RU2671616C1 - Methods for reducing matrix-bound nicotine-derived nitrosamine ketone in tobacco plant material - Google Patents

Abstract

FIELD: tobacco, cigars, cigarettes, smokables.SUBSTANCE: invention generally relates to methods for reducing the amount of nicotine-derived nitrosamine ketone or 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in plant tobacco material. Method of reducing the amount of matrix bound NNK in a dried tobacco plant material includes separating lignified tissue from non-lignified tissue, where the amount of lignin is reduced mechanically, where the lignified tissue is separated from the non-lignified tissue by removal of the vascular bundle, xylem or lignified sclerenchymatic tissue.EFFECT: technical result of the invention is efficient reduction the amount of matrix-bound NNK in dried tobacco plant material.15 cl, 6 ex, 2 tbl, 6 dwg

Description

FIELD OF TECHNOLOGY

The present invention relates generally to methods for reducing the amount of a nicotine derivative of nitrosamine ketone or 4- (methylnitrosamino) -1- (3-pyridyl) -1-butanone (NNK) in tobacco plant material.

BACKGROUND

During the manufacture and processing of tobacco products, by-products such as tobacco stems and leaf waste are obtained. Tobacco stalks and small particles of tobacco obtained during manufacturing methods are not suitable for direct use in the manufacture of tobacco products. Since stems and small particles are reflected in a significant amount of cash for raw materials, methods have been developed to further convert these stems and small particles into products such as reconstituted tobacco materials (e.g. reconstituted tobacco webs), which can then be used in relatively large quantities in mixtures with acceptable recycled tobacco leaf. Reconstituted tobacco can be made by a process involving the production of pulp or the production of canvases by reflux, in which the pulp from crushed tobacco stems and other parts of the tobacco sheet is crushed and mixed with a solution that may contain various additives. The resulting tobacco pulp is then sprayed to form a race film, dried, rolled and cut into strips that are added to the filler.

Nitrosamines are organic compounds found in many consumer products such as tobacco, food, and cosmetics. Nitrosamines have aroused great scientific interest, since some of the compounds of this class, as shown in laboratory animals, are carcinogenic. It has been reported that some varieties of dried tobacco contain tobacco-specific nitrosamines, which can be found in smokeless tobacco, mainstream smoke and sidestream smoke of cigarettes. In tobacco, at least four types of nitrosamines are formed in significant quantities. They are the nicotine derivative nitrosamine ketone or 4- (methylnitrosamino) -1- (3-pyridyl) -1-butanone (NNK), N-nitrosonornicotine (NNN), N-nitrosoanathabin (NAT) and N-nitrosoanabazine (NAB). Tobacco-specific nitrosamines are not believed to be present in significant quantities in growing tobacco plants or freshly cut tobacco (green tobacco), but are formed during the method of drying the tobacco. In addition to the formation of tobacco-specific nitrosamines during the drying process of green leaves, tobacco-specific nitrosamines can also be formed during the methods used to produce aqueous tobacco pulps, such as the methods used to produce reconstituted tobacco.

In an attempt to reduce the number of tobacco-specific nitrosamines, various treatments for tobacco plants or collected tobacco leaves have been proposed, including treatments for irradiation, chemical treatments and extraction. Other methods for reducing the amount of tobacco-specific nitrosamines have been proposed by MacKown et al. (1988) J. Agric. Food Chem. 36, 1031-1035. These methods include treatment using sterilization, microorganism inhibitors, bases to increase pH or ascorbic acid to reduce the accumulation of tobacco-specific nitrosamines during the manufacture of reconstituted tobacco canvases. WO 2012160133 describes a method for decreasing levels of tobacco-specific nitrosamines in tobacco homogenates by increasing their pH, especially when elevated levels of nitrosamines are caused by elevated levels of nitrites.

One problem when trying to reduce tobacco-specific nitrosamines in tobacco is that some of the nitrosamines in air-dried tobacco, including NNK, are in matrix bound or matrix form, the removal or extraction of which may be difficult. Associated with the matrix NNK can be extracted using 0.1 N. KOH solution from a water-washed Burley filler. This alkaline treatment also reduces NNK levels in smoke (Keene, S.K., 1992, The Effect of Base Digestion on TSNA in Extractables-Depleted Fillers. Legacy Tobacco Documents). However, as a result of the treatment, compounds significant in terms of toxicology can be introduced into tobacco, and the quality of the tobacco can be significantly impaired, which is a significant problem for the tobacco industry.

WO 2010/021809 describes a method for reducing the amount of nitrogen-containing compounds and lignin in tobacco. Nitrogen-containing compounds are removed by solvent extraction; lignin is removed in a separate step.

There is a need for an effective and cost-effective way to reduce the amount of matrix-bound NNK that is formed during the drying of tobacco. In particular, a cost-effective and simple way to reduce matrix-bound NNK in dried tobacco is particularly desirable, which does not cause toxic or potentially toxic compounds and does not degrade the quality of the tobacco product.

SUMMARY OF THE INVENTION

The present invention is based at least in part on the unexpected discovery that matrix-bound NNK is colocalized with lignin in tobacco plants. In particular, the authors of the present invention have found that in the dried portions of a tobacco plant, such as stems and middle veins, the NNK matrix bound is predominantly colocalized or colocalized, or colocalized exclusively with lignin, such as in lignified tissue, in particular in a vascular bundle (e.g. xylem), but not in surrounding tissues such as the cortical layer. Thus, by removing lignin (for example, by separating lignified tissue from non-lignified tissue), the amount of matrix-bound NNK and its metabolites in the plant material can be reduced. Matrix-linked NNKs may be covalently or non-covalently bound to lignin. Plant material is expected to produce smoke with reduced NNK levels and potentially improved organoleptic properties. The present disclosure may be applicable to such plant materials that accumulate matrix-linked NNK or potentially accumulate matrix-linked NNK. In particular, the present disclosure may be applicable to low-value plant material containing a matrix associated with NNK, which is used in certain methods of processing tobacco. The methods described in this document can be carried out without the use of any additives, and thus they do not provide for the introduction of additional compounds into the plant material that are significant from the point of view of toxicology. Lignin removal can be performed during or after drying of the tobacco plant material. Lignin can be removed prior to drying to prevent, reduce, or inhibit the colocalization of matrix-bound NNK with lignin.

One main objective of the present disclosure is to significantly reduce the amount of matrix-bound NNK and its metabolites in tobacco intended for smoking or other consumption. Another main goal is to reduce the carcinogenic potential of tobacco products, including cigarettes, cigars, chewing tobacco, snuff, as well as tobacco-containing chewing gum and lozenges. Another main objective is to reduce or decrease the amount of matrix-bound NNK and its metabolites in the tobacco plant material and in tobacco products. Another main objective is to reduce the amount of matrix-bound NNK and its metabolites in dried, such as partially or fully dried, tobacco plant material. Another main goal is to reduce the amount of NNK and its metabolites in aerosol, including smoke. Another objective of the present disclosure is to reduce the amount of NNK or its metabolites in people who smoke, consume or otherwise ingest tobacco in any form by producing a human-acceptable tobacco product containing a reduced amount of NNK or its metabolites, which, thus, it reduces the carcinogenic potential of such a product.

According to one aspect, a method is provided for reducing the amount of NNK matrix bound in a dried tobacco plant or in a dried tobacco plant material, comprising reducing the amount of lignin therein by separating the lignified tissue from the non-lignified one, where the amount of lignin is preferably reduced chemically and / or mechanically.

In one embodiment, the tobacco plant or tobacco plant material is treated to expand non-lignified plant tissue. The amount of lignified tissue is reduced by separating the expanded and unexpanded plant tissue based on various indicators of their density (for example, indicators of floating density), and / or various indicators of their strength, and / or their various sizes, and / or their different weights. Expanded plant tissue can be harvested for subsequent tobacco processing.

It was found that a significant reduction in NNK and associated TSNA levels can be achieved by fractionation of lignified tissues. Separate chemical extraction of nitrogenous compounds and / or lignin is not necessary.

In one embodiment, the amount of lignin is reduced by removing the vascular bundle, or xylem, or lignified sclerenchymal tissue, or a combination of two or more of them from a plant or plant material. Lignin can be localized in the vascular bundle. Lignin can be localized exclusively in the vascular bundle. Lignin can be localized exclusively in the vascular bundle, but not in the surrounding tissue. Lignin can be localized in xylem. Lignin can be localized exclusively in xylem. Lignin can be localized exclusively in xylem, but not in the surrounding tissue. Lignin can be localized in sclerenchymal tissue. Lignin can be localized exclusively in sclerenchymal tissue. Lignin can be localized exclusively in sclerenchymal tissue, but not in the surrounding tissue. In the outer layer of the middle veins of plants lignified tissue, as a rule, is absent.

In one embodiment, a plant or plant material that is treated in accordance with the present disclosure comprises middle veins of plants, or stems of plants, or petioles of plants, or a combination of two or more of them, or consists of them, or consists mainly of them.

In one embodiment, the amount of lignin is reduced by removing from the plant or plant material the cortical layer, for example, the outer cortical layer.

In one embodiment, the method comprises the steps of: (a) preparing a dried tobacco plant or dried tobacco plant material; (b) reducing the amount of lignin in a dried tobacco plant or dried tobacco plant material by fractionating the tobacco plant material; and (c) obtaining a dried tobacco plant or dried tobacco plant material in which the amount of lignin is reduced and the amount of matrix-bound NNK is reduced compared to dried a tobacco plant or dried tobacco plant material obtained in step (a).

In one embodiment, step (a) is followed by an additional step of measuring the amount of free NNK or matrix bound NNK or a combination thereof, and step (b) is optionally followed by an additional step of measuring the amount of free NNK or matrix bound NNK or a combination thereof.

In one embodiment, the method includes an additional step (d) of comparing the level of at least matrix-bound NNK measured after step (a) with the level of NNK measured after step (b), wherein reducing the amount of matrix-bound NNK in the tobacco material, obtained in stage (b), compared with the tobacco material obtained in stage (a), indicates that the amount of matrix-bound NNK in the tobacco material is reduced.

According to a further aspect, there is provided a method of reducing the formation of a matrix-bound NNK during the drying of a tobacco plant or tobacco plant material, comprising reducing the amount of lignin therein before drying.

In one embodiment, the method includes the steps of: (a) preparing an unsaturated tobacco plant or unsaturated tobacco plant material; (b) reducing the amount of lignin in an unsaturated tobacco plant or an uns dried tobacco plant material before drying; (c) drying the tobacco plant or tobacco plant material obtained in step (b); and (d) obtaining a dried tobacco plant or dried tobacco plant material in which the amount of matrix-bound NNK is reduced compared to a control in which the amount of lignin was not reduced.

In one embodiment, step (a) is followed by an additional step of measuring the amount of free NNK or matrix bound NNK or a combination thereof, and step (b) is optionally followed by an additional step of measuring the amount of free NNK or matrix bound NNK or a combination thereof, and wherein step (c) does not necessarily follow an additional step of measuring the amount of free NNK or matrix NNK or a combination thereof.

In one embodiment, after step (c) or step (d), the method comprises an additional step of comparing the level of at least the matrix NNK measured after step (a) with the level of NNK measured after step (b) and / or step (c) wherein a reduction in the amount of matrix-bound NNK in the tobacco material obtained in step (b) or step (c), compared with the tobacco material obtained in step (a), indicates that the amount bound to the matrix NNK in tobacco material is reduced.

According to a further aspect, there is provided tobacco plant material obtained or obtained by the method (s) described (described) herein.

According to a further aspect, there is provided the use of a tobacco plant or tobacco plant material in which the amount of lignin has been reduced compared to a control tobacco plant or control tobacco plant material to produce tobacco with reduced levels of NNK matrix bound, wherein said levels of NNK matrix bound are reduced by compared to control.

According to a further aspect, there is provided a method for producing reconstituted tobacco, comprising the steps of: (a) implementing the method (s) described (described) herein; (b) obtaining reconstituted tobacco from tobacco material obtained in step (a); and (c) optionally including reconstituted tobacco in the composition of the tobacco product.

According to a further aspect, there is provided reconstituted tobacco obtained or obtained by the method described herein.

According to a further aspect, there is provided a method for producing tobacco for use as a cut tobacco filler, comprising the steps of: (a) implementing the method (methods) described (described) herein and (b) rolling and slicing the tobacco material for use as a cut tobacco filler.

According to a further aspect, a dried tobacco plant material is provided having a reduced level of lignin compared to a control tobacco plant material in which the amount of lignin has not been reduced, wherein the amount of matrix-bound NNK is about 3,500 ng / g or less.

In one embodiment, the average particle size is more than about 0.5 millimeters.

In one embodiment, the amount of free NNK is less than about 330 ng / g, where the optional NNN content is less than about 1700 ng / g and where the nicotine content is less than about 2610 μg / g.

In one embodiment, the dried plant tobacco material comprises a plant cortex, such as, for example, the outer cortex of a plant, consists of or consists essentially of it.

In one embodiment, in a dried plant tobacco material, a vascular bundle or xylem, or lignified sclerenchymal tissue, or a combination of two or more of them is practically absent.

In one embodiment, the dried plant tobacco material comprises a cortex of a plant, such as, for example, the outer cortex of a plant, consists of or consists mainly of it, and the vascular bundle, or xylem, or lignified sclerenchymal tissue, or a combination of two or more of they are practically absent in it.

In one embodiment, the dried plant tobacco material is obtained or can be obtained from medium veins of plants, or plant stems, or petioles, or a combination of two or more of them.

In one embodiment, the average particle size is more than about 0.5 millimeters.

In one embodiment, the amount of free NNK is less than about 330 ng / g.

In one embodiment, the NNN content is less than about 1700 ng / g.

In one embodiment, the nicotine content is less than about 2610 μg / g.

According to a further aspect, there is provided a tobacco product or a reconstituted tobacco product containing the plant material or dried plant material described herein, consisting of or consisting essentially of it.

According to a further aspect, there is provided a method for producing an aerosol in which the amount of NNK is reduced compared to a control aerosol, the method comprising the steps of: (a) obtaining a dried tobacco plant or dried tobacco plant material; (b) reducing the amount of lignin in a dried tobacco plant or dried tobacco plant material; (c) obtaining a dried tobacco plant or dried tobacco plant material in which the amount of lignin is reduced and the amount of matrix-bound NNK is reduced compared to a dried tobacco plant or dried tobacco plant material obtained in step (a); and (d) heating the dried tobacco plant or dried tobacco tobacco material from step (c) to obtain an aerosol.

According to a further aspect, there is provided a method for producing an aerosol in which the amount of NNK is reduced compared to a control aerosol, the method comprising the steps of: (a) obtaining an unsaturated tobacco plant or an uns dried tobacco plant material; (b) reducing the amount of lignin in an unsaturated tobacco plant or an uns dried tobacco plant material before drying; (c) drying the tobacco plant or tobacco plant material obtained in step (b); (d) obtaining a dried tobacco plant or dried tobacco plant material in which the amount of matrix-bound NNK is reduced compared to a control in which the amount of lignin was not reduced; and (e) heating the dried tobacco plant or dried tobacco plant material from step (d) to obtain an aerosol.

According to a further aspect, there is provided a method for producing an aerosol in which the amount of NNK is reduced compared to a control aerosol, the process comprising the steps of: (a) preparing a tobacco product or a reconstituted tobacco product containing tobacco plant material or dried plant material obtained or obtained as described herein by methods consisting of or consisting mainly of it; and (b) heating the tobacco product or the reconstituted tobacco product to produce an aerosol.

According to a further aspect, there is provided an aerosol obtained or obtained by the method (s) described (described) herein.

According to a further aspect, dried tobacco plant material is provided, consisting mainly of the cortex of the tobacco plant, while the amount of matrix-bound NNK is reduced as described herein.

According to a further aspect, there is provided a method for blending tobacco, wherein at least two different types of tobacco are blended to form a tobacco bag, comprising the steps of: (a) preparing a first dried tobacco plant material and reducing the amount of lignin therein; (b) measuring the content of total and / or matrix-bound NNK in the first dried tobacco plant material and selecting dried tobacco plant material in which the content of total and / or matrix-related NNK is reduced compared to the first dried plant tobacco material obtained in the step (but); (c) preparing a second dried tobacco plant material having a higher total and / or matrix NNK content than the total and / or matrix NNK matrix in the first dried tobacco plant material obtained in step (b), and optionally measuring the content of total and / or matrix-bound NNK in the second dried tobacco plant material; (d) mixing the first and second dried tobacco plant material from steps (b) and (c) and optionally measuring the total and / or matrix NNK content of the mixed tobacco plant material; and (e) obtaining a mixed tobacco plant material in which the total and / or matrix-associated NNK in the mixed tobacco plant material is lower than in the second dried tobacco plant material obtained in step (c), optionally steps (a) and (b) are carried out after step (c).

According to a further aspect, provided is a mixed tobacco plant material obtained or obtained by the method (s) described (described) herein.

Each of the embodiments discussed above is disclosed as embodiments of each of the aspects of the present invention. Combinations of one or more embodiments are contemplated.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph showing the distribution of free NNK bound to the NNK matrix and lignin in lignified (L) and non-lignified (NL) tissue in dried middle Burley tobacco veins or dried Burley tobacco stems.

Figure 2 is a cross-sectional view of a hydrated dried Burley tobacco stem showing lignified (L) and non-lignified (NL) tissue. The lignified tissue is stained red with phloroglucin.

Figure 3 is a graph showing the distribution of free NNK bound to the NNK matrix and lignin in the lignified (L) and non-lignified (NL) tissue of Burley's green middle veins after nitrosation with sodium nitrite solution (1.5 ml (10 mg / ml) in water) for 4 hours at room temperature with shaking).

Figure 4 is a graph showing the free and matrix-bound NNK in fractions after sieving crushed freeze-dried stems of Burley tobacco plants.

Figure 5 is a graph showing the correlation between matrix bound NNK and lignin in the fractions after sieving the crushed freeze-dried stems of Burley tobacco plants.

Figure 6 is a graph showing the concentration of matrix-bound NNK (μg / g) in the sclerenchymal tissue (S) and in the outer layers of the middle veins (NS) after nitrosation and washing of the green medium veins of tobacco TN90. The levels of pseudooxy nicotine (PON) (μg / g) and nicotine (μg / g) are also shown. Matrix NNK (ng / g) bound levels in the lignified (CS) and non-lignified (CNS) portions of Burley’s dried commercial tobacco stem are also shown. The levels of NNN (ng / g) and nicotine (μg / g) are also shown.

DEFINITIONS

The technical terms and expressions used in this document should be given the meaning that usually applies to them in the field of plant biology and molecular biology. All of the following definitions of terms apply to the entire content of this disclosure.

The word “comprising” does not exclude other elements or stages, and singular forms do not exclude the plural.

The terms “significantly”, “approximately”, “approximately”, etc. in relation to the parameter or value, in particular, the exact parameter or exact value is also determined, respectively.

The term "plant" refers to any plant or any part thereof at any stage of its life cycle or development, as well as to its offspring. In one embodiment, the plant is a tobacco plant, which refers to a plant belonging to the genus Nicotiana. Preferred species of tobacco plant are described herein.

“Plant cell” refers to the structural and physiological unit of a plant. A plant cell can be in the form of a protoplast without a cell wall, an isolated cell or a cultured cell, or present as part of a more highly organized unit, such as, but not limited to, plant tissue, plant organ, or the whole plant. In one embodiment, the plant cell is a tobacco plant cell.

The term “plant material” refers to any part of a plant or a mixture of different parts of a plant and includes, without limitation, plant tissues, leaf waste, green leaf waste, stems, tobacco dust generated during plant processing, and broken leaf blades collected as they mature, as well as their combinations. In some embodiments, the plant material will comprise a plant part or a mixture of plant parts containing lignin, such as medium veins of plants, or plant stems, or petioles, or a combination of two or more of them, or consist of them, or consist mainly of them. The tobacco plant material may be in the form of processed parts or pieces of tobacco, uncured, dried or aged tobacco in the essentially natural form of a leaf blade or stem, in the form of a tobacco extract or a mixture of the above, for example, a mixture in which the extracted tobacco pulp is combined with granular dried and aged leaf blades of natural tobacco. The plant material may be in solid form, liquid form, in semi-liquid form, in crushed form, in crushed form, in sifted form or in particulate form, and the like. or may otherwise be processed to reduce particle size. The plant material may be in the form of a homogenate that has been subjected to homogenization, including, without limitation, cutting or grinding, or a combination thereof. The homogenate can be obtained from whole plants or from mixtures of plant components, such as from a mixture of plant parts containing lignin, such as medium veins, or stems, or petioles, or a combination of two or more of them that have been homogenized. The plant material may be in the form of a pulp comprising a suspension of plant material or a plant homogenate in an aqueous solution or solvent. The pulp may be 5% (weight / volume), 10% (weight / volume), 15% (weight / volume), 20% (weight / volume), or 25% (weight / volume), or a more concentrated mixture of vegetable material in an aqueous solution or solvent. In one embodiment, the plant material is plant material that contains, consists of, or consists primarily of, lignin, such as lignified tissue. In one embodiment, the plant material is plant material that contains, consists of, or consists essentially of, a vascular bundle. In one embodiment, the plant material is plant material that contains, consists of, or consists primarily of xylem. In one embodiment, the plant material is a plant material that contains, consists of, or consists mainly of, lignified sclerenic tissue. In one embodiment, the plant material comprises middle veins of plants, or plant stems, or petioles, or a combination of two or more of them, consists of or consists mainly of them. In one embodiment, the plant material is tobacco plant material.

The term “tobacco product” includes smoking articles or products for smoking and smokeless tobacco products.

The term “free NNK” refers to the concentration of NNK calculated from the NNK content in extracts obtained by extracting said plant material with an aqueous (aqueous) buffer (s) solution (s) at room temperature. The content of free NNK in such extracts can be determined using ultra-high performance liquid chromatography with tandem mass spectrometry (UPLC-MS / MS).

The term "total NNK" refers to the concentration of NNK calculated after exposure to the extraction mixtures in accordance with the methods described herein (for example, by heating to approximately 130 ° C for approximately 4 hours) and filtering aliquots of the extracts. The total NNK content in such extracts can be determined using UPLC-MS / MS.

As used herein, the term “coupled NNK” or “coupled to a NNK matrix” is the difference between the concentrations of “total NNK” and “free NNK”.

The terms “reduced lignin content” or “reduced lignin content” or “non-lignified” or their grammatical variants refer to a measurable quantitative decrease in the amount of lignin in a plant compared to the amount of lignin in the compared control plant. The quantitative reduction of lignin can be easily determined using assays known in the art, including Klason analysis of lignin (Method in Enzymol, 161: 87-101 (1988)), acetyl bromide analysis (Wood Sci. Technol., 22 : 271-280 1988)) or a photometric method based on thioglycolic acid derivatization (J. Chem. Ecol, 28, 2483-2501 (2002)). In non-lignified tissue, the amount of lignin is reduced relative to the compared control plant, and the amount of lignin can be completely, practically or partially removed. In non-lignified tissue, a detectable amount of lignin may be present provided that there is a measurable quantitative decrease in the amount of lignin relative to the control plant being compared in which the amount of lignin has not been reduced. In non-lignified tissue, it may not be possible to detect any amount of lignin. Non-lignified tissue may have a lignin content in the plant or part of the plant from which it is removed, comprising less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the total dry matter weight.

“Control plant” or “control plant cell” refers to a plant or plant cell, such as a natural or naturally occurring plant or plant cell, with a lignin content and / or NNK content that has not been manipulated or modified. Control plant material includes plant material obtained, derived from or obtained from a control plant or a control plant cell, or a combination thereof. A control plant or a control plant cell may be of the same type of plant or plant cell, for example, the same type of plant or plant cell as the plant or plant cell with which they are compared. A control plant or a control plant cell may correspond to a wild-type plant or a wild-type plant cell.

The terms "reduce" or "reduce" or their grammatical variations refer to a decrease of from about 10% to about 99% or a decrease of at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, but at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% or more of the content, amount or activity.

The term "inhibit" or its grammatical variants refer to a decrease of from about 98% to about 100% or a decrease of at least 98%, at least 99%, in particular 100%, of the content, amount or activity.

The term "increase" or its grammatical variations refer to an increase of from about 5% to about 99% or an increase of at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% or more of the content, amount or activity.

The term "approximately" in the context of a given digital value or range refers to a value or range that is within 20%, within 10%, or within 5% of the specified value or range.

The term “at least a portion” or grammatical variants thereof include at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98% or at least approx itelno 99% content, amount or activity.

DETAILED DESCRIPTION OF THE INVENTION

In general, the present disclosure can be applied to any form of tobacco plant material in which NNK or its metabolites, or a combination thereof, can be formed or have been formed. Suitably, at least a portion of the NNK is in bound form. At least a portion of the matrix-bound form is colocalized with lignin, as, for example, in lignified tissue. Methods for measuring free (free) nitrosamine (nitrosamines) and bound (bound) to a matrix of nitrosamine (nitrosamines) are well known in the art and are described herein. In short, aliquots of tobacco samples can be taken and nitrosamine content can be analyzed using ultra-high performance liquid chromatography with tandem mass spectrometry (UPLC-MS / MS). Typically, one or more standards corresponding to one or more nitrosamines, the amount of which is determined, will be included in aliquots of tobacco samples. The concentration in the sample calculated from the extracts corresponds to the concentration of “free (free) nitrosamine (nitrosamines)” in the sample. After processing the extraction mixtures in accordance with the methods described herein (for example, by heating to approximately 130 ° C for approximately 4 hours), the concentrations of nitrosamines can again be measured using UPLC-MS / MS. Based on these values, the concentration of “total NNK” in the samples can be calculated. The concentration of matrix-bound NNK is the difference between the concentrations of total NNK and free NNK.

A lot of research has been done on tobacco, especially with regard to tobacco-specific nitrosamines. Freshly picked tobacco leaves are called "green tobacco" and they are believed to be nitrosamine free, but green tobacco is not suitable for human consumption. The method of drying green tobacco depends on the type of tobacco collected. For example, Virginia smoke-dried (light) tobacco is typically dried by smoke-drying, while Burley and some darker varieties are usually air-dried. Smoke drying of tobacco, as a rule, takes place over a period of five to seven days, in contrast to one or two months of air drying. Many important chemical and biochemical changes occur during the drying method and continue in the early phases of the sheet drying itself. Changing the color of tobacco from yellow to brown usually results in the formation and significant accumulation of nitrosamines, including NNK, and an increased content of microorganisms. The exact mechanism by which tobacco-specific nitrosamines are formed, including NNK, is not clear, but it is believed to be intensified by the activity of microorganisms, which involves the participation of nitrate reductases of microorganisms in the formation of nitrites during the drying process.

As described above, it was found that the matrix-bound NNK is colocalized with lignin in tobacco plants or in tobacco plant material. Lignin is a general term for a large group of aromatic polymers resulting from the combinatorial oxidative combination of 4-hydroxyphenylpropanoids. These polymers accumulate mainly in the cell walls of cells with secondary thickening, as, for example, in fibers and vascular elements, which makes them stiff and impermeable. The mechanical stiffness due to lignin makes these tissues strong, so that the vascular elements can withstand the negative pressure created during transpiration without losing tissue stability. In addition to providing mechanical strength, lignin has protective functions. For example, the physical strength and chemical resistance of lignin can interfere with eating by herbivores. Lignification is a common response to infection or injury that can provide a physical barrier to block the penetration of pathogens. The main structural elements of lignin are hydroxycinnamic alcohols (or monolignols), coniferyl alcohol and synapyl alcohol, while the amounts of n-coumaril alcohol are usually insignificant. Monolignols are synthesized from Phe by common pathways specific for phenylpropanoids and monolignols. Phe is formed in plastids via the shikimate metabolic pathway of biosynthesis. Some enzymes involved in the metabolic pathway of lignin biosynthesis, namely enzymes of the cinnamic acid cytochrome P450 4-hydroxylase (C4H), n-coumarate-3-hydroxylase (C3H), and ferulate-5-hydroxylase (F5H), are membrane proteins, which are believed to be active on the cytosolic side of the endoplasmic reticulum. Although the formation of metabolic channels between phenylalanine ammonia lyase (PAL) and C4H has been shown, it remains unknown whether the enzymes of other metabolic pathways are also part of the metabolic complexes within the endoplasmic reticulum. Structural units derived from monolignols, when incorporated into a lignin polymer, are called guaiacil (G), syringyl (S), and n-hydroxyphenyl (H) structural units.

Lignin is usually found, for example, in medium veins of plants, plant stems or petioles. Thus, material for use in accordance with the present disclosure may include middle veins of plants, or plant stems, or petioles, or a mixture thereof, or a combination of two or more of them, and can be removed. Lignin is localized, for example, in the vascular bundle of a tobacco plant, which can be found in medium veins of plants, plant stems, petioles, etc. The vascular bundle consists of many relatively rigid structural elements of cellulose, tightly interconnected by means of fibrous connective plant tissue. This vascular-fibrous bundle surrounds the cortical layer formed by a relatively spongy plant tissue, or cover, which makes up the majority of the stem and part that is close in characteristics and properties to the tobacco leaf. Lignin is usually localized in the vascular bundle. The vascular bundle is part of the vascular plant transport system. Transport as such occurs in the vascular tissue, which exists in two forms, in the form of xylem and phloem. Both of these tissues are present in the vascular bundle, which, in addition, will include supporting and protective tissues. Of these vascular tissues, lignin can only be found in xylem.

The amount of lignin can be significantly reduced in medium veins of plants, plant stems or petioles, etc. or mixtures or combinations of two or more of them. Lignin can be practically removed from medium veins of plants, stems of plants or petioles, etc. or mixtures or combinations of two or more of them. In a suitable case, the volume of the vascular bundle or xylem or a combination thereof is significantly reduced in medium veins of plants, plant stems or petioles, etc. or mixtures or combinations of two or more of them. In a suitable case, the vascular bundle or xylem, or a combination thereof, is practically removed from the middle veins of plants, plant stems or petioles, etc. or mixtures or combinations of two or more of them. In a suitable case, the lignified sclerenchymal tissue is practically removed from the middle veins of plants, plant stems or petioles, etc. or mixtures or combinations of two or more of them.

The fact that the NNK associated with the matrix is colocalized with lignin is an advantage because lignin can be easily separated from other parts of the plant or other plant tissues. In one embodiment, the amount of lignin is reduced by separating the lignified tissue from the non-lignified tissue. For example, the outer cortical layer can be easily separated from the vascular bundle containing lignin, thereby obtaining plant material with reduced levels of lignin. Plant material containing reduced levels of lignin or virtually no lignin can be used in the manufacture of tobacco materials or tobacco products with reduced levels of matrix-bound NNK, as described herein. Optionally, the separated plant material containing lignin colocalized with the associated NNK matrix can be discarded or used in other methods.

According to a further aspect, there is provided a method of reducing or decreasing the amount of matrix-linked NNK in a dried tobacco plant or in a dried tobacco plant material, comprising reducing the amount of lignin therein. According to this method, a dried tobacco plant or dried tobacco plant material is provided. The amount of lignin in a dried tobacco plant or dried tobacco plant material is reduced. Lignin can be completely removed or partially removed. A dried tobacco plant or dried tobacco plant material is then obtained in which the amount of lignin is reduced and the amount of NNK bound to the matrix is also reduced compared to the original dried tobacco plant or dried tobacco plant material or compared to the control.

Lignin, subject to complete or partial removal, can be localized in the vascular bundle. Lignin, subject to complete or partial removal, can be localized exclusively in the vascular bundle. Lignin, subject to complete or partial removal, can be localized exclusively in the vascular bundle, but not in the surrounding tissue. Thus, the amount of matrix-bound NNK in a dried tobacco plant or in a dried tobacco plant material is reduced due to a decrease in the vascular bundle volume in a dried tobacco plant or in a dried tobacco plant material.

Lignin, subject to complete or partial removal, can be localized in xylem. Lignin, subject to complete or partial removal, can be localized exclusively in xylem. Lignin, subject to complete or partial removal, can be localized exclusively in xylem, but not in the surrounding tissue. Thus, the amount of matrix-bound NNK in a dried tobacco plant or in a dried tobacco plant material is reduced due to a reduction in xylem volume in a dried tobacco plant or in a dried tobacco plant material.

Lignin, subject to complete or partial removal, can be localized in the lignified sclerenchymal tissue. Lignin, subject to complete or partial removal, can be localized exclusively in lignified sclerenchymal tissue. Lignin, subject to complete or partial removal, can be localized exclusively in the lignified sclerenchymal tissue, but not in the surrounding tissue, as, for example, in the outer layer of the middle veins. Thus, the amount of matrix-bound NNK in a dried tobacco plant or in a dried tobacco plant material is reduced due to a decrease in the volume of lignified sclerenchymic tissue in a dried tobacco plant or in a dried tobacco plant material.

According to another aspect, there is provided a method of reducing, decreasing, preventing or inhibiting the formation of matrix NNK during drying of a tobacco plant or tobacco plant material, comprising reducing the amount of lignin therein before drying. At least initially, the tobacco plant or the tobacco plant material may be dried or substantially dried. The method can be used to reduce, reduce, prevent or inhibit the colocalization of NNK with lignin, which would otherwise occur during the subsequent drying method. According to this aspect, there is provided an uncooked tobacco plant, or an uncooked tobacco plant material, or an almost uncooked tobacco plant, or an almost uncooked tobacco plant material, wherein the amount of lignin therein is reduced before drying or during drying. Lignin can be completely removed or partially removed. Lignin can be localized in the vascular bundle. Lignin can be localized exclusively in the vascular bundle. Lignin can be localized exclusively in the vascular bundle, but not in the surrounding tissue. Thus, the vascular bundle can be completely removed or partially removed. Lignin can be localized in xylem. Lignin can be localized exclusively in xylem. Lignin can be localized exclusively in xylem, but not in the surrounding tissue. Thus, the xylem can be completely removed or partially removed. Lignin can be localized in lignified sclerenchymal tissue. Lignin can be localized exclusively in lignified sclerenchymal tissue. Lignin can be localized exclusively in lignified sclerenchymal tissue, but not in the surrounding tissue, as. for example, in the outer layer of the middle veins. Thus, the lignified sclerenchymal tissue can be completely removed or partially removed. Lignin can be completely removed or partially removed. After exposure of the tobacco plant or the tobacco plant material to drying using methods known in the art, a dried tobacco plant or dried tobacco plant material can be obtained in which the amount of NNK bound to the matrix and the amount of lignin are reduced compared to the starting material or compared to the control.

The amount of lignin in a tobacco plant or in a tobacco plant material can be reduced using a number of methods well known in the art. According to one method, a batch of stems, etc. can be moistened or soaked in a liquid, such as water, which causes softening and expansion or swelling of the cortical layer, while the lignin remains in the unexpanded state. The cortical layer can be removed manually (for example, by hand) and stored, and the vascular bundle containing lignin can be discarded or applied elsewhere. Therefore, the cortical layer (for example, the outer cortical layer) can be separated from the lignified tissue and stored, and the unexpanded lignified tissue can be discarded. The cortical layer can then be used for further processing of tobacco. Also disclosed is a plant cortical layer (e.g., the outer plant cortex) or an expanded plant cortex (e.g., the expanded plant outer cortex), in which the amount of NNK bound to the matrix is below detectable levels. Therefore, non-lignified plant tissue can be expanded to separate lignified and non-lignified tissue. Suitably, the non-lignified tissue is selectively or predominantly expanded compared to the lignified tissue.

As an additional example, lignin can be separated using a suitable mechanism for decortication. The decorator is a device for removing bark, wood and petioles of a plant, etc.

In another method, mechanical separation can be used. For example, a batch of stems is steeped or the like. followed by freeze-drying. In another example, a batch of stems or the like is soaked. followed by freeze-drying, grinding and sieving. Suitably, the plant material of the tobacco can be pulverized to powder form using equipment and techniques for grinding, grinding, and the like. Suitably, the plant material of the tobacco is in a relatively dry form during grinding or grinding using various equipment, such as hammer mills, cutting heads, pneumatically controlled mills, and the like.

The plant material can be reduced in size to form particles or particulate material using various methods known in the art. Particles or particulate material can be sized to produce fractions with reduced levels of lignin and reduced levels of matrix bound NNK. In one suitable method, the plant material is processed by impact, such as, for example, by impact with one or more objects harder than the plant material to be processed. In one embodiment, a collision with a metal is used, such as, for example, with metal balls. Impact can be achieved in various ways, such as by shaking. For example, plant material can be impacted with steel balls (2 steel balls, diameter 2 cm) with shaking at 300 rpm. for 15 minutes. Particles or material in the form of particles can be separated by size into fractions with different particle sizes using a sieve shaker. Suitably, the average particle size is more than about 0.5 millimeters, more than about 0.85 millimeters, or more than about 1 millimeter. These size fractions may be characterized by reduced levels of lignin and reduced levels of matrix-bound NNK.

The plant material can be crushed or ground if its moisture content is from less than about 15 percent by weight to less than about 5 percent by weight. The plant material of tobacco can be finely ground. Fine tobacco material is typically characterized by a particle size of from about 30 to 600 microns.

In one embodiment, the method includes expanding, for example, by contacting with a liquid (for example, by soaking in water), followed by freeze-drying, which will lead to expansion of plant material that does not contain lignin or contains only low levels of lignin. The lignified plant material will retain a higher density, higher physical strength, and a smaller particle size than the expanded plant tissue, which thus allows size separation. In another embodiment, the method comprises expanding, followed by freeze-drying, followed by grinding (e.g., by collision, discussed above), followed by sorting (e.g., sorting by size) of the resulting fragments and selecting fragments with reduced levels of lignin and reduced levels of bound with NNK matrix. Screening can be used for this purpose. According to another embodiment, the method comprises expanding, followed by grinding or grinding, or a combination thereof (e.g., by impact, discussed above), followed by size sorting (e.g., by sieving or based on density and / or mechanical strength) of the particles obtained and particle selection with reduced levels of lignin and reduced levels of matrix-bound NNK. Fractions with different sizes can also differ in their free NNK content, or their NNN content, or their nicotine content, or a combination of two or more of them.

After complete or partial removal of lignin, the plant material may optionally be further processed for use in a tobacco product. As an example, this material can be obtained in the form of aqueous pulp. The resulting pulp may contain a significant proportion of the colloidal particles of the cortical layer dispersed in it. The conversion of the cortical layer of tobacco into aqueous pulp can be carried out using a mill of the appropriate type, such as, for example, a ball mill or a colloid mill. Subsequent treatment of the cortical layer and aqueous pulp is described herein.

In some embodiments, plant material obtained or obtained by methods described herein comprises tissue surrounding the vascular bundle, or tissue surrounding the xylem, or tissue surrounding the lignified sclerenic tissue, or a combination of two or more of them, consists of or consists of mainly from them, while the vascular bundle, or xylem, or lignified sclerenchymal tissue, or a combination of two or more of them is practically absent. In some embodiments, the plant material comprises tissue surrounding the vascular bundle, or xylem, or lignified sclerenchymal tissue, or a combination of two or more of them, consists of or consists mainly of them and practically does not contain the vascular bundle, or xylem, or lignified sclerenchymal tissue, or a combination of two or more of them. In some embodiments, the plant material comprises tissue surrounding the vascular bundle, or xylem, or lignified sclerenchymal tissue, or a combination of two or more of them, consists of or consists mainly of them and does not contain a vascular bundle, or xylem, or lignified sclerenchymal fabric, or a combination of two or more of them. In some embodiments, the plant material comprises, consists of, or consists primarily of, the outer cortex of tobacco. In some embodiments, the implementation of the plant material contains the outer layers of the middle veins of plants, consists of them or consists mainly of them.

The methods described herein may include one or more additional steps of measuring and optionally comparing the levels of free NNK or matrix NNK or a combination thereof. Methods for measuring free NNK and matrix-bound NNK are described herein. In one embodiment, the amount of free NNK or matrix-bound NNK or a combination thereof is determined in a dried tobacco plant or in a dried tobacco plant material. After reducing the amount of lignin in a dried tobacco plant or dried tobacco plant material, the amount of free NNK or matrix bound NNK or a combination thereof can be measured again. Level at least associated with the NNK matrix. can be compared with the level in the original source material to determine whether the level associated with the matrix NNK has been reduced. At this stage, the pre-measured level of at least the matrix bound NNK can be compared with the NNK level measured after the lignin has been reduced. A decrease in the amount of matrix-bound NNK in the tobacco material obtained after a decrease in the amount of lignin compared to the originally obtained tobacco material indicates that the amount of matrix-bound NNK has been reduced.

The methods described herein may include one or more additional steps of measuring and optionally comparing the levels of free NNK or matrix-associated NNK or combinations thereof. In one embodiment, the amount of free NNK or matrix-bound NNK, or combinations thereof, are measured in an uncooked tobacco plant or in an uncooked tobacco plant material. Optionally, after decreasing the amount of lignin in the dried tobacco plant or the dried tobacco plant material before drying, the amount of free NNK or matrix-bound NNK or a combination thereof can be measured again. The method may include one or more stages of comparison. As an example, the method may include an additional step of comparing the initially measured level of at least NNK matrix bound, as discussed above, with the NNK level measured later, while reducing the amount of matrix bound NNK in the tobacco material compared to the originally produced tobacco material indicates that the amount of matrix-bound NNK in the tobacco material is reduced.

Free NNK or matrix NNK or a combination thereof can be measured at the beginning of the method and / or at the end of the method and / or during the method. Free NNK or matrix NNK or a combination thereof can be measured periodically or at specific intervals. Intervals can be fixed intervals or arbitrary intervals. Free NNK or matrix-linked NNKs or a combination thereof can be measured at the end of the method to check if free NNK or matrix-linked NNKs or a combination thereof is present within the required amount, concentration or range.

Lignin may be covalently or non-covalently bound lignin. A complex containing lignin bonded to NNK covalently or non-covalently is described. Also disclosed are a plant cell, plant tissue, or plant or plant material containing this complex. Also described is a method of reducing the amount of matrix-bound NNK in a dried tobacco plant or in a dried tobacco plant material, comprising reducing the amount of this complex therein.

According to a further aspect, a dried plant tissue is provided having a reduced level of lignin compared to a control plant tissue in which the amount of lignin has not been reduced, while the amount of matrix-bound NNK is approximately 3500 ng / g or less. The amount of matrix-bound NNK may be about 3000 ng / g or less, about 2500 ng / g or less, about 2000 ng / g or less, about 2000 ng / g or less, about 1500 ng / g or less, about 1000 ng / g or less or approximately 500 ng / g or less. Suitably, the average particle size of this dried plant tissue may be more than about 0.5 millimeters, more than about 0.85 millimeters, or more than about 1 millimeter. Suitably, the amount of free NNK in this dried plant tissue may be about 330 ng / g or less, about 300 ng / g or less, about 250 ng / g or less, about 200 ng / g or less, about 150 ng / g or less, about 100 ng / g or less, or about 50 ng / g or less. Suitably, the amount of NNN in this dried plant tissue may be about 1700 ng / g or less, about 1500 ng / g or less, about 1300 ng / g or less, about 1100 ng / g or less, about 1000 ng / g, or less than or approximately 500 ng / g or less. Suitably, the amount of nicotine in this dried plant tissue may be approximately 2600 μg or less, approximately 2300 μg or less, or approximately 2100 μg or less. Suitably, the amount of lignin in this dried plant tissue may correspond to a content of about 6.5% or less of the total dry matter weight of the dried plant tissue, a content of about 6% of the total dry matter mass of the dried plant tissue, and a content of about 5% of the total dry weight dried plant tissue, about 4% of the total dry matter of dried plant tissue, or about 3% of the total dry matter of dried plant tissue tissue.

In one embodiment, a dried plant tissue is provided having a reduced level of lignin compared to a control plant tissue in which the amount of lignin has not been reduced, wherein the amount of NNK bound to the matrix is about 3,500 ng / g or less and the average particle size is about 0 5 mm or more. Suitably, the amount of free NNK is about 300 ng / g or less. Suitably, the amount of NNN is about 1700 ng / g or less. Suitably, the amount of lignin in this dried plant tissue is about 6.4% or less of the total dry matter weight of the dried plant tissue. Suitably, the amount of nicotine is about 2600 mcg or less.

In another embodiment, a dried plant tissue is provided having a reduced level of lignin compared to a control plant tissue in which the amount of lignin has not been reduced, wherein the amount of matrix-bound NNK is about 1900 ng / g or less and the average particle size is from about 0.85 mm to about 1 mm. Suitably, the amount of free NNK is about 250 ng / g or less. Suitably, the amount of NNN is about 1270 ng / g or less. Suitably, the amount of lignin in this dried plant tissue is about 4.4% or less of the total dry matter weight of the dried plant tissue. Suitably, the amount of nicotine is approximately 2300 mcg or less.

In another embodiment, a dried plant tissue is provided having a reduced level of lignin compared to a control plant tissue in which the amount of lignin has not been reduced, wherein the amount of NNK bound to the matrix is about 1600 ng / g or less and the average particle size is more than approximately 1 mm. Suitably, the amount of free NNK is about 200 ng / g or less. Suitably, the amount of NNN is about 1100 ng / g or less. Suitably, the amount of lignin in this dried plant tissue corresponds to a content of about 3% or less of the total dry matter weight of the dried plant tissue. Suitably, the amount of nicotine is about 2100 mcg or less.

The tobacco plant or tobacco plant material used at the beginning of the method (s) described herein may comprise, or consist of, or consist of, an unhardened tobacco plant material, or a dried tobacco plant material, or a dried tobacco plant material, or consist mainly of them. Methods for drying tobacco, such as tobacco leaves, in particular green tobacco leaves, are well known to those skilled in the art and include, but are not limited to, air drying, fire drying, smoke drying and solar drying. The method of drying the tobacco depends on the type of tobacco collected. For example, Virginia smoke-dried (light) tobacco is typically dried by smoke drying, Burley and some darker varieties are usually air-dried, and pipe tobacco, chewing tobacco, and snuff are usually dried by fire-drying. Although tobacco plants or tobacco plant material belonging to any type of tobacco can be used, certain types of tobacco are preferred. Particularly preferred tobacco materials are selected from the group consisting of smoke-dried tobacco, Turkish tobacco, Burley tobacco, Virginia tobacco, Maryland tobacco, Oriental tobacco, or any combination of two or more of them. The shape of the tobacco material is generally not limited. It may be in the form of a homogenized tobacco material. Tobacco homogenates, such as, without limitation, dried tobacco homogenates, can be obtained from tobacco material using various methods known in the art, for example, tobacco can be in a crumbled, granulated, crushed or powder form. In some embodiments, it is desirable not to start with the tobacco material in powdered or powdered form, as certain mechanical separation methods that can be used to separate lignin may require grinding and / or sieving steps.

The tobacco material used or obtained may contain additives that include, without limitation, one or more of the following components, as well as combinations thereof: flavorings, organic and inorganic fillers (e.g., grain, processed grain, bloated grain, maltodextrin, dextrose, calcium carbonate, phosphate calcium, corn starch, lactose, mannitol, xylitol, sorbitol, fine cellulose, etc.), binders (e.g. povidone, sodium carboxymethyl cellulose and other types of binders based on my certified cellulose, sodium alginate, xanthan gum, starch-based binders, Arabian gum, lecithin, etc.), coloring materials (e.g. dyes and pigments, including caramel coloring matter and titanium dioxide, etc.) moisturizers (e.g. glycerin, propylene glycol, etc.), oral care additives, preservatives (e.g. potassium sorbate, etc.), syrups (e.g. honey, high fructose corn syrup and the like products used as flavorings) and loose additives extenders (e.g. microcrystalline cellulose, croscarmellose sodium, crospovidone, sodium starch glycolate, pregelatinized corn starch, etc.). Such additives are known to those skilled in the art and may be present in amounts and forms known in the art.

Tobacco can be produced as reconstituted tobacco. Thus, in one embodiment, the methods described herein can be used to produce reconstituted tobacco, such as, for example, reconstituted tobacco (leaf) webs. These canvases are paper-like material that can be obtained from recycled small tobacco particles, tobacco stalks and “varietal tobacco”, which consists of tobacco particles with a size typically less than 30 mesh collected at any stage of tobacco processing. Reconstituted tobacco can be obtained by extracting soluble chemicals from tobacco by-products, processing the remainder after extracting the tobacco fibers into paper, and then re-applying the extracted materials in concentrated form to the paper. Reconstituted tobacco, as a rule, can be obtained in various ways. For example, in one embodiment, tape casting can be used to produce reconstituted tobacco. When tape casting, as a rule, pulp from finely divided parts of tobacco and a binder are used, which are applied to a steel strip and then dried. After drying, the canvas is mixed with broken natural tobacco or crumbled and used in various tobacco products, including as a cigarette filler. Some examples of methods for producing reconstituted tobacco are described in US 3353541, US 3420241, US 3386449, US 3760815 and 4674519. Reconstituted tobacco can also be obtained using a paper making method. Some examples of methods for producing reconstituted tobacco in accordance with this method are described in US 3428053, US 3415253, US 3561451, US 3467109, US 3483874, US 3860012, US 3847164, US 4182349, US 5715844, US 5724998 and US 5765570. For example, the production of reconstituted tobacco using papermaking techniques may include the steps of mixing tobacco with water, extracting soluble ingredients from it, concentrating the soluble ingredients, cleaning the tobacco, forming a canvas, reapplying the concentrated soluble ingredients, drying and scrubbing. Various ingredients, such as flavoring or coloring agents, may be applied to the canvas.

From tobacco obtained or obtained using the methods described herein, a tobacco web, such as a reconstituted tobacco web, can be obtained. In accordance with this embodiment, the method may include the steps of: (a) preparing tobacco material, such as tobacco homogenate, in accordance with the methods described herein; (b) obtaining pulp of tobacco homogenate; (c) ejecting the pulp of the tobacco homogenate; and (d) drying the pulp of the tobacco homogenate to form a reconstituted tobacco web. In accordance with another embodiment, the method may include the steps of: (a) producing tobacco material, such as tobacco homogenate, in accordance with the methods described herein, and producing tobacco pulp; (b) refluxing the pulp of the tobacco homogenate; and (c) drying the pulp of the tobacco homogenate to form a tobacco web.

The pulp casting step of the tobacco homogenate can be carried out using any of the casting methods or paper making known in the art. As an example, reflux methods are described in US 5,724,998 and US 5,584,306; paper making methods are described in US 4,341,228; US 5584306 and US 6216706. Methods of casting, as a rule, include casting the pulp on a continuous stainless steel conveyor belt, drying the molded pulp with the formation of a fabric from reconstituted tobacco and removing the specified fabric. Methods for making paper typically include casting water pulp from a headbox onto a wire mesh to form the desired web. The aqueous pulp can be divided into a soluble part and a fibrous part. Water is removed from the fiber portion, and the web thus formed is then treated and dried.

Tobacco pulps may additionally contain one or more binders, such as gums and pectins. As described above, tobacco pulps that are used to produce reconstituted tobacco cans may further comprise conventional additives that include, without limitation, one or more of the following components, as well as combinations thereof: wood pulp fibers, aerosol forming substances, sugars, and flavors, and binders. Additives from the list described above are known to those skilled in the art and may be present in these aqueous pulps in quantities and forms known from the prior art.

After the reconstituted tobacco webs are described, this document can be cut like whole leaf tobacco to form a tobacco filler suitable for cigarettes and other tobacco products. The reconstituted tobacco canvases described herein can be further trimmed or peeled using mechanical finger-shaped elements to obtain pieces sorted by size similar to broken leaf sheets of natural tobacco, or cut into diamond-shaped pieces with a side of about 50 to 100 mm. The reconstituted tobacco slices described herein may be further blended with other types of tobacco, such as smoke-dried tobacco, Burley tobacco, Maryland tobacco, oriental tobacco, rare tobacco, specialty tobacco, blasted tobacco, and the like. The exact amount of each type of tobacco in the tobacco blend used to make a particular brand of cigarette varies between brands. See, for example, Tobacco Encyclopaedia, Voges (Ed.) P. 44-45 (1984), Browne, The Design of Cigarettes, 3rd Ed., P. 43 (1990) and Tobacco Production, Chemistry and Technology, Davis et al. (Eds.) P. 346 (1999). The whole mixture can then be crumbled to obtain a cut filler and incorporated into the tobacco product.

According to a further aspect, there is provided a method for blending tobacco, wherein at least two different types of tobacco are blended to form tobacco bags. Different tobacco bags have different formulations for mixing different types of tobacco. By way of example, types of tobacco may be, for example. Burley, smoke-dried tobacco, oriental, light and reconstituted tobacco. Burley, smoke-dried tobacco and oriental tobacco are specific types of tobacco, while light tobacco is a pre-prepared mixture of smoke-dried tobacco and oriental tobacco. According to this method, a first (of types) dried tobacco plant material is provided, and the amount of lignin therein is reduced. To reduce the amount of lignin, any of the methods described herein can be used. The content of total and / or matrix-bound NNK in the first dried plant tobacco material can be measured, and dried tobacco material in which the total and / or matrix-related NNK is reduced compared to the first dried plant material originally obtained can be selected for subsequent use. tobacco. Next, a second dried tobacco plant material is obtained having a higher content of total and / or matrix-bound NNK than the content of total and / or matrix-linked NNK in the first dried plant tobacco material. In some embodiments, the content of the total and / or matrix-associated NNK in this material may already be known, therefore, measurement of these values will not be required. In other embodiments, the content of the total and / or matrix-bound NNK in this material may be unknown, and therefore, measurement of these values will be required. Thus, in this method, measuring the content of total and / or matrix-bound NNK in the second dried plant material is an optional step. The first and second dried plant tobacco materials obtained in these steps can be mixed using methods well known in the art. Optionally, the content of total and / or matrix-bound NNK in the final blended tobacco plant material can be measured. According to this method, mixed tobacco plant material can be obtained in which the total and / or matrix-associated NNK content in the final mixed tobacco plant material is lower than in the second dried tobacco plant material. This method can mainly be used to obtain a mixture of tobacco material, in which the total content of NNK in the mixture is reduced. Tobacco material in which the amount of lignin has been reduced is mainly used to dilute or reduce the total NNK content in the blended tobacco material.

Tobacco material obtained or obtained in accordance with the present disclosure can also be used in a cut tobacco filling and in a smoking article formed by a cut tobacco core. Traditionally, tobacco products with cut filler for smoking products are obtained predominantly from the lamellar portion of the tobacco sheet, which is separated from the stem portion of the sheet during the beating process. A significant number of stem parts that remain after removal and separation of the leaf blade are not used. To increase the amount of tobacco material that can be used commercially, some tobacco stalks can be added back to the cut filler along with leaf blades. To improve the taste and burning characteristics of the tobacco stem for use in the cut excipient, the stem is often first subjected to one or more processing procedures, which may include the procedures described herein. The rolling step may be performed with respect to tobacco stems that have been subjected to the method of the present disclosure. The stems can be rolled to obtain the desired thickness, such as, for example, an average thickness of from about 0.6 mm to 0.8 mm. During the subsequent stages of processing and storage, the stems can expand to a final thickness of from about 0.8 mm to about 1.0 mm. After rolling, the stems are dried and transferred to a tobacco factory, where they are cut and added to the cut tobacco filling. In some cases, the rolling step may alternatively be included as part of a method for producing a cut filler in an online mode. Typically, the moisture content of the tobacco stems is from about 28% to about 34% of the volatiles removed in the furnace before rolling to prevent damage to the structure of the stems. If necessary, tobacco stems can be conditioned before rolling to increase the moisture content to this level. Known methods for conditioning tobacco stems include bringing the stems into contact with water, steam, or a mixture of water and steam. In methods where the rolling step is activated online and dried stems are used, the conditioning step will generally last longer and may require a soaking step in which the stems are soaked in water for several hours before rolling. Tobacco stems can be rolled using a one-step rolling method to reduce the thickness of the stems to the desired average thickness. After rolling, the stems can be cut to a slice width of 0.1 mm to 0.2 mm. The cut rolled stems are then optionally expanded using known stalk expansion techniques, and then dried. In the case when the stems are pre-milled and dried, as a rule, it will be necessary to condition the stems before cutting to increase the moisture content in the tobacco stems back to a value of 28% to 34% of the volatiles removed in the oven. This increases the elasticity of the tobacco stems in order to limit damage or breaking of the stems during cutting. Finally, the cut rolled stems are combined with the cut tobacco leaflets and any additional tobacco materials to form a cut filler containing at least 5% by weight of the cut rolled tobacco stems.

Thus, in a further aspect, there is provided a method for producing tobacco for use as a cut tobacco filler, comprising the steps of: (a) implementing the method (methods) described (described) herein and (b) rolling and slicing the tobacco material for use as a cut tobacco filler. A method for processing tobacco material, such as tobacco stems, for use in a cut tobacco filling is also described, the method comprising the steps of: (a) implementing the method described herein; (b) rolling of the tobacco material; (c) slicing the tobacco material; and (d) optionally drying the cut rolled stems. Rolled tobacco stalks can be combined with tobacco leaf blades, so that the steps are performed with respect to the combined tobacco stem and leaf blades. The cutting step may include cutting the rolled stems to a cut width of from about 0.3 mm to 1.3 mm. The method may include the steps of separating the stems from the tobacco leaf; slicing the stems to an average length of from about 15 mm to 80 mm; and rolling the stems to a thickness of 0.1 mm to 0.5 mm. Also provided is a method for producing a cut filler containing rolled tobacco stems, the method comprising treating tobacco stems using the method described herein; and mixing the treated stems with at least one type of tobacco leaf blade, expanded tobacco or reconstituted tobacco to produce a cut filler.

The cut tobacco filler obtained or obtained by this method may contain at least 60% and preferably at least 80% by weight of tobacco leaf blades with an average slice width of from 0.8 mm to 1.1 mm, suitably approximately 0.9 mm, and an average thickness of approximately 0.2 mm. The shredded tobacco filler may contain up to 95% by weight tobacco leaf blades with an average slice width of about 0.8 mm to 1.1 mm, more preferably about 0.9 mm, and an average thickness of about 0.2 mm. The particles of tobacco leaf blades in the cut excipient, therefore, are of the same size as the tobacco stem particles. By themselves, tobacco stalks do not visually differ from tobacco leaf blades even with a high inclusion rate. In addition, the mixture of tobacco stems and leaf blades can advantageously be transported and processed efficiently without significant sedimentation of the stems. Preferably, the average cut width of the cut rolled tobacco stems is in the range of about 0.1 mm, more suitably in the range of about 0.05 mm from the average thickness of the tobacco leaf blade in the cut filler. Cut fillers may be included in a variety of smoking articles. For example, cut filler can be used in the tobacco rod of a combustible smoking article, such as a filter cigarette, cigarilla or cigar. Alternatively, the cut filler can be used to produce an aerosol forming tobacco substrate in a sublimation smoking article or an electrically heated smoking system. Alternatively, the cut filler may be used as a self-roll product or a loose tobacco product, for example, for use in a pipe.

Tobacco material may be included in various consumer products, such as tobacco products. Methods for making such tobacco products are also covered. Tobacco products include, but are not limited to, smoking or smoking products and smokeless tobacco products, including non-combustible products, heated products, and aerosol forming products. Non-limiting examples of smoking or smoking articles include cigarettes, cigarillos, cigars, and pipe tobacco varieties. Non-limiting examples of smokeless tobacco products include chewing tobacco, snuff, and substrates for use in aerosol forming products. Smokeless tobacco products may contain tobacco in any form, including dried particles, scraps, granules, powders or pulp, coated on other ingredients, mixed with, surrounded by or otherwise combined with them in any format, such as cereals, films, tablets , foam materials or balls. The liquid contents of smokeless tobacco products may be contained in the device or may be enclosed in a form, such as beads, to prevent interaction with a water-soluble wrapper. The wrapper may be in the form of a pouch for partially or completely closing the tobacco-containing compositions or for performing the function of an adhesive for holding together several tablets, balls or flakes of tobacco. Illustrative wrapper materials include film compositions containing HPMC, CMC, pectin, alginates, pullulan, and other commercial food-grade film-forming polymers. Other wrapping materials may also include preformed capsules derived from gelatin, HPMC, starch / carrageenan or other commercially available materials. Such wrapping materials may include tobacco as an ingredient. Wrappers that do not collapse in the oral cavity may consist of woven or non-woven material, coated or uncoated paper, or perforated or other porous plastic films. Wrappers may contain flavoring or coloring agents. Smokeless products can be packaged together using a wrapper using any method known to those skilled in the art of serial packaging, including methods such as blister packaging in which a small package can be obtained using a continuous vertical packaging machine.

The amount of NNK associated with the matrix in these smoking articles, smokeless products and aerosols, etc. may be at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75 %, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% and 100% lower, such as, for example, approximately 200% or 300% lower compared to consumer products obtained from the control plant material of tobacco. The amount of free NNK can be almost unchanged.

The amount of NNK associated with the matrix in these smoking articles, smokeless products and aerosols, etc. may be about 3500 ng / g or less, or about 3000 ng / g or less, or about 2500 ng / g or less, or about 2000 ng / g or less, or about 1500 ng / g or less, or about 1000 ng / g or less, or approximately 500 ng / g or less.

The amount of free NNK in these products for smoking and smokeless products, etc. may be approximately 330 ng / g or less, approximately 300 ng / g or less, approximately 250 ng / g or less, approximately 200 ng / g or less, approximately 150 ng / g or less, approximately 100 ng / g or less, or approximately 50 ng / g or less.

The amount of NNN in these products for smoking, smokeless products and aerosols, etc. may be about 1700 ng / g or less, about 1500 ng / g or less, about 1300 ng / g or less, about 1100 ng / g or less, about 1000 ng / g or less, or about 500 ng / g or less.

The amount of nicotine in these products for smoking and smokeless products, etc. may be approximately 2600 μg or less, approximately 2300 μg or less, or approximately 2100 μg or less, or approximately 2000 μg or less, 1900 μg or less, or approximately 1800 μg or less.

The amount of lignin in these products for smoking and smokeless products, etc. may correspond to a content of approximately 6.5% or less of the total dry matter weight of dried plant tissue, a content of approximately 6% of the total dry matter mass of dried plant tissue, a content of approximately 5% of the total dry matter mass of dried plant tissue, a content of approximately 4% of the total dry matter weight of dried plant tissue or a content of about 3% of the total dry matter weight of dried plant tissue.

In one embodiment, the amount of matrix-bound NNK in these smoking articles and smokeless products and the like. is approximately 3500 ng / g or less, the amount of free NNK is approximately 300 ng / g or less, the amount of NNN is approximately 1700 ng / g or less, the amount of lignin corresponds to approximately 6.4% or less of the total dry matter weight of dried vegetable tissue, and the amount of nicotine is approximately 2600 mcg or less.

In one embodiment, smoking articles or smokeless products and the like. contain approximately 3,500 ng / g or less of matrix-bound NNK. Suitably, the amount of free NNK is about 300 ng / g or less. Suitably, the amount of NNN is about 1700 ng / g or less. Suitably, the amount of lignin corresponds to a content of about 6.4% or less of the total dry matter weight of the dried plant tissue. Suitably, the amount of nicotine is about 2600 mcg or less. Suitably, the average particle size is about 0.5 mm or more.

In another embodiment, smoking articles or smokeless products and the like. contain approximately 1900 ng / g or less of matrix-bound NNK. Suitably, the amount of free NNK is about 250 ng / g or less. Suitably, the amount of NNN is about 1270 ng / g or less. Suitably, the amount of lignin in this dried plant tissue is about 4.4% or less of the total dry matter weight of the dried plant tissue. Suitably, the amount of nicotine is approximately 2300 mcg or less. Suitably, the average particle size is from about 0.85 mm to about 1 mm.

In another embodiment, smoking articles or smokeless products and the like. contain approximately 1600 ng / g or less of matrix-bound NNK. Suitably, the amount of free NNK is about 200 ng / g or less. Suitably, the amount of NNN is about 1100 ng / g or less. Suitably, the amount of lignin in this dried plant tissue corresponds to a content of about 3% or less of the total dry matter weight of the dried plant tissue. Suitably, the amount of nicotine is about 2100 mcg or less. Suitably, the average particle size is more than about 1 mm.

Tobacco material can be obtained from tobacco plants, which include plants of the genus Nicotiana, various species of Nicotiana, including N. rustica and N. tabacum. Tobacco material can be obtained from various varieties of Nicotiana species, commonly known as smoke-dried varieties or light varieties, Burley varieties, dark varieties, and Oriental / Turkish varieties. In some embodiments, the tobacco material is derived from a plant that produces Burley, Virginia, smoke-dried, air-dried, fire-dried, oriental or dark tobacco. In some embodiments, the tobacco material is obtained, for example, from one or more of the following varieties: N. tabacum AA 37-1, N. tabacum B 13P, N. tabacum Xanthi (Mitchell-Mor), N. tabacum CT D # 3, hybrid 107, N. tabacum Bel-W3, N. tabacum 79-615, N. tabacum Samsun Holmes NN, F4 from crossing N. tabacum BU21 x N. tabacum Hoja Parado, line 97, N. tabacum KTRDC # 2, hybrid 49, N. tabacum KTRDC # 4, hybrid 110, N. tabacum Burley 21, N. tabacum KTRDC # 5 KY 160 SI, N. tabacum KTRDC # 7 FCA, N. tabacum KTRDC # 6 TN 86 SI, N tabacum PM021, N. tabacum K 149, N. tabacum K 326, N. tabacum K 346, N. tabacum K 358, N. tabacum K 394, N. tabacum K 399, N. tabacum K 730, N. tabacum KY 10, N. tabacum KY 14, N. tabacum KY 160, N. tabacum KY 17, N. tabacum KY 8959, N. tabacum KY 9, N. tabacum KY 907, N. tabacum MD 609, N. taba cum McNair 373, N. tabacum NC 2000, N. tabacum PG 01, N. tabacum PG 04, N. tabacum P01, N. tabacum P02, N. tabacum P03, N. tabacum RG 11, N. tabacum RG 17, N tabacum RG 8, N. tabacum Speight G-28, N. tabacum TN 86, N. tabacum TN 90, N. tabacum VA 509, N. tabacum AS44, N. tabacum Banket A1, N. tabacum Basma Drama B84 / 31 , N. tabacum Basma I Ziehna XP4 / B, N. tabacum Basma Xanthi BX 2A, N. tabacum Batek, N. tabacum Besuki Jember, N. tabacum C104, N. tabacum Coker 319, N. tabacum Coker 347, N. tabacum Criollo Misionero, N. tabacum PM092, N. tabacum Delcrest, N. tabacum Djebel 81, N. tabacum DVH 405, N. tabacum Galpao Comum, N. tabacum HB04P, N. tabacum Hicks Broadleaf, N. tabacum Kabakulak Elassona, N. tabacum PM102, N. tabacum Kutsage E1, N. tabacum KY 14xL8, N. tabacum KY 171, N. tabacum LA BU 21, N. tabacum McNair 944, N. tabacum NC 2326, N. tabacum NC 71, N. tabacum NC 297, N. tabacum NC 3, N. tabacum PVH 03, N. tabacum PVH 09, N. tabacum PVH 19, N. tabacum PVH 2110, N. tabacum Red Ru ssian, N. tabacum Samsun, N. tabacum Saplak, N. tabacum Simmaba, N. tabacum Talgar 28, N. tabacum PM132, N. tabacum Wislica, N. tabacum Yayaldag, N. tabacum NC 4, N. tabacum TR Madole, N. tabacum Prilep HC-72, N. tabacum Prilep P23, N. tabacum Prilep PB 156/1, N. tabacum Prilep P12-2 / 1, N. tabacum Yaka JK-48, N. tabacum Yaka JB 125/3, N. tabacum TI-1068, N. tabacum KDH-960, N. tabacum TI-1070, N. tabacum TW136, N. tabacum PM204, N. tabacum PM205, N. tabacum Basma, N. tabacum TKF 4028, N. tabacum L8, N. tabacum TKF 2002, N. tabacum TN90, N. tabacum GR141, N. tabacum Basma Xanthi, N. tabacum GR149, N. tabacum GR153 and N. tabacum Petit Havana. The use of any species of the genus Nicotiana, including N. ruslica and N. tabacum (e.g., LA B21, LN KY171, TI 1406, Basma, Galpao, Perique, Beinhart 1000-1 and Petico) is disclosed. Other species include N. acaulis, N. acuminate, N. acuminate var. multiflora, N. alata, N. amplexicaulis, N. arentsii, N. benavidesii, N. benthamiana, N. bigelovii, N. bonariensis, N. cavicola, N clevelandii, N. cordifolia, N. corymbosa, N. debneyi, N excelsior, N. forgetiana, N. fragrans, N. glauca, N. glutinosa, N. goodspeedii, N. gossei, N. hybrid, N. ingulba, N. kawakamii, N. knightiana, N. langsdorffii, N. linearis , N. longiflora, N. megalosiphon, N. miersii, N. noctiflora, N. nudicaulis, N. obtusifolia, N. occidentalis, N. occidentalis sub, hesperis, N. otophora, N. paniculata, N. puuciflora, N. petunioides, N. plumbaginifolia, N. quadrivalvis, N. raimondii, N. repanda, N. rosulata, N. rosulata sub, ingulba, N. rotundifolia, N. setchellii, N. simulans, N. solanifolia, N. spegazzinii, N Stocktonii, N. suaveolens, N. sylvestris, N. thyrsiflora, N. tomentosa, N. tomentosiformis, N. trigonophylla, N. umbratica, N. velutina, N. wigandioides and N. x sanderae.

The use of tobacco cultivars and elite tobacco cultivars is also contemplated herein. Particularly suitable varieties of Nicotiana tabacum include tobacco of the Burley type, dark type, smoke-dried type and Oriental type. Non-limiting examples of varieties or varieties are: BD 64, SS 101, SS 200, SS 27, SS 301, SS 400, SS 500, SS 600, SS 700, SS 800, SS 900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CD 263, DF911, DT 538 LC, tobacco Galpao, GL 26H, GL 350, GL 600, GL 737, GL 939, GL 973, HB 04P, HB 04P LC, HB3307PLC, hybrid 403LC, hybrid 404LC, hybrid 501 LC, K 149, K 326, K 346, K 358, K394, K 399, K 730, KDH 959, KT 200, KT204LC, KY10, KY24, KY 160, KY 17, KY 171, KY 907, KY907LC, KTY14xL8 LC , Little Crittenden, McNair 373, McNair 944, msKY 14xL8, Narrow Leaf Madole, Narrow Leaf Madole LC, NBH 98, N-126, N-777LC, N-7371LC, NC 100, NC 102, NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC 606, NC 71, NC 72, NC 810, NC BH 129, NC 2002, Neal Smith Madole, OXFORD 207, PD 7302 LC, PD 7309 LC, PD 7312 LC, tobacco 'Periq'e', PVH03, PVH09, PVH 19, PVH50, PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81, RG H51, RGH 4, RGH 51, RS 1410, Speight 168, Speight 172, Speight 179, Speight 210, Speight 220, Speight 225, Speight 227 , Speight 234, Speight G-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, TI 1406, TI 1269, TN 86, TN86LC, TN 90, TN 97, TN97LC, TN D94, TN D950, TR (Tom Rosson) Madole, VA 309, VA359, AA 37-1, B 13P, Xanthi (Mitchell-Mor), Bel-W3, 79-615, Samsun Holmes NN, KTRDC No. 2, Hybrid 49, Burley 21, KY 8959, KY 9, MD 609, PG 01, PG 04, PO1, PO2, PO3, RG 11, RG 8, VA 509, AS44, Banket A1, Basma Drama B84 / 31, Basma I Zichna ZP4 / B, Basma Xanthi BX 2A, Batek, Besuki Jember, C104, Coker 347, Criollo Misionero, Delcrest, Djebel 81, DVH 405, Galpro Comum, HB04P, Hicks Broadleaf, Kabakulak Elassona, Kutsage E1, LA BU 21, NC 2326, NC 297, PVH 2110, Red Russian, Samsun, Saplak, Simmaba, Talgar 28, Wislica, Yayaldag, Prilep HC-72, Prilep P23, Prilep PB 156/1, Prilep P12-2 / 1, Yaka JK-48, Yaka JB 125/3, TI- 1068, KDH-960, TI-10 70, TW136, Basma, TKF 4028, L8, TKF 2002, GR141, Basma Xanthi, GR149, GR153, Petit Havana. Also subspecies of the aforementioned with a low conversion of nicotine to nornicotin are also contemplated, even if they are not specifically indicated in this document.

The following examples are provided as an illustration, but not as a limitation. Unless otherwise indicated, the present invention uses traditional techniques and methods of molecular biology and plant biology.

EXAMPLES

Example 1

Method for analysis of free and matrix-bound NNK in tobacco

Aliquots of tobacco samples (e.g., approximately 750 mg) are extracted with approximately 30 ml Tris-HCl buffer (50 mm; pH 7.4) by shaking for approximately one hour at approximately room temperature. Add internal standards (100 ng / ml NNK-d ₄ ). Samples (0.4 ml) of the extracts were filtered using a 0.2 μm filter, and the NNK content was analyzed by ultra-high performance liquid chromatography with tandem mass spectrometry (UPLC-MS / MS). The concentrations in the samples calculated from the concentrations in these extracts correspond to the concentrations of “free NNK” in the sample. After processing the extraction mixtures (for example, by heating to about 130 ° C. for about 4 hours) and filtering aliquots of the extracts, the NNK concentrations were again measured using UPLC-MS / MS. Based on these values, the concentration of “total NNK” in the samples can be calculated. The concentration of matrix-bound NNK is the difference between the concentrations of total NNK and free NNK.

An alternative “total NNK” extraction method involves acidifying the extraction mixtures with concentrated HCl (for example 3 ml of 37% HCl added to 30 ml) and incubating for 48 hours at 80 ° C. Acidic extracts are neutralized before filtration and UPLC analysis by adding NaOH solution (6 N, 40 μl) and a suspension of magnesium hydroxide (10%; 40 μl) to 320 μl of extract.

Example 2

UPLC analysis

A Waters Acquity BEN C18 column, 1.7 μM, 2.1 × 50 mm, is used. The following eluents are used: (A) ammonium bicarbonate (10 mM; adjusted to 9.8 with ammonia) + 2% (v / v) acetonitrile; (B) acetonitrile. Apply the following gradient: 0 min. - 5% B; 0.5 min - 5% B; 3.3 minutes - 18.3% B. The applied flow rate is 0.5 ml / min. The column temperature used is 50 ° C.

Example 3

MS / MS Technique

This analysis is performed on a Waters TQ spectrometer using the following MRM transitions: NNK: 208.2 → 122.2; exposure time 100 ms; NNK-d4: 212.2 → 126.2; exposure time 100 ms; capillary voltage: 0.6 kV; cone voltage: 25 V; collision energy: 11 eV; source temperature: 120 ° C; temperature on the desolvation line: 400 ° С; desolvation gas flow: 800 l / h.

Example 4

Matrix NNK distribution in lignified and non-lignified tissues of Burley tobacco stems

Approximately 2 grams of medium veins of dried Burley tobacco leaves are manually divided into internal lignified tissue (36% of the total dry matter weight) and external non-lignified tissue (64% of the total dry matter weight). In each of these samples, the concentration of free NNK and total NNK was analyzed using the UPLC-MS described above. The concentration of matrix-bound NNK is calculated as the difference between the concentrations of free NNK and total NNK. Quantification of lignin content is carried out using a photometric method based on thioglycolic acid derivatization (see Brinkmann et al. (2002) J. Chem. Ecol., 28, 2483-2501).

The results in Figure 1 show the distribution of free NNK bound to the NNK matrix and lignin in the lignified (L) and non-lignified (NL) tissues of dried Burley tobacco stems. Figure 2 is a cross-sectional view of a hydrated dried Burley tobacco stem, showing lignified (L) and non-lignified (NL) tissue. The lignified tissue is stained red with phloroglucin. The results in FIG. 3 show the content of matrix-bound NNK in the lignified and non-lignified tissues of the green midrib after nitrosation with a sodium nitrite solution.

These results demonstrate that matrix-bound NNK is distributed mainly in the lignified tissues of Burley tobacco stem and midrib veins.

Example 5

Enrichment of the fraction of the stems of Burley tobacco with a low content of bound NNK by freeze-drying, grinding and size separation

A sample of Burley tobacco stems (52 g) is moistened with water (350 ml) and freeze-dried. A part of the obtained material (12 g) is crushed by shaking with steel balls (2 balls, diameter 2 cm; 300 rpm; 15 minutes) and using a shaker for sieves are divided into fractions with different particle sizes in the range from exceeding 1 mm, to a value of less than 0.25 mm.

Figure 4 and table 1 shows the levels of free and matrix-bound NNK, NNN, lignin and nicotine in the fractions after sifting by freeze-dried chopped Burley tobacco stalks. The results of the analysis of free and matrix-bound NNKs shown in FIG. 5 and Table 1 indicate that both the lignin content (as% dry weight of each fraction) and the matrix-bound NNK content are reduced in fractions with particle sizes greater than 0.5 mm, including in fractions with a particle size of from 0.5 mm to 0.85 mm, from 0.85 mm to 1 mm and more than 1 mm. Figure 5 shows that the lignin content correlates well with the content of matrix-bound NNK, which thus confirms the colocalization of lignin and matrix-bound NNK.

Example 6

Localization of the NNK Matrix Precursor in TN90 Green Middle Veins of Tobacco and Burley Tobacco Stems

The relative distribution of matrix-bound NNK is measured in the sclerenchymal tissue and non-sclerenchymal tissue of the middle veins of tobacco TN90. The assumption that the matrix bound NNK is bound to lignin suggests a higher concentration of its precursor in the lignified sclerenchymal tissue. In a second experiment, the relative distribution of free and matrix-bound NNK in the sclerachymal and non-sclerachymal tissue of Burley tobacco (dried) stems is examined.

Materials and methods

Green medium veins

Middle veins (proximal halves only) 15 mature TN90 leaves are manually divided into sclerenchymal tissue (S) (the “center” of the middle vein) and tissue other than sclerenchymal (NS). Both fabrics are freeze-dried and finely ground. The water-insoluble fraction of the two materials is determined by extracting 1 g each of them three times with 40 ml of methanol / water 1: 3 (at room temperature for 1 hour) and weighing the insoluble material (designated as SW and NSW) after freeze-drying. Measurements in the analysis of pseudooxy nicotine (PON) and nicotine in S and NS (n = 5) are carried out using the following methods. Fine plant material (~ 20 mg) was extracted by shaking at room temperature for 45 minutes with a mixture of methanol / water (4: 1) containing PON-methyl-d ₃ as the internal standard (200 ng / ml). After filtration (0.2 μm), the samples were analyzed by LC-MS using the following conditions: column: Acquity UPLC BEN C18 column (1.7 μm, 50 × 2.1 mm; Waters); column temperature: 50 C; eluents: Aqueous ammonium bicarbonate, adjusted to pH 9.8 using NH ₃ with acetonitrile (98: 2, v / v; eluent A); acetonitrile (eluent B); gradient: 0 min - 0% B, 0.5 ml / min .; 0.5 min - 0% B, 0.5 ml / min .; 6 minutes - 30% B, 0.5 ml / min .; MS detection: PON - from mass / charge 179.2 to mass / charge 106.1; PON-methyl-d ₃ - from mass / charge 182.2 to mass / charge 106.1; UV detection: 260 nm. PON and nicotine elute after 2.6 and 4.1 minutes, respectively. To quantify nicotine, a peak area at 260 nm and an external calibration are used. In order to evaluate the content of the matrix bound NNK precursor in S, NS, SW, and NSW, aliquots (~ 20 mg; n = 5) of these materials are nitrosated by incubation in a NaNO2 solution (1.5 ml (10 mg / ml in water)) for 4 hours at room temperature with shaking, centrifuged and washed / centrifuged four times with 10 ml of water. Then the precipitate after centrifugation of each nitrosated sample is absorbed in 4 ml of Tris-Hcl buffer (50 mM, pH 7.5; with NNK-d4 and NNN-d4 at 100 ng / ml), autoclaved (for 4 hours at 130 ° C) and analyzed for NNK content using the methods described herein.

Dried Burley Tobacco Stalks

A Burley tobacco stem sample (2 g = four pieces per ~ 5 cm) is manually divided into the outer (non-lignified) tissue (CNS) and the inner (lignified) portion (CS). Both samples were subjected to fine grinding in a vibratory mill (TissueLyzer from Retsch for 2.5 minutes, 50 s-1). The result is respectively 1183 mg and 651 mg of CNS and CS powder. The amount of free NNK in two samples was determined after extraction with ~ 50 mg aliquots (n = 5) of 1.5 ml Tris buffer (+ IS) for 1 hour at room temperature. The total NNK content was determined after autoclave extraction (130 ° C for 4 hours) ˜50 mg aliquots (n = 5) in 5 ml Tris buffer (+ IS).

results

The results of this experiment are shown in table 2 and figure 6. After nitrosation and washing in the sclerenchymal tissue (S), a 7-fold higher concentration of matrix-bound NNK is found in comparison with the outer layers of the middle veins (NS). The concentration of both PON and nicotine is two times higher in NS than in S. The results of experiments with this artificial nitrosation are confirmed by the levels of bound NNK in the lignified (CS) and non-lignified (CNS) portions of Burley's dried commercial tobacco stem sample. Although the concentration of free NNK is two times higher in CS, the concentration of matrix-bound NNK is 7 times higher in CS. Nicotine and NNN levels are higher in CS than in CNS, and lower than NNK.

findings

The presence of a precursor of matrix-bound NNK in high concentrations in the lignified sclerenchymal tissue of green middle veins indicates that the matrix-linked NNK is covalently or non-covalently bound to lignin.

Any publication cited or described in this document provides relevant information disclosed prior to the filing date of this application. The statements made in this document should not be construed as an assumption that the authors of the present invention are not entitled to date such disclosures earlier. All publications mentioned in the above description are incorporated herein by reference. Various modifications and variations of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the present invention, as claimed, should not be unduly limited to such specific embodiments. In fact, various modifications of the described methods of carrying out the invention, which are obvious to experts in cell, molecular biology and plant biology or related fields, are intended to be within the scope of the attached claims.

Claims (36)

1. A method of reducing the amount of matrix-bound NNK in dried tobacco plant material, comprising separating the lignified tissue from the non-lignified tissue, where the amount of lignin is reduced mechanically, where the lignified tissue is separated from the non-lignified tissue by removing a vascular bundle, xylem or lignified sclerenic tissue. 2. The method of claim 1, comprising the steps of: (a) obtaining dried plant tobacco material; (b) separating lignified tissue from non-lignified tobacco in dried plant material, and (c) obtaining dried tobacco plant material in which the amount of lignin is reduced and the amount of NNK bound to the matrix is reduced compared to the dried plant tobacco material obtained in step (a). 3. The method of claim 2, wherein step (a) is followed by an additional step of measuring the amount of at least an NNK matrix associated with it, and where step (b) is optionally followed by an additional step of measuring the amount of at least an NNK associated with the matrix. 4. The method according to p. 3, where the specified method includes an additional step (d) comparing the level of at least the matrix bound NNK measured after stage (a) with the level associated with the matrix NNK measured after stage (b), where the amount of matrix-bound NNK in the tobacco material obtained in step (b), compared with the tobacco material obtained in step (a), indicates that the amount of matrix-bound NNK in the tobacco material is reduced. 5. A method of reducing the formation of matrix-bound NNK during drying of tobacco plant material, comprising reducing the amount of lignin thereto before drying, comprising the steps of: (a) obtaining dried vegetable tobacco material; (b) mechanically separating the lignified tissue from the non-lignified tobacco in the non-dried plant material before drying; (c) drying the plant tobacco material obtained in step (b); and (d) obtaining dried plant tobacco material in which the amount of matrix-bound NNK is reduced compared to a control in which the amount of lignin is not reduced, where the lignified tissue is separated from the non-lignified tissue by removing a vascular bundle, xylem or lignified sclerenchymal tissue.              6. The method of claim 5, wherein step (a) is followed by an additional step of measuring the amount of at least an NNK matrix, and where step (b) is optionally followed by an additional step of measuring the amount of at least an NNK matrix, and where step (c) optionally is followed by an additional step of measuring the amount of at least the NNK associated with the matrix, preferably where after step (c) or step (d), the method includes an additional step of comparing the level of at least the matrix NNK measured after step (a) with the level of at least the matrix NNK measured after step (b) and / or step (c), where a decrease in the amount of matrix-bound NNK in the tobacco material obtained in step (b) or step (c), compared with tobacco material obtained in step (a), indicates that the amount of bound with matrix NNK in tobacco material is reduced. 7. The method according to any one of the preceding paragraphs, where the tobacco plant material is treated to expand non-lignified plant tissue, where the amount of lignin is reduced by separating the expanded and unexpanded plant tissue based on various indicators of their density and / or various indicators of their strength, and / or different particle sizes, and / or where the plant material obtained in stage (a) contains medium veins of plants, or stems of plants, or petioles of plants, or a combination of two or more of them, or consists of them, or consists mainly of them. 8. Plant tobacco material obtained or obtained using the method according to any one of paragraphs. 1-7. 9. The use of tobacco plant material in which the amount of lignin has been reduced compared to the control plant tobacco material by mechanically separating the lignified tissue from the non-lignified tissue to produce tobacco with reduced levels of NNK matrix bound, wherein said levels of NNK matrix bound are reduced compared to with a control where lignified tissue is separated from non-lignified tissue by removal of a vascular bundle, xylem or lignified sclerenchymal tissue.            10. A method of producing reconstituted tobacco, comprising the steps of: (a) implementing the method according to any one of paragraphs. 1-7; (b) obtaining reconstituted tobacco from tobacco material obtained in step (a); and (c) optionally including reconstituted tobacco in the tobacco product. 11. Reconstituted tobacco obtained or obtained using the method according to p. 10. 12. A method of producing tobacco for use as a shredded tobacco filler, comprising the steps of: (a) implementing the method according to any one of paragraphs. 1-8 and (b) rolling and slicing of tobacco material for use as a shredded tobacco filler. 13. Dried tobacco plant material having a reduced lignin level compared to a control tobacco plant material in which the amount of lignin was not reduced, where the amount of lignin in the dried tobacco plant material was reduced by mechanically separating the lignified tissue from the non-lignified tissue, where the vascular bundle or xylem, or lignified sclerenchymic tissue, is practically absent in dried plant tissue, and where the amount of matrix-bound NNK is approximately but 3500 ng / g or less, preferably where the amount of free NNK is less than about 330 ng / g, where the optional NNN content is less than about 1700 ng / g, and where the optional nicotine content is less than about 2610 μg / g; and / or where the dried plant tobacco material contains a cortical layer of a plant, such as, for example, the outer cortical layer of a plant, consists of or consists mainly of it. 14. A tobacco product or a reconstituted tobacco product containing the tobacco plant material of claim 8 or the dried plant material of claim 13, consisting of or consisting essentially of it. 15. A method of mixing tobacco, in which at least two different types of tobacco are mixed to produce a tobacco bag, the method comprising the steps of: (a) obtaining a first dried plant tobacco material and reducing the amount of lignin therein by mechanically separating the lignified tissue from the non-lignified tissue; (b) measuring the content of total and / or matrix-bound NNK in the first dried tobacco plant material and selecting dried tobacco plant material in which the content of total and / or matrix-related NNK is reduced compared to the first dried plant tobacco material obtained in the step (but); (c) preparing a second dried tobacco plant material having a higher total and / or matrix-bound NNK content than the total and / or matrix-linked NNK in the first dried tobacco plant material obtained in step (b), and optionally measuring the content of total and / or matrix-bound NNK in the second dried tobacco plant material; (d) mixing the first and second dried tobacco plant material from steps (b) and (c) and optionally measuring the total and / or matrix NNK content of the mixed tobacco plant material; and (e) preparing a mixed tobacco plant material in which the content of total and / or matrix-linked NNK in the mixed tobacco plant material is lower than in the second dried tobacco plant material obtained in step (c), where the lignified tissue is separated from the non-lignified tissue by removal of a vascular bundle, xylem or lignified sclerenchymal tissue, where optional steps (a) and (b) are carried out after step (c).

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