GB2203022A - Smoking material - Google Patents

Description

1 tci/8501 SMOKING MATERIAL AND PROCESS FOR MAKING TEE SAME.

220"029 U i-i The present invention relates to a smoking material or product containing suspension-cultured tobacco cells, tobacco leaf material or other fibrous material in association with various fillers, and to a process for making the material. In particular it relates to a process for culturing tobacco cells having an enhanced level of nicotine.

A smoking material according to this invention provides an improvement over conventional tobacco smoking material in that it offers greater scope for altering smoke composition and has more acceptable smoke flavour and aroma properties in comparison with prior art tobacco substitutes.

It has been proposed in British Patent 1353505 to obtain tobacco cells from the tissue culture of plants of the genus Nicotiana. In that prop6sal tobacco cells obtained by growth in a nutrient medium were harvested and partially dried to reduce the moisture content to 30-60% by weight and the resulting moist powder was formed into a sheet-like material by passage between extrusion rollers.

In British Patent 1355570 associated with British Patent 1353505 a process was proposed for producing a smoking material in which cultured tobacco cells were filtered from the nutrient medium on a gauze or filter cloth and the resulting sheet-like cake was dried. This patent also described a process for producing a rod-like smoking article in which the tobacco cells were formed into a paste which was then cast into rod-form and freeze-dried.

2 In view of the above mentioned British Patents 1353505 and 1355570, a known process for producing tobacco cells will be described briefly.

Small fragments (explan'ts) of tobacco leaf, stem or other organs or tissue are surface-sterilized and placed on a sterile agar nutrient medium. The explants are incubated typically at 251C and cells proliferate from the explants to form an aggregated amorphous mass of cells called a callus. Portions of the callus are inoculated aseptically into a liquid nutrient medium contained in 250 ml. conical flasks which are shaken on an orbital shaker at 251C. After several days a dense suspension of cells is formed and this may be used for successive inoculations into similar or larger vessels. The massed of cells acquired in this way are then transferred to stirred and aerated seed fermenters the contents of which, after the required cell density is achieved, are inoculated into production scale fermenters.

The cells are harvested and dewatered by pumping through rotary vacuum filters and the liquid which is removed may be recycled with fresh medium. The dewatered cells which normally contain 87-95% of moisture are wet milled or macerated and either spray-dried prior to extruded filament or flake production or used directly along with other fibrous materials such as tobacco plant parts or cellulose in the preparation of slurries for sheet making by a casting process. The processes whereby sheet, flake and filamentous smoking materials are manufactured are well known in the art.

According to a first aspect of the present invention'there is provided a smoking material including a substrate selected from tobacco cells, tobacco plant material, and cellulosic material, and including a crystallisable 3 sugar selected from the group of mono- and disaccharides, the total amount of said sug-ar in the material being at least 20% by weight of the material.

According to a second aspect of the present invention there is provided a process for producing tobacco cells containing at least 5% nicotine by dry weight, the process comprising preparing a tobacco callus, inoculating portions of the callus aseptically into a liquid nutrient medium containing 0.1 to 0.3 M of a saccharide and permitting in a growth cycle a dense suspension culture of cells to form containing said nicotine content, and separating the nicotine-rich cells so produced.

According to a third aspect of the invention there is provided a method of providing a high-yielding suspension culture of living cells by inoculating a nutrient medium with said living cells and immediately storing the inoculated medium at a temperature of O-WC.

According to a fourth aspect of the present invention there is provided a method of non-destructively extracting an alkaloid from suspensioncultured living plant cells comprising placing the cells in a solution of one or more inorganic salts and subsequently extracting the alkaloid from the solution.

According to a fifth aspect of.the present invention there is provided a method of making a smoking material comprising the steps of (a) preparing a mixture of cellulosic fibres, a burnmodifying agent, at least 20% by weight of a crystallizable sugar, a humectant, and water, (b) milling the mixture to give a slurry, and (c) further treating the slurry to give a smokeable product.

4 Objects of the present invention are, (1) to maximise productivity in terms of specific growth rates and nicotine yields. (2) to produce smoking materials in which less expensive components are extensively used and in which the use of cultured tobacco cells is minimised. (3) to overcome any problems associated with the flavour and aroma of the smoke derived from cultured tobacco cells which, being different to the corresponding properties of the smoke from conventional tobacco, may be unacceptable to some tobacco smokers.

The feature of the invention whereby relatively large proportions of simple sugars, e.g. more than about 20% by weight, are incorporated in the smoking material has advantages with regard to smoke flavour. These relatively large proportions hitherto not been achieved because of the undesirable stickiness (or tackiness) that such sugars impart.

We have found that the combination of a highly efficient cell selection technique which allows growth of cells at low densities and thereby the isolation of true clones, and the optimisation of cultural conditions by the process of the invention results in the acquisition of.cell lines containing 5 to 10% nicotine in the tobacco cells expressed as a percentage of dry weight. Cell lines containing at least 30% nicotine may be obtained by feeding exogenous nicotine to the cultures. These levels of nicotine are considerably hiiher than any hitherto disclosed in the prior art of tobacco cell tissue culture.

1 We have developed a method of preventing instability in these cell lines, which may be generally applicable to other high yielding cell lines. Maintenance of higli-yielding callus and suspension cultures by serial subculture is expensive and time-consuming and presents the risk of loss by error or by microbial contamination. Also, cytological changes may occur when cells are maintained under growth conditions for extensive periods often leading to a loss or deterioration of secondary metabolite production. The method of the present invention is surprisingly simple, effective and inexpensive, and overcomes these problems. It involves storage of suspension cultures at a temperature of 0-6C, preferably at CC. Storage may be continued for a period of several months. A pool of stable cell material is thereby provided which may be used either for experimental purposes or as inocula when production cultures become unstable.

Whereas successful regrowth of callus cultures following storage at a temperature of around CC is known in the art, we are not aware of any prior art which indicates not only that cell suspensions can be stored in this way, but also that their capacity for secondary product synthesis is unaffected by the storage procedure.

It is believed that previous attempts by other workers in the field to store suspension cultures at temperatures at around CC have probably been made but have not been successful. Further. it is believed that the main reason for this lack of success in earlier attempts is probably that cultures were grown for various periods of time prior to storage rather than placed in storage at low temperature immediately following inoculation. It is worthy of note, in this respect,,that we have found cell suspensions aged from 7 to 28 days, unlike freshly inoculated cells (i.e. aged 0 days), to have very poor growth and nicotine production

6 following storage at a temperature of CC.

In addition to its simplicity and ease of operation, the method of the present invention does not present the problems associated with conventional cryopreservation techniques such as the requirement for expensive equipment or cryoprotectants, the necessity for rapid yet precise preparation procedures and the development of a suitable and specific freezing protocol for each cell type concerned. Indeed, assuming general applicability. we propose that our technique which we have shown to be most effective for storing suspensions of Nicotiana tabacum cells, constitutes an inexpensive and effective method for preserving highyielding suspension cultures of a variety of plant species and perhaps also other material such as animal cell cultures.

We have found that nicotine production is substantially increased by using specific concentrations of naphthaleneacetic acid (1 microM), kinetin (0.1 microM) and sucrose (0.25 M) in the medium. Further increases in growth rate and/or nicotine production are obtained by replacing sucrose_by glucose, by increasing the dissolved oxygen concentration i.e. by increasing aeration of fermenters or flask shaker speed and by adding spermidine (0.1 milliM), spermine -(0.1 milliM) and supplementary vitamins.

We have found that fermenter productivity could he increased substantially by reducing cell moisture content. This was achieved both by clonal selection in which cell lines with less than 90% moisture were isolated by the cell plating technique described above and by adding extra amounts of sucrose to suspension cultures at periods throughout'the growth cycle. Adding extra amounts of sucrose throughout the growth cycle also resulted in cells in which the concentration of nitrate was less than the 7 concentration found in cells grown without extra sucrose.

Several processes for producing material for smoking are known and of these we have found that the cast sheet, flake and filament extrusion processes are the most appropriate.

Slurries prepared from substrates for casting on a band drier or pastes prepared for extrusion normally contain dewatered tobacco cells. However, other substrates such as tobacco materials, non-tobacco fibrous materials or combinations of these may also be used. The preferred tobacco materials are lamina or leaf petiole of Nicotiana tabacum or Nicotiana rustica and suitable fibrous materials are vegetable fibres such as refined cellulose, wood pulp and sugar beet pulp.

Whereas fillers well known in the art such as metal oxides, carbonates and silicates may be added, our preference is not to use such fillers to any great extent because they are known to have an adverse effect on burning coal retention and ash cohesion in cigarettes. Of the many burn control and ash improvement agents known to the art, we have found that in smoking materials of this invention calcium and potassium salts of organic acids are particularly useful additives by which burning and ash- forming properties may be controlled. Insoluble calcium salts may be formed in situ, as is known in the art, by combining soluble forms of the metal such as.calcium acetate with citric, malic or tartaric acids or with other acids.

Although the use of mono- and disaccharide sugars in'smoking materials is known, concentration levels have necessarily been restricted by the tendency of these highly water soluble and hygroscopic compounds to cause 8 unwanted stickiness or tackiness in the materials in which they are incorporated. We have found, however, that by careful choice of sugar, by careful selection of relative proportions when more than one type is used and with due regard to the relative concentrations of the other components, that surprisingly high proportions of certain sugars may be incorporated. This is not without considerable benefits both for physical strength of resulting smoking materials and for the organoleptic properties of their smokes.

While we do not wish to be constrained by theoretical considerations, it is our belief that the unexpectedly good physical strength and lack of stickiness exhibitedby the cast smoking materials of this invention are accounted for by the ability of the contained sugars to crystallise after a certain lapse of time following film formation. We have found that the disaccharide lactose and the monosaccharide glucose and combinations thereof in which the proportion of glucose is between 15 and 45% by weight are particularly useful in this respect, and glucose in particular is beneficial in sweetening smoke taste. Galactose and xylose are also effective monosaccharides but are relatively more expensive. Sucrose and fructose do not crystallise under the conditions which obtain in sheet manufacture, therefore use of these two sugars is best restricted to lower levels where they both have a role as smoke sweetening agents.

Binders and film-forming agents are believed to have an adverse effect on smoke flavour. It is therefore an advantage of the present invention that such compounds are not normally required to be added to the slurries from which the cast sheet materials of this invention are,Prepared and only in low concentrations to the pastes from which the filaments and sheets are extruded. When binders are required there is a range of materials known to 9 the art from which to choose. We have found that sodium carboxymethyl cellulose is the preferred material.

We have found that by incorporating crystallisable sugars in our sheet formulations it becomes possible to incorporate relatively large quantities of compounds which are normally liquid at ambient temperatures. Thus unexpectedly high levels of polyhydric alcohol humectants such as glycerol. glycerol monoacetate and propylene glycol may be added without causing stickiness, loss of physical strength or reduced filling power. Likewise, polyhydric alcohols such as sorbitol which are normally solids at ambient temperatures and which distil with telatively little modification into smoke, may also be incorporated. These considerations also apply to the extruded sheet and filaments of the invention.

Lipoidal and lipophilic compounds are also useful additives. Fatty acids, fatty alcohols and fatty esters, diand triglycerides such as palmitic acid, dodecanol, methyl palmitate, dipalmitin and triolein respectively may be added singly or in combination. Natural lipid mixtures such as olive oil, corn oil, arachis oil, coconut oil, linseed oil, jojoba or cocoa butter may also be utilised.

The components of smoke generated from smoking materials are generally considered to be formed either by direct distillation of volatile components or by pyrolysis or by pyrosynthesis. It is a distinct advantage in materials of the present invention that, if required, greater emphasis may be placed on the direct distillation mechanism in the smoke-forming process.

1 Tobacco aroma and smoke flavour character may be imparted to materials of this invention by including tobacco leaf or stem material in the slurries or pastes prior to casting or extruding respectively. Alternatively tobacco extracts, resinoids or oils may be used for this purpose. Flavour and aroma character of a type which is associated with tobacco products may be developed by adding flavour and aroma components well known in the tobacco flav'ourist's art. For example, vanillin, coumarin, menthol, essential oils such as flouve and foin, resinoids, plant and fruit extracts such as licorice, prune, raisin and apple extracts may be used. Discrete chemical compounds such as esters, lactones and ketones or mixtures thereof may also be used. A large array of flavouring and fragrance materials is available and this offers considerable scope to impart subtle and attractive flavour and fragrance properties particularly since the smoking materials of the invention are designed not to display overstrong intrinsic background smoke aroma and flavour character. A wide range of consumer preferences may thus be catered for.

The nicotine contents of smoking materials of this invention are primarily derived from the tobacco cells obtained by suspension culture. Nicotine concentrations may be supplemented if required by addition of tobacco plant materials, extracts or distillates. The nicotine contents of variants which do not contain cultured tobacco cells may be derived from tobacco plant materials which have relatively high levels of nicotine compared with most conventional smoking tobaccos. Nicotiana rustica lamina and stem or extracts or distillates thereof are particularly useful in this respect.

Alternatively, nicotine which has been isolated from tobacco plant or cultured tobacco cell material may be incorporated into the slurries or pastes from which these smoking materials are derived either in semi-pure 1 or in concentrated liquid form or as salts with organic or inorganic acids 1 11 or in mixtures containing other ingredients of the slurries or pastes. This option is particularly appropriate for variants which contain neither cultured tobacco cells nor tobacco plant material. In this regard we found surprisingly that filtered tobacco cells, on placement into fresh medium or a solution of one or more of the inorganic salt components of the medium, especially ammonium nitrate, expelled their nicotine alkaloid content almost entirely within a few hours. Alkaloid fractions obtained in this way, unlike those obtained by methods involving cell destruction, were substantially free of cell debris and other extractives, thereby facilitating filtration and improving purity. Also, cells thus treated retained high levels of viability, aliowing them to be regrown following nicotine removal. It is envisaged that this method could be generally applied to isolate alkaloids non-destructively from suspension-cultured plant cell material (i.e. without killing a substantial proportion of the cells).

It is important with regard to the stability of nicotine incorporated in smoking materials to ensure that the pH is maintained on the acid side of neutrality. pH is readily adjusted downwards when necessary, preferably to levels between 4 and 6 by adding water soluble acids to the slurries and pastes. Citric or malic acids are preferred acids to perform this function but there are many acids known to the art which would serve equally well.

The colour of the sheets, flakes and filaments of this invention depends on the nature of the added fibrous materials. Products which incorporate tobacco materials adopt the natural colour of tobacco. Products containing tobacco cells, being brown in colour, resemble aircured tobacco in appearance. Flue-cured tobacco appearance is readily produced and air-cured tobacco appearance is improved by adding appropriate quantities of yellow 12 pigments to the slurries or pastes prior to casting or extruding. Food dyes or any suitable colorant known in the art may be used but preference is given to 13-carotene which is one of the major yellow pigments of natural tobacco. 13carotene is preferably dissolved in or emulsified with non-polar carriers such as olive oil or triacetin prior to addition.

Gradual loss of colour which may otherwise occur particularly in variants which do not contain tobacco leaf material, may be prevented by incorporating antioxidants, preferably a-tocopherol, a tobacco leaf component but also ascorbic acid or butylated hydroxytoluene (BHT). Slurries and pastes prepared with colourless or lightly coloured fibrous materials such as cellulose or sugar beet pulp may be coloured to the required hue using a combination of caramel browning and yellow pigment.

The processes of casting or extruding slurries or pastes are well known in the art and need not be described in detail. Mixtures are prepared for casting sheet material by intimately mixing the components in the required proportions with water. Where cultured tobacco cells are included, addition of extra water may not be necessary. The mixtures are milled to give a homogeneous slurry containing 5-25% preferably 10-20% solids. Solids contents outside this range are possible but increased processing difficulties may result.

Small scale trials of the sheet-forming performance of a plurality of slurry types are effectively conducted using a laboratory spreader with a 1 mm. gap and casting the slurries onto flat surfaces covered with plastic film. Drying to 15% or less moisture is achieved using portable hot air driers, and sheets are separated from the plastic film in large pieces. The dried sheets are broken up into small irregularly-shaped pieces, shredded j 13 and stored (cured) for the period, normally 3 days to 3 weeks, required to effect complete or partial crystallisation of the incorporated sugars. The appropriate curing period depends mainly on the nature and concentration levels of the sugar and humectant components. Compositions in which the sugar component is represented solely by lactose are quick to cure. On the other hand those in which the sugar is solely glucose or galactose require a longer curing period.

We have found that a combination of 40 parts of glucose and 60 parts of lactose whilst being relatively inexpensive requires a satisfactorily short curing period and also provides a desirable background smoke aroma and flavour. The curing period may be shortened by increasing the proportion of lactose towards 100% but in doing this the burnt sugar sweet character is reduced. Greater proportions of glucose result in longer curing times or failure to cure. Products which contain relatively low levels (0.1-5%) of glycerol or other polyhydric alcohol cure more quickly than those containing higher levels (5-2M). In this respect not only the sugar composition but also the absorptive power, hygroscopicity and concentration of the fibrous material is important.

Bulk quantities of sheet material are prepared by casting slurries on to a band drier in the normal way. Following rewetting and doctoring the sheet off the band, the material is preferably dried back to less than 10% moisture. The sheet may then be cut into suitably sized pieces for blending with tobacco materials or stored in bulk to cure without adhesion of the separate pieces becoming a problem. Alternatively, the sheet may be shredded prior to being stored for curing.

1 Pastes which are suitable for extrusion into filaments, flakes or sheets 14 require high solids contents, typically of the order of 70-80%. When the intention is to incorporate significant quantities of tobacco cell suspension cultures in such pastes it is therefore necessary to reduce their moisture content from around 90-95% to a much lower level. We have found that spray-dried material has superior physical form compared with that produced by hot air, vacuum or microwave drying. Tobacco cell cultures are harvested, dewatered and wet milled. pH is adjusted if necessary down to 4-5 using a water soluble acid such as citric or malic acid. A combination of low pH and a spray drier outlet temperature of 6080'C results in a powder containing around 10% moisture from which nicotine loss is zero or minimal. The resulting powder or dry-milled tobacco or dry-milled cellulose or any combination of these is mixed thoroughly with the other components mentioned above and a paste is produced by adding water to the required consistency. The paste is extruded either in a screw extruder fitted with a die containing a plurality of rectangular orifices or between the rollers of a roll mill. The filaments and sheet are cut, or cut and shredded, respectively prior to manufacturing into cigarettes. Alternatively, when it is intended to blend the extruded material with tobacco, flakes are produced or the sheet material is cut into small shapes by conventional procedures.

Where blending with tobacco leaf material at the pre-shredding stage is not intended we have found that a particular advantage is to be gained in connection with the cast sheet product by shredding it immediately after manufacture at a moisture content of less than 10% and storing the shreds in bulk for at least 1 day. In this way the shreds come to rest in random and irregular formations and such formations are perpetuated by the subsequent crystallisation of the contained sugars whereupon moisture may be raised to normal levels. Smoking material thus formed closely resembles is conventional tobacco shreds in phsical form and this not only expedites its manufacture into cigarettes using conventional cigarette making machinery but also is a beneficial feature with regard to ash cohesion.

The invention will now be described by way of example only with reference to the following nonlimiting examples and Figures 1-12 which are graphs illustrating results obtained from the examples. The figures will be referred to and described in the examples as the occasion arises. In the examples all composition percentages and ratios are to be taken as being with reference to weight unless otherwise stated.

EXAMPLE 1: Selection of Cell lines with Reduced Moisture Contents.

(i) Preparation of Plating Medium.

A semisolid medium for cell-plating was prepared by filteringactively growing cell suspensions through a mesh (0.5 mm. mesh size) to obtain a filtrate consisting of conditioned medium and cells. The filtrate was mixed with an equal volume of agar medium (Table 1) at double normal nutrient strength which had been sterilised by autoclaving at 1.05 kg. per cm.2 gauge for 15 minutes and then cooled to 400C. Plates were poured immediately after mixing. When the plates were cool, a second layer of sterilised agar medium of normal strength was poured over the first layer and allowed to set.

16 TABLE 1

Formulation of growth medium (modified Murashige and COMPOUND ammonium nitrate; NH4NO3 potassium nitrate; KM3 anhydrous calcium chloride; CaC12 anhydrous magnesium sulphate; MgS04 potassium dihydrogen phosphate; KH2P04 sodium EDTA; Na2.EDITA ferrous sulphate; FeS04.7H20 manganese sulphate; MnS04.H20 zinc sulphate; ZnS04.7H20 boric acid; H3B03 potassium iodide; KI sodium molybdate; Na2Mo04.2H20 copper sulphate; CuS04.5H20 cobalt chloride; CoC12.6H20 i-inositol; thiamine hydrochloride; thiamine. HCl nicotinic acid pyridoxine hydrochloride; pyroxidine.HCl glycine naphthaleneacetic acid kinetin sucrose agar in semi-solid media = A is ethylene diamine tetra-acetate.

Skoog medium) mg/litre 1650.000 1900.000 332.200 180.700 170.000 37.250 27.850 16.900 8.600 6.200 0.830 0.250 0.025 0.025 100.000 0.500 0.500 0.500 2.000 0. 000001 m 0.0000001 m 34000.000 6000.0001 17 i (ii) Cell Plating..

Actively growing cell suspensions were filtered through two layers of muslin. The filtrate was diluted with sterile liquid medium (Table 1) in a ratio of 1:40 and 1 ml. portions of the mixture spread onto the surface of the plating medium. After 3-4 weeks, discrete colonies of cells were removed from the plating medium and transferred to fresh semi-solid medium. After a further period of growth, 1 g. portions of callus were transferred to 100 ml. of sterilised liquid medium (Table 1) contained in 250 ml. conical flasks. The cultures were orbitally shaken at 150 r.p.m. in the dark at 25'C for 28 days prior to being analysed for moisture content. The results are shown in Figure 1 wherein, as can be seen, three cell lines of the eighty-six selected by cell plating contained less than 91% moisture.

EXAMPLE 2: Selection of Cell Lines with Increased Nicotine Content.

Cell lines were selected and grown as described in Example 1. Freezedried cells were analysed for nicotine after 28 days growth in liquid medium (Figure 2).

As can he seen in Figure 2, cell lines containing up to 7.3% nicotine were selected by cell plating.

EXAMPLE 3: Improved Method of Selecting Cell Lines with Increased Nicotine Content.

While cell lines with high levels of nicotine can be isolated by cell plating (Example 2), the method is labour intensive and time consuming. 18 Attempts were therefore made to improve the selection technique by

plating out a moderately dense suspension of cells and imposing a selection pressure WV light) under which only those cells with a high capacity for nicotine synthesis might be expected to grow. Irradiation of plated cells with UV light for 40 minutes reduced the number of clones which grew from approximately 1000 (control plates) to 11 (UV treatment). Analysis of the 11 cell lines for nicotine content showed that one of them contained 6.3% nicotine (Figure 3). The nicotine content of this cell line compared favourably with that of the highest yielding cell lines isolated from 758 clones produced by the standard procedure described in Example 1 (Figure 4).

EXAMPLE 4: Increasing Nicotine Content by Replacing Sucrose with Glucose.

Suspension cultured cells (27. w/v) were added to 100 ml. of sterilised liquid medium (Table 1) contained in 250 ml. conical flasks or to a similar medium in which 0.1M sucrose was replaced by 0.2M glucose. The cultures were orbitally shaken at 150 r.p.m. in the dark at 251C. Samples were removed at intervals and analysed for dry weight and nicotine content (Figure 5).

As can be seen in Figure 5, the nicotine content of cells grown in the presence of 0.2M glucose was more than double that of cells grown in the presence of 0.1M sucrose.

EXAMPLE 5: Increasing Nicotine Content by Modification of Shaking Speed.

Suspension cultured cells (2% w/v) were added to 100 ml. of sterilised liquid medium (Table 1) contained in 250 ml. conical flasks. The cultures 19 were orbitally shaken at either 100 or 150 r.p.m. in the dark at 25'C. Samples were removed at intervals and analysed for dry weight and nicotine content (Figure 6).

As can be seen in Figure 6, an increase in shaking speed from 100 to 150 r.p.m. caused over a four-fold increase in the nicotine content of the cells.

EXAMPLE 6: Increasing Nicotine Content by the Addition of Spermine.

Suspension cultured cells (2% w/v) were added to 100 ml. of sterilised liquid medium (Table 1) contained in 250 ml. conical flasks (control) or to a similar medium which contained 0.1 mM spermine. Cultures were incubated and analysed as described in Example 4.

Figure 7 shows that the nicotine content of the cells was increased by about 507. in the presence of 0.1 mM spermine.

EXAMPLE 7: Increasing Nicotine Content by the Addition of Supplementary Vitamins.

Suspension cultured cells (2% w/v) were added to 100 ml. of sterilised liquid medium (Table 1) contained in 250 ml. conical flasks (control) or to a similar medium which contained 4.92 mg./L nicotinic acid, 1.23 mg./L. pyroxidine-HCl, 13.49 mg./L. thiamine-HCl, 0.24 mg./L. biotin, 0.88 mg. /L. folic acid, 1.20 mg./L. D-calcium pantothenate and 1.98 mg./L. ascorbate. Cultures were inoculated and analysed as described in Example 4.

1 Figure 8 shows that the nicotine content of cells grown in the presence of supplementary vitamins was increased by about 37% after 27 days growth.

EXAMPLE 8: Increasing Nicotine Content by the Addition of Exogenous Nicotine.

Stispension cultured cells (2% w/v) were added to 100 ml. of sterilised liquid medium (Table 1) contained in 250 ml. conical flasks (control) or to the same medium containing 80, 160, 320 or 640 mg. of nicotine per 100 ml. Cultures were incubated as described in Example 4 and analysed for dry weight and nicotine content after 34 days (Figure g).

As Figure 9 shows, exogenous nicotine did not reduce dry weight yield 'but did increase the nicotine content of the cells. At the highest concentration of exogenous nicotine (640 mgj100 ml.) the nicotine content of the cells was increased to almost 30% of dry weight. Only small amounts of nicotine were present in the medium 'after 34 days.

EXAMPLE 9: Increasing Productivity by the Supplementary Addition of Sucrose.

Suspension cultured cells (10% w/v) were added to 100 ml. of sterilised liquid medium (Table 1) contained in 250 ml. conical flasks. Cultures were incubated and analysed as described in Example 4. After 11 days, 10 ml. of distilled water was added to half of the flasks (controls) and 10 ml. of a concentrated sucrose solution added to the other flasks to give a final concentration of 0.2M.

Figure 10 shows that the supplementary addition of 0.2M sucrose reduced the 1 moisture content of the cells from 97% to 88%, increased the dry weight A 21 yield approximately threefold and increased the total amount of nicotine by about 66% after 18 days growth.

EXAMPLE 10: Growth Curve of a High Nicotine Yielding Cell Line.

Suspension cultured cells (2% w/v) were added to 100 ml. of sterilised liquid medium (Table 1) contained in 250 ml. conical flasks. Cultures were incubated and analysed as described in Example 4.

Figure 11 shows the growth and nicotine content of a high yielding cell line of Nicotiana tabacum.

EXAMPLE ll: Cryopreservation by Storage of Suspension Cultures at 4C.

Suspension cultured cells (2% w/v) were added to 100 ml. of sterile liquid medium (Table 1) contained in 250 ml. conical flasks. The flasks were immediately placed in a cold store at 40C and removed at intervals of 1, 2, 3 and 4 months when they were incubated under normal conditions as described in Example 4. Control cultures were maintained under normal conditions throughout the duration of the experiment and subcultured at monthly intervals. Both control and treated cultures were analysed for growth and nicotine production after 28 days under normal incubation conditions.

Figure 12 shows that the dry weight yields of the treated cultures were similar to those of the controls when they were grown after storage at 41C for 1, 2, 3 or 4 months. In contrast to control cultures which showed a slight decline in nicotine production during the 4 month period, the treated cultures surprisingly tended to show an increase in nicotine 22 production. The results clearly show that cultures of Nicotiana tabacum can be stored at 4'C for at least 4 months without losing their capacity for growth or nicotine synthesis.

EXAMPLE 12: Rapid Release of Nicotine from Nicotiana tabacum Cells.

Suspension cultured cells (2Y. w/v) were added to 100 ml. of sterile distilled water, sterile liquid medium (Table 1) or to sterile ammonium nitrate solution (20mM) contained in 250 ml. conical flasks. Cultures were incubated as described in Example 4 and, after six hours, the cells were filtered from the medium, freeze-dried and analysed for nicotine. The amount of nicotine released from the cells was expressed as a percentage of that in the inoculum.

As can be seen in Table 2, cells which were incubated in distilled water for 6 hours released 66% of their nicotine. Cells which were incubated in medium or in a simplified medium consisting only of 20mM ammonium nitrate released 96% of their nicotine over the six hour incubation period.

TABLE 2

The effect of medium constituents on the release of nicotine from Nicotiana tabacum cells (Example 12).

Medium Constituents Amount of nicotine released from the cells distilled water 66 ammonium nitrate (20mM) complete medium 96 96 23 Examples 13, 14 and 16-21 concern the preparation of a smoking material or product from suspension-cultured tobacco cells prepared according to the method described in Examples 1 to 12, and containing 5- 30% by dry weight of nicotine. Example 15 does not contain suspension- cultured tobacco cells.

The required quantities of suspension-cultured tobacco cells were obtained using either 90 L. or 20 L. fermenters or 5 L. conical flasks orbitally shaken at 150 r.p.m. Tobacco cells were separated from the nutrient media either by filtration or by centrifugation.

EXAMPLE 13

A tobacco cell substrate (700 g.; 95% moisture), a cellulose fibre substrate (14 g.; dry-milled Whatman No. 1 filter paper sheets), calcium g. 1 PO g.), glucose (25 g.), glycerol (4.2 g.) and spray-dried licorice powder (4.2 g.) were. mixed and passed through a carborundum stone mill several times until a slurry of fine consistency was obtained. The slurry contained 17% solids.

4+".+ 11A -4+"-+ (A 9 1QA A mixture of olive oil (0.7 g.), arachis oil (0,7 g.) and linseed oil (0. 1 g.) in which was dissolved 13-carotene (0.25 g.) was mixed with the slurry and blending was completed by further passes through the stone mill. The slurry was cast onto a polyethylene coated band and hot air dried. The resulting sheet material was removed and cut into random pieces approximately 1 inch square.

The approximate proportions of components in the resulting smoking material, expressed on a dry weight basis, are shown in Table 3.

24 TABLE 3

Proportions of components in smoking material of Example 13 (dry weight basis).

25.3 10i2 10.2 3.0 26.1 18.1 3.0 3.0 0.5 0.5 0.03 0.07 tobacco cells cellulose fibres calcium citrate potassium citrate lactose glucose glycerol licorice olive oil arachis oil linseed oil 13-carotene The dried product was shredded, stored in bulk at 28% relative humidity for one week and then dried at ambient relative humidity for 4 weeks to complete curing prior to making into cigarettes.

King-size cigarettes made from this material burned well with good smoulder, puffing and ashing properties.

EXAMPLE 14

A shredded tobacco substrate (250 g.; standard flue-cured blend), a cellulose fibre substrate (100 g.), calcium citrate (100-g.), potassium citrate (30 g.), glycerol (50 g.), olive oil (5 g.), glucose (195 g.), lactose (270 g.) End water ( 5 L.) were mixed and milled as in Example 13 giving a slurry of pH 5.6 and 16.7% solids content. The slurry was cast, dried, shredded and cured as before.

The approximate composition of the light brown product, expressed on a dry weight basis, is shown in Table 4.

TABLE 4

Composition of product of Example 14 (% dry weight basis).

tobacco 25O 10.0 10.0 3.0 5.0 0.5 19.5 27.0 cellulose fibres calcium citrate potassium citrate glycerol olive oil glucose lactose King-size cigarettes made from the material burned well with good smoulder, puffing and ashing properties, and with mild mainstream and caramellic sidestream smoke character.

EXAMPLE 15

A cellulose fibre substrate (300 g.), calcium citrate (100 g.), potassium citrate (100 g.), glycerol (100 g.), glucose (120 g.), lactose (280 g.), caramel (5 g.) and water (9 L.) were mixed and milled as in Example 13 26 giving a slurry containing 10.1% solids. The resulting slurry was cast, dried and shredded as before.

Prior to completion of curing, the shredded material was sprayed with a solution of palmitic acid (2 g.), tripalmitin (2 g.), methyl palmitate (2 g.), 13-carotene (0.2 g.) and a-tocopherol (2 g.) dissolved in a 1:1 mixture (600 ml.) of ethyl acetate and n-hexane to give, following evaporation of solvent, an approximately 1% loading of the lipid mixture.

The approximate composition of the yellow/brown product, expressed on a dry weight basis, is shown in Table 5.

TABLE 5

Composition of product of Example 15 (% dry weight basis) cellulose fibres 29.6 calcium citrate 9.9 potassium citrate glycerol glucose lactose caramel lipids 9.9 9.9 11.8 27.6 0.5 0.8 After a total curing period of 7 days, king-size cigarettes were made from the resulting smoking material. These burned well with good smoulder, puffing and ashing properties. Unflavoured cigarettes exhibited mild mainstream smoke with some burning paper character and sidestream smoke 1 with sweet burnt sugar notes. Subjective quality was significantly 27 improved when cigarettes were treated with vanillic/fruity-type pipe tobacco flavour formulations.

ECWLE 16 A shredded tobacco substrate (250 g.; standard flue-cured blend), a tobacco cell substrate (5 kg.; 95% moisture), calcium citrate (100 g.), potassium citrate (50 g.), lactose (200 g.), glucose (130 g.) and glycerol (20 g.) were mixed and milled as in Example 13 giving a slurry containing 17.4% solids. The slurry was cast, dried, shredded and cured as before.

The approximate proportions of components in the resulting smoking material, expressed on a dry weight basis, are shown in Table 6.

TABLE 6

Composition of smoking material of Example 16 (% dry weight basis).

tobacco 25 tobacco cells calcium citrate potassium citrate lactose glucose glycerol 10 5 20 13 2 King-size cigarettes made from this material burned well with good smoulder, puffing and ashing properties, and exhibited mild tobacco character in both the mainstream and sidestream smoke.

1 28 E-CWLE 17 Whole fermenter broth (12.9 kg.; i.e. cells plus cell debris plus spent nutrient medium) cellulose fibres (100 g.), lactose (280 g.), glucose (55. 5 g.), calcium citrate (75 g.) potassium citrate (75 g.) glycerol (100 g. ) and sodium carboxymethyl cellulose (10 g.) were mixed and milled as in Example 13 giving a slurry containing 8V. solids.

The slurry was cast, dried and shredded and, after curing for 2 weeks, the shreds were coated by immersion in a solution of palmitic acid (2.4 g. ), methyl palmitate (2.4 g.), tripalmitin (2.4 g.), olive oil (2.4 g.), 0carotene (0.2 g.), atocopherol (0.2 g.) in a 1:1 mixture (600 ml.) of ethyl acetate and n-hexane giving a loading of approximately 1% following removal of excess lipid solution and evaporation of residual solvent.

The approximate composition of the yellow brown product, expressed on a dry weight basis, is shown in Table 7.

29 TABLE 7

Composition of product of Example 17 (% dry weight basis). tobacco cells spent nutrient solids cellulose fibres lactose glucose calcium citrate potassium citrate glycerol sodium carboxymethyl cellulose lipids 24.6 6.3 9.8 27.5 5.4 7.3 7.3 9.8 1.0 1.0 King-size cigarettes made from the finished smoking material burned well with good smoulder, puffing and ashing properties.

EXAMPLE 18

A tobacco cell substrate (5 kg.; 95% moisture), a cellulose fibre substrate (100 g.), calcium citrate (100 g.), potassium citrate (30 g.), lactose (280 g.), glucose (180 g.), sorbitol (50 g.) and olive oil (5 g.) were mixed with water (2 L.) and the mixture was milled as in Example 13 giving a slurry containing 12.8% solids. The slurry was cast, dried, shredded and cured for several weeks.

The approximate composition of the brown product, expressed on a dry weight basis, is shown in Table 8.

TABLE 8

Composition of product of Example 18 (% dry weight basis) tobacco cells 25.1 cellulose fibres 10.1 calcium citrate 10.1 potassium citrate lactose glucose sorbitol olive oil 3.0 28.1 18.1 5.0 0.5 King-size cigarettes made from this material burned well with good smoulder, puffing and ashing properties.

EXAMPLE 19

Nicotiana rustica tobacco strips (250 g.), cellulose fibres (100 g.), calcium citrate (100 g.), potassium citrate (30 g.), glycerol (50 g.), lactose (270 g.), glucose (195 g.), and olive oil (5 g.) were mixed with water (5 L.) and the mixture was milled as in Example 13. The slurry was cast, dried, shredded and cured for several weeks.

The approximate composition of the dark brown product at 8.1% moisture is shown in Table 9.

31 TABLE 9

Composition of product of Example 19 at 8.1% moisture.

Nicotiana rustica tobacco 23.0 cellulose fibres 9.2 calcium citrate 9.2 potassium citrate glycerol lactose glucose olive oil water Nicotine content was 1.7% at 8.1% moisture.

2.8 4.6 24.8 17.9 0.4 8.1 King-size cigarettes made from this material burned well with good smoulder, puffing and ashing properties. The mainstream smoke exhibited sweet and cigar-like notes and the sidestream smoke had oriental tobacco- like character.

EXAMPLE 20

Tobacco cells (1.2 kg.; high nicotine cell line; nicotine 7.8% on a dry weight basis), cellulose fibres (24 g.), lactose (70 g.), glucose (26 g.), glycerol (16.8 g.), calcium citrate (21.6 g.) and potassium citrate (21. 6 g.) were mixed and milled as in Example 13.

The resulting slurry was cast, dried, shredded and cured for several weeks.

32 The approximate composition of the brown product, expressed on a dry weight basis, is shown in Table 10.

TABLE 10

Composition of product of Example 20 (% dry weight basis) tobacco cells 25.0 cellulose 10.0 lactose 29.2 glucose glycerol calcium citrate potassium citrate 10.8 7.0 9.0 9.0 Nicotine content was 1.9% on a dry weight basis.

King-size cigarettes made from this material burned well with good smoulder, puffing and ashing properties.

EXAMPLE 21

Tobacco cell substrate (1.6 kg.; very high nicotine; cf. Example 8; nicotine 1.5% on a fresh weight basis), cellulose fibre substrate (220 g. ), calcium citrate (100 g.), potassium citrate (100 g.), glycerol (100 g. ), glucose (120 g.), lactose (280 g.), caramel (5 g.) and water (7 L.) were mixed and milled as in Example 13. The resulting slurry was cast, dried and shredded as before. After 3 days, the semi-cured shredded material was coated with lipids as in Example 15.

33 The approximate composition of the yellow/brown product, expressed on a dry weight basis, is shown in Table 11.

TABLE 11

Composition of product of Example 21 (% dry weight basis).

tobacco cells 7.9 cellulose fibres calcium citrate potassium citrate glycerol glucose lactose caramel lipids 21.7 9.8 9.8 9.8 11.8 27.6 0.5 1.1 Nicotine content was 2.3% on a dry weight basis. King-size cigarettes made from this material burned well with good smoulder, puffing and ashing properties.

34 CLA ISIS 1. A smoking material including a substrate selected from tobacco cells, tobacco plant material, and cellulosic material, and including a crystallisable sugar selected from the group of mono- and disaccharides, the total amount of said sugar in the material being at least 20% by weight of the material.

2. A smoking material as claimed in 1 wherein the disaccharide is lactose and the monosaccharide is selected from glucose, galactose and xylose.

3. A smoking material as claimed in 2 wherein said sugar comprises a mixture of lactose and glucose.

4. A smoking material as claimed in 3 wherein the percentage of glucose in the mixture is at least 15% by weight.

5. A smoking material as claimed in 1 wherein the smoking material contains nicotine.

6. A smoking material as claimed in 5 wherein the nicotine is provided by the inclusion in the smoking material of cultured tobacco cells containing an enhanced level of nicotine.

7. A smoking material as claimed in 6 wherein the cultured tobacco cells contain 5-30% nicotine by dry weight.

8. A smoking material as claimed in 5 wherein the nicotine is provided by 1 the inclusion of tobacco plant material in the smoking material.

1 9. A smoking material as claimed in 5 wherein the nicotine is provided in the smoking material as free nicotine or as a salt or as a tobacco extract.

10. A smoking material as claimed in 9 wherein the smoking material includes a nicoine stabiliser.

11. A smoking material as claimed in 10 wherein the nicotine stabiliser is provided by a water-soluble acid.

12. A smoking material as claimed in 11 wherein the water-soluble acid is citric acid or malic acid.

13. A smoking material as claimed in 1 wherein the smoking material contains a burn-modifying agent.

14. A smoking material as claimed in 13 wherein the burn-modifying agent is a calcium or potassium salt of an organic acid.

15. A smoking material as claimed in 14 wherein the organic acid is selected from the group comprising citric, malic, and tartaric acids.

16. A smoking material as claimed in 1 wherein the tobacco plant material is selected from tobacco lamina, and tobacco leaf petiole of Nicotiana tabacum or Nicotiana rustica.

17. A sTaoking material as claimed in 1 wherein the cellulosic material is a vegetable fibre or a cellulose fibre.

36 18. A smoking material as claimed in 1 wherein the smoking material contains a humectant.

19. A smoking material as claimed in 18 wherein the humectant is a polyhydric alcohol or an ester of a polyhydric alcohol.

20. A smoking material as claimed in 19 wherein the humectant is selected from the group comprising glycerol, glycerol monoacetate, propylene glycol, and sorbitol.

21. A smoking material as claimed in 19 wherein the humectant comprises 0. 1-20% by weight of the smoking material.

22. A smoking material as claimed in 1 wherein the smoking material contains lipoidal or lipophilic compounds.

23. A smoking material as claimed in 22 wherein the lipoidal and lipophilic compounds are selected from the group consisting of fatty acids, fatty alcohols, fatty esters, di-and tri- glycerides, and natural lipid mixtures.

24. A smoking material as claimed-in 1 when prepared by an extrusion process and further containing a binder.

25. A smoking material as claimed in 24 wherein the binder is sodium carboxymethyl cellulose.

26. A smoking material as claimed in 1 wherein the smoking material contains flavour and aroma components.

37 27. A smoking material as claimed in 26 wherein the components include tobacco leaf or stem material to impart tobacco smoke aroma and amoke flavour character to the smoking material.

28. A smoking material as claimed in 26 wherein the components are selected from vanillin, coumarin, menthol, essential oils such as flouve and foin, resinoids, plant and fruit extracts such as licorice, prune, raisin and apple extracts and esters, lactones and ketones or mixtures thereof.

29. A process for producing tobacco cells containing at least 5% nicotine by dry weight, the process comprising preparing a tobacco callus, inoculating portions of the callus aseptically into a liquid nutrient medium containing 0.1 to 0.3 M of a saccharide and permitting in a growth cycle a dense suspension culture of cells to form containing said nicotine content, and separating the nicotine-rich cells so produced.

30. A process as claimed in claim 29 including clonal selection by a cell plating technique to produce tobacco cell lines with said nicotine content.

31. A process as claimed in claim 30 wherein the clonal selection technique comprises irradiating the suspension with ultra violet light so as to reduce the number of clones produced to those with a high capacity for nicotine synthesis.

32. A process as claimed in claim 29 comprising increasing the nicotine content of the cells by adding nicotine to the liquid nutrient medium at the beginning of the growth cygle.

38 33. A process as claimed in claim 32 comprising increasing the nicotine content of the cells to between about 5% to about 30% dry weight by adding the nicotine in a concentration up to 640 mg/100 ml to the liquid nutrient medium at the beginning of the growth cycle.

34. A process as claimed in claim 32 or 33 wherein the nicotine is provided by placing filtered tobacco cells in & solution of one or more inorganic salts and subsequently extracting nicotine alkaloid from the solution.

35. A process as claimed in claim 34 wherein the inorganic salts include ammonium nitrate and potassium nitrate.

36. A process as claimed in claim 29 wherein the saccharide is a mono- or di- saccharide selected from the group consisting of lactose, glucose, sucrose, fructose, galactose, and xylose.

37. A process as claimed in claim 36 wherein the saccharide is sucrose.

38. A process as claimed in claim 29 comprising increasing the nicotine content of the cells by adding to the medium additives selected from the group consisting of naphthaleneacetic acid, kinetin, spermidine, and spermine.

39. A process as claimed in claim 29 comprising increasing the nicotine content of the cells by adding supplementary vitamins to the medium.

39 40. A process as claimed in claim 39 wherein the supplementary vitamins are selected from the.group consisting of nicotinic acid, pyroxidine hydrochloride, thiamine hydrochloride, biotin, folic acid, D- calcium pantothenate and D-calcium ascorbate.

41. A process as claimed in claim 29 wherein the process includes storing the culture immediately after inoculation at a temperature of 0-60C.

42. A process as claimed in claim 41 wherein the temperature is 4C.

43. A process as claimed in claim 42 wherein the storage is for a period of 1 to 4 months.

44. A cultured cell line of tobacco containing 5-30% by weight of nicotine on a dry weight basis.

45. A method of providing a high-yielding suspension culture of living cells by inoculating a nutrient medium with said living cells and 1 immediately storing the inoculated medium at a temperature of 0-60C.

46. A method of non-destructively extracting an alkaloid from suspensioncultured living plant cells comprising placing the cells in a solution of one or more inorganic salts and subsequently extracting the alkaloid from the solution.

47. A process as claimed in claim 46 wherein the inorganic salts include ammonium nitrate and potassium nitrate.

48. A process as claimed in claim 29 including the further steps of harvesting, dewatering, and wet-milling the cells at a pH in the range 46, and subsequently spray drying the cells at a temperature of 60-80C.

49. A method of making a smoking material comprising the steps of (a) preparing a mixture of cellulosic fibres, a burn-modifyingagent, at least 20% by weight of a crystallizable sugar, a humectant, and water, (b) milling the mixture to give a slurry, and (c) further treating the slurry to give a smokeable product.

50. A method as claimed in claim 49 wherein the further treatment includes casting or extruding the slurry, drying the cast or extruded slurry to form a dried product, and curing the dried product.

51. A method as claimed in claim 49 including adding cultured tobacco cells containing at least 5% nicotine to the components of the mixture at step (a).

52. A method as claimed in claim 50 comprising treating the cured product with a lipid or lipids.

5a. A method as claimed in claim 49 wherein the sugar is a mixture of lactose and glucose.

54. A method as claimed in claim 49 wherein the burn-modifying agent is selected from the group comprising calcium citrate and potassium citrate.

1 1 W 41 55. A method as claimed in claim 49 wherein the humectant is selected from the group comprising glycerol, glycerol monoacetate, propylene glycol and sorbitol, 56. A method as claimed in claim 49 including the addition of tobacco plant material to the mixture at step (a).

57. A smoking material containing a cultured cell line of tobacco containing 5-30% nicotine, substantially as hereinbefore described with reference to any one of Examples 13, 14 and 16 to 21.

58. A smoking material substantially as hereinbefore described with reference to Example 15.

59. A process for producing tobacco cells containing at least 5% nicotine by dry weight, substantially as hereinbefore described with reference to any one of Examples 1 to 12.

Published 1988 at The Patent Office, State House, 66!71 High Holborn, London WC1R 4TP. Further copies may be obtained from The Patent Office, Sales Branch, St Mary Cray, Orpington, Kent BR5 3RD. Printed by Multiplex techniques ltd, St Mary Cray, Kent. Con. 1187.