Note: Descriptions are shown in the official language in which they were submitted.
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METHODS FOR REDUCING MATRIX-BOUND NICOTINE-DERIVED NITROSAMINE KETONE
IN TOBACCO PLANT MATERIAL
FIELD OF THE INVENTION
The present invention relates, in general, to methods for reducing the amount
of nicotine-derived
nitrosamine ketone or 4-(methylnitrosamino)-1-(3-pyridyI)-1-butanone (NNK) in
tobacco plant
material.
BACKGROUND OF THE INVENTION
During the manufacture and processing of tobacco products, by-products - such
as tobacco stems,
and leaf scraps - are produced. Tobacco stems and tobacco fines from
manufacturing processes
are unsuitable for use directly in the manufacturing of tobacco products.
Since the stems and fines
represent a substantial amount of raw material investment, processes have been
developed to
further convert these stems and fines into products - such as reconstituted
tobacco materials (eg.
reconstituted tobacco sheets) - which are then useable in relatively large
amounts in a mixture with
acceptable processed tobacco leaf. Reconstituted tobacco can be manufactured
in a slurry or cast
sheet process wherein pulp of mashed tobacco stems and other parts of the
tobacco leaf are
ground and mixed with a solution that might contain different additives. The
resulting tobacco slurry
is then sprayed to form a thin film, dried, rolled and diced into strips which
are added to a filler.
Nitrosamines are organic compounds found in many consumer products - such as
tobacco, food
products and cosmetics. Nitrosamines have drawn intense scientific interest
because some of the
compounds in this class have been shown to be carcinogenic in laboratory
animals. It has been
reported that some cured tobaccos contain tobacco specific nitrosamines that
can be found in
smokeless tobacco, mainstream smoke and side stream smoke of cigarettes. In
tobacco, at least
four species of nitrosamines are produced at appreciable quantity. These are
nicotine-derived
nitrosamine ketone or 4-(methylnitrosamino)-1-(3-pyridyI)-1-butanone (NNK), N-
nitrosonornicotine
(NNN), N-nitrosoanatabine (NAT), and N-nitrosoanabasine (NAB). Tobacco
specific nitrosamines
are not considered to be present in significant quantities in growing tobacco
plants or fresh cut
tobacco (green tobacco), but are formed during the tobacco curing process. In
addition to the
formation of tobacco specific nitrosamines during the curing process of green
leaves, tobacco
specific nitrosamines may also be formed during processes used to prepare
aqueous tobacco
slurries - such as processes used to prepare reconstituted tobacco.
In an attempt to reduce tobacco specific nitrosamines, various treatments of
tobacco plants or
harvested tobacco leaves have been suggested, including radiation treatments,
chemical
treatments and extractions. Other methods for reducing tobacco specific
nitrosamines have been
suggested by MacKown etal. (1988) J. Agric. Food Chem. 36, 1031-1035. These
methods involve
treatment using sterilization, microbial inhibitors, bases to increase pH, or
ascorbic acid to
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decrease the accumulation of tobacco specific nitrosamines during the
production of reconstituted
tobacco sheets. W02012160133 describes a process for decreasing the levels of
tobacco specific
nitrosamines in tobacco homogenates by increasing the pH thereof, especially
when elevated
levels of nitrosamines are created by elevated nitrite levels.
One problem with trying to reduce the levels of tobacco specific nitrosamines
in tobacco is that
some of the nitrosamines in air-cured tobacco, including NNK, exist in a bound
or matrix-bound
form, which can be difficult to remove or extract. Matrix-bound NNK can be
extracted with a 0.1N
KOH solution from water-washed Burley filler. This alkaline treatment also
decreases NNK levels
in smoke (Keene, O.K., 1992, The Effect of Base Digestion on TSNA in
Extractables-Depleted
Fillers. Legacy Tobacco Documents). However, the treatment can introduce
toxicologically
relevant compounds into tobacco and can significantly deteriorate the quality
of the tobacco, which
is highly problematic for the tobacco industry.
W02010/021809 describes a method for recuing nitrogen compounds and lignin in
tobacco.
Nitrogen compounds are removed by solvent extraction; lignin is removed in a
separate step.
A need remains for an effective and cost efficient method for reducing matrix-
bound NNK that is
formed during the curing of tobacco. In particular, a cost-effective and
simple method for reducing
the levels of matrix-bound NNK in cured tobacco that does not introduce toxic
or potentially toxic
compounds and does not deteriorate the quality of the tobacco product is
particularly desirable.
SUMMARY OF THE INVENTION
The present invention is based, at least in part, on the surprising finding
that matrix-bound NNK co-
localises with lignin in tobacco plants. In particular, the present inventors
have observed that
within cured parts of a tobacco plant ¨ such as in the stems and midribs -
matrix-bound NNK co-
localises or co-localises predominantly or co-localises exclusively with
lignin ¨ such as lignified
tissue, particularly in the vascular bundle (for example, the xylem) and not
the surrounding tissues
¨ such as the cortex. Therefore removal of lignin (for example, by separating
lignified from non-
lignified tissue) can reduce the amount of matrix-bound NNK and metabolites
thereof in plant
material. Matrix-bound NNK can be covalently or non-covalently linked to
lignin. The plant
material is expected to deliver smoke with reduced NNK levels and potentially
improved sensory
properties. The present disclosure can be applied to those plant materials
which accumulate
matrix-bound NNK or have the potential to accumulate matrix-bound NNK. In
particular, the
disclosure can be applied to low-value plant material comprising matrix-bound
NNK that is used in
certain tobacco processes. The methods described herein can be carried out
without the use of
any additives and thereby do not introduce additional toxicologically relevant
compounds into the
plant material. The removal of lignin can take place during or after curing of
tobacco plant material.
Lignin can be removed before curing to prevent, reduce or inhibit matrix-bound
NNK co-localising
with lignin.
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One general object of this disclosure is to substantially decrease the amount
of matrix-bound NNK
and metabolites thereof in tobacco intended for smoking or consumption by
other means. Another
general object is to reduce the carcinogenic potential of tobacco products,
including cigarettes,
cigars, chewing tobacco, snuff and tobacco-containing gum and lozenges. Still
another general
object is to decrease or reduce the amount of matrix-bound NNK and metabolites
thereof in
tobacco plant material and in tobacco products. Another general object is to
reduce the amount of
matrix-bound NNK and metabolites thereof in cured ¨ such as partially or fully
cured - tobacco
plant material. Another general object is to reduce the amount of NNK and
metabolites thereof in
aerosol, including smoke. Yet another object of this disclosure is to reduce
the amount of NNK or
metabolites thereof in humans who smoke, consume or otherwise ingest tobacco
in some form, by
providing a tobacco product suitable for human consumption which contains a
reduced amount of
NNK or metabolites thereof, thereby lowering the carcinogenic potential of
such product.
In one aspect, there is provided a method of reducing the amount of matrix-
bound NNK in a cured
tobacco plant or in cured tobacco plant material comprising reducing the
amount of lignin therein
by separating lignified from non-lignified tissue, preferably, wherein the
amount of lignin is reduced
chemically and/or mechanically.
In one embodiment, the tobacco plant or the tobacco plant material is treated
to expand non-
lignified plant tissue. The amount of lignified tissue is reduced by
separating the expanded and
non-expanded plant tissue based on their different densities (for example,
buoyant densities)
and/or their different strengths and/or their different sizes and/or their
different weight. The
expanded plant tissue can be collected for further tobacco processing.
It has been observed that significant reduction in levels of NNK and
associated TSNAs can be
obtained by fractionating the 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 sclerenchymatic tissue or a combination of two or more thereof from
the plant or plant
material. The lignin can be located in the vascular bundle. The lignin can be
located exclusively in
the vascular bundle. The lignin can be located exclusively in the vascular
bundle and not the
surrounding tissue. The lignin can be located in the xylem. The lignin can be
located exclusively
in the xylem. The lignin can be located exclusively in the xylem and not the
surrounding tissue.
The lignin can be located in sclerenchymatic tissue. The lignin can be located
exclusively in
sclerenchymatic tissue. The lignin can be located exclusively in the
sclerenchymatic tissue and not
the surrounding tissue. Lignified tissue is generally absent from the outer
layer of plant midribs.
In one embodiment, the plant or plant material that is treated according to
the present disclosure
comprises or consists or consists essentially of plant midribs or plant stems
or plant stalks or a
combination of two or more thereof.
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In one embodiment, the amount of lignin is reduced by harvesting the cortex ¨
such as the outer
cortex - from the plant or plant material.
In one embodiment, the method comprises the steps of: (a) providing a cured
tobacco plant or
cured tobacco plant material; (b) reducing the amount of lignin in the cured
tobacco plant or the
cured tobacco plant material by fractionating the tobacco plant material; and
(c) obtaining a cured
tobacco plant or cured tobacco plant material in which the amount of lignin is
reduced and the
amount of matrix-bound NNK is reduced as compared to the cured tobacco plant
or the cured
tobacco plant material provided in step (a).
In one embodiment, following step (a) there is a further step of measuring the
amount of free NNK
or matrix-bound NNK or a combination thereof, and optionally, wherein
following step (b) there is a
further step of measuring the amount of free NNK or matrix-bound NNK or a
combination thereof.
In one embodiment, the method comprises the further step (d) of comparing the
level of at least
matrix-bound NNK measured following step (a) with the level of NNK measured
following step (b),
wherein a reduction in the amount of matrix-bound NNK in the tobacco material
obtained in step
(b) as compared to the tobacco material provided in step (a) is indicative
that the amount of matrix-
bound NNK in the tobacco material is reduced.
In a further aspect, there is provided a method of reducing the formation of
matrix-bound NNK
during the curing of a tobacco plant or tobacco plant material comprising
reducing the amount of
lignin therein prior to curing.
In one embodiment, the method comprises the steps of: (a) providing an uncured
tobacco plant or
uncured tobacco plant material; (b) reducing the amount of lignin in the
uncured tobacco plant or
the uncured tobacco plant material prior to curing; (c) curing the tobacco
plant or the tobacco plant
material provided in step (b); and (d) obtaining a cured tobacco plant or
cured tobacco plant
material in which the amount of matrix-bound NNK is reduced as compared to a
control in which
the amount of lignin has not been reduced.
In one embodiment, following step (a) there is a further step of measuring the
amount of free NNK
or matrix-bound NNK or a combination thereof, and optionally, wherein
following step (b) there is a
further step of measuring the amount of free NNK or matrix-bound NNK or a
combination thereof
and optionally, wherein following step (c) there is a further step of
measuring the amount of free
NNK or matrix-bound NNK or a combination thereof.
In one embodiment, following step (c) or step (d) said method comprises the
further step of
comparing the level of at least matrix-bound NNK measured following step (a)
with the level of
NNK measured following 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) as compared
to the tobacco
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material provided in step (a) is indicative that the amount of matrix-bound
NNK in the tobacco
material is reduced.
In a further aspect, there is provided tobacco plant material obtained or
obtainable by the
method(s) described herein.
In a further aspect, there is provided the use of a tobacco plant or tobacco
plant material in which
the amount of lignin therein has been reduced as compared to control tobacco
plant or control
tobacco plant material for manufacturing tobacco with reduced levels of matrix-
bound NNK,
wherein said levels of matrix-bound NNK are reduced as compared to the
control.
In a further aspect, there is provided a method for producing reconstituted
tobacco comprising the
steps of: (a) performing the method(s) described herein; (b) manufacturing the
tobacco material
obtained in step (a) into reconstituted tobacco; and (c) optionally
incorporating the reconstituted
tobacco into a tobacco product.
In a further aspect, there is provided reconstituted tobacco obtained or
obtainable by the method
described herein.
In a further aspect, there is provided a method for preparing tobacco for use
as a tobacco cut filler
comprising the steps of: (a) performing the method(s) described herein; and
(b) rolling and cutting
the tobacco material for use as a tobacco cut filler.
In a further aspect, there is provided cured tobacco plant material containing
a reduced level of
lignin as compared to control tobacco plant material in which the amount of
lignin has not been
reduced, and wherein the amount of matrix-bound NNK is about 3500 ng/g or
less.
In one embodiment, the average particle size is greater than about 0.5
millimetres.
In one embodiment, the amount of free NNK is less than about 330 ng/g,
optionally wherein the
NNN content is less than about 1700 ng/g and optionally wherein the nicotine
content is less than
about 2610 pg/g.
In one embodiment, the cured tobacco plant material comprises, consists of
consists essentially of
plant cortex ¨ such as outer plant cortex.
In one embodiment, vascular bundle or xylem or lignified sclerenchymatic
tissue or a combination
of two or more thereof is substantially absent from the cured tobacco plant
material.
In one embodiment, the cured tobacco plant material comprises, consists of
consists essentially of
plant cortex ¨ such as outer plant cortex ¨ and vascular bundle or xylem or
lignified
sclerenchymatic tissue or a combination of two or more thereof is
substantially absent therefrom.
In one embodiment, the cured tobacco plant material is obtained or obtainable
from plant midribs
or plant stems or plant stalks or a combination of two or more thereof.
In one embodiment, the average particle size is greater than about 0.5
millimetres.
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 pg/g.
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In a further aspect, there is provided a tobacco product or a reconstituted
tobacco product
comprising, consisting or consisting essentially of the plant material or the
cured plant material
described herein.
In a further aspect, there is provided a method of producing an aerosol in
which the amount of
NNK is reduced as compared to a control aerosol comprising the steps of: (a)
providing a cured
tobacco plant or cured tobacco plant material; (b) reducing the amount of
lignin in the cured
tobacco plant or the cured tobacco plant material; (c) obtaining a cured
tobacco plant or cured
tobacco plant material in which the amount of lignin is reduced and the amount
of matrix-bound
NNK is reduced as compared to the cured tobacco plant or the cured tobacco
plant material
provided in step (a); and (d) heating the cured tobacco plant or cured tobacco
plant material from
step (c) to produce an aerosol.
In a further aspect, there is provided a method of producing an aerosol in
which the amount of
NNK is reduced as compared to a control aerosol comprising the steps of: (a)
providing an uncured
tobacco plant or uncured tobacco plant material; (b) reducing the amount of
lignin in the uncured
tobacco plant or the uncured tobacco plant material prior to curing; (c)
curing the tobacco plant or
the tobacco plant material provided in step (b); (d) obtaining a cured tobacco
plant or cured
tobacco plant material in which the amount of matrix-bound NNK is reduced as
compared to a
control in which the amount of lignin has not been reduced; and (e) heating
the cured tobacco plant
or cured tobacco plant material from step (d) to produce an aerosol.
In a further aspect, there is provided a method of producing an aerosol in
which the amount of
NNK is reduced as compared to a control aerosol comprising the step of: (a)
providing a tobacco
product or a reconstituted tobacco product comprising, consisting or
consisting essentially of the
tobacco plant material or the cured plant material obtained or obtainable by
the methods described
herein; and (b) heating the tobacco product or the reconstituted tobacco
product to produce an
aerosol.
In a further aspect, there is provided an aerosol obtained or obtainable by
the method(s) described
herein.
In a further aspect, there is provided cured tobacco plant material consisting
essentially of tobacco
plant cortex and wherein the amount of matrix-bound NNK is reduced as
described herein.
In a further aspect, there is provided a method for blending tobacco in which
at least two different
types of tobacco are blended so as to form a tobacco blend comprising the
steps of: (a) providing a
first cured tobacco plant material and reducing the amount of lignin therein;
(b) measuring the total
and/or matrix-bound NNK content of the first cured tobacco plant material and
selecting cured
tobacco plant material in which the total and/or matrix-bound NNK content is
reduced as compared
to the first cured tobacco plant material provided in step (a); (c) providing
a second cured tobacco
plant material which has a higher total and/or matrix-bound NNK content than
the total and/or
matrix-bound NNK of the first cured tobacco plant material obtained in step
(b), and optionally
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measuring the total and/or matrix-bound NNK content in the second cured
tobacco plant material;
(d) blending together the first and second cured tobacco plant materials from
steps (b) and (c) and
optionally measuring the total and/or matrix-bound NNK content in the blended
tobacco plant
material; and (e) obtaining a blended tobacco plant material in which the
total and/or matrix-bound
NNK content of the blended tobacco plant material is lower than the second
cured tobacco plant
material provided in step (c), optionally wherein steps (a) and (b) are
performed after step (c).
In a further aspect, there is provided blended tobacco plant material obtained
or obtainable by the
method(s) described herein.
Each of the embodiments discussed above are disclosed as embodiments of each
of the aspects
of the invention. Combinations of one or of the embodiments are contemplated.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a graph illustrating the distribution of free NNK, matrix-bound
NNK and lignin in lignified
(L) and non-lignified (NL) tissues in cured Burley midribs or cured Burley
stems.
Figure 2 is a cross-section of a hydrated cured Burley stem showing lignified
(L) and non-lignified
(NL) tissues. Lignified tissue is stained red with phloroglucinol.
Figure 3 is a graph illustrating the distribution of free NNK, matrix-bound
NNK and lignin in lignified
(L) and non-lignified (NL) tissues of green Burley midribs 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 illustrating free and matrix-bound NNK in sieving
fractions of ground freeze-
dried Burley tobacco plant stems.
Figure 5 is graph showing the correlation between matrix-bound NNK and lignin
in sieving
fractions of ground freeze-dried Burley tobacco plant stems.
Figure 6 is a graph showing the concentration of matrix-bound NNK (pg/g) in
sclerenchymatic
tissue (S) and in the outer layers of the midribs (NS) after nitrosation and
washing in green midribs
of TN90. Levels of pseudo-oxynictoine (PON) (pg/g) and nicotine (pg/g) are
also shown. Matrix-
bound NNK levels (ng/g) in lignified (CS) and non-lignified (CNS) parts of a
commercial cured
Burley stem sample are also shown. Levels of NNN (ng/g) and nicotine (pg/g)
are also shown.
DEFINITIONS
The technical terms and expressions used herein are generally to be given the
meaning commonly
applied to them in the pertinent art of plant and molecular biology. All of
the following term
definitions apply to the complete content of this disclosure.
The word "comprising" does not exclude other elements or steps, and the
indefinite article "a" or
"an" does not exclude a plurality.
The terms "essentially", "about", "approximately" and the like in connection
with an attribute or a
value particularly also define exactly the attribute or exactly the value,
respectively.
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The term "plant" refers to any plant or any part thereof at any stage of its
life cycle or development,
and its progenies. 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.
A "plant cell" refers to a structural and physiological unit of a plant. The
plant cell may be in the
form of a protoplast without a cell wall, an isolated single cell or a
cultured cell, or as a part of
higher organized unit such as but not limited to, plant tissue, a plant organ,
or a 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 plant or a
mixture of different plants and includes without limitation plant tissues,
leaf scraps, green leaf
scraps, stems, dust created during plant processing, and leaf prime lamina
strip and combinations
thereof. In certain embodiments, the plant material will comprise, consist or
consist essentially of a
plant part or a mixture of plant parts containing lignin ¨ such as plant
midribs or plant stems or
plant stalks or a combination or two or more thereof. Tobacco plant material
can have the form of
processed tobacco parts or pieces, uncured, cured or aged tobacco in
essentially natural lamina or
stem form, a tobacco extract or a mixture of the foregoing, for example, a
mixture that combines
extracted tobacco pulp with granulated cured and aged natural tobacco lamina.
The plant material
can be in solid form, in liquid form, in semi-solid form, in ground form, in
crushed form, in sieved
form, or in particulate form or the like or otherwise treated to reduce
particle size. The plant
material can be in the form of a homogenate that has been subjected to
homogenization, including,
but not limited to cutting or grinding or a combination thereof. The
homogenate may be prepared
from whole plants or from mixtures of plant components - such as a mixture of
plant parts
containing lignin, for example, midribs or stems or stalks or a combination of
two or more thereof -
that have been subjected to homogenisation. The plant material can be in the
form of a slurry,
including a suspension of plant material or a plant homogenate in an aqueous
solution or solvent.
The slurry can be a 5% (w/v), 10% (w/v), 15% (w/v), 20% (w/v) or 25% (w/v) or
more mixture of
plant material in an aqueous solution or solvent. In one embodiment, the plant
material is that
plant material which comprises, consists or consists essentially of lignin ¨
such as lignified tissue.
In one embodiment, the plant material is that plant material which comprises,
consists or consists
essentially of the vascular bundle. In one embodiment, the plant material is
that plant material
which comprises, consists or consists essentially of xylem. In one embodiment,
the plant material
is that plant material which comprises, consists or consists essentially of
lignified sclerenchymatic
tissue. In one embodiment, the plant material comprises, consists or consists
essentially of plant
midribs or plant stems or plant stalks or a combination of two or more
thereof. In one embodiment,
the plant material is tobacco plant material.
The term "tobacco product" includes smoking or smokable articles, and
smokeless tobacco
products.
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The term "free NNK" refers to the NNK concentration calculated from the NNK
content of extracts
prepared by extracting said plant material with aqueous buffer(s) at room
temperature. The free
NNK content in such extracts can be determined using ultra performance liquid
chromatography-
tandem mass spectrometry (UP LC-MS/MS).
The term "total NNK" refers to the NNK concentration calculated after
subjecting the extraction
mixtures to the methods described herein (for example, by heating to about 130
C for about 4
hours) and filtering aliquots of the extracts. The total NNK content in such
extracts can be
determined using UPLC-MS/MS.
The term "bound NNK" or "matrix-bound NNK" as used herein represents the
difference between
the "total NNK" and the "free NNK" concentration.
The terms "reduced lignin content" or "decreased lignin content" or "non-
lignified" grammatical
variations thereof refers to a measurable quantitative reduction in the amount
of lignin in a plant
when compared to the amount of lignin in a comparable control plant. A
quantitative reduction of
lignin can be readily ascertained by assays that are known in the art and
include the Klason lignin
assay (Method in Enzymol., 161:87-101 (1988)), the acetyl bromide assay (Wood
Sci. Technol.,
22:271-280 1988)) or the photometric method based on derivatisation with
thioglycolic acid (J.
Chem. Ecol., 28, 2483-2501 (2002)). In non-lignified tissue, the amount of
lignin is decreased as
compared to a comparable control plant and the amount of lignin can be
completely, substantially
or partially removed. In non-lignified tissue, a detectable amount of lignin
can be present provided
that there is a measurable quantitative reduction in the amount of lignin when
compared to a
comparable control plant 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 can contain less
than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the total dry weight content
of lignin in the
plant or plant part from which it is removed.
A "control plant" or "control plant cell" refers to a plant or a plant cell -
such as a native or naturally
occurring plant or plant cell - having a lignin content and/or a NNK content
that has not been
manipulated or modified. Control plant material includes plant material
obtained from, derived from
or derivable from the control plant or the control plant cell or a combination
thereof. The control
plant or control plant cell can be the same type of plant or plant cell, for
example, the same species
of plant or plant cell as the plant or plant cell that it is being compared
to. The control plant or
control plant cell may correspond to a wild-type plant or wild-type plant
cell.
The term "reduce" or "decrease" or grammatical variations thereof refers to a
reduction of from
about 10% to about 99%, or a reduction 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%, at least 90%, at
least 95%, at least 98%, at least 99%, or at least 100% or more of a quantity,
amount or activity.
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The term "inhibit" or grammatical variations thereof, refers to a reduction of
from about 98% to
about 100%, or a reduction of at least 98%, at least 99%, but particularly of
100%, of a quantity,
amount or activity.
The term "increase" or grammatical variations thereof refers 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 least 90%, at
least 95%, at least 98%, at least 99%, or at least 100% or more of a quantity,
amount or activity.
The term "about" in the context of a given numerate value or range refers to a
value or range that
is within 20%, within 10%, or within 5% of the given value or range.
The term "at least a portion" or grammatical variations thereof includes 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 about 99% of a quantity,
amount or activity.
DETAILED DESCRIPTION
Generally speaking, the present disclosure can be applied to any form of
tobacco plant material in
which NNK or metabolites thereof or a combination thereof can form or have
formed. Suitably, at
least a portion of the NNK is in the bound form. At least a portion of the
matrix-bound form co-
localises with lignin - such as lignified tissue. Methods for measuring free
nitrosamine(s) and
matrix-bound nitrosamine(s) are well known in the art and described herein.
Briefly, aliquots of
tobacco samples can be extracted and the nitrosamine content therein can be
analysed using ultra
performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).
Typically, one or
more standards corresponding to the one or more nitrosamines that are being
quantified will be
incorporated into the aliquots of the tobacco samples. The sample
concentration calculated from
the extract corresponds to the "free nitrosamine(s)" concentration in the
sample. After treating the
extraction mixtures to the methods described herein (for example, by heating
to about 130 C for
about 4 hours) nitrosamine concentrations can again be measured by UPLC-MS/MS.
From these
values, the "total NNK" concentration in the samples can be calculated. The
"matrix-bound NNK"
concentration is the difference between the "total NNK" and the "free NNK"
concentration.
Much research has been performed on tobacco, especially in relation to tobacco-
specific
nitrosamines. Freshly harvested tobacco leaves are referred to as "green
tobacco" and are
believed to contain no nitrosamines, but green tobacco is not suitable for
human consumption.
The process of curing green tobacco depends on the type of tobacco harvested.
For example,
Virginia flue (bright) tobacco is typically flue-cured, whereas Burley and
certain dark strains are
usually air-cured. The flue-curing of tobacco typically takes place over a
period of five to seven
days compared to one to two months for air-curing. Many major chemical and
biochemical
changes occur during the curing process and continue through the early phases
of leaf drying.
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The conversion of the tobacco from its yellow to brown colour generally
results in formation and
substantial accumulation of nitrosamines, including NNK, and an increased
microbial content. The
exact mechanism by which tobacco-specific nitrosamines, including NNK, are
formed is not clear,
but is believed to be enhanced by microbial activity, involving microbial
nitrate reductases in the
generation of nitrite during the curing process.
As described above, matrix-bound NNK has been found to co-localise with lignin
in tobacco plants
or in tobacco plant material. Lignin is the generic term for a large group of
aromatic polymers
resulting from the oxidative combinatorial coupling of 4-
hydroxyphenylpropanoids. These polymers
are deposited predominantly in the walls of secondarily thickened cells - such
as fibers and
tracheary elements - making them rigid and impervious. The mechanical rigidity
of lignin
strengthens these tissues so that the tracheary elements can endure the
negative pressure
generated from transpiration without collapse of the tissue. In addition to
providing mechanical
strength, lignin has protective functions. For example, the physical toughness
and chemical
durability of lignin may deter feeding by herbivores. Lignification is a
frequent response to infection
or wounding, which may provide a physical barrier to block the penetration of
pathogens. The
main building blocks of lignin are the hydroxycinnamyl alcohols (or
monolignols) coniferyl alcohol
and sinapyl alcohol, with typically minor amounts of p-coumaryl alcohol. The
monolignols are
synthesized from Phe through the general phenylpropanoid and monolignol-
specific pathways.
Phe is derived from the shikimate biosynthetic pathway in the plastid. Certain
enzymes of the lignin
biosynthetic pathway, namely the cytochrome P450 enzymes cinnamate 4-
hydroxylase (C4H), p-
coumar ate 3-hydroxylase (C3H), and ferulate 5-hydroxylase (F5H), are membrane
proteins
thought to be active at the cytosolic side of the endoplasmic reticulum.
Although metabolic
channeling has been shown between phenylalanine ammonia-lyase (PAL) and C4H,
it remains
unknown whether the other pathway enzymes are also part of metabolic complexes
at the
endoplasmic reticulum. The units resulting from the monolignols, when
incorporated into the lignin
polymer, are called guaiacyl (G), syringyl (S), and p-hydroxyphenyl (H) units.
Lignin is commonly found in, for example, plant midribs, plant stems or plant
stalks. Thus, the
material for use in the present disclosure can include plant midribs or plant
stems or plant stalks or
a mixture thereof or a combination of two or more thereof and can be removed.
Lignin is located
in, for example, the vascular bundle of a tobacco plant which can be found in
plant midribs, plant
stems, plant stalks and the like. The vascular bundle is composed of a
plurality of relatively hard,
cellulose members closely secured together by fibrous vegetable connecting
tissue. Surrounding
this fibro-vascular bundle is the cortex which is formed of a relatively
sponge-like vegetable tissue
or covering constituting the larger portion of the stem and the portion which
is closer in
characteristics and properties to the lamina of the tobacco leaf. Lignin is
generally located in the
vascular bundle. The vascular bundle is a part of the transport system in
vascular plants. The
transport itself happens in vascular tissue, which exists in two forms, the
xylem and phloem. Both
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these tissues are present in a vascular bundle, which in addition will include
supporting and
protective tissues. Of these vascular tissues, lignin is to be found only in
the xylem.
The amount of lignin can be substantially reduced in plant midribs, plant
stems or plant stalks and
the like or a mixture or a combination of two or more thereof. Lignin can be
substantially removed
from plant midribs, plant stems or plant stalks and the like or a mixture or a
combination of two or
more thereof. Suitably, the vascular bundle or the xylem or a combination
thereof is substantially
reduced in plant midribs, plant stems or plant stalks and the like or a
mixture or a combination of
two or more thereof. Suitably, the vascular bundle or the xylem or a
combination thereof is
substantially removed from plant midribs, plant stems or plant stalks and the
like or a mixture or a
combination of two or more thereof. Suitably, lignified sclerenchymatic tissue
is substantially
removed from plant midribs, plant stems or plant stalks and the like or a
mixture or a combination
of two or more thereof.
It is an advantage that matrix-bound NNK co-localises with lignin since lignin
can be readily
separated from other parts of the plant or other plant tissues. In one
embodiment, the amount of
lignin is reduced by separating lignified from non-lignified tissue. For
example, the outer cortex can
be readily separated from the vascular bundle containing lignin, thus
obtaining plant material with
reduced levels of lignin. The plant material containing reduced levels of
lignin or substantially 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 co-localised with matrix-bound NNK can be discarded or used in other
processes.
In one aspect, there is provided a method of reducing or decreasing the amount
of matrix-bound
NNK in a cured tobacco plant or in cured tobacco plant material comprising
reducing the amount of
lignin therein. According to this method, a cured tobacco plant or cured
tobacco plant material is
provided. The amount of lignin in the cured tobacco plant or the cured tobacco
plant material is
reduced. The lignin can be completely removed or partially removed. A cured
tobacco plant or
cured tobacco plant material is then obtained in which the amount of lignin is
reduced and the
amount of matrix-bound NNK is also reduced as compared to the cured tobacco
plant or the cured
tobacco plant material initially provided or as compared to a control.
The lignin to be completely or partially removed can be located in the
vascular bundle. The lignin
to be completely or partially removed can be located exclusively in the
vascular bundle. The lignin
to be completely or partially removed can be located exclusively in the
vascular bundle and not the
surrounding tissue. Thus, the amount of matrix-bound NNK in a cured tobacco
plant or in cured
tobacco plant material is reduced by reducing the amount of the vascular
bundle in the cured
tobacco plant or in the cured tobacco plant material.
The lignin to be completely or partially removed can be located in the xylem.
The lignin to be
completely or partially removed can be located exclusively in the xylem. The
lignin to be
completely or partially removed can be located exclusively in the xylem and
not the surrounding
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tissue. Thus, the amount of matrix-bound NNK in a cured tobacco plant or in
cured tobacco plant
material is reduced by reducing the amount of xylem in the cured tobacco plant
or in the cured
tobacco plant material.
The lignin to be completely or partially removed can be located in lignified
sclerenchymatic tissue.
The lignin to be completely or partially removed can be located exclusively in
lignified
sclerenchymatic tissue. The lignin to be completely or partially removed can
be located exclusively
in the lignified sclerenchymatic tissue and not the surrounding tissue ¨ such
as the outer layer of
midribs. Thus, the amount of matrix-bound NNK in a cured tobacco plant or in
cured tobacco plant
material is reduced by reducing the amount of the lignified sclerenchymatic
tissue in the cured
tobacco plant or in the cured tobacco plant material.
In another aspect, there is provided a method of reducing, decreasing,
preventing or inhibiting the
formation of matrix-bound NNK during the curing of a tobacco plant or tobacco
plant material
comprising reducing the amount of lignin therein prior to curing. At least
initially, the tobacco plant
or tobacco plant material can be uncured or substantially uncured. The method
can be used to
reduce, decrease, prevent or inhibit the co-localisation of NNK with lignin
that would otherwise
occur during the subsequent curing process. According to this aspect, an
uncured tobacco plant or
uncured tobacco plant material or substantially uncured tobacco plant or
substantially uncured
tobacco plant material is provided and the amount of lignin therein is reduced
prior to curing or
during curing. The lignin can be completely removed or partially removed. The
lignin can be
located in the vascular bundle. The lignin can be located exclusively in the
vascular bundle. The
lignin can be located exclusively in the vascular bundle and not the
surrounding tissue. Thus, the
vascular bundle can be completely removed or partially removed. The lignin can
be located in the
xylem. The lignin can be located exclusively in the xylem. The lignin can be
located exclusively in
the xylem and not the surrounding tissue. Thus, the xylem can be completely
removed or partially
removed. The lignin can be located in the lignified sclerenchymatic tissue.
The lignin can be
located exclusively in the lignified sclerenchymatic tissue. The lignin can be
located exclusively in
the lignified sclerenchymatic tissue and not the surrounding tissue ¨ such as
the outer layer of
midribs. Thus, the lignified sclerenchymatic tissue can be completely removed
or partially
removed. The lignin can be completely removed or partially removed.
After subjecting the
tobacco plant or the tobacco plant material to curing, using methods that are
well known in the art,
a cured tobacco plant or cured tobacco plant material in which the amount of
matrix-bound NNK
and the amount of lignin is reduced as compared to the starting material or as
compared to a
control can be obtained.
The amount of lignin in a tobacco plant or in tobacco plant material can be
reduced using various
methods that are well known in the art. In one method, a batch of stems or the
like can be
moistened or soaked in fluid, for example, water, which causes the cortex to
soften and expand or
swell whilst leaving the lignin in an unexpanded state. The cortex can be
removed manually (eg.
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by hand) and retained and the vascular bundle containing lignin can be
discarded or used
elsewhere. Accordingly, the cortex (for example, the outer cortex) can be
separated from lignified
tissue and retained and the unexpanded lignified tissue discarded. The cortex
can then be used
for further tobacco processing. There is also disclosed plant cortex (for
example, plant outer
cortex) or expanded plant cortex (for example, the expanded plant outer
cortex) in which the
amount of matrix-bound NNK is below detectable levels. Accordingly, non-
lignified plant tissue can
be expanded in order to separate lignified and non-lignified tissue. Suitably,
the non-lignified tissue
is selectively or preferentially expanded over the lignified tissue.
By way of further example, lignin can be separated using suitable
decorticating machinery. A
decorticator is a machine for stripping the bark, wood and plant stalks and
the like.
In another method, mechanical separation can be used. For example, soaking of
the batch of
stems or the like is followed by freeze drying. In another example, soaking of
the batch of stems or
the like is followed by freeze drying, grinding and sieving. Suitably, tobacco
plant material can be
ground into a powder form using equipment and techniques for grinding,
milling, or the like.
Suitably, the tobacco plant material is relatively dry in form during grinding
or milling, using various
equipment - such as hammer mills, cutter heads, air control mills and the
like.
The plant material can be reduced in size to form particles or particulate
matter using various
methods that are known in the art. The particles or particulate matter can be
separated by size to
obtain fractions with reduced levels of lignin and reduced levels of matrix-
bound NNK. In one
suitable method, plant material is treated by impact ¨ such as by impact with
one or more objects
that are harder than the plant material to be treated. In one embodiment,
impact with metal ¨ such
as metal balls - is used. The impact can be delivered using various methods,
such as 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. The particles or particulate matter can be
separated by size
using a sieve shaker into fractions of different particle size(s). Suitably,
the average particle is
greater than about 0.5 millimetres, greater than about 0.85 millimetres or
greater than about 1
millimetre. These size fractions can have reduced levels of lignin and reduced
levels of matrix-
bound NNK.
The plant material can be ground or milled when the moisture content thereof
is less than about 15
weight percent to less than about 5 weight percent. The tobacco plant material
can be finely
ground. Finely ground tobacco material typically has a particle size of from
about 30 to 600
microns.
In one embodiment, the method comprises expansion, for example by contacting
with a fluid (eg.
by water soaking), followed by freeze drying, which will result in the
expansion of plant material -
which does not contain lignin or contains only low levels of lignin. Lignified
plant material will retain
a higher density, higher physical strength and a smaller particle size than
the expanded plant
tissue thereby permitting separation by size. In another embodiment, the
method comprises
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expansion, followed by freeze drying, followed by grinding (for example, by
impact as discussed
above), followed by sorting (for example, sorting by size) of the resulting
fragments and selection
of the fragments with reduced levels of lignin and reduced levels of matrix-
bound NNK. Sieving
may be used for this purpose. In another embodiment, the method comprises
expansion, followed
by grinding or crushing or a combination thereof, (for example, by impact, as
described above),
followed by sorting by size (using sieving or based upon density and/or
mechanical strength, for
example)the resulting particles and selection of the particles with reduced
levels of lignin and
reduced levels of matrix-bound NNK. The different sized fractions may also
differ in their free-NNK
content or their NNN content or their nicotine content or a combination of two
of more thereof.
Following the complete or partial removal of lignin, the plant material can
optionally be further
processed for use in a tobacco product. By way of example, this material can
be formed into an
aqueous slurry. The resulting slurry can contain a substantial proportion of
colloidal cortex
particles dispersed therein. The conversion of the tobacco cortex into an
aqueous slurry can be
accomplished using a suitable type of mill - such as a ball mill or a colloid
mill. The further
processing of the cortex and aqueous slurry is described herein.
In certain embodiments, the plant material obtained or obtainable by the
methods described herein
comprises, consists or consists essentially of tissue surrounding the vascular
bundle or the tissue
surrounding the xylem or the tissue surrounding the lignified sclerenchymatic
tissue or a
combination of two or more thereof with the vascular bundle or the xylem or
the lignified
sclerenchymatic tissue or a combination of two or more thereof substantially
absent. In certain
embodiments, the plant material comprises, consists or consists essentially of
the tissue
surrounding the vascular bundle or the xylem or lignified sclerenchymatic
tissue or a combination
of two or more thereof and substantially no vascular bundle or no xylem or no
lignified
sclerenchymatic tissue or a combination of two or more thereof. In certain
embodiments, the plant
material comprises, consists or consists essentially of the tissue surrounding
the vascular bundle
or the xylem or the lignified sclerenchymatic tissue or a combination of two
or more thereof and no
vascular bundle or no xylem or no lignified sclerenchymatic tissue or a
combination of two or more
thereof. In certain embodiments, the plant material comprises, consists or
consists essentially of
the outer tobacco cortex. In certain embodiments, the plant material
comprises, consists or
consists essentially of the outer layers of plant midribs.
The methods described herein may comprise one or more further steps of
measuring and
optionally comparing the levels of free NNK or matrix-bound 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 cured tobacco plant or in cured tobacco plant material. After reducing
the amount of lignin in
the cured tobacco plant or the cured tobacco plant material the amount of free
NNK or matrix-
bound NNK or a combination thereof can be measured again. The level of at
least matrix-bound
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NNK can be compared with the initial starting material to ascertain if the
level of matrix-bound NNK
has been reduced. In this step, the level of at least matrix-bound NNK
previously measured can
be compared with the level of NNK measured following the reduction of the
amount of lignin. A
reduction in the amount of matrix-bound NNK in the tobacco material obtained
following the
reduction of the amount of lignin as compared to the tobacco material
initially provided is indicative
that the amount of matrix-bound NNK has been reduced.
The methods described herein may comprise one or more further steps of
measuring and
optionally comparing the levels of free NNK or matrix-bound NNK or
combinations thereof. In one
embodiment, the amount of free NNK or matrix-bound NNK or a combination
thereof is measured
in the uncured tobacco plant or uncured tobacco plant material. Optionally,
after reducing the
amount of lignin in the uncured tobacco plant or the uncured tobacco plant
material prior to curing
the amount of free NNK or matrix-bound NNK or a combination thereof can be
measured again.
The method may include one or more comparison steps. By way of example, the
method may
comprise the further step of comparing the level of at least matrix-bound NNK
initially measured,
as discussed above, with the level of NNK later measured, wherein a reduction
in the amount of
matrix-bound NNK in the tobacco material as compared to the tobacco material
initially provided is
indicative that the amount of matrix-bound NNK in the tobacco material is
reduced.
Free NNK or matrix-bound NNK or a combination thereof can be measured at the
start of the
method and/or at the end of the method and/or during the method. Free NNK or
matrix-bound
NNK or a combination thereof may be measured intermittently or at intervals.
The intervals may be
fixed intervals or random intervals. Free NNK or matrix-bound NNK or a
combination thereof can
measured at the end of the method to check that the free NNK or matrix-bound
NNK or a
combination thereof is present within a desired amount, concentration or
range.
Lignin can be covalently or non-covalently bound to lignin. A complex
comprising lignin covalently
or non-covalently bound to NNK is described. A plant cell, plant tissue or
plant or plant material
comprising the complex is also disclosed. A method for reducing the amount of
matrix-bound NNK
in a cured tobacco plant or in cured tobacco plant material is also described
comprising reducing
the amount of the complex therein.
In a further aspect, there is provided cured plant tissue containing a reduced
level of lignin, as
compared to control plant tissue in which the amount of lignin has not been
reduced, and wherein
the amount of matrix-bound NNK is about 3500 ng/g or less. The amount of
matrix-bound NNK
can 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 about 500 ng/g or
less. Suitably, the
average particle size of this cured plant tissue can be greater than about 0.5
millimetres, greater
than about 0.85 millimetres or greater than about 1 millimetre. Suitably, the
amount of free NNK in
this cured plant tissue can 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
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less. Suitably, the amount of NNN in this cured plant tissue can 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. Suitably, the amount of nicotine in this cured plant
tissue can be about
2600 pg or less, about 2300 pg or less or about 2100 pg or less. Suitably, the
amount of lignin in
this cured plant tissue can be about 6.5 % or less of the total dry weight
content of the cured plant
tissue, about 6 % of the total dry weight content of the cured plant tissue,
about 5 % of the total dry
weight content of the cured plant tissue, about 4 % of the total dry weight
content of the cured plant
tissue or about 3 % of the total dry weight content of the cured plant tissue.
In one embodiment, there is provided cured plant tissue containing a reduced
level of lignin, as
compared to control plant tissue in which the amount of lignin has not been
reduced, and wherein
the amount of matrix-bound NNK is about 3500 ng/g or less and the average
particle size is about
0.5 mm or greater. 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 cured plant tissue
is about 6.4 % or less of the total dry weight content of the cured plant
tissue. Suitably, the amount
of nicotine is about 2600 pg or less.
In another embodiment, there is provided cured plant tissue containing a
reduced level of lignin, as
compared to control plant tissue in which the amount of lignin has not been
reduced, and wherein
the amount of matrix-bound NNK is about 1900 ng/g or less and the average
particle size is
between about 0.85 mm and 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
cured plant tissue is about 4.4 % or less of the total dry weight content of
the cured plant tissue.
Suitably, the amount of nicotine is about 2300 pg or less.
In another embodiment, there is provided cured plant tissue containing a
reduced level of lignin, as
compared to control plant tissue in which the amount of lignin has not been
reduced, and wherein
the amount of matrix-bound NNK is about 1600 ng/g or less and the average
particle size is
greater than about 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 cured plant
tissue is about 3 % or less of the total dry weight content of the cured plant
tissue. Suitably, the
amount of nicotine is about 2100 pg or less.
The tobacco plant or the tobacco plant material that is used at the start of
the method(s) described
herein can comprise or consist or consist essentially of uncured tobacco plant
or uncured tobacco
plant material or cured tobacco plant or cured tobacco plant material.
Processes of curing tobacco
¨ such as tobacco leaves, especially, green tobacco leaves are well known to
those skilled in the
art and include without limitation air-curing, fire-curing, flue-curing and
sun-curing. The process of
curing tobacco depends on the type of tobacco harvested. For example, Virginia
flue (bright)
tobacco is typically flue-cured, Burley and certain dark strains are usually
air-cured, and pipe
tobacco, chewing tobacco, and snuff are usually fire-cured. Although tobacco
plants or tobacco
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plant material from any type of tobacco may be used, certain types of tobacco
are preferred.
Particularly preferred tobacco materials are selected from the group
consisting of: flue-Cured,
Turkish, Burley, Virginia, Maryland, Oriental, or any combination of two or
more thereof. The
shape of the tobacco material is in general not limited. It can be in the form
of homogenised
tobacco material. Tobacco homogenates - such as but not limited to cured
tobacco homogenates -
may be prepared from tobacco material using various methods known in the art,
for example, the
tobacco may be in a shredded, granulated, ground or powder form. In certain
embodiments, it is
desirable not to begin with tobacco material in the ground or powder form
since certain mechanical
separation methods that can be used to separate lignin can require grinding
and/or sieving steps.
The tobacco material used or obtained may comprise additives that include, but
are not limited to,
one or more of the following components as well as combinations thereof:
flavourants, organic and
inorganic fillers (for example, grains, processed grains, puffed grains,
maltodextrin, dextrose,
calcium carbonate, calcium phosphate, corn starch, lactose, manitol, xylitol,
sorbitol, finely divided
cellulose, and the like), binders (for example, povidone, sodium
carboxymethylcellulose and other
modified cellulosic types of binders, sodium alginate, xanthan gum, starch-
based binders, gum
arabic, lecithin, and the like), colorants (for example, dyes and pigments,
including caramel
colouring and titanium dioxide, and the like), humectants (for example,
glycerin, propylene glycol,
and the like), oral care additives, preservatives (for example, potassium
sorbate, and the like),
syrups (for example, honey, high fructose corn syrup, and the like used as
flavourants), and
disintegration aids (for example, microcrystalline cellulose, croscarmellose
sodium, crospovidone,
sodium starch glycolate, pregelatinized corn starch, and the like). Such
additives are known to
those having skill in the art and may be present in amounts and in forms known
in the art.
The tobacco can be formed into reconstituted tobacco. Thus, in one embodiment,
the methods
described herein can be used in the preparation of reconstituted tobacco, such
as reconstituted
tobacco (leaf) sheets. These sheets are paper-like material that can be made
from recycled
tobacco fines, tobacco stems and "class tobacco", which consists of tobacco
particles generally
less than 30 mesh in size that are collected at any stage of tobacco
processing. The reconstituted
tobacco can be made by extracting the soluble chemicals in the tobacco by-
products, processing
the leftover tobacco fibers from the extraction into a paper, and then
reapplying the extracted
materials in concentrated form onto the paper. Reconstituted tobacco can
generally be formed in a
variety of ways. For instance, in one embodiment, band casting can be utilised
to form the
reconstituted tobacco. Band casting typically employs a slurry of finely
divided tobacco parts and a
binder that is coated onto a steel band and then dried. After drying, the
sheet is blended with
natural tobacco strips or shredded and used in various tobacco products,
including as a cigarette
filler. Some examples of processes for producing reconstituted tobacco are
described in US
3,353,541, US 3,420,241, US 3,386,449, US 3,760,815 and 4,674,519.
Reconstituted tobacco can
also be formed by a papermaking process. Some examples of processes for
forming reconstituted
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tobacco according to this process are described in US 3,428,053, US 3,415,253,
US 3,561,451,
US 3,467,109, US 3,483,874, US 3,860,012, US 3,847,164, US 4,182,349, US
5,715,844, US
5,724,998; and US 5,765,570. For example, the formation of reconstituted
tobacco using
papermaking techniques can involve the steps of mixing tobacco with water,
extracting the soluble
ingredients therefrom, concentrating the soluble ingredients, refining the
tobacco, forming a web,
reapplying the concentrated soluble ingredients, drying, and threshing.
Various ingredients - such
as flavour or colour treatments - can be applied to the web.
The tobacco obtained or obtainable by the methods described herein may be
formed into a
tobacco sheet - such as a reconstituted tobacco sheet. According to this
embodiment, the method
may comprise the steps of: (a) obtaining tobacco material ¨ such as a tobacco
homogenate ¨
according to the methods described herein; (b) preparing a slurry of tobacco
homogenate; (c)
casting the slurry of the tobacco homogenate; and (d) drying the slurry of the
tobacco homogenate
to form a reconstituted tobacco sheet. According to another embodiment, the
method may
comprise the steps of: (a) obtaining tobacco material ¨ such as a tobacco
homogenate ¨ according
to the methods described herein and preparing a tobacco slurry; (b) casting
the slurry of the
tobacco homogenate; and (c) drying the slurry of the tobacco homogenate to
form a tobacco sheet.
The step of casting the slurry of the tobacco homogenate may be performed
using any of the
casting or paper making processes that are known in the art. By way of
example, casting
processes are described in US 5,724,998 and US 5,584,306; paper-making
processes are
described in US 4,341,228; US 5,584,306 and US 6,216,706. Casting processes
typically include
casting the slurry onto a continuous stainless steel belt, drying the cast
slurry to form a
reconstituted tobacco sheet and removing said sheet. Paper-making processes
typically include
casting the aqueous slurry from a head box onto a wire screen for forming the
desired sheet. The
aqueous slurry may be separated into a soluble portion and a fibrous portion.
Water is drained
from the fibrous portion and a sheet is so-formed is subsequently treated and
dried.
The tobacco slurries may further comprise one or more binders - such as gums
and pectins. As
described above, tobacco slurries that are used to prepare reconstituted
tobacco sheets may
further comprise common additives that include, but are not limited to, one or
more of the following
components as well as combinations of these: wood cellulose fibers, aerosol
formers, sugars, and
flavourants and binders. Additives of the list described above are known to
those having skill in the
art and may be present in these aqueous slurries in amounts and in forms known
in the art.
Once prepared, the reconstituted tobacco sheets described herein may be cut in
a similar fashion
as whole leaf tobacco to produce tobacco filler suitable for cigarettes and
other tobacco products.
The reconstituted tobacco sheets described herein may be further trashed or
flayed with
mechanical fingers into sized pieces similar to natural tobacco lamina strips
or cut into diamond
shaped pieces, between about 50 to 100 mm on a side. The reconstituted tobacco
sheet pieces
described herein may be further blended with other tobaccos such as flue-cured
tobacco, Burley
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WO 2015/091880 PCT/EP2014/078607
tobacco, Maryland tobacco, Oriental tobacco, rare tobacco, specialty tobacco,
expanded tobacco
and the like. The precise amount of each type of tobacco within a tobacco
blend used for the
manufacture of a particular cigarette brand varies from brand to brand. See,
for example, Tobacco
Encyclopaedia, Voges (Ed.) p. 44-45 (1984), Browne, The Design of Cigarettes,
3rd Ed., p.43
(1990) and Tobacco Production, Chemistry and Technology, Davis et al. (Eds.)
p. 346 (1999). The
entire blend may then be shredded into a cut filler and incorporated into a
tobacco product.
According to a further aspect, there is provided a method for blending tobacco
in which at least two
different types of tobacco are blended so as to form a tobacco blend. The
various tobacco blends
have different recipes for blending different tobacco types. Tobacco types can
be, by way of
example, Burley, Flue Cured, Oriental, Bright and Reconstituted tobacco.
Burley, Flue Cured and
Oriental tobacco are specific types of tobacco, while Bright tobacco is a pre-
blend of Flue Cured
and Oriental tobacco. According to the method, a first (type of) cured tobacco
plant material is
provided and the amount of lignin therein is reduced. Any of the methods
described herein can be
used to reduce the amount of lignin. The total and/or matrix-bound NNK content
of the first cured
tobacco plant material can be measured and cured tobacco plant material in
which the total and/or
matrix-bound NNK content is reduced as compared to first cured tobacco plant
material initially
provided can be selected for further use. A second cured tobacco plant
material which has a
higher total and/or matrix-bound NNK content than the total and/or matrix-
bound NNK of the first
cured tobacco plant material is next provided. In some embodiments, the total
and/or matrix-
bound NNK content of this material may already be known so measurement of
these values will
not be required. In other embodiments, the total and/or matrix-bound NNK
content of this material
may not be known and so measurement will be required. Thus, measuring the
total and/or matrix-
bound NNK content in the second cured tobacco plant material is an optional
step in this method.
The first and second cured tobacco plant materials obtained from these steps
can be blended
together using processes that are well known in the art. Optionally, the total
and/or matrix-bound
NNK content in the final blended tobacco plant material can be measured.
According to this
method, a blended tobacco plant material can be obtained in which the total
and/or matrix-bound
NNK content of the final blended tobacco plant material is lower than the
second cured tobacco
plant material. Advantageously, this method can be used to provide a blend of
tobacco material in
which the overall NNK content of the blend is reduced. Essentially, the
tobacco material in which
the amount of lignin therein has been reduced is used to dilute or reduce the
overall NNK content
in the blended tobacco material.
The tobacco material obtained or obtainable according to this disclosure can
also be used in
tobacco cut filler and in a smoking article formed from a tobacco rod of the
cut filler.
Conventionally, cut filler tobacco products for smoking articles are formed
predominantly from the
lamina portion of the tobacco leaf, which is separated from the stem portion
of the leaf during a
threshing process. Much of the stem portion that remains after the lamina has
been removed and
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separated is not used. In order to increase the amount of the tobacco material
that can be used
commercially, some tobacco stems can be added back into the cut filler
together with the lamina.
In order to improve the taste and burning characteristics of the tobacco stem
for use in the cut filler,
the stems are often first subjected to one or more treatment procedures, which
can include the
procedures described herein. The rolling step can be carried out on tobacco
stems that have been
subjected to the method of the present disclosure. The stems can be rolled to
a desired thickness
¨ such as a mean thickness of about 0.6 mm to 0.8 mm. During subsequent
processing and
storage steps, the stems can expand to a final thickness of about 0.8 mm to
about 1.0 mm. After
rolling, the stems are dried and transferred to the tobacco production plant,
where they are cut and
added to the tobacco cut filler. In some cases, the rolling step may
alternatively be incorporated as
part of the on-line production process for cut filler. Typically the moisture
content of the tobacco
stems is about 28 % to about 34 % oven volatiles prior to rolling in order to
prevent damage to the
structure of the stems. If necessary, the tobacco stems can be conditioned
prior to rolling in order
to increase the moisture content to this level. Known processes for
conditioning tobacco stems
involve contacting the stems with water, steam or a mixture of water and
steam. In methods where
the rolling step is incorporated on-line and dried stems are used, the
conditioning step will typically
take longer and may require a soaking step in which the stems are soaked in
water for a number of
hours prior to rolling. The tobacco stems can be rolled using a one-step
rolling process to reduce
the thickness of the stems to the desired mean thickness. After rolling, the
stems can be cut to a
cut width of between 0.1 mm and 0.2 mm. The cut rolled stems are then
optionally expanded using
known stem expansion techniques, and then dried. Where the stems are pre-
rolled and dried, it
will typically be necessary to condition the stems prior to cutting in order
to increase the moisture
content of the tobacco stems back to between 28 % and 34 % oven volatiles.
This increases the
pliability of the tobacco stems in order to limit damage or breakage of the
stems during cutting.
Finally, the cut rolled stems are combined with tobacco cut lamina and any
additional tobacco
materials in order to form cut filler having at least 5 % by weight of the cut
rolled tobacco stems.
Thus, in a further aspect, there is provided a method for preparing tobacco
for use as a tobacco cut
filler comprising the steps of: (a) performing the method(s) as described
herein; and (b) rolling and
cutting the tobacco material for use as a tobacco cut filler. There is also
described a method of
treating tobacco material ¨ such as tobacco stems - for use in tobacco cut
filler, the method
comprising the steps of: (a) performing the method as described herein; (b)
rolling the tobacco
material; (c) cutting the rolled tobacco material; and (d) optionally drying
the cut rolled stems. The
rolled tobacco stems can be combined with tobacco lamina such that the steps
are carried out on
the combined tobacco stems and lamina. The cutting step can comprise cutting
the rolled stems to
a cut width of between about 0.3 mm and 1.3 mm. The method can comprise the
steps of:
removing stems from the tobacco leaf; cutting the stems to an average length
of between about 15
mm and 80 mm; and rolling the stems to a thickness of between 0.1 mm and 0.5
mm. A method of
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producing cut filler comprising rolled tobacco stems is also provided, the
method comprising:
treating tobacco stems using the method described herein; and blending the
treated stems with at
least one type of tobacco lamina, expanded tobacco or reconstituted tobacco to
produce cut filler.
The tobacco cut filler obtained or obtainable by this method can comprise at
least 60 %, and
preferably at least 80 % by weight tobacco lamina having a mean cut width
between 0.8 mm and
1.1 mm, suitably, about 0.9 mm, and a mean thickness of about 0.2 mm. The
tobacco cut filler can
comprise up to 95 % by weight tobacco lamina with a mean cut width between
about 0.8 mm and
1.1 mm, more suitably about 0.9 mm, and a mean thickness of about 0.2 mm. The
particles of
tobacco lamina in the cut filler are therefore of similar dimensions to the
particles of tobacco stem.
As such, the tobacco stems are not visually distinct from the tobacco lamina,
even at a high
inclusion rate. In addition, the blend of tobacco stems and lamina can
advantageously be
transported and processed effectively without significant settling of the
stems. Suitably, the mean
cut width of the cut rolled tobacco stems is within about 0.1 mm, more
suitably within about 0.05
mm of the mean thickness of the tobacco lamina in the cut filler. Cut fillers
may be incorporated
into a variety of smoking articles. For example, the cut filler may be used in
the tobacco rod of a
combustible smoking article, such as a filter cigarette, cigarillo or cigar.
Alternatively, the cut filler
may be used to provide the tobacco aerosol generating substrate in a
distillation based smoking
article, or an electrically heated smoking system. Alternatively, the cut
filler may be used as a roll-
your-own product, or loose tobacco product for example, for use in a pipe.
The tobacco material can be incorporated into various consumable products -
such as tobacco
products. Also encompassed are methods for making such tobacco products.
Tobacco products
include without limitation smoking articles or smokable articles and smokeless
tobacco products,
including non-combustible products, heated products, and aerosol-generating
products. Non-
limiting examples of smoking or smokable articles include cigarettes,
cigarillos, cigars and pipe
tobaccos. Non-limiting examples of smokeless tobacco products include chewing
tobaccos, snuffs,
and substrates for use in aerosol-generating products. Smokeless tobacco
products may comprise
tobacco in any form, including as dried particles, shreds, granules, powders,
or a slurry, deposited
on, mixed in, surrounded by, or otherwise combined with other ingredients in
any format, such as
flakes, films, tabs, foams, or beads. Liquid contents of smokeless tobacco
products can be
contained in a device or enclosed in a form, such as beads, to preclude
interaction with a water-
soluble wrapper. The wrapper may be shaped as a pouch to partially or
completely enclose
tobacco-incorporating compositions, or to function as an adhesive to hold
together a plurality of
tabs, beads, or flakes of tobacco. Exemplary materials for constructing a
wrapper include film
compositions comprising HPMC, CMC, pectin, alginates, pullulan, and other
commercially viable,
edible film-forming polymers. Other wrapping materials may include pre-formed
capsules produced
from gelatin, HPMC, starch/carrageenan, or other commercially available
materials. Such wrapping
materials may include tobacco as an ingredient. Wrappers that are not orally
disintegrable may be
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composed of woven or nonwoven fabrics, of coated or uncoated paper, or of
perforated or
otherwise porous plastic films. Wrappers may incorporate flavouring or
colouring agents.
Smokeless products can be assembled together with a wrapper utilizing any
method known to
persons skilled in the art of commercial packaging, including methods such as
blister packing, in
which a small package can be formed by a vertical form/fill/seal packaging
machine.
The amount of matrix-bound NNK in these smokable articles, smokeless products
and aerosols
and the like 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
about 200% or 300% lower - when compared to consumable products derived from
control tobacco
plant material. The amount of free-NNK may be substantially unchanged.
The amount of matrix-bound NNK in these smokable articles, smokeless products
and aerosols
and the like 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 about
500 ng/g or less.
The amount of free NNK in these smokable articles and smokeless products and
the like 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.
The amount of NNN in these smokable articles, smokeless products and aerosols
and the like 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 smokable articles and smokeless products and
the like may be
about 2600 pg or less, about 2300 pg or less, or about 2100 pg or less, or
about 2000 pg or less
1900 pg or less, or about 1800 pg or less.
The amount of lignin in these smokable articles and smokeless products and the
like may about
6.5 % or less of the total dry weight content of the cured plant tissue, about
6 % of the total dry
weight content of the cured plant tissue, about 5 % of the total dry weight
content of the cured plant
tissue, about 4 % of the total dry weight content of the cured plant tissue or
about 3 % of the total
dry weight content of the cured plant tissue.
In one embodiment, the amount of matrix-bound NNK in these smokable articles
and smokeless
products and the like is about 3500 ng/g or less, the amount of free NNK is
about 300 ng/g or less,
the amount of NNN is about 1700 ng/g or less, the amount of lignin is about
6.4 % or less of the
total dry weight content of the cured plant tissue, and the amount of nicotine
is about 2600 pg or
less.
In one embodiment, the smokable articles or smokeless products and the like
comprise about 3500
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 in is about
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WO 2015/091880 PCT/EP2014/078607
6.4 % or less of the total dry weight content of the cured plant tissue.
Suitably, the amount of
nicotine is about 2600 pg or less. Suitably, the average particle size is
about 0.5 mm or greater.
In another embodiment, the smokable articles or smokeless products and the
like comprise about
1900 ng/g or less 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 cured
plant tissue is about 4.4 % or less of the total dry weight content of the
cured plant tissue. Suitably,
the amount of nicotine is about 2300 pg or less. Suitably, the average
particle size is between
about 0.85 mm and about 1 mm.
In another embodiment, the smokable articles or smokeless products and the
like comprise about
1600 ng/g or less 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 cured
plant tissue is about 3 % or less of the total dry weight content of the cured
plant tissue. Suitably,
the amount of nicotine is about 2100 pg or less. Suitably, the average
particle is greater than
about 1 mm.
The tobacco material can be derived from tobacco plants, which include plants
of the genus
Nicotiana, various species of Nicotiana, including N. rustica and N. tabacum.
The tobacco material
can be derived from varieties of Nicotiana species, commonly known as flue or
bright varieties,
Burley varieties, dark varieties and oriental/Turkish varieties. In some
embodiments, the tobacco
material is derived from a Burley, Virginia, flue-cured, air-cured, fire-
cured, Oriental, or a dark
tobacco plant. In some embodiments, the tobacco material is derived, 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 KT D#3 Hybrid 107, N. tabacum Bel-W3, N. tabacum 79-
615, N.
tabacum Samsun Holmes NN, F4 from cross N. tabacum BU21 x N. tabacum Hoja
Parado, line 97,
N. tabacum KTRDC#2 Hybrid 49, N. tabacum KTRDC#4 Hybrid 1 10, N. tabacum
Burley 21, N.
tabacum PM016, 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. tabacum 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 A544, N. tabacum Banket Al
, N.
tabacum Basma Drama B84/31 , N. tabacum Basma I Zichna ZP4/B, N. tabacum Basma
Xanthi
BX 2A, N. tabacum Batek, N. tabacum Besuki Jember, N. tabacum 0104, N. tabacum
Coker 319,
N. tabacum Coker 347, N. tabacum Criollo Misionero, N. tabacum PM092, N.
tabacum De!crest, N.
tabacum Djebel 81, N. tabacum DVH 405, N. tabacum Galpao Comum, N. tabacum
HBO4P, N.
tabacum Hicks Broadleaf, N. tabacum Kabakulak Elassona, N. tabacum PM102, N.
tabacum
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Kutsage El , 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 Russian, 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 is
disclosed, including N. rustica and N. tabacum (for example, LA B21, LN KY171,
TI 1406, Basma,
Galpao, Perique, Beinhart 1000-1, and Petico). Other species include N.
acaulis, N. acuminate, N.
acuminate var. multffiora, N. alata, N. amplexicaulis, N. arentsii, N.
benavidesii, N. benthamiana, N.
bigelo vii, 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.
longffiora, N. megalosiphon,
N. miersii, N. noctiflora, N. nudicaulis, N. obtusifolia, N. occidentalis, N.
occidentalis subsp.
hesperis, N. otophora, N. paniculata, N. pauciflora, N. petunioides, N.
plumbaginifolia, N.
quadrivalvis, N. raimondii, N. repanda, N. rosulata, N. rosulata subsp.
ingulba, N. rotundifolia, N.
setcheffii, N. simulans, N. solanifolia, N. spegazzinii, N. stocktonii, N.
suaveolens, N. sylvestris, N.
thyrsiflora, N. tomentosa, N. tomentosiformis, N. trigonophylla, N. umbra
tica, N. velutina, N.
wigandioides, and N. x sanderae.
The use of tobacco cultivars and elite tobacco cultivars is also contemplated
herein. Particularly
useful Nicotiana tabacum varieties include Burley type, dark type, flue-cured
type, and Oriental
type tobaccos. Non-limiting examples of varieties or cultivars are: BD 64, CC
101, CC 200, CC 27,
CC 301, 00 400, 00 500, CC 600, CC 700, 00 800, CC 900, Coker 176, Coker 319,
Coker 371
Gold, Coker 48, CD 263, DF911, DT 538 LC Galpao tobacco, 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, KY14, 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' Periq'e' tobacco, PVH03, PVH09, PVH19, 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,
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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 number 2 Hybrid 49,
Burley 21, KY 8959, KY 9, MD 609, PG 01, PG 04, P01, P02, P03, RG 11, RG 8, VA
509, AS44, Banket Al, Basma Drama B84/31, Basma I Zichna ZP4/B, Basma Xanthi
BX 2A,
Batek, Besuki Jember, 0104, Coker 347, Criollo Misionero, Delcrest, Djebel 81,
DVH 405,
Galpao Comum, HBO4P, Hicks Broadleaf, Kabakulak Elassona,
Kutsage El, 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-1070, TW136, Basma, TKF 4028, L8, TKF
2002,
GR141, Basma xanthi, GR149, GR153, Petit Havana. Low converter subvarieties of
the
above, even if not specifically identified herein, are also contemplated.
The following examples are provided as an illustration and not as a
limitation. Unless otherwise
indicated, the present invention employs conventional techniques and methods
of molecular
biology and plant biology.
EXAMPLES
Example 1
Method for analysis of free and matrix-bound NNK in tobacco
Aliquots of tobacco samples (for example, about 750 mg) are extracted with
about 30 mL of Tris-
HCI buffer (50 mM; pH 7.4) by shaking for about one hour at approximately room
temperature.
Internals standard (100 ng/mL NNK-d4) are added. Samples (0.4 mL) of the
extracts are filtered
using a 0.2 pM filter and the NNK content is analysed using ultra performance
liquid
chromatography-tandem mass spectrometry (UP LC-MS/MS).
The sample concentrations
calculated from these extract concentrations correspond to the "free NNK"
concentrations in the
sample. After treating the extraction mixtures (for example, by heating to
about 130 C for about 4
hours) and filtering aliquots of the extracts, NNK concentrations are again
measured by UPLC-
MS/MS. From these values, the "total NNK" concentration in the samples can be
calculated. The
"matrix-bound NNK" concentration is the difference between the "total NNK" and
the "free NNK"
concentrations.
An alternative method for "total-NNK" extraction comprises acidification of
the extraction mixtures
with concentrated HCI (for example, 3 mL of 37% HCI added to 30 mL) and
incubation for 48 hours
at 80 C. The acidic extracts are neutralised before filtration and UPLC
analysis by adding NaOH
solution (6N, 40 pL) and magnesium hydroxide suspension (10%; 40 pL) to 320 pL
of extract.
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Example 2
UPLC analysis
The column used is Waters Acquity BEH 018, 1.7 pm, 2.1 x 50 mm. The eluents
used are: (A)
ammonium bicarbonate (10mM; adjusted to pH 9.8 with ammonia) + 2% (v/v)
acetonitrile; (B)
acetonitrile. The gradient used is 0 min ¨ 5 % B; 0.5 min ¨ 5% B; 3.3 min ¨
18.3 % B. The flow
that is used is 0.5 mL/min. The column temperature that is used is 50 C.
Example 3
MS/MS methodology
This analysis is carried out on a Waters TQ spectrometer using the following
MRM transitions:
NNK: 208.2 4 122.2; dwell time 100 ms; NNK-d4: 212.2 4 126.2; dwell time 100
ms; Capillary
voltage: 0.6 kV; Cone voltage: 25 V; Collision energy: 11 eV; Source
temperature: 120 C;
Desolvation temperature: 400 C; Desolvation gas flow: 800 L/h.
Example 4
Distribution of matrix-bound NNK in lignified and non-lignified tissues of
Burley stems
About 2 grams of midribs of cured Burley tobacco leaves are separated by hand
into inner lignified
tissue (36 % of total dry weight) and outer non-lignified tissue (64 % of
total dry weight). In each of
these samples the concentration of free NNK and total NNK is analysed by UPLC-
MS, as
described above. Matrix-bound NNK is calculated as the difference between free
NNK and total
NNK concentration. Lignin content is quantified using a photometric method
based on
derivatisation with thioglycolic acid (see Brinkmann etal. (2002) J. Chem.
Ecol., 28, 2483-2501).
The results in Figure 1 show the distribution of free NNK, matrix-bound NNK
and lignin in lignified
(L) and non-lignified (NL) tissues of cured Burley stems. Figure 2 is a cross-
section of a hydrated
cured Burley stem showing lignified (L) and non-lignified (NL) tissues.
Lignified tissue is stained
red with phloroglucinol. The results in Figure 3 shows the matrix-bound NNK
content of lignified
and non-lignified tissues of green midribs after nitrosating with sodium
nitrite solution.
These results show that matrix-bound NNK is distributed principally in
lignified tissues of Burley
tobacco stems and midribs.
Example 5
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Enrichment of a fraction with low bound-NNK content from Burley Stem by freeze-
drying, grinding
and size separation
A sample of Burley Stems (52 g) is humidified with water (350 mL) and freeze-
dried. A part of the
resulting material (12 g) is ground by shaking with steel balls (2 balls,
diameter 2 cm; 300 rpm; 15
min) and separated with a sieve shaker into fractions of different particle
size ranging from greater
than 1 mm to less than 0.25 mm.
Figure 4 and Table 1 show the free- and matrix-bound NNK, NNN, lignin and
nicotine levels in
sieving fractions of ground freeze-dried Burley stems. The analysis of free-
and matrix-bound NNK
in Figure 5 and Table 1 indicates that both the lignin content (as % dry
weight of each fraction) and
the matrix-bound NNK content is decreased in the fractions with particle size
of greater than 0.5
mm, including fractions with particle size of 0.5 mm to 0.85 mm, 0.85 mm to 1
mm and greater than
1 mm. Figure 5 shows that lignin content correlates well with matrix-bound NNK
thereby
confirming the co-localisation of lignin and matrix-bound NNK.
Example 6
Localization of matrix-bound NNK precursor in green TN90 midribs and Burley
stems
The relative distribution of matrix-bound NNK is measured in sclerenchymatic
and non-
sclerenchymatic tissue of TN90 midribs. The prediction of matrix-bound NNK
being bound to lignin
predicts a higher concentration of this precursor in the lignified
sclerenchymatic tissue. In a second
experiment the relative distribution of free and matrix-bound NNK in
sclerenchymatic and non-
sclerenchymatic tissue of (cured) Burley stems is investigated.
Materials & Methods
Green midribs
The midribs (only the proximal halves) of 15 mature TN90 leaves are manually
separated in
sclerenchymatic tissue (S) (the "center" of the midrib) and non-
sclerenchymatic tissue (NS). Both
are freeze-dried and finely ground. The water-insoluble fraction of the two
materials is determined
by extracting 1g, each, three times with 40 mL of methanol/water 1:3 (room
temperature for 1 hour)
and weighing the insoluble material (designated SW and NSW) after freeze-
drying. Pseudo-
oxynictoine (PON) and nicotine analysis in S and NS (n=5) is measured using
the follow methods.
Finely powdered plant material (-20 mg) is extracted by shaking at room
temperature for 45
minutes with methanol/water (4:1) containing PON-methyl-d3 as an internal
standard (200 ng/mL).
After filtration (0.2 pm) samples are subjected to LC-MS analysis using the
following conditions:
Column: Acquity UPLC BEH C18 column (1.7 pm, 50 x 2.1 mm; Waters); Column
temperature:
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50 C; Eluents: Aqueous ammonium bicarbonate adjusted to pH 9.8 using NH3 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 min ¨ 30 % B, 0.5 mL/min; MS detection: PON m/z 179.2 a m/z
106.1; PON-methyl-
d3 m/z 182.2 a m/z 106.1; UV detection: 260 nm. PON and nicotine are eluted
after 2.6 and
4.1 minutes, respectively. For the quantification of nicotine the peak area at
260 nm and external
calibration is used. In order to estimate the content of matrix-bound NNK
precursor in S, NS, SW
and NSW, aliquots (-20 mg; n=5) of these materials are nitrosated by
incubation in 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 water. Then, the centrifugation
sediment of each
nitrosated sample is taken up in 4 mL Tris-HCI buffer (50mM pH 7.5; with NNK-
d4 and NNN-d4 at
100 ng/mL), autoclaved (for 4 hours at 130 C) and analysed for NNK content
using the methods
described herein.
Cured Burley stems
A Burley stem sample (2g = four ¨5 cm pieces) is manually separated into the
outer (non-lignified)
tissue (CNS) and the inner (lignified) part (CS). Both samples are finely
ground in a mixer mill
(Retsch "Tissuelyzer" for 2.5 minutes, 50 s-1). This results in 1183 mg and
651 mg of CNS and CS
powder, respectively. Free NNK in the two samples is determined after
extraction of ¨50 mg
aliquots (n=5) with 1.5 mL Tris-buffer (+IS) for 1 hour at room temperature.
Total NNK is
determined after autoclave extraction (130 C for 4 hours) of ¨50 mg aliquots
(n=5) in 5 mL Tris-
buffer (+ IS).
Results
The results of this experiment are shown in Table 2 and in Figure 6. After
nitrosation and washing,
a 7-fold higher concentration of matrix-bound NNK is found in the
sclerenchymatic tissue (S)
compared to the outer layers of the midribs (NS). PON and nicotine are both
two-fold higher in NS
than in S. The results of these artificial nitrosation experiments are
corroborated by the bound-
NNK levels in the lignified (CS) and non-lignified (CNS) parts of a
commercially cured Burley stem
sample. While free NNK is two-fold higher in CS, matrix-bound NNK is 7-fold
higher in CS.
Nicotine and NNN levels are higher in CS than CNS and less than NNK.
Conclusions
The presence of high-concentrations of matrix-bound NNK precursor in the
lignified,
sclerenchymatic tissue of green midribs indicates that matrix-bound NNK is
covalently or non-
covalently linked to lignin.
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Any publication cited or described herein provides relevant information
disclosed prior to the filing
date of the present application. Statements herein are not to be construed as
an admission that
the inventors are not entitled to antedate such disclosures. All publications
mentioned in the above
specification are herein incorporated by reference. Various modifications and
variations of the
invention will be apparent to those skilled in the art without departing from
the scope and spirit of
the invention. Although the invention has been described in connection with
specific preferred
embodiments, it should be understood that the invention as claimed should not
be unduly limited to
such specific embodiments. Indeed, various modifications of the described
modes for carrying out
the invention which are obvious to those skilled in cellular, molecular and
plant biology or related
fields are intended to be within the scope of the following claims.
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TABLE 1
Fraction of total weight freeNNN NIC bound NNK Lignin
Sieving fraction NNK
Plol [ng/g] [ug/g] [ng/g] [% d.w.]
[ng/g]
A: > 1mm 16 200 1098 2091 1606 2.92
A: 0.85 - 1 mm 4 251 1269 2307 1905 4.41
A: 0.5 - 0.85 mm 16 328 1690 2608 3500 6.33
A: 0.25 - 0.5 mm 27 463 2305 2937 6218 11.31
A: <0.25 mm 38 297 1732 3237 2936 4.81
total 329 1760 2839 3651 6.47
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TABLE 2
water- water-
dry dry before after
fresh weight insolubles insolubles
weight matter washing washing
CM [% dry [% fresh
[g] rol [ffig] (mg] weight] weight]
S 25 3.1 12.4 1010 668 66 8.2
NS 171 11 6.4 1010 414 41 2.6
CS 0.65
CNS 1.18
NNK after Free
PON NIC PON/NIC NNN
Sample autoclaving NNK
[ugig] Rigig] *1000 [ng/g)
ing/g] ing/g)
S 265 13245 1660
NS 116 184 111
SW 331 17610 1595
NSW 109 326 272
PON-S 6.8 784 8.63
PON-NS 14.0 1909 7.32
CS-free 9496 1356 120
CNS-free 7985 895 64
CS-total 8459 2038
CNS-tota I 7057 310
32
