GB2548543A - Fermentation method for tobacco slurry - Google Patents

Abstract

A method of treating a slurry of tobacco material comprising the steps: providing a slurry of tobacco material; in the presence of homolactic acid bacteria and limited oxygen, fermenting the slurry of tobacco material; and obtaining a slurry of fermented tobacco material in which the lactic acid content therein is increased and the malic acid content therein is decreased. The slurry of tobacco is fermented between 20ºC and 40ºC and is performed under microaerophilic or anaerobic conditions and the homolactic acid bacteria would comprise members of the genus Lactobacillus and Pediococcus. One or more enzymes that have cellulose or cellobiose hydrolase activity are added prior or during the fermentation step. Also provided are methods of forming reconstituted tobacco sheets from the fermented slurry.

Description

FERMENTATION METHOD FOR TOBACCO SLURRY

The present invention relates, in general, to methods, compositions and uses for improving the properties of tobacco material for use in the tobacco industry.

During the production and processing of tobacco products, tobacco by-products - such as tobacco stems, leaf scraps, and tobacco dust - produced during the manufacturing process (i.e., stemming, aging, blending, cutting, drying, cooling, screening, shaping and packaging) can be recycled to reclaim their useful tobacco content. In the past, such tobacco byproducts have been formed into what is known in the industry as reconstituted tobacco. The reconstituted tobacco can be formed into sheets which may be cut in a similar fashion as whole leaf tobacco to produce tobacco filler suitable for cigarettes and other smoking articles.

In some instances it is desirable to reduce or avoid the use of chemical additives - such as flavours - in tobacco products. In some situations, the chemical additive may be banned by government regulation. However, avoiding the use of additives can have a negative impact on the tobacco products, for example, the sensory properties.

The present invention seeks to address the problem of how to improve the properties, for example, the sensory properties of tobacco, whilst reducing or avoiding the use of one or more additives in the tobacco.

This present invention relates, in one aspect, to a process for improving the sensory qualities of tobacco by a bacterial fermentation process. In this process, lactic acid is produced in the tobacco slurry by the conversion of fermentable sugars (such as glucose), and organic acids (such as malic acid) that are naturally present in tobacco into lactic acid by bacterial fermentation.

The biological fermentation is suitably homolactic fermentation meaning that lactic acid is the main or substantially the only fermentation product. The biological fermentation can also include malolactic fermentation meaning that malic acid is converted into lactic acid and C02. Through this process, the level of organic acids in the tobacco can be modulated. Specifically, the level of lactic acid can be increased. The level of malic acid can be decreased. In addition, the water retention capability of tobacco can be improved by the in-situ formation of lactic acid which is a naturally occurring humectant. In addition, the stability of tobacco 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 material without the formation of alcohols or acetic acid which would otherwise detract from the sensory properties of the tobacco.

In one aspect, there is provided 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 provided in step (a).

Suitably, the method further comprises measuring the levels of lactic acid and/or malic acid in the slurry before, during, and/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 reach a predetermined value. Alternatively or additionally, the pH of the slurry may be monitored during the fermentation. In embodiments, the fermentation is continued until the pH of the slurry reaches a predetermined value.

Suitably, the slurry of tobacco material has a dry matter content of between about 10% and about 30%.

Suitably, the method of treating the slurry of tobacco material improves the sensory properties thereof.

Suitably, step (b) comprises inoculating or contacting the slurry of tobacco material with one or more homolactic acid bacteria to ferment the slurry of tobacco material.

Suitably, the slurry of tobacco material is fermented for between about 24 hours and 48 hours. Suitably, the slurry of tobacco is fermented at a temperature of between about 20 °C and about 45 °C, preferably between about 30 °C and about 40 °C.

Suitably, step (b) is performed under microaerophilic or anaerobic conditions.

Suitably, one or more enzymes are added prior to step (b) or during step (b) or a combination thereof.

Suitably, the one or more enzymes have cellulolytic activity, suitably, wherein said cellulolytic activity comprises cellulase activity, cellobiose hydrolase activity, or a combination thereof

Suitably the bacteria are homolactic acid bacteria. Suitably, the homolactic acid bacteria are members of the genus Lactobacillus or Pediococcus. Suitably the bacteria are lactic acid bacteria or homolactic acid bacteria that are obtainable or obtained from natural or fermented tobacco plant materials. Suitably, the lactic acid bacteria are Lactobacillus crispatus, Lactobacillus plantarum, Pediococcus pentosaceus, Pediococcus acidilactici, or a combination of two or more of the foregoing.

Suitably, the tobacco material includes tobacco by-products of cigarette manufacturing, including but not limited to tobacco dust, tobacco stem powder, tobacco stalk powder or a mixture of two or more of the foregoing. Typically, a mixture of such materials of various origins are used. The origin of the tobacco material can be any type of tobacco, including but not limited to Virginia tobacco, Burley tobacco, or Oriental tobacco. Suitably, the tobacco has been cured, including flue-cured tobacco, air-cured tobacco, fire-cured tobacco, or sun-cured tobacco.

Suitably, the bacteria produce fermentation products that consist essentially 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.

In a further aspect, there is disclosed a slurry of fermented tobacco material obtained or obtainable by the present method.

In a further aspect, there is provided a method of forming a reconstituted tobacco sheet comprising the steps of: (a) treating a slurry of tobacco material as described herein to obtain a fermenting or fermented tobacco slurry; (b) adding one or more binding agents, flavorings, humectants, and cellulosic fibers, or a combination of at least two of the foregoing additives to the fermenting or fermented tobacco slurry, wherein steps (a) and (b) are carried out in any order; followed by (c) casting and drying the fermenting or fermented tobacco slurry to obtain a reconstituted tobacco sheet.

In a further aspect, there is disclosed a reconstituted tobacco sheet obtained or obtainable by the present method.

In a further aspect, there is provided a method of forming a tobacco product comprising the steps of: (a) preparing a reconstituted tobacco sheet according to the method described herein; (b) cutting the reconstituted tobacco sheet into cut filler; and (c) incorporating the cut filler into a tobacco product.

In a further aspect, there is disclosed a tobacco product obtained or obtainable by the present method.

In a further aspect, there is provided a slurry of tobacco material having a dry matter content of between about 10% and 30% (w/v) tobacco and comprising one or more homolactic acid bacteria and having a malic acid concentration of less than 25 mg/g dry mass of tobacco. Suitably, the slurry according to this aspect is obtained or obtainable by a method according to the first aspect of the invention.

In a further aspect, there is provided the use of a slurry of tobacco material having a dry matter content of between about 10% and 30% (w/v) tobacco and comprising one or more homolactic acid bacteria and having a malic acid concentration of less than 25 mg/g dry mass of tobacco for improving the sensory properties or for increasing the water retention capability of tobacco or for reducing the spoilage of tobacco. Suitably, the slurry according to this aspect is obtained or obtainable by a method according to the first aspect of the invention.

In certain embodiments according to any of the above aspects, the slurry of tobacco material can have lactic acid in the amount of about 50 to about 100 mg/g, about 75 to about 125 mg/g, about 100 to about 200 mg/g, about 150 to about 225 mg/g and up to about 250 mg/g dry mass of tobacco.

The invention will now be described in more detail with reference to the accompanying drawings, in which:

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.

In one aspect, there is provided a method of treating a slurry of tobacco material comprising the steps of: (a) preparing a suspension or a slurry of tobacco material; (b) in the presence of homolactic acid bacteria and limited oxygen, fermenting the slurry of tobacco material; (c) optionally measuring the levels of lactic acid and malic acid in the slurry of tobacco material; and (d) 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).

In the first step of the disclosed method, tobacco materials and water are mixed to form a tobacco slurry. The tobacco materials used to make the slurry can include tobacco leaf scraps, tobacco stems, tobacco dust, ground tobacco, or tobacco leaf prime lamina strip. Combinations of these material can be used. Tobacco materials can include cellulosic fibers of different origins to improve the mechanical strength of the final product. The term "tobacco slurry" refers to a suspension of a tobacco homogenate in an aqueous solution, for example without limitation, in water alone. The slurry can have a dry matter content of about 5% (w/v), about 10% (w/v), about 15% (w/v), about 20% (w/v), about 25% (w/v) or about 30% (w/v) comprising a mixture of tobacco material (for example, tobacco homogenate) in an aqueous solution. Suitably, the slurry has a dry matter content of between about 10% (w/v) to about 30% (w/v). Suitably, the slurry is mixed in order to ensure homogeneity.

The tobacco slurry is then treated in order to convert one or more fermentable sugars (for example, glucose) and one or more organic acids (for example, malic acid) that are naturally present in the tobacco slurry into lactic acid. Suitably this conversion is achieved by bacterial fermentation. The bacteria used are lactic acid bacteria (LAB). The LAB comprise a clade of Gram-positive, 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, Carnobactehum, 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 C02 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. 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 lindneri, 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 parabuchrteri, Lactobacillus paracollinoides, Lactobacillus parafarraginis, Lactobacillus parakefiri, Lactobacillus paralimentarius, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus perolens, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus psittaci, Lactobacillus rennini, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus rimae, Lactobacillus rogosae, Lactobacillus rossiae, Lactobacillus ruminis, Lactobacillus saerimneri, Lactobacillus sakei, Lactobacillus salivarius, Lactobacillus sanfranciscertsis, 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 slurry of tobacco material is fermented in the presence of one or more homolactic acid bacteria and limited oxygen. The fermentation can be carried out in a suitable fermentation vessel - such as a bioreactor - that is well known to the person skilled in the art. The fermentation vessel may be formed of plastic, metal, and/or other materials. The fermentation vessel may include one or more coatings. The coating may inhibit corrosion and/or facilitate removal of solids. The footprint of the vessel may be substantially square, substantially circular, substantially oval, substantially rectangular, and/or irregularly shaped. The vessel may have a shape configured to minimise stagnant regions in the bioreactor. In certain embodiments, the shape of the inner surface of the vessel may minimise stagnant regions in the vessel during mixing. The inner surfaces of the vessel may be rounded instead of meeting at an edge. For example, the inner surface of the vessel may have a shape substantially similar to an oval or a circle to minimize the presence of stagnant regions in the vessel, during use. The vessel may include one or more stirrers to agitate the contents. One or more stirrers may be positioned to reduce dead mixing zones in the vessel. 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; pH; flow rates, volume 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 and/or modify parameters of the system continuously or periodically. The vessel can be sealed such that atmospheric oxygen cannot enter. The vessel can also be equipped with a valve to depressurize.

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 slurry. However, if no additional homolactic acid bacteria are added to the slurry then a longer fermentation time will be required, and there is a greater risk of spoilage or unwanted microbial actions.

Suitably, therefore the tobacco slurry is contacted with or inoculated with exogenous homolactic acid bacteria since this will shorten the lag phase and effectively decrease the fermentation time by about 50%, for example from about 40 hours to about 24 hours. As used herein the term “exogenous homolactic acid bacteria” refers to homolactic acid bacteria that are supplied by a source external to the tobacco slurry as opposed to homolactic acid bacteria bacteria that are naturally occurring in the tobacco slurry. 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 slurry may be the same genera or species of homolactic acid bacteria that naturally occur in the tobacco slurry such that the exogenous addition of the bacteria will increase the quantities thereof in the tobacco slurry and thereby shorten fermentation times. The homolactic acid bacteria that are used to contact or inoculate the tobacco slurry may be a different genera or species of homolactic acid bacteria than those that naturally occur in the tobacco slurry.

It is also contemplated that homolactic acid bacteria can be used 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 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. A specific example of such a composition is Bonsilage (Schaumann GmbH). Suitably, the tobacco slurry is inoculated with the fermentation bacteria in amounts of from about 104 to about 10s, for example about 105 to about 107 or about 106 to about 107 colony forming units (cfu)/gram.

The tobacco slurry can be fermented under conditions of gentle stirring which may be facilitated by manual or automated means. 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.

As discussed, the fermentation process occurs under conditions of limited 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 sealed conditions then the fermentation process will eventually become substantially anaerobic or even anaerobic as oxygen becomes depleted over time and C02 resulting from the conversion of malic acid into lactic acid is released. In certain embodiments, it is preferred that the tobacco slurry is contacted with the LAB under substantially anaerobic or microaerophilic or anaerobic conditions. When anaerobic conditions are used, no increase in suspension temperature is generally observed during the fermentation.

As used herein, the term “limited oxygen” means that the oxygen levels 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. The term “oxygen level” refers to the partial pressure of the oxygen, i.e. the chemical activity of the 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 oxygen.

Limited oxygen conditions can be achieved in various ways. By way of example, this 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. Anaerobic conditions can also be achieved by minimising the stirring in the fermentation vessel to reduce or prevent oxygen from being incorporated into the tobacco slurry. In this case, 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 20°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 about 40°C, or at about 37°C.

Suitably, the fermentation is conducted for between about 20 and about 60 hours, between about 24 and about 48 hours, or between about 24 and about 40 hours or until such times as when sufficient malic acid is consumed, or when sufficient lactic acid is generated, in the tobacco slurry.

The following is an exemplary set of changes that can be observed with in the tobacco slurry after the fermentation. The amount of lactic acid increases during the fermentation and after about 40 hours fermentation. The amount of malic acid decreases during the fermentation and after about 40 hours fermentation. The amount of succinic acid increases during the fermentation and after about 40 hours fermentation. The amount of glucose decreases during the fermentation and after about 40 hours fermentation. The amount of fructose decreases during the fermentation and after about 40 hours fermentation. The amount of sucrose decreases during the fermentation and after about 40 hours fermentation. The amount of malonic acid decreases during the fermentation and after about 40 hours fermentation. The amount of citric acid generally decreases during the fermentation and after about 40 hours fermentation.

The amount of lactic acid generated after fermentation, for example after about 40 hours of fermentation, depending on the type of tobacco used in the slurry, can be greater than or equal to 50 mg/g dry mass tobacco, greater than or equal to 100 mg/g dry mass tobacco, greater than or equal to 150 mg/g dry mass tobacco, greater than or equal to 200 mg/g dry mass tobacco, or greater than or equal to 250 mg/g dry mass tobacco - such as greater than or equal to 152 mg/g dry mass tobacco or greater than or equal to 159 mg/g dry mass tobacco. Other changes also occur. For example, the amount of malic acid after fermentation, for example after about 40 hours of fermentation, can be reduced to less than 40 mg/g, less than 30 mg/g, less than 25 mg/g, less than 20 mg/g, or less than 10mg/g dry mass tobacco. For example, the initial amount of malic acid present in the slurry can vary from about 100 mg/g to about 50 mg/g. The amount of acetic acid after fermentation, for example after about 40 hours of fermentation, is generally no greater than 5 mg/g dry mass tobacco. The amount of succinic acid after fermentation, for example after about 40 hours of fermentation, can increase from about 1 mg/g dry mass tobacco to greater than 3 mg/g dry mass tobacco. The amount of glucose after fermentation, for example after about 40 hours of fermentation, can decrease from about 60 mg/g dry mass tobacco to 1 mg/g or less dry mass tobacco. The amount of fructose after fermentation, for example after about 40 hours of fermentation, can decrease from about 85 mg/g dry mass tobacco to less than 40 mg/g dry mass tobacco. The amount of sucrose after fermentation, for example after about 40 hours of fermentation, can decrease from about 1 mg/g dry mass tobacco to less than 1 mg/g dry mass tobacco. The amount of malonic acid after fermentation, for example after about 40 hours of fermentation, can decrease from about 92 mg/g dry mass tobacco to less than 20 mg/g dry mass tobacco. The amount of citric acid after fermentation, for example after about 40 hours of fermentation, can decrease from about 7 mg/g dry mass tobacco to less than 7 mg/g dry mass tobacco - such as 4 mg/g dry mass tobacco.

In various exemplary embodiments, the fermentation process can result in the following changes in the tobacco slurry: lactic acid is increased from about 1 mg/g dry mass tobacco up to about 200 mg/g dry mass tobacco; malic acid is depleted by at least 70% or from about 70% to about 80% compared to the level in the starting tobacco slurry; glucose is depleted by about 80%, compared to the level in the unfermented tobacco slurry and fructose is decreased by about 20% compared to the level in the unfermented tobacco slurry.

There is also disclosed a (fermenting or fermented) slurry of tobacco material having a dry matter content of between about 10% and 30% (w/v) tobacco, one or more homolactic acid bacteria, increased levels of lactic acid and decreased levels of malic acid as compared to a non-fermenting or non-fermented slurry of tobacco material. In one aspect, a slurry of tobacco material having a dry matter content of between about 10% and 30% (w/v) tobacco and comprising one or more homolactic acid bacteria and having a lactic acid concentration of at least about 150 mg/g dry mass of tobacco and a malic acid concentration of less than 25 mg/g dry mass of tobacco is disclosed. The lactic acid concentration can be between about 150 and 160 mg/g dry mass of tobacco and the malic acid concentration can be between about 15 and 20 mg/g dry mass of tobacco. The tobacco slurry can have a glucose concentration of less than 1 mg/g dry mass tobacco. The tobacco slurry can have a fructose concentration of less than 40 mg/g dry mass tobacco. The fructose concentration can be between 35 and 40 mg/g dry mass tobacco. The tobacco slurry can have a sucrose concentration of less than 1 mg/g dry mass tobacco. The tobacco slurry can have a malonic acid concentration of less than 80 mg/g dry mass tobacco. The malonic acid concentration can be between about 60 and 80 mg/g dry mass tobacco - such as between about 65 and 75 mg/g dry mass tobacco.

In one embodiment, the tobacco slurry comprises a lactic acid concentration of at least about 150 mg/g dry mass of tobacco - such as between about 150 and 160 mg/g dry mass of tobacco, a malic acid concentration of less than 20 mg/g dry mass of tobacco - such as between about 15 and 20 mg/g dry mass of tobacco, a glucose concentration of less than 1 mg/g dry mass tobacco, a fructose concentration of less than 40 mg/g dry mass tobacco -such as between 35 and 40 mg/g dry mass tobacco, a sucrose concentration of less than 1 mg/g dry mass tobacco and a malonic acid concentration of less than 80 mg/g dry mass tobacco - such as between about 60 and 80 mg/g dry mass tobacco or between about 65 and 75 mg/g dry mass tobacco. Suitably, the slurry is obtained or obtainable by the method of the invention.

Either before or at the end of the fermentation process, one or more further components may be added to the tobacco slurry. These components may be added at the same levels or at lower levels than would otherwise be added if the tobacco slurry had not been 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, organic and inorganic fillers (for example, grains, processed grains, puffed grains, maltodextrin, dextrose, calcium carbonate, calcium phosphate, corn starch, invert sugar, lactose, manitol, 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), cellulosic fibers from stem, stalks and woody parts of plants, 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 exogenously added lactic acid - are added at reduced levels or are omitted altogether. As used herein the term “exogenously added lactic acid” refers to lactic acid from an external source that is added to the tobacco as opposed to being a naturally occurring lactic acid that is formed in the tobacco slurry. Thus, the exogenously added lactic acid may be, for example, chemically synthesized 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 slurry that has not been treated using the methods of the present disclosure. In one embodiment, the tobacco slurry or the tobacco slurry 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. 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 slurry at a level ranging from 2-6% (w/w dry weight basis) of binding agent, and from 0 % to 5%, 2% to 10%, 5% to 15% or 0% to 20% (w/w dry weight basis) of humectant.

Cellulosic fibers that may be added to the slurry as a pulp suspension comprising cellulosic fibers. The fibers can be generated by a variety of mechanical methods well known in the art. For example, stem, such as tobacco stem, may be fibrillated, extruded or refined to produce cellulosic fibers that are pliable, have greater surface area and a length of less than about 1200 micrometers. For example, stems can be processed in a twin screw extruder or a disc refiner to obtain a pulp suspension comprising cellulosic fibers having a length of less than about 1000 micrometers or a length from about 300 micrometers to about 600 micrometers. Suitably, the stem material is be processed in an extruder or a refiner to obtain a pulp suspension having a freeness (drainability) of at least about 30 degrees Schopper-Riegler, or at least about 50 degrees Schopper-Riegler. The term “freeness” refers to the drainability of a suspension or pulp product. The “freeness” is defined by International Standard ISO 5267-1 entitled: Determination of Drainability - Part 1: Schopper-Riegler Method. The Schopper-Riegler test is designed to provide a measure of the rate at which a dilute suspension of pulp may be dewatered. In specific embodiments, the cellulosic fibers are produced in a twin screw extruder wherein different sections of the extruder may be operated at different temperatures ranging from at least 80°C to about 110°C. In specific embodiments, the cellulosic fibers are produced in a disc refiner wherein the discs are separated by gaps ranging from at least 50 micrometers to about 500 micrometers.

The tobacco slurry can be subjected to a homogenisation step using a high shear mixer for sufficient time to disperse and hydrate the additive(s). The step of casting the tobacco slurry may be performed using any of the casting or paper making processes that are known in the art. By way of example, casting processes are described in US 5,724,998 and US 5,584,306; paper-making processes are described in US 4,341,228; US 5,584,306 and US 6,216,706. Casting processes typically include casting the slurry onto a continuous stainless steel belt, drying the cast slurry to form a reconstituted tobacco sheet and removing said sheet. Paper-making processes typically include casting the aqueous slurry from a head box onto a wire screen for forming the desired sheet. The aqueous slurry may be separated into a soluble portion and a fibrous portion. Water is drained from the fibrous portion and a sheet is formed which is subsequently treated and dried. The tobacco slurry can be dried to reach final a moisture content of about 10-14% dry matter.

In other known methods, the tobacco slurry is subjected to a step in which the non-soluble portion of the tobacco slurry is separated from the liquid phase by any technique known in the art. By way of example, the slurry may be compressed or centrifuged to remove the tobacco-flavoured liquid phase containing the water-soluble components. The non-soluble portion is then subjected to a paper-making process, for example, by using a Fourdrinier machine, to form a base web. As is known, a Fourdrinier machine typically includes a forming section, a press section and a drying section. In the forming section, which comprises a conveyor belt made with plastic fabric mesh, often referred to as a “wire”, as it was once woven from bronze, the pulp is drained to create a continuous paper web. Subsequently, this wet web is fed onwards to the press section, where the excess water is squeezed out of the web. Finally, the pressed web is conveyed onto a heated drying section. Separately, the tobacco-flavoured liquid is subjected to an evaporation process to form a concentrated liquor, which is then added back to the base web in order to restore at least partially the original flavours that would otherwise be lost.

In a further aspect, a method of forming a reconstituted tobacco sheet is disclosed comprising the steps of: (a) treating a slurry of tobacco material as described herein to obtain a fermented tobacco slurry; (b) adding one or more binding agents or one or more humectants or a combination thereof to the fermented tobacco slurry, wherein steps (a) and (b) are carried out in any order; followed by (c) casting and drying the fermented tobacco slurry to obtain a reconstituted tobacco sheet. A reconstituted tobacco sheet obtained or obtainable by this method is also disclosed.

In a variation of the process described herein, one or more enzymes or enzyme preparations can be added to or contacted with the tobacco slurry during the method of the present disclosure. For example, one or more enzymes or enzyme preparations can be added to or contacted with the tobacco slurry 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 slurry 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 cellulase (endo- and exo-) and cellobiose hydrolase.Methods well known in the art may be applied to inactivate the enzyme after the treatment, thus the method may further comprise a step of inactivating any added enzymes. A method of forming a tobacco product is provided comprising the steps of: (a) preparing a reconstituted tobacco sheet as described herein; (b) cutting the reconstituted tobacco sheet into cut filler; and (c) incorporating the cut filler into a tobacco product. A tobacco product obtained or obtainable by this method is also disclosed. The tobacco - such as tobacco sheets - produced can be cut into tobacco shreds similar to cut filler and incorporated into tobacco products. 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. Non-limiting examples of smokeless tobacco products include chewing tobaccos, snuffs, and substrates for use in aerosol-generating products. Smokeless tobacco products may comprise tobacco in any form, including as dried particles, shreds, granules, powders, or a slurry, deposited on, mixed in, surrounded by, or otherwise combined with other ingredients in any format, such as flakes, films, tabs, foams, or beads. Liquid contents of smokeless tobacco products can be contained in a device or enclosed in a form, such as beads, to preclude interaction with a water-soluble wrapper. The wrapper may be shaped as a pouch to partially or completely enclose tobacco-incorporating compositions, or to function as an adhesive to hold together a plurality of tabs, beads, or flakes of tobacco. Exemplary materials for constructing a wrapper include film compositions comprising HPMC, CMC, pectin, alginates, pullulan, and other commercially viable, edible film-forming polymers. Other wrapping materials may include pre-formed capsules produced from gelatin, HPMC, starch/carrageenan, or other commercially available materials. Such wrapping materials may include tobacco as an ingredient. Wrappers that are not orally disintegrable may be composed of woven or nonwoven fabrics, of coated or uncoated paper, or of perforated or otherwise porous plastic films. Wrappers may incorporate flavouring or colouring agents. Smokeless products can be assembled together with a wrapper utilizing any method known to persons skilled in the art of commercial packaging, including methods such as blister packing, in which a small package can be formed by a vertical form/fill/seal packaging machine.

Once prepared, the reconstituted tobacco sheets described herein may be cut in a similar fashion as whole leaf tobacco to produce tobacco filler suitable for cigarettes and other tobacco products. The reconstituted tobacco sheets described herein may be further trashed or flayed with mechanical fingers into sized pieces similar to natural tobacco lamina strips or cut into diamond shaped pieces, between about 50 to 100 mm on a side. The reconstituted tobacco sheet pieces described herein may be further blended with other 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 entire blend may then be shredded into a cut filler and incorporated into a tobacco product. Accordingly, methods are provided for making a tobacco product comprising tobacco homogenate (for example, reconstituted tobacco sheet) with reduced amounts of tobacco specific nitrosamines.

As discussed herein, the level of at least lactic acid can be increased and the level of malic acid can be decreased. In addition, the water retention capability of tobacco can be improved by the in-situ formation of lactic acid which is a naturally occurring humectant. In addition, the stability of tobacco against spoilage by molds and other undesirable bacteria can be improved through the use of lactic acid bacteria.

In one further aspect, there is provided a method for increasing the water retention capability of tobacco comprising the steps of: (a) preparing a slurry of tobacco material; (b) in the presence of homolactic acid bacteria and limited oxygen, fermenting the slurry of tobacco material; (c) optionally measuring the levels of lactic acid in the slurry of tobacco material; and (d) obtaining a slurry of fermented tobacco material in which the lactic acid content therein is increased and the water retention capability of the tobacco material has increased as compared to the slurry of tobacco material prepared in step (a).

In a further aspect, there is disclosed a slurry of fermented tobacco material obtained or obtainable by any method according to the present invention.

In one still further aspect, there is provided a method for reducing the spoilage of tobacco (for example, by fungi, bacteria or the like) comprising the steps of: (a) preparing a slurry of tobacco material; (b) in the presence of homolactic acid bacteria and limited oxygen, fermenting the slurry of tobacco material; (c) optionally measuring the levels of lactic acid in the slurry of tobacco material; and (d) obtaining a slurry of fermented tobacco material in which the lactic acid content therein is increased as compared to the slurry of tobacco material prepared in step (a).

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. In particular, it will be noted that any feature or element that is disclosed in relation to any one aspect may also be present in any other aspect of the invention.

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

The following example demonstrates that high levels of lactic acid were produced in slurry made with cured Virginia tobacco by anaerobic fermentation using lactic acid bacteria (LAB). Lactic acid was the major fermentation product in fermented tobacco, with yields of 140 to 200 g/kg dry matter. Two different types of fermentation appeared to occur during the fermentation of tobacco, namely lactic acid fermentation with glucose and fructose as substrates and malolactic fermentation with malic acid as substrate. With the exception of carbon dioxide, which was formed in the conversion of malic acid to lactic acid, only minor amounts of other fermentation products were formed. Cultures of many different Lactobacillus species were demonstrated as being suitable for fermentation of tobacco. Although the results described below were obtained using Lactobacillus cultures isolated from natural sources, the following freeze-dried commercial cultures were also used successfully: Ecosyl® 100 (Ecosyl Products, Stokesley UK); freeze-dried culture of L. plantarum MTD/1 (NCIMB 40027); Bonsilage® (Schaumann, Pinneberg, Germany); 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).

Tobacco slurry was prepared by suspending ground tobacco in water to 20% (w/w), equivalent to a moisture level of 82%. Freshly prepared slurry (150 ml in one set of experiments, and 2.5 I in a second set of experiments) was inoculated with concentrated LAB culture suspensions in peptone physiological salt solution to a concentration of 5x106 colony forming units (cfu)/g and subsequently incubated in a water bath at 37°C. The pH of the slurries was determined continuously by means of pH probes inserted in the slurry and coupled to a Cinac® multi-channel pH-meter (Unity Scientific, Brookfield, UK). The moisture level and water activity of tobacco samples were determined with a Kern DBS infrared moisture analyser (sample size approximately 2 g) and a Aqualab/Decagon 3TE Water activity meter (sample size approximately 1 g), respectively.

Table 1 shows the concentrations of sugars and organic acids in the 20 selected fermented slurry samples and a control sample of slurry incubated without added culture after 40 h incubation, as well as in the unfermented ground tobacco that was used as substrate. Lactic acid was the major organic acid in all fermented slurry samples, including the uninoculated control slurry, with an average concentration of 177 g/kg dry matter. Low amounts of acetic acid and succinic acid were detected (acetic acid in all samples, succinic in some samples). A low amount of citric acid, equal to or less than the amount in unfermented tobacco, was detected. Other organic acids and alcohols that were analysed (formic acid, propionic acid, butyric acid, pyruvic acid, oxaloic acid, ethanol, 1-propanol and 2-propanol) were below the detection limit.

Glucose was completely or almost completely used during fermentation, whereas, on average, 70% of fructose was used (range 18% to 98%). Also malic acid and malonic acid were partly degraded during fermentation (on average about 75%). Conversion of malic acid into lactic acid and C02 is a known fermentation pathway in various LAB, known as the malolactic fermentation. For malonic acid no general fermentation pathway by LAB is known. Based on the data of Table 1, fermentation of glucose and fructose could account for about 65% of the amount of lactic acid formed. Assuming a mixed type of fermentation with both homolactic fermentation (using glucose and fructose) and malolactic fermentation (using malic acid), on average 108% (range 90% to 151%) of the amount of lactic acid could be accounted for. Based on these results we consider it likely that malic acid was, at least partly, used as a substrate for lactic acid production by most of the tested LAB strains. It is notable that slurries inoculated with heterofermentative LAB (L brevis, L. fructivorans, L. kimchi and W. confusa) did not show a heterolactic fermentation profile, i.e. a profile with acetic acid or ethanol as major products. Based on these results, it is reasonable to conclude that LAB can be used generally (even including heterofermentative LAB species) to produce the desired fermentation products.

Table 1: Concentration (g/kg dry mass) of glucose and organic acids after 40 hours of fermentation, and pH after 24 and 40 hours

Example 2

This example demonstrates that lactic acid bacteria can be obtained from fermented cut cured Virginia tobacco. Prior to fermentation, the moisture content of the material was increased to 60%. The tobacco materials were packed in air-tight glass jars, which allowed overpressure to escape, and incubated for 14 or 28 days at ambient temperature. Viable bacteria were isolated and grown in three culture media which differ in degree of selectivity: Brain-Heart Infusion (BHI) is a non-selective medium, MRS a semi-selective medium for lactic acid bacteria (Sigma-Aldrich, J.D. DeMan et al. J. Appl. Bacteriol. 23, 130, (1960)) and Rogosa® SL (Sigma-Aldrich, Journal of Bacteriology, 62(1), 132-133 (1951) a selective medium for lactobacilli. The number of bacteria (in log10cfu/g) present in the 14 and 28-day fermentation were very similar.

Table 2

The results indicate that lactobacilli were the predominant bacterial population. To identify the predominant species occurring in the naturally fermented tobacco silage, five colonies were isolated from plates of each of the three media. All fifteen isolates were identified later as L. plantarum by means of partial sequencing of the 16S rRNA gene.

Example 3

This example illustrates the treatment at a large scale of tobacco slurry. Anaerobic fermentation was conducted as described above with ground tobacco as the substrate, a moisture level of 82%, three different cultures (Bonsilage®, L. crispatus and L. plantarum), an incubation time of 40 h at 37°C and a quantity of 2.5 kg per treatment. Fermented slurry samples were frozen prior to analysis. As shown in Table 3 the slurry samples produced lactic acid at 152 to 159 g/kg dry matter) and contained malic acid below 20 g/kg dry matter.

Table 3 Concentration of sugar and organic acids, and final pH of frozen tobacco slurry made by a large - scale process as described above.

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. Various modifications and variations of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. 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 obvious to those skilled in cellular biology, microbiology and plant biology or related fields are intended to be within the scope of the following claims.

Claims (15)

1. 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).

2. The method according to claim 1, wherein step (b) comprises inoculating or contacting the slurry of tobacco material with one or more homolactic acid bacteria to ferment the slurry of tobacco material.

3. The method according to claim 1 or claim 2, wherein the slurry of tobacco material is fermented for between about 20°C to about 45°C .

4. The method according to any of the preceding claims, wherein step (b) is performed under microaerophilic conditions or anaerobic conditions.

5. The method according to any of the preceding claims, wherein one or more enzymes are added prior to step (b) or during step (b) or a combination thereof.

6. The method according to claim 5, wherein the one or more enzymes have cellulase or cellobiose hydrolase activity.

7. The method according to any of the preceding claims, wherein the homolactic acid bacteria comprise members of the genus Lactobacillus and Pediococcus, suitably, wherein the homolactic acid bacteria comprise Lactobacillus plantarum, Lactobacillus crispatus, Pediococcus acidilactici, Pediococcus pentosaceus or a combination of two or more of the foregoing species.

8. The method according to any of the preceding claims, wherein the homolactic acid bacteria is obtainable or obtained from tobacco.

9. The method according to any of the preceding claims, wherein the tobacco material includes tobacco dust, tobacco stem powder, tobacco stalks powder or a combination thereof.

10. The method according to any of the preceding claims, wherein step (b) comprises fermenting the slurry of tobacco material until the concentration of malic acid is less than 25mg/g dry mass of tobacco.

11. The method according to any of the preceding claims, wherein step (b) comprises fermenting the slurry of tobacco material until the ratio of malic acid concentration to lactic acid concentration is less than about 0.2.

12. A method of forming a reconstituted tobacco sheet comprising the steps of (a) treating a slurry of tobacco material according to any of claims 1 to 11 to obtain a fermented tobacco slurry; and (b) adding one or more binding agents, humectants, flavorings, and cellulosic fibers, or a combination of at least two of the additives, to the tobacco slurry, wherein steps (a) and (b) are carried out in any order; followed by the step of (c) casting and drying the fermented tobacco slurry to obtain a reconstituted tobacco sheet.

13. A method of forming a tobacco product comprising the steps of: (a) preparing a reconstituted tobacco sheet according to the method of claim 12; (b) cutting the reconstituted tobacco sheet into cut filler; and (c) incorporating the cut filler into a tobacco product.

14. A reconstituted tobacco sheet comprising fermented tobacco made according to claim 12.

15. Use of a slurry of fermented tobacco material having a dry matter content of between about 10% and 30% (w/v) tobacco and comprising one or more homolactic acid bacteria and having a malic acid concentration of less than 25 mg/g dry mass of tobacco for improving the sensory properties of tobacco or for increasing the water retention capability of tobacco or for reducing the spoilage of tobacco.