprises measuring the pH of the tobacco stem material during the fermentation step. The fermentation causes a change in pH that provides a convenient marker for monitoring the progress of the fermentation. Suitably, the fermentation step is terminated when the pH reaches a predetermined value indicative of the desired degree of fermentation, e.g., 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, or 3.5. The pH can suitably be measured by liquidizing the tobacco stem material at 30% dry matter content in deionized water in a blender and measuring the pH of the resulting slurry.

The tobacco stems are fermented for between about 4 days and about 42 days, for example from about 6 days to about 10 days, typically from about 4 days to about 21 days, about 21 days to about 42 days, at a temperature of between about 15°C and about 45°C, suitably between about 20°C and about 40°C, and suitably at about 37°C.

Suitably, the fermentation is performed under microaerophilic or anaerobic conditions.

The fermentation conditions, in particular the selection of homolactic acid bacteria and the short time and low temperature fermentation, will not result in significant degradation of cellulosic or pectin components of the tobacco stems. In embodiments where such degradation is desired, one or more suitable enzymes can be added at one or more time points prior to, during or after fermentation. Suitably, the enzymes comprise, consist essentially of, or consist of a mixture containing one or more enzymes having cellulolytic activity, suitably, wherein said cellulolytic activities comprises cellulase and cellobiose hydrolase or a combination thereof. In these embodiments, the bacterial fermentation of glucose is combined with the enzyme saccharification of cellulose.

The bacteria that carry out the fermentation step are mostly homolactic acid bacteria and suitably consist essentially of such bacteria. That is to say, bacteria producing fermentation products that consist essentially of, or consist of, carbon dioxide and lactic acid. Suitably, the fermentation does not result in the bulk production or accumulation of one or more alcohols, such as ethanol, or acetic acid.

Suitably, the homolactic acid bacteria are members of the genus Lactobacillus. Suitably, the lactic acid bacteria are Lactobacillus crispatus, Lactobacillus plantarum, Pediococcus pentosaceus, Pediococcus acidilacti or a combination of any two or more of the foregoing species.

Suitably, the fermentation step lowers the malic acid content of the tobacco stem material by at least about 50%, suitably by at least about 60%, more suitably by at least about 75%. Suitably, the malic acid content of the tobacco stem material after treatment is less than about 20 mg/g dry basis. Suitably, the fermentation step increases the lactic acid content of the tobacco stem material up to at least about 2%, more suitably at least about 5%, for example about 6% to about 10% dry weight basis. Suitably, the fermentation step lowers the reducing sugar content of the tobacco stem material by at least about 10%, Suitably by at least about 20%, for example by about 25% to about 50%.

Suitably, the method of the invention comprises optionally adding further ingredients such as flavorings, to raw stem material, fermented stem material, or processed stem material, such that it can be used in blending to make cut filler for a cigarette.

In a further aspect, there is provided a fermented tobacco stem material having a malic acid concentration of less than 25 mg/g dry mass basis. Suitably, the fermented tobacco stem material according to this aspect of the invention are obtained or obtainable by a method according to the invention.

In a further aspect, there is provided a method of forming a reconstituted tobacco sheet comprising combining fermented tobacco stem material and by-products of tobacco manufacturing, such as tobacco dust, to form a slurry, casting the slurry, and drying the slurry to form a reconstituted tobacco sheet. Also provided is a reconstituted tobacco sheet comprising fermented tobacco stem material obtained or obtainable by this method.

In a further aspect, there is provided a method of forming a tobacco product comprising the steps of providing fermented and processed tobacco stem material according to the method described herein; and incorporating the fermented processed tobacco stem material into a tobacco product. Suitably, the fermented processed tobacco stems in such tobacco product are not subjected to expansion using steam, in particular puffing such as steam puffing, before or after the fermentation step. Suitably, the fermented processed tobacco stems in such tobacco product may be subjected to expansion using a fluid other than steam, in particular such as carbon dioxide or dry ice, before or after the fermentation step. For example, the tobacco product may be a cigarette having a filler made up of cut leaf blended with the fermented processed tobacco stem material.

In a further aspect, the present invention provides a tobacco product comprising fermented tobacco stem material according to the present invention. Suitably, the tobacco product is a cigarette.

Figure 1 is a schematic overview of a first exemplary method of the invention; and Figure 2 is a schematic overview of a second exemplary method of the invention.

The technical terms and expressions used herein are generally to be given the meaning commonly applied to them in the pertinent arts of microbiology, chemistry and plant biology. The term definitions apply to the complete content of this disclosure.

The word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality.

The term “stem” is used herein to refer to the structural portion of the tobacco plant connecting the lamina to the stalk, and also to the veins or ribs that extend through the leaves between the lamina portions. Unless it is stated, the term as used herein does not include any portion of the tobacco stalk.

In methods according to the invention, both tobacco stems and tobacco stalks can be used effectively. Thus, the expression “tobacco stem material” is used throughout this specification to refer to a composition to which an operation in the method can equally be applied whether it comprises tobacco stems only, tobacco stalks only or a mixture of tobacco stems and tobacco stalks. The methods of the present invention are suitably performed on materials comprising at least about 10% by dry weight of tobacco stems, more suitably at least about 20% or 25% or 50% by dry weight of tobacco stems, still more suitably at least about 75% or about 90% by dry weight of tobacco stems, or consisting essentially of tobacco stems. The methods of the present invention are suitably performed on materials comprising at least about 10% by dry weight of tobacco stalks, more suitably at least about 20% or 25% or 50% by dry weight of tobacco stalks, still more suitably at least about 75% or about 90% by dry weight of tobacco stalks, or consisting essentially of tobacco stalks. The methods of the present invention are suitably performed on materials comprising at least about 10% by dry weight of tobacco stems and stalks, more suitably at least about 20% or 25% or 50% by dry weight of tobacco stems and stalks, still more suitably at least about 75% or about 90% by dry weight of tobacco stems and stalks, or consisting essentially of tobacco stems and stalks. The remainder of the material suitably comprises, consists essentially of, or consists of, other tobacco plant material such as tobacco lamina or tobacco dust, or tobacco stem or stalk where applicable.

The term “raw” is used throughout this specification to mean that the tobacco stems or tobacco stalks have not undergone any treatment. In particular, they have not been subjected to any rolling, flaking, refining, extruding or cutting operation. Nor have they been subjected to bacterial fermentation. Thus, the dimensions of the “raw tobacco stems” have not been reduced in any way by any mechanical processing step. By the term “length of the raw tobacco stems”, reference is made throughout this specification to a major longitudinal dimension of the raw tobacco stems. Further, by the term “thickness of the raw tobacco stems”, reference is made to the maximum thickness of a raw tobacco stem as measured in a plane substantially perpendicular to the longitudinal direction along which the length of the raw stem or stalk is measured. The thickness of raw tobacco stems ranges from 2 mm to 10 mm.

The term “moisture content” throughout the present specification refers to the percentage of “Oven Volatiles” (% OV or percent OV) of the tobacco stems or other tobacco materials. It is determined by measuring the percentage weight loss from the stems upon drying a sample of the stem material in an oven at 100 ± 1 degrees Centigrade (°C) for 3 hours ± 0.5 minutes. In practice, it is assumed that a significant majority of the weight loss from the stems results from the evaporation of moisture. It should be noted that, on an absolute basis, the values of moisture content determined by oven drying may be greater than the results of water content analysis when using a specific method such as ISO 6488 (Karl Fischer method). The difference is sample-type dependent and is due to the loss of volatile materials other than water from the tobacco material during oven drying. The terms “% dry mass basis” and “% dry weight basis” in the present specification likewise refers to the percentage by weight of a component based on the weight of the sample after oven drying, i.e. based on the weight of the sample minus the weight of the oven volatiles in the sample.

The present inventive process is applicable to all tobacco types, i.e. flue-cured, burley or oriental. Depending on the tobacco type, different substrates may be metabolized by the bacteria, i.e. glucose and malic acid in flue-cured and oriental stems, mainly malic acid in burley type stems. Suitably, the tobacco stems in the starting material have not been expanded or puffed, and in particular they have not been subjected to a steam puffing process.

Suitably, the tobacco stem starting material is provided at, or adjusted to, a moisture content of at least about 15% by weight, at least about 20% by weight, at least about 25% by weight, at least about 30% by weight, at least about 35% by weight, at least about 40% by weight, at least about 45% by weight, or at least about 50% by weight, or at least about 60% by weight before fermentation. The moisture adjustment can be performed by conventional conditioning methods, for example by water spraying, stirring and equilibration.

In various embodiments, the tobacco stem materials are mechanically processed before or after the fermentation step. In suitable embodiments, the tobacco stems may be rolled, before or after the fermentation step. The term “rolled” as used herein include any action whereby the stem material is passed between two surfaces, at least one of which is rotating, and separated by a gap that is smaller than the thickness of the stem. The thickness of rolled stems ranges from about 0.6 mm to about 1.2 mm, preferably from about 0.7 mm to about 1.1 mm, or about 1.1 mm. In suitable embodiments, the tobacco stems may be cut before or after the fermentation step. In a specifc embodiment, the rolled moist tobacco stems are cut transversely into strips of about 0.05 mm to about 0.3 mm, of about 0.1 mm to about 0.2 mm, and preferably about 0.15 mm.

In suitable embodiments, the tobacco stems may be flaked before or after fermentation. The term “flaked” is used herein to mean that the stem material is rolled between two rotating surfaces which rotate at different speeds (e.g., a differential of about 10% rpm) and separated by a gap that is smaller than the thickness of the stem (e.g., 0.06 to 0.08 mm, preferably 0.07 mm), resulting in an abrasive and bruising action on the tobacco stem such that they are deformed yet not so damaged that they lose too much of their strength. In a specifc embodiment, the flaked moist tobacco stems are cut transversely into strips of about 0.5 mm to about 1.5 mm, of about 0.75 mm to about 1.2 mm, and preferably about 0.9 mm.

In suitable embodiments, the tobacco stems may be extruded or processed in a screw extruder, particularly in a twin screw extruder, before or after fermentation. In suitable embodiments, the tobacco stems may be refined or processed in a refiner, particularly in a disc refiner or cone refiner, before or after fermentation.

In various embodiments, the tobacco stem material is processed mechanically by cutting, rolling, flaking, extruding, or refining to obtain a tobacco stem particle wherein the major dimension is in the range of about 3 mm to about 30 mm, at least about 5 mm, and about 5 mm to about 20 mm. In various embodiments, the tobacco stem particle that is obtained or obtainable by mechanical processing has a second largest dimension that is in the range of about 0.5 mm to about 2 mm, about 0.7 mm to about 1.5 mm, about 0.9 mm to about 1.1 mm, about 1.1 mm, and about 0.9 mm, and that is traverse to the major dimension. Moreover, the tobacco stem particle that is obtained or obtainable by mechanical processing has a third largest dimension, that is in the range of, about 0.05 mm to about 0.3 mm, about 0.1 mm to about 0.2 mm, about 0.13 to about 0.15 mm, about 0.13 mm, and about 0.15 mm. Accordingly, the mechanical process step of the methods of the invention result in tobacco stem particles or a composition comprising tobacco stem particles, such a composition also interchangeably referred to herein as processed tobacco stem materials. The stem particle dimensions can be measured by means of sieves or with optical means.

In a particular embodiment, the raw tobacco stems may be disc-refined. In more detail, the step of refining the tobacco stems may comprise a first step of conditioning raw tobacco stem materials to a moisture content of at least about 40 % and a second step of refining the tobacco stems between discs separated by a first gap. Suitably, a third step of refining the tobacco stems between discs separated by a second gap smaller than the first gap is performed at least once. The first gap may be less than about 1000 micrometers. Suitably, the first gap is less than about 750 micrometers. More suitably, the first gap is less than about 500 micrometers. The second gap may be less than about 500 micrometers. Suitably, the second gap is less than about 350 micrometers. More suitably, the second gap is less than about 200 micrometers. In addition, or as an alternative, the second gap may be more than about 50 micrometers. Suitably, the second gap may be more than about 100 micrometers. More suitably, the second gap may be more than about 200 micrometers. In a preferred embodiment, the first gap is about 500 micrometers and the second gap is about 50 micrometers.

The composition produced by the above refining methods may be used as or in the tobacco stem material in the fermentation processes of the present invention.

Suitably, the fermentation step comprises inoculating or contacting the tobacco stem material with one or more homolactic acid bacteria to ferment the tobacco stem material. This may be done by spraying the material with a composition comprising the bacteria. The inoculation may be performed at a level of from about 104 to about 108, for example about 106 to about 107 colony forming units (cfu)/g.

The tobacco stem materials are treated by bacterial fermentation to convert into lactic acid at least one substrate selected from (a) one or more fermentable sugars (for example, glucose and other 6-carbon sugars), and (b) one or more organic acids (for example, malic acid) that are naturally present in the tobacco stems. The bacteria used are lactic acid bacteria (LAB). The LAB comprise a clade of Grampositive, low-GC, acid-tolerant, generally non-sporulating, non-respiring rod or cocci that are associated by their common metabolic and physiological characteristics. The LAB produce lactic acid as the major metabolic end-product of carbohydrate fermentation. The genera that comprise the LAB include Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Sporolactobacillus, Tetragenococcus, Vagococcus, and Weisella. In particular, the LAB that are used are homofermentative LAB which include species belonging to the genera Lactobacillus, Enterococcus, Lactococcus, Pediococcus, Streptococcus, Tetragenococcus, and Vagococcus. Homofermentative LAB utilise a homolactic fermentation pathway which produces two molecules of lactic acid for each molecule of hexose via the Embden-Meyerhof pathway under conditions of limited oxygen. Homofermentative LAB are also known as homolactic acid bacteria. Heterofermentative or heterolactic fermentation, in contrast, yields carbon dioxide and ethanol in addition to lactic acid, via the phosphoketolase pathway. One skilled in the art would recognize that among the LAB, some homolactic species can also produce lactic acid, ethanol and CO2 as fermentation end products, and thus producing a similar final result. Use of certain homolactic acid bacteria, such as L. plantarum, which is also a facultative heterolactic acid bacteria, is within the scope of the invention.

Homolactic acid bacteria used in accordance with the present disclosure can be a single species within a single genera, a mixture of multiple different species within a single genera or multiple different species within multiple different genera. The homolactic acid bacteria used in the invention can be present in a mixture of bacteria that is naturally occurring and obtainable from the tobacco plant. There is no requirement that homolactic acid bacteria of any specific genus or species be separated, isolated or enriched so long as the desired fermentation end products are obtained. I In one embodiment, the use of homolactic acid bacteria belonging to the genera Lactobacillus are used. Exemplary species of this genera include Lactobacillus acetotolerans, Lactobacillus acidifarinae, Lactobacillus acidipiscis, Lactobacillus acidophilus (Doderlein bacillus), Lactobacillus agilis, Lactobacillus algidus, Lactobacillus alimentarius, Lactobacillus amylolyticus, Lactobacillus amylophilus, Lactobacillus amylotrophicus, Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus antri, Lactobacillus apodemi, Lactobacillus aviarius, Lactobacillus bifermentans, Lactobacillus brevis, Lactobacillus buchneri,

Lactobacillus camelliae, Lactobacillus casei, Lactobacillus catenaformis, Lactobacillus ceti, Lactobacillus coleohominis, Lactobacillus collinoides,

Lactobacillus composti, Lactobacillus concavus, Lactobacillus coryniformis,

Lactobacillus crispatus, Lactobacillus crustorum, Lactobacillus curvatus,

Lactobacillus delbrueckii, Lactobacillus delbrueckii subsp. Bulgaricus, Lactobacillus delbrueckii subsp. Lactis, Lactobacillus diolivorans, Lactobacillus equi, Lactobacillus equigenerosi, Lactobacillus farraginis, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus formicalis, Lactobacillus fructivorans, Lactobacillus frumenti, Lactobacillus fuchuensis, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus gastricus, Lactobacillus ghanensis, Lactobacillus graminis,

Lactobacillus hammesii, Lactobacillus hamsteri, Lactobacillus harbinensis,

Lactobacillus hayakitensis, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus homohiochii, Lactobacillus iners, Lactobacillus ingluviei, Lactobacillus intestinalis, Lactobacillus jensenii, Lactobacillus johnsonii, Lactobacillus kalixensis,

Lactobacillus kefuranofaciens, Lactobacillus kefiri, Lactobacillus kimchii, Lactobacillus kitasatonis, Lactobacillus kunkeei, Lactobacillus leichmannii, Lactobacillus lindneii, Lactobacillus malefermentans, Lactobacillus mali, Lactobacillus manihotivorans, Lactobacillus mindensis, Lactobacillus mucosae, Lactobacillus murinus, Lactobacillus nagelii, Lactobacillus namurensis, Lactobacillus nantensis, Lactobacillus oligofermentans, Lactobacillus oris, Lactobacillus partis, Lactobacillus pantheris, Lactobacillus parabrevis, Lactobacillus parabuchneri, Lactobacillus paracollirtoides, Lactobacillus parafarragirtis, Lactobacillus parakefiri, Lactobacillus paralimentarius, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus perolerts, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus psittaci, Lactobacillus rermini, Lactobacillus reuteri, Lactobacillus rhamrtosus, Lactobacillus rimae, Lactobacillus rogosae, Lactobacillus rossiae, Lactobacillus ruminis, Lactobacillus saerimneri, Lactobacillus sakei, Lactobacillus salivarius, Lactobacillus sanfrartciscensis, Lactobacillus satsumensis, Lactobacillus secaliphilus, Lactobacillus sharpeae, Lactobacillus siliginis, Lactobacillus spicheri, Lactobacillus suebicus, Lactobacillus thailandensis, Lactobacillus ultunensis, Lactobacillus vaccinostercus, Lactobacillus vaginalis, Lactobacillus versmoldensis, Lactobacillus vini, Lactobacillus vitulinus, Lactobacillus zeae and Lactobacillus zymae. Lactobacillus species that are used in accordance with the present disclosure can be a single species or multiple different species. Many lactic acid bacteria species have been isolated, studied and deposited in public microorganism depository.

In one embodiment, Lactobacillus plantarum is used. In one embodiment, Lactobacillus crispatus is used. In one embodiment, Lactobacillus crispatus and Lactobacillus plantarum are used. In one embodiment, a composition comprising or consisting essentially of Lactobacillus crispatus, Lactobacillus plantarum, or both are used. In another embodiment, Pediococcus acidilactici, Pediococcus pentosaceus, or both are used. In yet another embodiment, a composition comprising or consisting essentially of Lactobacillus crispatus, Lactobacillus plantarum, Pediococcus acidilactici, Pediococcus pentosaceus, or a combination of two or more of the foregoing are used. Lactic acid bacteria can be readily obtained from natural sources, including tobacco plant materials. Accordingly, lactic acid bacteria or homolactic acid bacteria that are obtainable or obtained from tobacco plant materials can be used in the methods of the invention. Such bacteria can be obtained from tobacco that has been allowed to undergo anaerobic fermentation without adding any inoculant. For example, Lactobacillus plantarum can be isolated from tobacco by techniques well known in the art. However, there is no requirement to use exclusively one type of lactic acid bacteria.A combination of homo- and hetero-fermentative lactic acid bacteria can be used if desired and provided that the desired bacterial fermentation is achieved.

The use of a composition comprising homolactic acid bacteria while in the presence of other types of bacteria, yeast or fungi is also contemplated provided that the desired bacterial fermentation is achieved.To carry out the bacterial fermentation, the tobacco stems are fermented in the presence of the one or more homolactic acid bacteria and limited oxygen. The fermentation can be carried out in a suitable fermentation vessel. The fermentation vessel may be formed of plastic, metal, and combinations thereof, optionally including other materials. The fermentation vessel may include one or more coatings. The coating may inhibit corrosion and may also facilitate removal of solids. The footprint of the vessel may be substantially square, substantially circular, substantially oval, substantially rectangular, or irregularly shaped. The fermentation vessel can include a controller. The controller can be configured to automate the system. The controller can measure various parameters of the system, such as temperature, oxygen levels and the like. The controller may use measurements of the various parameters to modify values of one or more parameters of the system. The controller may measure parameters of the system continuously or periodically, and may also modify said parameters continuously or periodically. The vessel is suitably sealed such that atmospheric oxygen cannot enter. The vessel can be equipped with valves and inert gas inlets for gas flushing and control of the fermentation atmosphere.

It will be understood by the person skilled in the art that tobacco materials can naturally contain homolactic acid bacteria. Thus, under conditions of limited oxygen, fermentation can be achieved without the further addition of homolactic acid bacteria to the tobacco stem material. However, if no additional homolactic acid bacteria are added to the stem material then a longer fermentation time will be required, for example, more than 14 days to more than 42 days, and there is a greater risk of spoilage or unwanted microbial actions.

Suitably, therefore the tobacco stems are contacted with or inoculated with exogenous homolactic acid bacteria since this will significantly decrease the fermentation time to less than about 42 days, less than about 21 days or less than 14 days, for example to between about 4 and about 10 days, suitably to between about 6 and about 8 days. As used herein the term “exogenous homolactic acid bacteria” refers to homolactic acid bacteria that are supplied by a source external to the tobacco stem materials as opposed to homolactic acid bacteria bacteria that are naturally occurring in the tobacco stem materials. The exogenous homolactic acid bacteria can comprise the same species of homolactic acid bacteria that are naturally occurring on the tobacco. Thus, the exogenously added homolactic acid bacteria may be cultured bacteria - such as an in vitro culture of bacteria. The culture may be a homogenous or heterogeneous population of homolactic acid bacteria. For example, the culture may comprise or consist essentially of Lactobacillus species, such as L. plantarum. The homolactic acid bacteria that are used to contact or inoculate the tobacco stems may comprise the same genera or species of homolactic acid bacteria that naturally occur in the tobacco stems such that the addition of the exogenous homolactic bacteria will increase the quantities thereof in the tobacco stems and thereby shorten fermentation times. The homolactic acid bacteria that are used to contact or inoculate the tobacco stem materials may comprise a different genera or species of homolactic acid bacteria than those that naturally occur in the tobacco stems. Suitably, the bacteria added to the tobacco consist essentially of homolactic acid bacteria.

It is also contemplated that homolactic acid bacteria useful for the method can be provided in the form of a composition comprising the bacteria. By way of example, the composition can include one or more species or genera of homolactic acid bacteria. By way of further example, the composition can include various different types of bacteria, and even fungi such as yeast in addition to homolactic acid bacteria required for the purpose of converting sugars and organic acids into lactic acid. The composition can include one or more additives or stabilizers and the like. Example of such a composition include Bonsilage® (Schaumann GmbH), which is a formulation of homofermentative lactic acid bacteria; Ecosyl® 100 (Ecosyl Products, Stokesley UK); freeze-dried culture of L. plantarum MTD/1 (NCIMB 40027); freeze-dried culture of L. plantarum DSM 12836 and P. pentosaceus DSM 12834; Lalsil® CL (Lallemand, Blagnac, France); freeze-dried culture of L. plantarum MA18-5U and P. acidilactici MA18-5M; and Enoferm®V22 (Lallemand, Blagnac, France); freeze-dried culture of L. plantarum V22, for use in wine-making. The bacteria used in Ecosyl® 100, Bonsilage® and Lalsil® CL are authorised by the European Union (EU) as feed additives, in the group of silage additives. EU authorisation documents for the strains can be found in the EU register of feed additives (European Community, 2013).

As discussed, the fermentation process occurs under conditions of limited oxygen or no oxygen. Although LAB can grow anaerobically, unlike most anaerobes, they can grow in the presence of oxygen as aero-tolerant anaerobes. Moreover, if the fermentation process is carried out under conditions of isolation, such as in a sealed container, then the fermentation process will eventually become substantially anaerobic or even anaerobic as CO2 resulting from the conversion of malic acid into lactic acid is released. In certain embodiments, it is preferred that the tobacco stems are contacted with the LAB under substantially anaerobic or microaerophilic or anaerobic conditions. When anaerobic conditions are used, no consistent or sustained increase in temperature is generally observed during the fermentation.

As used herein, the term “limited oxygen” means that the oxygen levels (i.e. partial pressures, chemical activities) are reduced by at least 50% as compared to the levels of atmospheric oxygen. Oxygen is present in the atmosphere at 21% v/v oxygen. Suitably, the oxygen levels are reduced by at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93% or 94% as compared to the levels of atmospheric oxygen. As used herein, the term “substantially anaerobic” means that the oxygen levels are reduced by at least about 95%, 96%, 97%, 98% or 99% as compared to the levels of atmospheric oxygen. The term “microaerophilic” means that oxygen is present at a lower concentration than is present in the atmosphere. The term “anaerobic” assumes its normal meaning in the art as the 100% absence of atmospheric oxygen.

Limited oxygen conditions can be achieved in various ways. By way of example, they can be achieved by flushing the headspace of the fermentation vessel using, for example, inert gas, either incompletely or completely in which case anaerobic conditions will be created. In some cases anaerobic conditions will be reached through the release of C02 resulting from the conversion of malic acid into lactic acid.

Suitably, the fermentation is conducted at a temperature of between about 15°C and about 45°C, between about 20°C and about 40°C, between about 20°C and about 35°C, or between about 30°C and 40°C, or at about 37°C.

Suitably, the fermentation is conducted for between about 4 and about 14 days, between about 6 and about 10 days, or between about 6 and about 8 days, or until such times as sufficient lactic acid is generated in the tobacco stems, or sufficient malic acid has been consumed, or both, as determined by chemical analysis or monitoring of pH.

The amount of lactic acid increases during the fermentation. The amount of malic acid decreases during the fermentation. .

The initial and final amounts of the various fermentation substrates and products will depend on the tobacco stem material being treated. The fermentation process can result in the following changes in the tobacco stem material: lactic acid is increased up to at least about 10mg/g, for example about 20mg/g to about 80 mg/g dry mass basis; malic acid is depleted by at least about 40%, at least about 50%, at least about 60% or at least about 75% compared to the level in the starting tobacco stems. In addition, glucose may be depleted by at least about 50%, 65% or 80% compared to the level in the unfermented tobacco stems. Fructose may be decreased by at least about 5%, 10% or 20% compared to the level in the unfermented tobacco stems.

The material may be subjected to processing as hereinbefore described after fermentation if this has not already been done before the fermentation step. In certain embodiments, the tobacco stem material is not subjected to expansion or puffing by steam or other fluids, before or after fermentation. In other embodiments, the tobacco stem material may be subjected to expansion or puffing by fluids other than steam before or after fermentation.

In a variation of the process described herein, one or more enzymes or enzyme preparations can be added to or contacted with the tobacco stem material in the method of the present disclosure. For example, one or more enzymes or enzyme preparations can be added to or contacted with the tobacco stems either before the fermentation step, or in combination with the lactic acid bacteria. The enzymes will typically be in a substantially purified form although the specific nature of the enzymes or enzyme preparations is not critical provided that they are able to exert their desired activity. In one embodiment, one or more enzymes that degrade cellulose present in the tobacco stems to release glucose which is converted by the bacteria into lactic acid can be added. In one embodiment, the enzyme preparation used contains a mixture of cellulolytic activities, such as cellulose (endo- and exo-) and cellobiose hydrolyse. An enzyme inactivation step may be performed after the fermentation process to prevent further degradation of the material by the enzymes.

Either before or after the fermentation process, one or more further components may be added to the tobacco stem material. These components may be added at the same levels or at lower levels than would otherwise be added if the tobacco stems were not treated in accordance with the methods described herein. In particular, the present invention affords the advantage that components which modulate the sensory properties can be added at either lower levels or not added at all. The further components that can be added include, but are not limited to, sugars such as invert sugar, organic and inorganic fillers (for example, grains, processed grains, puffed grains, maltodextrin, dextrose, calcium carbonate, calcium phosphate, corn starch, lactose, mannitol, xylitol, sorbitol, finely divided cellulose, and the like), binders (for example, povidone, sodium carboxymethylcellulose and other modified cellulosic types of binders, sodium alginate, xanthan gum, starch-based binders, gum arabic, guar gum, lecithin, and the like), humectants (for example, glycerin, propylene glycol, and the like) and disintegration aids (for example, microcrystalline cellulose, croscarmellose sodium, crospovidone, sodium starch glycolate, pregelatinized corn starch, and the like). Preservatives (for example, potassium sorbate, and the like) may be included in certain embodiments or they be omitted. Colorants (for example, dyes and pigments, including caramel coloring and titanium dioxide, and the like) may be included in certain embodiments or they be omitted. One or more flavorings may be added in certain embodiments or they may be added at reduced levels or they may be omitted altogether. It is preferred that one or more artificial flavor ingredients - such as exogenous lactic acid - are added at reduced levels or are omitted altogether. As used herein the term “exogenous lactic acid” refers to lactic acid from an external source that is added to the tobacco stems as opposed to being a naturally occurring lactic acid that is formed in the tobacco stem materials. Thus, the exogenous lactic acid may be, for example, chemically synthesised lactic acid. If one or more flavorings are included then it is an object of the present disclosure that they can be added in reduced amounts - such as in amounts of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%, 90% or 100% less than would otherwise be included in the tobacco stems that has not been treated using the methods of the present disclosure. In one embodiment, the tobacco stems or the tobacco stems obtained or obtainable by the present disclosure is substantially free or free from artificial flavorings, or contains reduced amount of artificial flavorings, including exogenous lactic acid - such as exogenous lactic acid -that improve the sensory properties of tobacco stems. The components described are known to those having ordinary skill in the art and may be present in amounts and in forms that are known. In one embodiment, at least one binding agent and at least one humectant are added to the tobacco stems at a level ranging from about 1% to about 10% (w/w), for example about 2-6% (w/w) of binding agent, and about 0% to about 20% (w/w), for example about 1% to about 4% (w/w) of humectant.

After fermentation, the tobacco stem material is dried, suitably to a moisture content of about 10% to about 30% prior to storage or incorporation into tobacco products. Suitably, the fermented tobacco stem material is dried to a moisture content of about 12%OV to about 14% OV. The drying is suitably carried out rapidly using conventional methods.

In embodiments where the raw stem material has undergone fibrillation before or after the fermentation step, the fermented tobacco stem material may be processed into reconstituted tobacco sheet using conventional methods. Suitably, the fermented tobacco stem material may be used to form a pulp and then cast and dried to form the reconstituted tobacco sheet. For example, it may be cast onto a belt and dried to form the reconstituted tobacco sheet.

Suitably, the method of treating the tobacco stem material improves the sensory properties thereof. It has also been found that the process according to the invention improves the water retention capacity of the stems. OV% at equilibrium may increase by up to 1 percentage point. It has also been found that the process according to the invention increases the filling power of the tobacco. Corrected cylinder volume (CCV) increases by up to about 4%. The process of the invention does not change the nicotine level of the tobacco stems, since evaporation of nicotine is minimized by the low fermentation temperature, and the anaerobic conditions inhibit development of bacteria able to degrade nicotine. Further, the need to introduce non-tobacco cellulosic material is substantially eliminated altogether, because the tobacco stem fibres obtained by the processing step provide sufficiently strength. Accordingly, in another aspect the present invention provides a fermented tobacco stem material, wherein said fermented tobacco stem material contains less than about 20 mg/g dry mass basis of malic acid. Suitably, the fermented tobacco stem material is obtained or obtainable by fermentation of a tobacco stem material in accordance with the methods of the invention. The fermented tobacco stem material can be incorporated into tobacco products.

Suitably, the malic acid content of the tobacco stem material should be reduced by at least 60%, at least 50%, at least 40%, at least 30%, or at least 20% after fermentation.Suitably, the malic acid to lactic acid ratio can be less than 15, 10, 5 and preferably below 3, 2, or 1 after fermentation Suitably, the fermented tobacco stem material has a lactic acid concentration of at least about 10 mg/g dry mass of tobacco and a malic acid concentration of less than 20 mg/g dry mass basis. The lactic acid concentration can be between about 20 and 80 mg/g dry mass basis and the malic acid concentration can be between about 10 and 20 mg/g dry mass basis. The tobacco stem material after fermentation can have a glucose concentration of less than 1 mg/g dry mass basis. The tobacco stem material after fermentation can have a fructose concentration of less than 40 mg/g dry mass basis. The tobacco stem material after fermentation can have a sucrose concentration of less than 1 mg/g dry mass basis. The tobacco stem material after fermentation can have a malonic acid concentration of less than 80 mg/g dry mass basis. The malonic acid concentration can be between about 60 and 80 mg/g dry mass basis - such as between about 65 and 75 mg/g. The acetic acid concentration is suitably less than about 10 mg/g dry mass basis.

In one embodiment, the fermented tobacco stem materials comprise a lactic acid concentration of at least about 10 mg/g dry mass basis - such as between about 20 and 80 mg/g dry mass basis, a malic acid concentration of less than 20 mg/g dry mass basis - such as between about 10 and 20 mg/g dry mass basis, a glucose concentration of less than 1 mg/g dry mass basis, a fructose concentration of less than 40 mg/g dry mass basis, a sucrose concentration of less than 1 mg/g dry mass basis and a malonic acid concentration of less than 80 mg/g dry mass basis.

Once prepared, the fermented tobacco stem materials described herein may be processed in a similar fashion as unfermented tobacco stems to produce tobacco filler suitable for cigarettes and other tobacco products. The fermented tobacco stem materials may be mixed with unfermented tobacco stem materials to produce cut filler.

The fermented tobacco stem materials described herein may be further blended with materials derived from other types of tobaccos such as flue-cured tobacco, Burley tobacco, Maryland tobacco, Oriental tobacco, rare tobacco, specialty tobacco, expanded tobacco and the like. The precise amount of each type of tobacco within a tobacco blend used for the manufacture of a particular cigarette brand varies from brand to brand. See, for example, Tobacco Encyclopaedia, Voges (Ed.) p. 44-45 (1984), Browne, The Design of Cigarettes, 3rd Ed., p.43 (1990) and Tobacco Production, Chemistry and Technology, Davis et al. (Eds.) p. 346 (1999).

The fermented tobacco stem material can be combined with tobacco lamina to produce a blend for use in or as cut filler with at least 2 % by weight of the fermented stem material, more preferably at least 5 % by weight of the fermented stem material, still more preferably at least 6 % by weight of the fermented tobacco stem material. Due to the improved taste of the fermented tobacco stem material, significantly higher proportions of tobacco stem can be included in the blend while still providing an acceptable blend for use in cut filler. The proportion of fermented tobacco stem material in the blend may exceed 50 %. However, particularly preferably the blend contains between 2 % and 40 % by weight of the fermented tobacco stem material, more preferably between 6 % and 20 % by weight and still more preferably between 2 % and 20 % by weight. The blending of the fermented tobacco stem material with the other types of tobacco material may take place prior to the cutting and drying steps, so that the final blend is cut and dried together in the same batch. Alternatively, the blending may take place after the cutting and drying have been carried out on the Burley tobacco stems, so that the blending is the final step in the production of the cut filler.

Accordingly, in a further aspect, the present invention provides a tobacco product comprising a fermented tobacco stem material in accordance with the present invention. The term “tobacco product” encompasses compositions comprising or consisting essentially of the dried fermented tobacco stem material obtained by the processes of the invention, including tobacco blends and reconstituted tobacco sheet material. The term “tobacco product” includes without limitation smoking articles or smokable articles and smokeless tobacco products, including non-combustible products, heated products, and aerosol-generating products. Non-limiting examples of smoking or smokable articles include cigarettes, cigarillos, cigars and pipe tobaccos. Suitably, the tobacco product according to this aspect of the invention is a smoking article or smokable article, in particular a cigarette.

The following examples are provided as an illustration and not as a limitation. Unless otherwise indicated, the present invention employs conventional techniques and methods of microbiology and plant biology.

Example 1

Burley tobacco stems were conditioned to a moisture content of 55wt% moisture. The tobacco stems were inoculated with homofermentative lactic acid bacteria at approximately 106 CFU/g (dry basis) of a mixture of Lactobacillus plantarum and Pediococcus acidilactici (Lalsil®. Schaumann GmbH). The inoculated tobacco stems were packed into a container. The container was flushed with inert gas and sealed against oxygen ingress. The tobacco stems were left to ferment at ambient temperature for 14 days. The tobacco stems were then dried to about 20% moisture (w/w). After drying, the tobacco stems were processed for blending and incorporation into tobacco products.

Chemical characteristics of exemplary burley stems before and after fermentation were as follows (percentages by weight dry basis; LOQ = limit of quantitation):

Citric acid: 0.74% before fermentation 0.73% after fermentation

Malic acid: 4.63% before fermentation 1.94% after fermentation

Acetic acid: 0.24% before fermentation 0.74% after fermentation

Lactic acid: 0.03% before fermentation 2.22% after fermentation NH3: 0.11 % before fermentation <LOQ after fermentation

Nitric oxides: 1.48% before fermentation 1.42% after fermentation

Reducing Sugars: <LOQ before fermentation <LOQ after fermentation

Total Alkaloids: 0.70% before fermentation 0.64% after fermentation A change in pH was also observed during the fermentation. The measurement of pH may generally provide a convenient way to monitor the progress of the fermentation. In the fermented Burley tobacco stems, the amount of malic acid was reduced by 58% after fermentation. The amount of lactic acid was increased by about 74 times after fermentation. The ratio of malic acid to lactic acid was 154 before the fermentation and it dropped to 0.87 after the fermentation.

While the embodiments described above have been described in detail, it will be apparent to those skilled in the art that various changes and modifications can be made, and equivalents employed, without departing from the scope of the appended claims. All features and modifications that are disclosed in relation to any particular aspect of the invention may also be present as optional or preferred features for any other aspect of the invention. Any publication cited or described herein provides relevant information disclosed prior to the filing date of the present application. Statements herein are not to be construed as an admission that the inventors are not entitled to antedate such disclosures. All publications mentioned in the above specification are herein incorporated by reference. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are apparent to those skilled in tobacco manufacturing, biochemical engineering, material sciences or related fields are intended to be within the scope of the following claims.

Claims

1. 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.

2. The tobacco product according to claim 1, wherein the method further comprises, after the steps of processing and fermenting, the step of (c) drying the processed fermented tobacco stem material.

3. The tobacco product according to any preceding claim, wherein after the processing step of the method, the tobacco stem material have a thickness of between about 0.1 mm to about 1.2 mm.

4. The tobacco product according to any preceding claim, wherein the processing step of the method comprises conditioning the tobacco stem material such that the moisture content is greater than 20%.

5. The tobacco product according to any preceding claim, wherein the processing step of the method comprises rolling or flaking the tobacco stem material.

6. The tobacco product according to any preceding claim, wherein the processing step of the method comprises subjecting the tobacco stem material to extrusion in a twin screw extruder or refining in a disc refiner.

7. The tobacco product according to any preceding claim, wherein the fermenting step of the method comprises inoculating or contacting the tobacco stem material with one or more homolactic acid bacteria to ferment the tobacco stem material.

8. The tobacco product according to any preceding claim, wherein the fermenting step of the method comprises inoculating or contacting the tobacco stem material with bacteria from the genus Lactobacillus, Pediococcus, or both, to ferment the tobacco stem material.

9. The tobacco product according to any preceding claim, wherein after the fermenting step of the method, the amount of malic acid in the tobacco stem material has been reduced by more than 75%, and the amount of lactic acid has been increased to at least about 2% to about 5% dry weight of the tobacco stem material.

10. A method for the production of a tobacco product comprising tobacco stem material, said 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.

11. The method according to claim 10, wherein the tobacco stem material is fermented for between about six days and about eight days at a temperature of between about 20 °C and about 40 °C, suitably at about 30 °C.

12. The method according to claim 10 or 11, further comprising measuring the amount of malic acid in said tobacco stem material during said fermentation step, and terminating said fermentation step when the amount of malic acid has been reduced by more than about 75%.

13. The method according to any of claims 10 to 12, wherein the fermenting step is performed under microaerophilic conditions or anaerobic conditions.

14. The method according to any of claims 10 to 13, wherein one or more enzymes having cellulose or cellobiose hydrolase activity are added prior to the fermenting step.

15. The method according to any of claims 10 to 14, wherein the method further comprises measuring the level of malic acid, lactic acid and/or pH during said fermentation and continuing said fermentation until the level of lactic acid, malic acid, pH, and/or the ratio of lactic acid to malic acid reaches a predetermined value.