WO2014080227A1

WO2014080227A1 - Treatment of tobacco material

Info

Publication number: WO2014080227A1
Application number: PCT/GB2013/053105
Authority: WO
Inventors: Steve Coates; Jin Hu; Gemma CHOPE; Manuela DONETTI; Simon PENSON
Original assignee: British American Tobacco Investments Ltd IFI; British American Tobacco Co Ltd
Current assignee: British American Tobacco Investments Ltd IFI
Priority date: 2012-11-26
Filing date: 2013-11-25
Publication date: 2014-05-30
Anticipated expiration: 2015-05-26
Also published as: GB201221212D0

Abstract

A method is provided which comprises treating a tobacco material with an aqueous medium, wherein treating the tobacco material with aqueous medium includes the use of an aqueous medium : tobacco material ratio which is greater than or equal to 10 : 1 by weight, and/or adding an aqueous medium to the tobacco material multiple times, optionally in combination with at least one of: the aqueous medium having an alkaline p H; the aqueous medium comprising a surfactant; the aqueous medium having a temperature higher than ambient temperature; and the tobacco material being treated with aqueous medium for longer than or equal to two hours. Also provided is a tobacco material which has been treated by such a method, or a derivative thereof, and a smoking article which comprises a tobacco material treated by such a method.

Description

Treatment of Tobacco Material

Field of the Invention

The present invention relates to a method for the treatment of tobacco material.

Background

In some circumstances, it may be desirable to reduce the content of certain constituents from tobacco material before incorporating the tobacco material into a smoking article such as a cigarette.

Summary

According to a first aspect, there is provided a method for treating a tobacco material, wherein the method comprises treating the tobacco material with aqueous medium, and wherein treating the tobacco material with aqueous medium comprises the use of an aqueous medium : tobacco material ratio which is greater than or equal to 10:1 by weight, and/or adding an aqueous medium to the tobacco material multiple times, optionally in combination with at least one of: the aqueous medium having an alkaline pH; the aqueous medium comprising a surfactant; the aqueous medium having a temperature higher than ambient temperature; and the tobacco material being treated with aqueous medium for longer than or equal to two hours.

In some embodiments, the method comprises treating the tobacco material with aqueous medium, wherein treating the tobacco material with aqueous medium comprises the use of an aqueous medium : tobacco ratio which is greater than or equal to io: l by weight, and/ or adding an aqueous medium to the tobacco material multiple times, optionally in combination with two or more of, three or more of, or all of: the aqueous medium having an alkaline pH, the aqueous medium comprising a surfactant; the aqueous medium having a temperature higher than ambient temperature; and the tobacco material being treated with aqueous medium for longer than or equal to two hours.

In some embodiments, treating the tobacco material with an aqueous medium results in the removal of one or more chemical substances from the tobacco material. In some embodiments, one or more of the removed chemical substances are proteins. In some embodiments, the method of the invention does not substantially reduce the amount of nicotine in the tobacco material.

In some embodiments, the aqueous medium with which the tobacco material is treated comprises the surfactant SDS.

In some embodiments, the aqueous medium with which the tobacco material is treated has a temperature higher than or equal to 50°C. In some embodiments, the tobacco material treated with an aqueous medium is subsequently separated from the aqueous medium.

In some embodiments, the tobacco material is separated from the aqueous medium by filtration and/ or centrifugation.

In some embodiments, the tobacco material is washed with water following treatment with an aqueous medium.

In some embodiments, the method further comprises treating the tobacco material with: one or more enzymes; one or more adsorbents; and/or one or more non-aqueous media.

According to a second aspect, there is provided a tobacco material which has been treated by a method according to the first aspect, or a derivative thereof.

According to a third aspect, there is provided a smoking article which comprises the tobacco material according to the second aspect.

According to a fourth aspect, there is provided a use of an aqueous medium for removing protein from tobacco material, wherein the use involves an aqueous medium : tobacco material ratio which is greater than or equal to 10:1 by weight, and/ or adding an aqueous medium to the tobacco material multiple times

Brief Description of the Drawings

Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which: Figure 1 provides a summary of the four-stage experimental procedure used to determine the optimum set of conditions for extracting protein from cured whole leaf tobacco material.

Figure 2 illustrates the masses of protein which were removed from whole leaf and cut rag tobacco material in some of the experiments carried out in stage 4 of the

experimental procedure

Figure 3 shows the results obtained for the amount of protein extracted as a fraction of the mass of the original tobacco material, in each stage of the four-stage experimental procedure.

Figure 4 provides a graphical illustration of the quantity of protein removed from tobacco material as a fraction of the mass of original tobacco material and as a fraction of the dry mass of original tobacco material for each of the conducted experiments. Figure 5 is a schematic side view of a smoking article including treated tobacco material according to embodiments of the invention.

Detailed Description

There is provided a method of treating a tobacco material with an aqueous medium under certain conditions and under certain combinations of conditions. In the past, methods attempting to remove proteins have been proposed, although they have tended to be complex and expensive.

Tobacco material is commonly treated with aqueous media, and it is known that certain conditions may be favourable during the treatment process. In the current invention, however, there are provided certain combinations of conditions which may,

advantageously, result in the conditions having a synergistic effect and may, therefore, result in a more effective treatment process.

The treatment of a tobacco material with an aqueous medium comprises the use of an aqueous medium : tobacco material ratio greater than or equal to 10:1 by weight, and/ or adding an aqueous medium to the tobacco material multiple times.

The treatment of a tobacco material with an aqueous medium may further comprise at least one of: the aqueous medium having an alkaline pH; the aqueous medium comprising a surfactant; the aqueous medium having a temperature higher than ambient temperature; and the aqueous medium being treated with aqueous medium for longer than or equal to two hours.

The treatment of a tobacco material with an aqueous medium may be applied to any suitable tobacco material. The tobacco material may be derived from any suitable part of any suitable tobacco plant of the plant genus Nicotiana. The tobacco material may then be treated in any suitable way, and may be cured using any suitable method of curing, before being treated with an aqueous medium. In some embodiments the tobacco material treated with an aqueous medium is cured cut rag and/ or cured whole leaf tobacco. Examples of tobaccos which may be treated include, but are not limited to: Virginia, Burley, Maryland, Oriental and Rustica.

The treatment of a tobacco material with an aqueous medium may comprise any suitable steps, and any suitable number of steps, in order to modify the tobacco material in any suitable way. The tobacco material may be incorporated into a smoking article and, in this case, may be modified by the treatment process in order to provide it with the characteristics desirable for a tobacco material used in this way.

In some embodiments, it may be desirable for the treatment with an aqueous medium to modify the tobacco material in any suitable way while at the same time minimally affecting the integrity and/or structure of the tobacco material. In some embodiments, the treatment results in the removal of a large quantity of protein from the tobacco material while at the same time not significantly affecting the integrity and/ or structure of the tobacco material. For example, in embodiments wherein the tobacco material is cured whole leaf tobacco, it may be desirable for the treatment to remove a large quantity of protein from the leaf without significantly affecting the integrity and/or structure of the leaf.

The treatment of a tobacco material with an aqueous medium comprises at least one step in which the tobacco material is treated with an aqueous medium, and may comprise more than one step in which the tobacco material is treated with an aqueous medium. In embodiments wherein there is more than one step in which the tobacco material is treated with an aqueous medium, the same or different aqueous media and the same or different conditions maybe used in each step. Treating the tobacco material with an aqueous medium may be used for the purpose of modifying the tobacco material to remove of one or more chemical substances. In particular, in some embodiments, treatment with an aqueous medium leads to the removal of one or more chemical substances which are considered to be undesirable in the tobacco material in some circumstances. In some embodiments, the treatment leads to the removal of one or more proteins.

In some embodiments, the treatment of tobacco material with an aqueous medium results in a reduction in the protein content of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or at least 95%, based upon the protein content of the untreated tobacco material.

In some embodiments, the treatment of the tobacco material with an aqueous medium results in the extraction of protein in an amount of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or at least 95%, based upon the protein content of the untreated tobacco material.

In some embodiments, the treatment of a tobacco material with an aqueous medium, and in particular the step of treating tobacco material with an aqueous medium, does not lead to the removal of chemical substances whose removal would be undesirable, which could be the case for a variety of different reasons. One reason, for example, could be that the substance makes a positive contribution to the experience of smoking a smoking article which contains the treated tobacco material. Nicotine may be an example of such a substance, and for this reason in some embodiments it is undesirable to remove this molecule. In some embodiments, the treatment of a tobacco material with an aqueous medium removes less than 50%, 40%, 30%, 20%, 10%, or 5% of the nicotine from the tobacco material; in further

embodiments, the treatment removes less than 2%, 1%, 0.5%, or 0.1% of nicotine from the tobacco material; and, in further embodiments still, the treatment removes essentially no nicotine from the tobacco material.

An aqueous medium is a body of liquid water which may comprise any soluble species at any concentration in solution; any insoluble species at any ratio to the water, by weight, in suspension; and/or any miscible or immiscible non-aqueous liquids at any ratio to the water, by volume. Any suitable aqueous medium or aqueous media may be used in the treatment of a tobacco material with an aqueous medium. In some embodiments, the inclusion of one or more of the above in an aqueous medium used in the treatment of a tobacco material with an aqueous medium improves the ability of the aqueous medium to remove protein from tobacco material.

Treating tobacco material with an aqueous medium is likely to be advantageous because an aqueous medium may modify the tobacco material undergoing treatment in a number of different ways. An aqueous medium maybe used for the purpose of removing one or more chemical substances, for example, and in this case is likely to be particularly useful since, in addition to being a very good solvent for particular chemical substances, an aqueous medium is likely to be highly selective towards which chemical substances it can dissolve, and this selectivity is likely to be highly sensitive to the adopted treatment conditions. When tobacco material is treated with an aqueous medium, the aqueous medium may have any suitable weight and the tobacco material may have any suitable weight. In addition, the ratio of the aqueous medium to the tobacco material, by weight, may have any suitable value. In some embodiments, the ratio of aqueous medium to tobacco material results in the removal of a large quantity of protein from the tobacco material. In some embodiments, the ratio of aqueous medium to tobacco material does not result in the removal of a large quantity of substances whose removal would be undesirable, such as nicotine. In some embodiments, the ratio of aqueous medium to tobacco material has minimal effect on the integrity and/or structure of the tobacco material. In some embodiments, a large quantity of protein is likely to be removed when the weight of the aqueous medium is greater than the weight of the tobacco material. In some embodiments, therefore, the weight of the aqueous medium is greater than the weight of the tobacco material and, in further embodiments, the ratio may be 10:1, 15:1, 20:1, 25:1, 30:1, or any suitable higher ratio. In some embodiments, the ratio is 20:1 by weight.

In some embodiments, any one of the above ratios may be used in combination with one or more of the other specified conditions described herein. When tobacco material is treated with an aqueous medium, it may be treated with an aqueous medium once or more than once. In embodiments wherein the tobacco material is treated with an aqueous medium more than once, the tobacco material may be treated with aqueous medium any suitable number of times and, each time, the aqueous medium, duration of treatment, and/or adopted treatment conditions may be the same or may be different. Furthermore, on each occasion the tobacco material is treated with an aqueous medium, the tobacco material may be added to the aqueous medium or the aqueous medium may be added to the tobacco material. In some embodiments, the tobacco material may be separated from one aqueous medium before being treated with another and, in this case, the tobacco material may be separated using any suitable method of separation. For example, the tobacco material may be separated by filtration using any suitable filtration method, any suitable filtering medium pore size, and any suitable number of filtration steps, and/or may be separated by centrifugation using any suitable centrifuge system, any suitable angular velocity, and any suitable number of centrifugation steps.

Treating tobacco material with an aqueous medium multiple times is likely to be advantageous for a number of different reasons. Firstly, for example, it means that the tobacco material may be treated with different aqueous media, each of which may be more effective for the removal of different chemical substances. And secondly, for example, when an aqueous medium removes a particular chemical substance from tobacco material, the concentration of that chemical substance in solution increases and eventually reaches thermodynamic equilibrium, at which point no more of that substance may be removed, in which case, treating the tobacco material with another aqueous solution can facilitate the further removal of the chemical substance.

In some embodiments, the tobacco material may be treated with an aqueous medium multiple times in combination with one or more of the other specified conditions described herein. For example, the tobacco material may be treated more than once with an aqueous medium, and the ratio of aqueous medium to tobacco material may be at least 10: 1 by weight.

In some embodiments, the tobacco material is treated two, three, or four times with an aqueous medium, and the ratio of aqueous medium to tobacco material is 20:1 by weight. When tobacco material is treated with an aqueous medium, the aqueous medium may have any suitable pH, and the pH may be constant or variable. In some embodiments, having an aqueous medium at a suitable pH results in the removal of a large quantity of protein from the tobacco material. In some embodiments, having an aqueous medium at a suitable pH does not result in the removal of a large quantity of substances whose removal would be undesirable, such as nicotine. In some embodiments, the pH has minimal effect on the integrity and/or structure of the tobacco material. Experiments were conducted, as described later under "Experimental Work", which would suggest that an alkaline pH leads to the removal of a large quantity of proteins when used in combination with the other preferred conditions. In some embodiments, therefore, the tobacco material is treated with an aqueous medium with an alkaline pH and, in some embodiments, the pH may be 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13_> 13·5_> 14_> or any suitable higher pH. In some embodiments, the pH of the aqueous medium may be about 7.5.

In some embodiments, the tobacco material may be treated with an aqueous medium with an alkaline pH in combination with one or more of the other specified conditions described herein. For example, the tobacco material may be treated more than once with an aqueous medium, the ratio of aqueous medium to tobacco material may be at least 10: 1 by weight, and the pH of the aqueous medium may be alkaline.

In some embodiments, the tobacco material is treated two, three, or four times with an aqueous medium, the ratio of aqueous medium to tobacco material is about 20:1 by weight, and the pH of the aqueous medium is about 7.5.

The pH of an aqueous medium used in the treatment of tobacco material may be modified using any suitable means, for example by adding any suitable solute or solutes, by changing the temperature in any suitable way, and/ or by adding a chemical buffer or mixture of chemical buffers at any suitable concentration. Suitable chemical buffers include any suitable weak acids or weak bases, and specific examples of suitable buffers include, but are not limited to: phosphate salts, such as sodium phosphate; hydrogen phosphate salts, such as sodium hydrogen phosphate; dihydrogen phosphate salts, such as sodium dihydrogen; any suitable citrate-phosphate buffer system; and any suitable derivatives thereof. In some embodiments, when tobacco material is treated with an aqueous medium, the aqueous medium may comprise one or more suitable surfactants. In some

embodiments, the presence of one or more surfactants in the aqueous medium results in the removal of a large quantity of protein from the tobacco material. In some embodiments, the presence of one or more surfactants in the aqueous medium does not result in the removal of substances whose removal would be undesirable, such as nicotine. In some embodiments, the presence of one or more surfactants in the aqueous medium has minimal effect on the integrity and/or structure of the tobacco material.

One or more of the surfactants may be anionic, cationic, zwitterionic, or non-ionic. Suitable surfactants include, but are not limited to: sodium alkylsulfonates; sodium alkylsulfates, such as sodium dodecylsulfate (SDS); sodium alkylarylsulfonates, such as sodium dodecylbenezenesulfonate; sodium alkylsulfosuccinates, such as sodium dioctylsulfosuccinate (DSS); sodium or potassium carboxylates; Triton X-ioo;

N-dodecyl β-D-maltoside; 3-[(3-cholamidopropyl)dimethylammonio]-i-propane sulfonate (CHAPS); and any suitable derivatives thereof. In some embodiments, at least one of the surfactants used is SDS. In embodiments wherein the aqueous medium comprises one or more surfactants, the surfactants may be present in the aqueous medium at any suitable concentration. In some embodiments, the concentration of the surfactants results in the removal of a large quantity of proteins from the tobacco material. In some embodiments, the concentration of the surfactants does not result in the removal of substances whose removal would be undesirable, such as nicotine. In some embodiments, the

concentration of the surfactants has minimal effect on the integrity and/or structure of the tobacco material.

In the conducted experiments described later under "Experimental Work", the surfactant SDS was used at a molar concentration of 0.1 %, and it was found that the use of SDS at this concentration enhanced the extraction of protein from tobacco material. A suitable concentration of surfactant may therefore be a molar concentration that falls within the range of 0.75% to 0.125%, 0.5% to 0.15%, 0.25% to 0.175%, or 0.01% to 0.2%, for example. In some embodiments, the aqueous medium may comprise SDS at a molar concentration of about 0.1%. In some embodiments, the tobacco material may be treated with an aqueous medium comprising a surfactant in combination with one or more of the other specified conditions described herein. For example, the tobacco material may be treated more than once with an aqueous medium, the ratio of aqueous medium to tobacco material may be at least io: ι by weight, the pH of the aqueous medium may be alkaline, and the aqueous medium may comprise a surfactant.

In some embodiments, the tobacco material is treated two, three, or four times with an aqueous medium, the ratio of aqueous medium to tobacco material is about 20:1 by weight, the pH of the aqueous medium is about 7.5, and the aqueous medium comprises SDS at a molar concentration of about 0.1%.

When tobacco material is treated with an aqueous medium, the aqueous medium may be at any suitable temperature, and the temperature may be constant or variable. In some embodiments, having the aqueous medium at a suitable temperature results in the removal of a large quantity of proteins from the tobacco material. In some embodiments, having the aqueous medium at a suitable temperature does not result in the removal of a large quantity of substances whose removal would be undesirable, such as nicotine. In some embodiments, the temperature has minimal effect on the integrity and/ or structure of the tobacco material.

In some embodiments, a large quantity of protein is likely to be removed when the temperature of the aqueous medium is higher than ambient temperature. This is because the use of a higher temperature is likely to, firstly, promote the thermodynamic favourability of dissolution for chemical substances which dissolve with a positive change in enthalpy and, secondly, facilitate the occurrence of kinetically-disfavoured dissolution reactions and bring them closer to thermodynamic equilibrium. In some embodiments, therefore, tobacco material is treated with an aqueous medium at a temperature higher than ambient temperature and, in further embodiments, the temperature may be greater than or about 25°C, 30°C, 40°C, 50°C, 6o°C, 70°C, 8o°C, 90°C, ioo°C, or any suitable higher temperature, optionally with an upper temperature limit of about 8o°C, 90°C, ioo°C, iio°C or 120°C. In some embodiments, the temperature of the aqueous medium is about 70°C. The temperature of the aqueous medium may be increased above ioo°C by increasing the boiling point of the water using any suitable means, such as by increasing the surrounding pressure and/ or adding any suitable solute or solutes to the water. In some embodiments, the tobacco material may be treated with an aqueous medium at a temperature above ambient temperature in combination with one or more of the other specified conditions described herein. For example, the tobacco material may be treated more than once with an aqueous medium, the ratio of aqueous medium to tobacco material may be at least 10:1 by weight, the pH of the aqueous medium may be alkaline, the aqueous medium may comprise a surfactant, and the temperature may be above ambient temperature.

In some embodiments, the tobacco material is treated two, three, or four times with an aqueous medium, the ratio of aqueous medium to tobacco material is about 20:1 by weight, the pH of the aqueous medium is about 7.5, the aqueous medium comprises SDS at a molar concentration of about 0.1%, and the temperature is about 70°C.

When tobacco material is treated with an aqueous medium, the tobacco material may be treated with an aqueous medium for any suitable length of time before being separated using any suitable method of separation, such as filtration and/or centrifugation. In some embodiments, the length of time results in the removal of a large quantity of proteins from the tobacco material. In some embodiments, the length of time does not result in the removal of a large quantity of substances whose removal would be undesirable, such as nicotine. In some embodiments, the length of time has minimal effect on the integrity and/ or structure of the tobacco material.

In some embodiments, a large quantity of protein is likely to be removed when the tobacco material is treated with an aqueous medium for longer than or equal to 1 hour. This is because the chemical substances removed from the tobacco material begin in the tobacco material and so, by extending the reaction time and therefore bringing the reactions closer to thermodynamic equilibrium, the concentration of substances dissolved in aqueous medium can only increase. In some embodiments, therefore, the tobacco material is treated with an aqueous medium for longer than or equal to 1 hour and, in further embodiments, the tobacco material may be treated with an aqueous medium for at least or about 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, or any suitable longer length of time, optionally up to about 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours. In some embodiments, the tobacco material is treated with aqueous medium for about 4 hours. Treating the tobacco material for less than 4 hours is likely to lead to the removal of less protein, but, treating the tobacco material for longer than 4 hours is likely to significantly affect the integrity and/ or structure of the tobacco material.

In some embodiments, the tobacco material may be treated with aqueous medium for at least 2 hours in combination with one or more of the other specified conditions described herein. For example, the tobacco material may be treated more than once with an aqueous medium, the ratio of aqueous medium to tobacco material may be at least 10:1 by weight, the pH of the aqueous medium may be alkaline, the aqueous medium may comprise a surfactant, the temperature may be above ambient

temperature, and the tobacco material may be treated with aqueous medium for at least 2 hours.

In some embodiments, the tobacco material is treated two, three, or four times with an aqueous medium, the ratio of aqueous medium to tobacco material is about 20:1 by weight, the pH of the aqueous medium is about 7.5, the aqueous medium comprises SDS at a molar concentration of about 0.1%, the temperature is about 70°C, and the tobacco material is treated for about 4 hours.

In embodiments wherein the tobacco material is treated more than once with aqueous medium and is treated for a total time of 4 hours, the tobacco material may be treated with aqueous medium any suitable number of times and for any length of time on each occasion. For example, the tobacco material may be sequentially treated with two or three aqueous media over 4 hours, and in this case possible combinations of treatment times include, but are not limited to: 3 hours and 1 hour; 2 hours and 2 hours; and, 2 hours and 1 hour and 1 hour. When tobacco material is treated with an aqueous medium, the aqueous medium may comprise any suitable substances in addition to those previously described. In some embodiments, the presence of these substances, and the concentration at which they are added, results in the removal of a large quantity of proteins from the tobacco material. In some embodiments, the presence of these substances, and the

concentration at which they are added, does not result in the removal of substances whose removal would be undesirable, such as nicotine. In some embodiments, the presence of these substances, and the concentration at which they are added, has minimal effect on the integrity and/or structure of the tobacco material.

In some embodiments, one or more of any suitable ionic species may be added to the aqueous medium at any suitable concentration in order to modify the ionic strength of the aqueous medium in any suitable way. Suitable ionic species include, but are not limited to: sodium chloride, potassium chloride, and ammonium sulphate.

Alternatively or in addition, one or more of any suitable chaotropes, such as urea and thiourea, may be added to the aqueous medium at any suitable concentration.

Advantageously, these may help disrupt the native structure of proteins in the tobacco material, thereby facilitating the removal of these proteins by, for example, weakening the bonds by which they are held in the tobacco material. Alternatively or in addition, one or more of any suitable oxidising agents and/or reducing agents, such as DTT and β-mercaptoethanol, may be added to the aqueous medium at any suitable concentration. Advantageously, these agents may lead to the chemical modification of proteins in the tobacco material, and such chemical modification may result in the proteins being easier to remove.

When tobacco material is treated with an aqueous medium, the mixture may be agitated. This may be advantageous since it is likely to increase the rate at which protein compounds are removed from the tobacco material by providing mechanical disruption and increasing the rate of chemical reaction.

After the tobacco material has been treated with an aqueous medium, and in some cases treated with an aqueous medium multiple times, the resultant tobacco material may be separated from the aqueous medium. For example, the tobacco material may be filtered from the aqueous medium using any suitable filtration method, any suitable filtering medium pore size, and any suitable number of filtration steps. For example, the tobacco material may be filtered by nanofiltration, microfiltration, and/or ultrafiltration. Alternatively or in addition, the tobacco material may be separated from the aqueous medium by centrifugation using any suitable centrifuge system, any suitable angular velocity, and any suitable number of centrifugation steps. Once separated from the aqueous by filtration and/ or centrifugation, the tobacco material may be washed any suitable number of times using any suitable liquid or liquids, such as water.

In addition to treating tobacco material with an aqueous medium, the method may comprise any other suitable treatments. Suitable treatments include, but are not limited to: treating the tobacco material with one or more enzymes, such as phenol- oxidising and proteolytic enzymes; treating the tobacco material with one or more suitable adsorbent materials, such as polyvinylpolypyrrolidone (PVPP),

hydroxylapatite, bentonite, activated carbon, and attapulgite; and treating the tobacco material with one or more suitable non-aqueous media.

Additionally or alternatively, the tobacco material treated with an aqueous medium may be subsequently subjected to further extraction processes. Having undergone any of the previously-described treatment steps in accordance with the method of the invention, the tobacco material may be dried and further modified in any suitable way before being incorporated into a smoking article. For example, certain chemical substances may be added to the tobacco material, such as flavourants where local regulations permit, and the tobacco material may be cut and/or shredded before being incorporated into a smoking article using any suitable method of incorporation.

As used herein, the terms "flavour" and "flavourant" refer to materials which, where local regulations permit, may be used to create a desired taste or aroma in a product for adult consumers. They may include extracts (e.g., licorice, hydrangea, Japanese white bark magnolia leaf, chamomile, fenugreek, clove, menthol, Japanese mint, aniseed, cinnamon, herb, wintergreen, cherry, berry, peach, apple, Drambuie, bourbon, scotch, whiskey, spearmint, peppermint, lavender, cardamon, celery, cascarilla, nutmeg, sandalwood, bergamot, geranium, honey essence, rose oil, vanilla, lemon oil, orange oil, cassia, caraway, cognac, jasmine, ylang-ylang, sage, fennel, piment, ginger, anise, coriander, coffee, or a mint oil from any species of the genus Mentha), flavour enhancers, bitterness receptor site blockers, sensorial receptor site activators or stimulators, sugars and/or sugar substitutes (e.g., sucralose, acesulfame potassium, aspartame, saccharine, cyclamates, lactose, sucrose, glucose, fructose, sorbitol, or mannitol), and other additives such as charcoal, chlorophyll, minerals, botanicals, or breath freshening agents. They may be imitation, synthetic or natural ingredients or blends thereof. They may be in any suitable form, for example, oil, liquid, or powder. In one exemplary embodiment of the current invention, a sample of cured whole leaf tobacco is added to an aqueous medium at 70°C so that the ratio of aqueous medium to tobacco material is 20:1. The aqueous medium has a pH of 7.5 due to the presence of a phosphate buffer, and a molar concentration of 0.1% SDS. The tobacco material is left in the aqueous medium for 1 hour before being filtered and added to a fresh aqueous medium under the same conditions. This is repeated four times, before the tobacco material is filtered, dried, and modified in any suitable way before being incorporated into a smoking article.

As used herein, the term "smoking article" includes smokeable products such as cigarettes, cigars and cigarillos whether based on tobacco, tobacco derivatives, expanded tobacco, reconstituted tobacco or tobacco substitutes and also heat-not-burn products. The smoking article may be provided with a filter for the gaseous flow drawn by the smoker.

Referring to Figure 5, for purpose of illustration and not limitation, a smoking article 1 according to an exemplary embodiment of the invention comprises a filter 2 and a cylindrical rod of smokeable material 3, such as tobacco treated in accordance with the invention described herein, aligned with the filter 2 such that one end of the smokeable material rod 3 abuts the end of the filter 2. The filter 2 is wrapped in a plug wrap (not shown) and the smokeable material rod 3 is joined to the filter 2 by tipping paper (not shown) in a conventional manner. In some embodiments, the methods described herein may comprise one or more further steps to modify the tobacco material in any suitable way. For example, the tobacco material may be modified to provide it with one or more characteristics desirable for a tobacco material. For example, where the treated tobacco material is to be incorporated into a smoking article such as a cigarette, the tobacco material may be treated in order to modify the flavour it generates upon combustion, and/ or may be treated in order to remove one or more of its chemical substances.

Experimental Work

A series of experiments were carried out in order to determine the optimum set of conditions for removing protein from cured whole leaf tobacco when treating tobacco material with aqueous medium. The disclosed experimental work is not intended to limit the scope of the invention.

The experimental procedure consisted of four stages. In each stage, one or two parameters were tested and optimised before proceeding to the next stage and doing the same. In this way, by the end of the fourth stage, the optimum set of conditions for removing protein from cured whole leaf tobacco was obtained.

The variables tested in the experiments were: pH (using phosphate buffer compounds), temperature, presence/absence of detergent (SDS), presence/absence of salt (NaCl), aqueous solution to tobacco leaf ratio, duration of treatment, and the number of times tobacco material is treated with aqueous solution.

Experimental Procedure

The four-stage experimental procedure is summarised in Figure 1, and a more detailed overview of the four-stage experimental procedure is described below:

The mass of a cured intact leaf of tobacco was measured (with an approximate mass of 10 g), before being placed in a 250 ml conical flask. (A sample of leaf material was also collected for Karl Fischer moisture analysis.)

An aqueous solution comprising sodium phosphate buffer was added to the conical flask to give an aqueous solution to tobacco ratio (w/w) of 20:1 (in stages 1, 2, and 4), 15: 1 (in stage 3), and 25:1 (in stage 3). The sodium phosphate solution was made up by adding two sodium phosphate species— sodium phosphate monobasic (NaHP0₄) and sodium phosphate dibasic (NaH₂P0₄)— to ultrapure water, and different

concentrations of these two species were added in order to give the aqueous solution a pH of 7.0 (in stage 1), 7.5 (in stages 1, 2, 3, and 4), and 8.0 (in stage 1). Citric acid was also added to the sodium phosphate solution at the necessary concentration to provide a pH of 5.0 (in stage 2).

The effect of adding a detergent (0.1 % molar concentration SDS) and/or salt (0.5 % molar concentration NaCl) to the solution was tested (in stage 2). The samples were incubated in a shaking controlled-temperature water bath at 30°C (in stage 1), 50°C (in stage 1), and 70°C (in stages 1, 2, 3, and 4) for 1 hour (in stage 3), 4 hours (in stages 1 and 2), and 16 hours (in stage 3). Finally, in stage 4, three different sequential extraction combinations were tested over 4 hours at 70°C, these combinations being: 3 hours & 1 hour, 2 hours & 2 hours, and 2 hours & 1 hour & 1 hour. In each sequential extraction, the tobacco material was added to a new previously-warmed solution each time it was treated with another aqueous solution. In stage 4, the effects of aqueous solution treatment were tested for cured cut rag tobacco as well as for cured whole leaf tobacco.

Following this four-stage procedure, the residual tobacco leaf material was separated from the aqueous solution using Biichner filtration, before being spread thinly on filter paper and kept in a fume hood overnight to dry. The next morning, the samples were freeze-dried for a total of three days and, at the end of these three days, the total weight of the residual tobacco material was measured.

Meanwhile, the liquid extract obtained following Biichner filtration was divided into aliquots. A 1 ml aliquot sample was freeze-dried for three days before the weight of the aqueous solution was measured. The remaining extract was stored at 20°C and its protein concentration was determined using a modified BCA assay.

All experiments were carried out three times, except for the treatment of cut rag tobacco in stage 4, which was carried out two times. All BCA assays were carried out twice. The statistical significance of the data collected in these repeat experiments was tested using the AN OVA test on Minitab V16.2.0 software, and the determined p value was considered statistically significant when P «3.05.

Photographs were taken of the residual tobacco leaves and liquid extract using a DigiEye system after they had dried overnight.

Results

A summary of the results obtained in the experiments may be found in Figure 2, and a more detailed overview of the experimental results is provided below:

Stage 1: pH and temperature In stage 1, the tested variables were temperature and pH. Three pH values (7.0, 7.5, 8.0) and three temperature values (30°C, 50°C, 70°C) were tested.

In every experiment, it was found that the pH of the aqueous solution decreased, thereby suggesting that the tobacco material was acidic. The temperature had a statistically significant effect (P < 0.001) on the final pH value: the final pH was lower for 70°C compared to 50°C, and lower for 50°C compared to 30°C. The initial pH did not have a statistically significant effect on the final pH value: an initial pH of 8.0 did show a greater drop in pH than extracts with an initial pH of 7.5 and 7.0, however.

After the freeze-drying step, the masses of the residual tobacco materials were measured as a percentage of their initial masses. Temperature had a statistically significant effect (P = 0.022) on the weight loss of the tobacco material: weight loss was greater for 70°C compared to 50°C, and lower for 50°C than for 30°C. The pH of the solution did not have a statistically significant effect on the weight loss of the tobacco material: weight loss was greatest (for all temperatures) when the pH was 7.0, however. Weight loss was found to be 8.8% under certain conditions, thereby indicating that substances besides protein were removed by the treatment process because such a large reduction in mass could not be attributed to protein alone.

The concentration of protein was measured in each of the aqueous extracts using a modified BCA assay in order to provide a measure of the amount of protein removed from the tobacco material. Temperature had a statistically significant effect (P = 0.014) on the measured concentration of protein: the concentration of protein was greater for 70°C than for 50°C, and greater for 50°C than for 30°C. The pH of the solution did not have a statistically significant effect on the measured protein concentration: the measured protein concentration was higher at 70°C at pH 7.5 and 8.0 than at pH 7.0, however. Temperature had a greater effect than pH on tobacco weight loss and protein removal, and the results obtained when testing different temperatures were more statistically significant than those obtained when testing different pH values.

In view of the above, stage 1 suggested that the optimum temperature for protein extraction is 70°C since this temperature caused the greatest reduction in the protein content of tobacco material, and the optimum pH for protein extraction is 7.5 since this pH caused a greater reduction in protein content than pH 7.0 and 8.0.

Stage 2: Presence/ absence of detergent and/or salt; and test atpH 5.0

In stage 2, the tested variables were the presence or absence of detergent (SDS— Sodium Dodecyl Sulfate) and/or salt (NaCl), and the treatment of tobacco material at pH 5.0 (using a phosphate-citrate buffer system consisting of citric acid and dibasic sodium phosphate). In every experiment, it was found that the pH of the aqueous solution decreased, thereby suggesting that the tobacco material was acidic. For the sodium phosphate- buffered samples (pH 7.5), the presence of salt and/or detergent had a statistically significant effect (P < 0.001) on the final pH value: the presence of salt and/or detergent reduced the pH more than the absence of both, and the pH was reduced the most in the presence of NaCl. For the phosphate citrate-buffered samples, the temperature had a statistically significant effect (P = 0.017) on the final pH: the final pH was lower for 70°C than for 50°C, although the difference was small, 4.53 at 50°C and 4.49 at 70°C. After the freeze-drying step, the masses of the residual tobacco materials were measured as a percentage of their initial masses. For the sodium phosphate-buffered samples, the presence of salt and/ or detergent did not have a statistically significant effect on weight loss: weight loss was slightly lower in the presence of salt and/or detergent, however. For the phosphate citrate-buffered samples, weight loss was higher compared to the sodium phosphate-buffered samples, and temperature did not have a significant effect.

The concentration of protein was measured in each of the aqueous extracts using a modified BCA assay in order to provide a measure of the amount of protein removed from the tobacco material. The addition of SDS (0.1% molar concentration) to the sodium phosphate-buffered solution (pH 7.5, 70°C) resulted in the removal of a significantly more protein than when SDS was not added to aqueous solution. The presence of NaCl (0.05% molar concentration) was found to have no significant effect, however. Extraction using the phosphate-citrate buffer system (pH 5.0) resulted in the statistically significant (P = 0.05) removal of less protein than when using the sodium phosphate buffer (pH 7.5). For the extraction using the phosphate-citrate buffer system, temperature had no significant effect on the removal of protein.

In view of the above, stage 2 suggested that the optimum pH for protein extraction is still 7.5 since the phosphate-citrate buffer system resulted in the removal of less protein, that the addition of NaCl is unnecessary since its modification of the ionic strength of the aqueous solution did not result in the removal of more protein, and that the addition of SDS (0.1% molar concentration) improves the extraction process since the presence of SDS increased the removal of protein and had no effect on the weight loss of the tobacco material.

In view of the above, at the end of stage 2 it was therefore found that the most effective conditions for removing protein from tobacco material are a pH of 7.5 (using a sodium phosphate buffer), a temperature of 70°C, and the presence of SDS at a molar concentration of 0.1%.

Stage 3: Aqueous solution to tobacco leaf ratio and duration of treatment

In stage 3, the tested variables were aqueous solution to tobacco leaf ratio, and duration of treatment. Three ratios (15:1, 20:1, 25:1) and three time durations (1 hour, 4 hours, 16 hours) were tested. In addition, a small trial was carried out in which two intermediate time durations (6 hours, 8 hours) were tested for each ratio.

In every experiment, it was found that the pH of the aqueous solution decreased, thereby suggesting that the tobacco material was acidic. The ratio had a statistically significant effect (P < 0.001) on the final pH value: the final pH was lower for a ratio of 15:1 compared to a ratio of 20:1 and 25:1. The treatment duration had a significant effect (P < 0.001) on the final pH value: the longer the treatment duration, the lower the final pH. The smallest decrease in pH was observed when the ratio was 20:1 and the treatment duration was 1 hour.

After the freeze-drying step, the masses of the residual tobacco materials were measured as a percentage of their initial masses. The ratio had a statistically significant effect (P < 0.001) on the weight loss of the tobacco material: the greater the ratio the greater the weight loss of tobacco material. The treatment duration also had a statistically significant effect (P < 0.001) on the weight loss of the tobacco material: a treatment duration of 16 hours led to greater weight loss than a treatment duration of 1 or 4 hours. The greatest weight loss was observed when the ratio was 25:1 and the treatment duration was 16 hours. There was less variability in the weight loss of tobacco material between different ratios for treatment durations of 1 hour and 4 hours compared to 16 hours.

The concentration of protein was measured in each of the aqueous extracts using a modified BCA assay in order to provide a measure of the amount of protein removed from the tobacco material. The ratio had a statistically significant effect (P < 0.001) on the measured protein concentration: a higher concentration of protein was measured for a ratio of 15:1 than for a ratio of 25:1. The treatment duration also had a statistically significant effect (P < 0.001) on the measured protein concentration: a higher protein concentration was measured for treatment duration of 16 hours than a treatment duration of 1 hour. In view of the above, stage 3 suggested that the optimum ratio for protein extraction is 20:1 because a ratio of 25:1 led to the greatest weight loss of tobacco material and the most negative effect on tobacco leaf structure despite removing more protein than a ratio of 20:1, and the optimum treatment duration for protein extraction is 4 hours because a treatment duration of 16 hours led to the greatest weight loss and most negative effect on tobacco leaf structure despite removing more protein than treatment duration of 4 hours.

Stage 4: Number of sequential treatments and treatment of cut rag tobacco

In stage 4, the effect of treating tobacco material multiple times over a total duration of 4 hours was tested, and three different combinations of time were trialled over these fours hours: (a) 3 hours & 1 hour, (b) 2 hours & 2 hours, and (c) 2 hours & 1 hour & 1 hour. In between each treatment, the tobacco material was filtered from the aqueous solution before being added to a new, previously-warmed aqueous solution. In addition, the extraction process was tested on cured cut rag tobacco.

In every experiment, it was found that the pH of the aqueous solution decreased, thereby suggesting that the tobacco material was acidic. Sequential extraction had a statistically significant effect (P < 0.001) on the final pH value: in general, the pH was lower in each of the sequential washes compared to the initial wash, although this was not the case for the second wash of protocol (c). After the freeze-drying step, the masses of the residual tobacco materials were measured as a percentage of their initial masses. Sequential extraction had a statistically significant effect (P < o.ooi) on the weight loss of the tobacco material: the greater the number of washes, the greater the weight loss of the tobacco material. There was also a statistically significant (P < o.ooi) difference between the weight loss of cut rag and intact leaf tobacco: the weight loss of cut rag was found to be greater than the weight loss of intact leaf tobacco, which is to be expected by virtue of the greater surface area : volume ratio of cut rag tobacco. The concentration of protein was measured in each of the aqueous extracts using a modified BCA assay in order to provide a measure of the amount of protein removed from the tobacco material. Sequential extraction led to the removal of a greater amount of protein, and it was found that the greatest amount of protein was recovered for both intact leaf and cut rag tobacco when protocol sequential extraction (c) 2 hours & ι hour & l hour was used.

It was also found that a greater amount of protein was removed from cut rag than from intact leaf tobacco, which was to be expected because of its larger surface area. Figure 2 graphically illustrates this difference by depicting the mass of protein removed from the whole leaf tobacco and cut rag tobacco as a fraction of the total mass of the original tobacco material (mg/g) for whole leaf and cut rag tobacco.

The most important measurement to take in order to determine the optimum conditions for removing protein from tobacco material was, of course, the quantity of protein that was removed as a fraction of the original tobacco material.

The BCA assay was used to determine the quantity of protein in the aqueous extracts following filtration for this purpose, and the measured quantity was expressed in two ways: as a fraction of the mass of the original tobacco material (mg/g) and as a fraction of the dry mass of the original tobacco material (mg/g).

Figure 3 provides a flow chart showing the amount of protein which was removed in each of the experiments in each of the experimental stages as a fraction of the mass of the original tobacco material. In each stage, the optimum conditions for removing protein are highlighted, and the means and standard deviations for all repeated experiments are described. Figure 4 provides a bar graph showing, in addition to the same, the quantity of protein which was removed as a fraction of the dry mass of the original tobacco material. In addition to measuring protein extraction, it was important to measure the change in dry mass of the tobacco material. This was achieved by measuring the dry mass of the extracted tobacco material after freeze-drying. The accuracy of this measure was tested by also measuring the dry mass of removed tobacco material in the aqueous extract after freeze-drying, before summing these values together to give an estimate of the total dry mass of the original tobacco material, and comparing this to the dry mass of the original tobacco material calculated using Karl Fischer analysis.

It was found that the sum of the two values did indeed give a good approximation of the original total dry mass. Table 1 below conveys how accurate the measurement method was by showing the mean differences between the dry masses obtained by Karl Fischer analysis and the summation method, in addition to the standard deviation of the measured differences, and the number of samples which were measured in each experimental stage.

Table 1

The measurement method is actually likely to be more accurate than the above numbers suggest. This is because, in the method, the dry mass of a 1 ml sample of the extract was measured before being multiplied by the total amount of extract to give a measure of the dry mass of the total extract, thereby multiplying the magnitude of any practical errors in the process.

In order to address various issues and advance the art, the entirety of this disclosure shows by way of illustration various embodiments in which the claimed invention(s) maybe practiced and provide for superior tobacco treatment, tobacco material, and products incorporating tobacco material. The advantages and features of the disclosure are of a representative sample of embodiments only, and are not exhaustive and/or exclusive. They are presented only to assist in understanding and teach the claimed features. It is to be understood that advantages, embodiments, examples, functions, features, structures, and/ or other aspects of the disclosure are not to be considered limitations on the disclosure as defined by the claims or limitations on equivalents to the claims, and that other embodiments may be utilised and modifications may be made without departing from the scope and/or spirit of the disclosure. Various embodiments may suitably comprise, consist of, or consist essentially of, various combinations of the disclosed elements, components, features, parts, steps, means, etc. In addition, the disclosure includes other inventions not presently claimed, but which may be claimed in future.

Claims

1. A method for treating a tobacco material, wherein the method comprises treating the tobacco material with aqueous medium, and wherein treating the tobacco material with aqueous medium comprises the use of an aqueous medium : tobacco material ratio which is greater than or equal to 10: 1 by weight, and/or adding an aqueous medium to the tobacco material multiple times, optionally in combination with at least one of:

i) the aqueous medium having an alkaline pH;

ii) the aqueous medium comprising a surfactant;

iii) the aqueous medium having a temperature higher than ambient

temperature; and

iv) the tobacco material being treated with aqueous medium for longer than or equal to two hours.

A method according to claim 1, comprising two or more of (i) to (iv). A method according to claim 1, comprising three or more of (i) to (iv). 4. A method according to claim 1, comprising all of (i) to (iv).

5. A method according to any one of the preceding claims, wherein treating the tobacco material with an aqueous medium results in the removal of one or more chemical substances from the tobacco material.

6. A method according to claim 5, wherein one or more of the removed chemical substances are proteins.

7. A method according to any one of the preceding claims, wherein the method does not substantially reduce the amount of nicotine in the tobacco material.

8. A method according to any one of the preceding claims, wherein the aqueous medium comprises the surfactant SDS. 9. A method according to any one of the preceding claims, wherein the temperature of the aqueous medium is higher than or equal to 50°C.

10. A method according to any one of the preceding claims, wherein the tobacco material treated with an aqueous medium is subsequently separated from the aqueous medium, optionally by filtration and/or centrifugation.

11. A method according to any one of the preceding claims, wherein the tobacco material is washed with water following treatment with an aqueous medium.

12. A method according to any one of the preceding claims, wherein the method further comprises treating the tobacco material with: one or more enzymes; one or more adsorbents; and/or one or more non-aqueous media.

13. A tobacco material which has been treated by a method according to any one of the preceding claims, or a derivative thereof.

14. A smoking article which comprises a tobacco material according to claim 13, or a derivative thereof.

15. Use of an aqueous medium for removing protein from tobacco material, wherein the use involves an aqueous medium : tobacco material ratio which is greater than or equal to 10:1 by weight, and/or adding an aqueous medium to the tobacco material multiple times, optionally in combination with at least one of:

v) the aqueous medium having an alkaline pH;

vi) the aqueous medium comprising a surfactant;

vii) the aqueous medium having a temperature higher than ambient

temperature; and

viii) the tobacco material being treated with aqueous medium for longer than or equal to two hours.