Note: Descriptions are shown in the official language in which they were submitted.
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Treatment of Tobacco Material
Field
The present invention relates to a method for the treatment of tobacco
material.
Background
In some circumstances, it may be desirable to reduce the content of certain
constituents
from tobacco material before incorporating the tobacco material into a smoking
article
such as a cigarette.
Summary
According to a first aspect, there is provided a method for treating a tobacco
material,
which comprises using a combination of protease enzymes with different optimal
operating conditions and adjusting the operating conditions during the enzyme
/5 incubation period.
In some embodiments, the method results in a reduction in the protein content
of the
treated tobacco material compared to the protein content of the untreated
tobacco
material.
In some embodiments, adjusting the operating conditions comprises adjusting
the pH.
In some embodiments, adjusting the pH comprises decreasing the pH, optionally
by the
addition of an acid. In some embodiments, adjusting the pH comprises
increasing the
pH, optionally by the addition of an alkali.
In some embodiments, adjusting the operating conditions comprises adjusting
the
temperature. In some embodiments, adjusting the temperature comprises
adjusting
from a lower temperature to a higher temperature. In some embodiments,
adjusting
the temperature comprises a adjusting from a higher temperature to a lower
temperature.
In some embodiments, the combination of enzymes comprises enzymes with two
different optimal operating conditions. In some embodiments, the combination
of
enzymes comprises enzymes with three different optimal operating conditions.
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In some embodiments, the protein content of the treated tobacco material is
reduced by at
least 50%, at least 60%, at least 70%, at least 80%, and/or at least 90%,
compared to the
protein content of the untreated tobacco material.
According to a second aspect, there is provided a tobacco material which has
been treated by a
method according to the first aspect, or a derivative thereof.
According to a third aspect, there is provided a smoking article comprising a
tobacco material
according to the second aspect.
According to a fourth aspect, there is provided the use of a combination of
protease enzymes
with different optimal operating conditions for removing one or more proteins
from a tobacco
material by adjusting the operating conditions during the enzyme incubation
period.
According to another aspect, there is provided a method for treating a tobacco
material,
wherein the method comprises incubating the tobacco material for a period of
time with a
combination of protease enzymes with different optimal operating conditions
and adjusting
the operating conditions during the enzyme incubation period from the optimal
temperature
and/or pH of one protease enzyme to the optimal temperature and/or pH of
another protease
enzyme.
According to another aspect, there is provided use of a combination of
protease enzymes with
different optimal operating conditions for removing one or more proteins from
a tobacco
material by incubating the tobacco material with the protease enzymes for a
period of time
and adjusting the operating conditions during the enzyme incubation period
from the optimal
temperature and/or pH of one protease enzyme to the optimal temperature and/or
pH of
another protease enzyme.
Brief Description of the Drawings
Embodiments of the invention are described, by way of example only, with
reference to the
accompanying drawings, in which:
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Figure 1 is a flow diagram illustrating a method of treating tobacco material
in accordance
with an embodiment of the present invention.
Figure 2 is a flow diagram illustrating a method of treating tobacco material
in accordance
with an embodiment of the present invention.
Figure 3 is a table of the results of enzymatic treatment of unwashed tobacco
with two
proteases with different optimal operating conditions with a shift in pH value
and/or
temperature during enzyme incubation.
Figure 4 is a table of the results of enzymatic treatment of washed tobacco
with two proteases
with different optimal operating conditions with a shift in pH value and/or
temperature during
enzyme incubation.
Figure 5 is a table of the results of enzymatic treatment with a protease with
no shift in pH
value or temperature during enzyme incubation.
Figure 6 is a graph of the results of enzymatic treatment of washed tobacco
with two proteases
with different optimal operating conditions with a shift in pH value from pH
10 to 4 during
enzyme incubation.
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Figure 7 is a graph of the results of enzymatic treatment of washed tobacco
with two
proteases with different optimal operating conditions with a shift in pH value
from pH
4 to 10 during enzyme incubation.
Figure 8 is a schematic side view of a smoking article including treated
tobacco material
according to embodiments of the invention.
Detailed Description
The present invention relates to a method for treating a tobacco material, for
removing
proteins from tobacco material and/or reducing the protein content of tobacco
io material.
Treating tobacco material may be used to remove one or more other chemical
substances which are desirable to remove. Proteins in tobacco material are
often found
to be, and/or to be precursors of, such substances.
Alternatively or in addition, it may be desirable to reduce the protein
content of tobacco
material to improve the quality of the tobacco material.
Methods attempting to remove proteins have been proposed, although they have
tended to be expensive, lengthy and/or detrimental to the physical structure
of the
tobacco material, and/or not reduce the protein content to a desired level.
The method of the present invention removes proteins from tobacco material.
Exemplary proteins that may be removed from the tobacco material include
RuBisCO.
The method of the present invention results in a reduction in the protein
content of
tobacco material compared to the protein content of the untreated tobacco
material.
Figure 1 is a flow diagram illustrating a method of treating tobacco material
in
accordance with an embodiment. Tobacco material 1 undergoes protease enzyme
treatment 10. The solid and liquid components of the resulting suspension are
separated 20, resulting in a tobacco material with reduced protein content 30
and a
liquid component 31.
As used herein, the term "tobacco material" includes any part, such as for
example the
leaves or stems of any member of the genus Nicotiana and reconstituted
materials
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thereof. The tobacco material for use in the present invention is preferably
from the
species Nicotiana tabacum.
Any type of tobacco can be used in the present invention. Examples of tobacco
which
can be used include but are not limited to Virginia, Burley, Maryland,
Oriental and
Rustica tobaccos.
The tobacco material may be treated in any suitable way before being treated
according
to the method of the invention.
As used herein, the terms "incubation period" and "enzyme incubation period"
refer to
the period of time when the tobacco material is incubated with one or more
protease
enzyme(s).
/5 As used herein, the term "optimal operating conditions" refers to the
reaction
conditions under which the enzyme has optimal activity and/or activity
sufficient for
the reaction to proceed at a reasonable and/or desirable rate. The optimal
operating
conditions may comprise the pH and/or temperature ranges at which a protease
enzyme has optimal activity and/or activity sufficient for the reaction to
proceed at a
reasonable and/or desirable rate.
In some embodiments, the method of the present invention relates to a method
for
reducing the protein content of cured tobacco material. The tobacco material
may be
cured using any suitable method of curing before being treated according to
the method
of the invention.
The tobacco material may be any suitable form of tobacco. The tobacco material
may
comprise lamina, sheet, reconstituted, ground and/or milled tobacco material.
When
the tobacco material comprises lamina tobacco material, the lamina may be in
the form
of strip, cut or whole leaf tobacco. In some embodiments, the tobacco material
is in the
form of cut rag or whole leaf. In some embodiments, the tobacco material is
cured cut
rag and/or cured whole leaf tobacco.
In some embodiments, the tobacco material may be washed prior to protease
enzyme
treatment.
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Any suitable liquid medium may be used to wash the tobacco material. An
exemplary
washing medium is an aqueous solution.
In some embodiments, the tobacco material may be washed with water. The
tobacco
material may be washed with municipal water. Alternatively or in addition, the
tobacco
material may be washed with purified water. As used herein, "purified water"
relates to
water treated to remove contaminants and/or impurities. In some embodiments,
the
purified water is deionised water.
/o Washing the tobacco material may remove proteins from the tobacco
material. For
example, when the tobacco material is washed with water, water-soluble
proteins may
be removed from the tobacco material.
Washing the tobacco material may confer the advantage that the tobacco
material
/5 already has a reduced protein content before protease enzyme treatment.
Thus, the
reduction of the protein content using protease enzymes may be more efficient
and/or
more effective using washed tobacco material than unwashed tobacco material.
Alternatively or in addition, the enzyme incubation time required to remove a
desired
20 level of proteins may be shorter and/or the enzyme concentration
required to remove a
desired level of proteins may be less with the inclusion of a washing step
prior to
enzyme treatment.
The tobacco material may be washed by placing in a suitable washing medium for
up to
25 10 minutes, up to 15 minutes, up to 20 minutes, up to 25 minutes or up
to 30 minutes.
The amount of washing medium may be sufficient to remove any proteins that are
soluble in the washing medium from the tobacco material. The person skilled in
the art
will be aware that the extraction efficiency may be optimised by altering the
ratio of the
30 washing medium to tobacco material.
The temperature of the tobacco material and washing medium may be higher than
ambient temperature, as this may assist in the extraction process. The
temperature of
the tobacco material and washing medium may be up to 20 C, up to 30 C, up to
40 C,
35 up to 5o C, up to 60 C, up to 70 C or up to 80 C. Suitable temperatures
of the tobacco
material and washing medium will be known to the person skilled in the art.
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In some embodiments, the temperature of the tobacco material and washing
medium is
50 C and the tobacco material is washed for 25 minutes.
The tobacco material and washing medium may be agitated during washing, for
example by stirring and/or shaking.
Following washing, the tobacco material may be separated from the washing
medium.
Suitable means for separating the tobacco material from the washing medium
will be
io known to the person skilled in the art, and may comprise filtration
and/or
centrifugation.
Following contact with the tobacco material, the washing medium may comprise
components that are desirable to retain in tobacco material. For example, the
washing
/5 medium may comprise tobacco proteins that are soluble in the washing
medium. Such
proteins may confer particular organoleptic properties, such as tobacco
flavour.
Alternatively or in addition, the washing medium may comprise nicotine, sugars
and/or
other components that may be desirable to retain in tobacco material.
Therefore, in
some embodiments, following separation from the tobacco material the washing
20 medium is reapplied to tobacco material, for example, to tobacco
material that has
undergone protease enzyme treatment.
The method of the present invention reduces the protein content of the tobacco
material by using protease enzyme activity.
Protease enzymes degrade proteins into smaller sized peptides and amino acids.
Therefore, in the method of the present invention, protease enzyme activity
degrades
the proteins in the tobacco material, which are released from the tobacco
material,
resulting in the removal of proteins from the tobacco material and/or the
reduction in
protein content of the tobacco material.
Enzyme treatment is considered to be one of the most effective methods of
reducing the
protein content of tobacco material. Furthermore, the use of enzymes to reduce
the
protein content of the tobacco material is advantageous as they only catalyse
certain
transformations while leaving other material intact, and/or the use of enzymes
avoids
the use of hazardous chemicals. In contrast, alternative methods of protein
reduction
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may damage the physical structure of the tobacco material, for example by
requiring
conditions that damage the tobacco material.
The protease enzyme to be used in the present invention may be any type of
protease
enzyme. The protease enzyme to be used in the present invention may be
classified by
the International Union of Biochemistry and Molecular Biology as EC 3.4 (i.e.
enzymes
classified as peptidases).
Protease enzymes that are used commercially in the food and detergent
industries that
io are available at low cost may be suitable for the method of the present
invention.
The protease enzyme may be obtained from any suitable source. For example, the
protease enzyme may be of plant, animal, fungal, bacterial and/or viral
origin.
Alternatively or in addition, the protease enzyme may be synthesised.
In some embodiments in which the protease enzyme has a bacterial origin, the
protease
may be obtained from the Bacillus genus. Exemplary protease enzymes obtained
from
the Bacillus genus that are commercially available include Savinase , Esperase
,
Neutrase and Alcalase . In some embodiments, the protease is obtained from
the
species Bacillus licheniformis. Other suitable bacterial protease enzymes will
be known
to the person skilled in the art.
In some embodiments in which the protease enzyme has a fungal origin, the
protease
may be obtained from the Aspergillus genus. In some embodiments, the protease
is
obtained from the species Aspergillus oryzae. An exemplary protease enzyme
obtained
from Aspergillus oryzae that is commercially available is Enzobake . In some
embodiments, the protease is obtained from the species Aspergillus saitoi.
Other
suitable fungal protease enzymes will be known to the person skilled in the
art.
The protease enzyme may be an acidic enzyme. As used herein, the term "acidic
enzyme" refers to an enzyme whose optimal activity comprises activity at
acidic pH. An
exemplary acidic protease enzyme is protease obtained from Aspergillus oryzae,
which
has an optimal activity in the pH range 3.0 to 7.0, optionally in the pH range
3.0 to 5.0
or at a pH of about 7Ø
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The protease enzyme may be an alkaline enzyme. As used herein, the term
"alkaline
enzyme" refers to an enzyme whose optimal activity comprises activity at
alkaline pH.
Exemplary alkaline protease enzymes include protease obtained from Bacillus
licheniformis, which has an optimal activity in the pH range 3.0 to 11.5, and
Savinase ,
which has an optimal activity in the pH range 7.0 to 11Ø
Alternatively or in addition, the protease enzyme may be an enzyme whose
optimal
activity is at neutral pH.
/o The protease enzyme treatment of the tobacco material may be carried out
in solution.
In some embodiments, the protease enzyme treatment of the tobacco material is
carried out in aqueous solution.
The solution in which the protease enzyme treatment of the tobacco material is
carried
/5 out may be selected according to the particular protease enzyme used.
Suitable
solutions will be known to the person skilled in the art.
In some embodiments, protease enzyme is added to the selected solution after
the
addition of the tobacco material. In other words, in some embodiments,
protease
20 enzyme is added to the tobacco material in the selected solution.
The conditions under which the enzyme is added may be selected to avoid loss
of
enzyme activity.
25 In some embodiments, the pH of the solution is adjusted prior to the
addition of the
enzyme to the selected solution. The pH may correspond to a pH at which the
protease
enzyme has optimal activity.
In some embodiments, the pH of the tobacco material in the selected solution
30 corresponds to a pH at which the protease enzyme has optimal activity,
and therefore
the pH of the tobacco material in the selected solution may not need to be
adjusted.
When water is added to tobacco material, it may exhibit acidic or neutral
characteristics. Accordingly, in some embodiments in which the tobacco
material is
35 treated with acidic protease enzyme and the selected solution is water,
such as
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deionised water, the tobacco material in the selected solution may be acidic,
and the pH
may not require further adjustment.
In some embodiments in which the pH of the tobacco material in the selected
solution
does not correspond to a pH at which the protease enzyme has optimal activity,
the pH
may be adjusted to the desired pH.
The skilled person will be aware of suitable methods of adjusting the pH. In
some
embodiments, the pH is adjusted by the addition of an acid such as citric acid
and/or
/o an inorganic acid, or by the addition of an alkali such as sodium
hydroxide.
In some embodiments, a buffer may be added to maintain the pH of the solution.
Suitable buffers will be known to the person skilled in the art.
/5 In some embodiments, the temperature of the solution is adjusted prior
to the addition
of the enzyme to the selected solution. The temperature of the solution may be
adjusted
to a temperature at which the protease enzyme has optimal activity.
In some embodiments, the temperature of the selected solution corresponds to a
20 temperature at which the protease enzyme has optimal activity, and
therefore the
temperature of the selected solution may not need to be adjusted.
In some embodiments in which the temperature of the selected solution does not
correspond to a temperature at which the protease enzyme has optimal activity,
the
25 temperature may be adjusted to the desired temperature.
In some embodiments, the temperature of the selected solution is between about
15 C
and 75 C. In some embodiments, the temperature of the selected solution is up
to about
60 C, to minimise any negative effects on enzyme activity. In some embodiments
in
30 which the tobacco material is treated with protease enzyme obtained from
Aspergillus
oryzae, the temperature of the selected solution may be about 30 C. In some
embodiments in which the tobacco material is treated with Savinase , the
temperature
of the selected solution may be about 40 C.
35 Combining the tobacco material and protease enzyme may produce a
tobacco/enzyme
mixture.
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In some embodiments, the protease enzyme treatment comprises treating tobacco
material with one type of protease enzyme. In some alternative embodiments,
the
protease enzyme treatment comprises treating tobacco material with a
combination of
different protease enzymes. The combination of protease enzymes may comprise
at
least two enzymes with different optimal operating conditions. Alternatively
or in
addition, the combination of protease enzymes may comprise at least three
enzymes
with different optimal operating conditions.
In some embodiments in which a combination of protease enzymes with different
optimal operating conditions is used, the operating conditions may be adjusted
or
shifted during the enzyme incubation period. Adjusting the operating
conditions may
comprise adjusting the pH and/or the temperature.
/5 Treating the tobacco material with a combination of protease enzymes
with different
optimal operating conditions, which are added at the same time to the tobacco
material, confers the advantage that just one enzyme addition step is required
during
the method of reducing the protein content of the tobacco material, which may
be
suitable for carrying out the method on an industrial or commercial scale.
In some embodiments in which a combination of protease enzymes with different
optimal operating conditions is used, the protease enzymes may differ in terms
of their
optimal pH. The pH of the tobacco/enzyme mixture may therefore be adjusted
during
the enzyme incubation period, from the optimal pH of one protease enzyme to
the
optimal pH of another protease enzyme present in the tobacco/enzyme mixture.
The pH may be adjusted by decreasing or increasing the pH of the
tobacco/enzyme
mixture.
In some embodiments in which a combination of alkaline and acidic protease
enzymes
is used, the pH of the tobacco/enzyme mixture may be adjusted from alkaline to
acidic
pH or from acidic to alkaline pH. In other words, the pH may be adjusted from
above
7.0 to below 7.0 or from below 7.0 to above 7Ø
In some embodiments in which a combination of alkaline enzymes and enzymes
with
an optimal activity at neutral pH is used, the pH of the tobacco/enzyme
mixture may be
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adjusted from alkaline to neutral pH or from neutral to alkaline pH. In other
words, the
pH may be adjusted from above 7.0 to about 7.0 or from about 7.0 to above 7Ø
In some embodiments in which a combination of acidic enzymes and enzymes with
an
optimal activity at neutral pH is used, the pH of the tobacco/enzyme mixture
may be
adjusted from acidic to neutral pH or from neutral to acidic pH. In other
words, the pH
may be adjusted from below about 7.0 to 7.0 or from about 7.0 to below 7Ø
In some embodiments in which a combination of alkaline, acidic enzymes and
enzymes
with an optimal activity at neutral pH is used, the pH of the tobacco/enzyme
mixture
may be adjusted from the pH for the optimal activity of the first enzyme, to
the pH for
the optimal activity of the second enzyme, to the pH for the optimal activity
of the third
enzyme.
/5 The change in pH may take place during the course of the reaction. In
other words, the
pH change may not be caused by the active adjustment of the pH by the
operator.
Alternatively or in addition, the change in pH may be caused by the active
adjustment
of the pH.
To decrease the pH during the enzyme incubation period, an acid may be added.
Suitable acids will be known to the person skilled in the aft. In some
embodiments, the
pH is decreased by the addition of citric acid. Alternatively or in addition,
the pH is
decreased by bubbling CO, through the tobacco/enzyme mixture.
To increase the pH during the enzyme incubation period, an alkali may be
added.
Suitable alkalis will be known to the person skilled in the art. In some
embodiments,
the pH is increased by the addition of sodium hydroxide.
In some embodiments of the method in which a combination of protease enzymes
with
different optimal operating conditions is used, the protease enzymes may
differ in
terms of their optimal temperature. The temperature of the tobacco/enzyme
mixture
may therefore be adjusted during the enzyme incubation period, from the
optimal
temperature of one protease enzyme to the optimal temperature of another
protease
enzyme present in the tobacco/enzyme mixture.
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The temperature may be adjusted by decreasing or increasing the temperature of
the
tobacco/enzyme mixture.
In some embodiments of the method in which a combination of protease enzymes
with
different optimal operating conditions is used, the protease enzymes may
differ in
terms of their optimal pH and their optimal temperature. The pH and
temperature of
the tobacco/enzyme mixture may therefore be adjusted during the enzyme
incubation
period, from the optimal pH and temperature of one protease enzyme to the
optimal
pH and temperature of another protease enzyme present in the tobacco/enzyme
io mixture.
Using a combination of enzymes with different optimal operating conditions and
adjusting the operating conditions may make the enzyme treatment step more
effective
and/or more efficient at reducing the protein content of the tobacco material.
/5 Alternatively or in addition, using a combination of enzymes with
different optimal
operating conditions and adjusting the operating conditions may minimise the
effects
on the physical properties of the tobacco material, such as damage to the
structure of
the tobacco material.
20 The tobacco material may be incubated with protease enzyme for a period
of time
sufficient to reduce the protein content of the tobacco material to a desired
level.
In embodiments in which a combination of protease enzymes with different
optimal
operating conditions is used and the operating conditions are adjusted during
the
25 incubation period, the tobacco material may be incubated with protease
enzyme for up
to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, or up to 5 hours. In
some
embodiments in which a combination of protease enzymes with different optimal
operating conditions is used, the incubation period is up to 1 hour.
30 In embodiments in which a combination of protease enzymes with different
optimal
operating conditions is used, the operating conditions may be adjusted after a
proportion of the incubation period has passed. The operating conditions may
be
adjusted after about 25%, after about 50%, or after about 75% of the
incubation period
has passed. For example, in embodiments in which the incubation period is 1
hour and
35 the combination of protease enzymes comprises enzymes with two different
optimal
operating conditions, the operating conditions may be adjusted after about 50%
of the
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incubation period has passed, i.e. after about 30 minutes. In embodiments in
which the
incubation period is 1 hour and the combination of protease enzymes comprises
enzymes with three different optimal operating conditions, the operating
conditions
may firstly be adjusted after about 33% of the incubation period has passed,
i.e. after
about 20 minutes, and secondly after about 66% of the incubation period has
passed,
i.e. after about 40 minutes.
The tobacco material/enzyme mixture may be agitated, for example by stirring,
rocking
and/or shaking, during the enzyme incubation period. The tobacco
material/enzyme
io mixture may be agitated to provide adequate mixing without subjecting
the tobacco
fibres to mechanical stress that would break down the structure of the tobacco
material.
The protease enzyme concentration in the tobacco/enzyme mixture may be
sufficient to
reduce the protein content to a desired level within the desired incubation
time. At the
/5 same time, the amount of protease enzyme used during the method may be
sufficiently
low so that the method is cost-effective.
The amount of protease enzyme and solution added to the tobacco material may
be
determined according to the desired enzyme concentration.
The concentration of the protease enzyme may be between about 0.01 g/1 and
about 5.0
g/1 and/or between about 0.1 g/1 and about 2.0 g/l. In some embodiments, the
concentration of the protease enzyme is about 1.0 g/l. In some embodiments,
the
concentration of the protease enzyme is about 1.8 g/l. To clarify, as used
herein, the
concentration of the protease enzyme does not equate to enzyme activity.
In some embodiments, the tobacco material is incubated for 1 hour with two
protease
enzymes with different optimal operating conditions, both at a concentration
of 1.0 g/l.
The enzymatic treatment of the tobacco material results in the decomposition
of
protein fragments. Many of the resulting protein fragments are solubilised
and/or
dispersed in the liquid component of the tobacco/enzyme mixture, and are
therefore
easily separated from the tobacco material.
Following incubation of the tobacco material with the protease enzyme, the
insoluble
tobacco material may be separated from the liquid component of the
tobacco/enzyme
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mixture. The tobacco material and the liquid component may be separated with
any
suitable apparatus. In some embodiments, the tobacco material and the liquid
component are separated using filtration. Suitable alternative methods of
separating
the tobacco material and liquid component will be known to the person skilled
in the
are, and may include centrifugation and/or using a press, sieve and/or belt
filter.
In some embodiments, the tobacco material and the liquid component are
separated
using one or more filtration step(s). For example, the tobacco/enzyme mixture
may be
passed through a coarse filter and/or a fine filter. Suitable filters will be
known to the
io person skilled in the aft.
Figure 2 is a flow diagram illustrating a method of treating tobacco material
in
accordance with an embodiment. Whole leaf tobacco material 2 undergoes an
optional
washing step 3. The tobacco material is then added to the selected solution 4
and the
pH and/or temperature of the tobacco material in solution is optionally
adjusted 5
prior to the addition of protease enzyme. The tobacco material then undergoes
protease
enzyme treatment 10. The tobacco material and the liquid component of the
resulting
tobacco/enzyme mixture are separated 20, resulting in a tobacco material with
reduced
protein content 30 and a liquid component 31.
Following separation from the tobacco material, the liquid component may be
discarded. Alternatively, the liquid component may be retained. For example,
the liquid
component may be retained to recover the solution, which may be reused.
Following separation from the liquid component of the tobacco/enzyme mixture,
the
tobacco material may be washed to remove any protein fragments and/or enzymes.
The
tobacco material may be washed with any suitable medium. In some embodiments,
the
washing medium is heated. In some embodiments, the washing medium is salt
water.
The tobacco material may be treated to deactivate any enzyme remaining in the
tobacco
fibre. Suitable deactivation methods will be known to the person skilled in
the art. In
some embodiments, the tobacco material may be heat-treated to deactivate any
enzyme
remaining in the tobacco fibre.
The treatment of the tobacco material according to a method which comprises
using a
combination of protease enzymes with different optimal operating conditions
and
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adjusting the operating conditions during the enzyme incubation period may
result in a
reduction in the protein content of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%,
50%, 60%, 70%, 75%, 80%, 85%, 90% or at least 95%, based upon the protein
content
of the untreated tobacco material. In some embodiments, a reduction in protein
content of at least 50% and/or at least 75% is achieved.
Alternatively or in addition, the treatment of the tobacco material according
to a
method which comprises using a combination of protease enzymes with different
optimal operating conditions and adjusting the operating conditions during the
enzyme
io incubation period may result in the removal of at least 5%, 10%, 15%,
20%, 25%, 30%,
35%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or at least 95% of the proteins
from
the tobacco material.
In some embodiments, the treatment of the tobacco material according to a
method
/5 which comprises using a combination of protease enzymes with different
optimal
operating conditions and adjusting the operating conditions during the enzyme
incubation period results in the extraction of protein in an amount of at
least 5%, 10%,
15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or at least
95%,
based upon the protein content of the untreated tobacco material.
The method of the invention may comprise one or more further treatment steps.
Suitable additional treatment steps include, but are not limited to: treating
the tobacco
material with one or more suitable non-ionic liquids, such as water; treating
the
tobacco material with one or more additional enzymes, which may be enzymes
which
catalyse the modification of polyphenols, such as phenol-oxidising enzymes;
treating
the tobacco material with one or more suitable surfactants, such as sodium
dodecylsulfate (SDS), in any suitable solvent; treating the tobacco material
with one or
more suitable adsorbent materials, such as polyvinyl polypyrrolidone (PVPP),
hydroxylapatite, bentonite, activated carbon or attapulgite, in any suitable
solvent if
appropriate; and treating the tobacco material with one or more suitable non-
aqueous
liquids, such as ionic liquids.
Alternatively or in addition, the tobacco material treated according to the
method of the
present invention may be subsequently subjected to further extraction
processes.
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Following treatment according to the method of the present invention, the
tobacco
material with reduced protein content may be incorporated into a smoking
article.
As used herein, the term "smoking article" includes smokeable products such as
cigarettes, cigars and cigarillos whether based on tobacco, tobacco
derivatives,
expanded tobacco, reconstituted tobacco or tobacco substitutes and also heat-
not-burn
products. The smoking article may be provided with a filter for the gaseous
flow drawn
by the smoker.
/o Following treatment according to the method of the present invention,
the tobacco
material may be further modified in any suitable way before being incorporated
into a
smoking article. For example, the tobacco material may be dried, certain
chemical
substances may be added to the tobacco material, such as flavourants where
local
regulations permit, and/or the tobacco material may be cut and/or shredded
before
/5 being incorporated into a smoking article using any suitable method of
incorporation.
As used herein, the terms "flavour" and "flavourant" refer to materials which,
where
local regulations permit, may be used to create a desired taste or aroma in a
product for
adult consumers. They may include extracts (e.g., licorice, hydrangea,
Japanese white
20 bark magnolia leaf, chamomile, fenugreek, clove, menthol, Japanese mint,
aniseed,
cinnamon, herb, wintergreen, cherry, berry, peach, apple, Drambuie, bourbon,
scotch,
whiskey, spearmint, peppermint, lavender, cardamon, celery, cascarilla,
nutmeg,
sandalwood, bergamot, geranium, honey essence, rose oil, vanilla, lemon oil,
orange oil,
cassia, caraway, cognac, jasmine, ylang-ylang, sage, fennel, piment, ginger,
anise,
25 coriander, coffee, or a mint oil from any species of the genus Mentha),
flavour
enhancers, bitterness receptor site blockers, sensorial receptor site
activators or
stimulators, sugars and/or sugar substitutes (e.g., sucralose, acesulfame
potassium,
aspartame, saccharine, cyclamates, lactose, sucrose, glucose, fructose,
sorbitol, or
mannitol), and other additives such as charcoal, chlorophyll, minerals,
botanicals, or
30 breath freshening agents. They may be imitation, synthetic or natural
ingredients or
blends thereof. They may be in any suitable form, for example, oil, liquid, or
powder.
Referring to Figure 8, for purpose of illustration and not limitation, a
smoking article
101 according to an exemplary embodiment of the invention comprises a filter
102 and
35 a cylindrical rod of smokeable material 1433, such as tobacco treated in
accordance with
the invention described herein, aligned with the filter 102 such that one end
of the
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smokeable material rod 103 abuts the end of the filter 102. The filter 102 is
wrapped
in a plug wrap (not shown) and the smokeable material rod 103 is joined to the
filter
102 by tipping paper (not shown) in a conventional manner.
In some embodiments, the methods described herein may comprise one or more
further steps to modify the tobacco material in any suitable way. For example,
the
tobacco material may be modified to provide it with one or more
characteristics
desirable for a tobacco material. For example, where the treated tobacco
material is to
be incorporated into a smoking article such as a cigarette, the tobacco
material may be
io treated in order to modify the flavour it generates upon combustion,
and/or may be
treated in order to remove one or more of its chemical substances.
Experimental Work
A series of experiments were carried out in order to investigate the removal
of proteins
/5 from tobacco material by using a combination of protease enzymes with
different
optimal operating conditions and adjusting the operating conditions during the
enzyme
incubation period. The disclosed experimental work is not intended to limit
the scope
of the invention.
20 Example 1 ¨ Unwashed tobacco material treatment with two proteases with
different
optimal operating conditions
Treatment of Tobacco Material
Whole tobacco leaves were dried to constant weight at 45 C in a drying chamber
and
were then crushed, to enable tobacco treatment on a laboratory scale. 25 g of
the
25 crushed dried tobacco was placed in 500 ml of deionised water or buffer,
selected
according to the enzyme combination used (the enzymes were obtained from Sigma
Aldrich ). Referring to Figure 3, tobacco treated with enzyme groups 2 and 4
was
placed in deionised water, the alkaline buffer 0.1 M sodium phosphate pH 8.0
was used
for tobacco treatment starting with an alkaline pH and the acidic buffer 0.1 M
sodium
30 acetate pH 4.0 was used for tobacco treatment starting with an acidic
pH. Following the
addition of two enzymes with different optimal operating conditions, each at a
concentration of 1.0 g/l, the resulting suspension of tobacco material and
enzymes was
incubated in an incubation shaker at 120 revolutions per minute (rpm) for 1
hour. The
temperature of the incubation shaker was set according to the optimal working
35 temperature of the enzymes, and in some cases the temperature was
changed following
30 minutes of incubation. The pH value was set according to the optimal
working pH of
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the enzymes, and was monitored during incubation with a pH meter, and in some
cases
the pH was changed following 30 minutes of incubation by the addition of 40%
acetic
acid (to make the pH more acidic) or by the addition of 2 M sodium hydroxide
solution
(to make the pH more alkaline). The pH of the tobacco treated with enzyme
groups 2 or
4 decreased during the course of the reaction and was not adjusted by the
addition of
an acid. Following incubation with the enzymes, the tobacco material was
separated
from the liquid component by two filtration steps, the first filtration step
was carried
out using coarse paper filter, and the filtrate from the paper filter was
filtered with a
0.45 vtrn hydrophilic PVDF membrane syringe filter (Rotilabo , diameter 25 mm,
Carl
Roth, Germany).
The parameters used for the treatment of the tobacco material are outlined in
Figure 3.
Analysis of Protein Removal
The resulting liquid component of the samples prepared according to Figure 3
was
analysed for the nitrogen content. Total nitrogen was measured with a TOC-N
Analyzer
(Shimadzu) and/or a total nitrogen test kit (Spectroquant Nitrogen total
photometric
test, DMP 10 ¨ 150 mg/1, Merck).
The nitrogen content of the liquid component was corrected for the nitrogen
present
that was contributed by nicotine and enzyme proteins, taking the approximate
proportion of nitrogen in nicotine to be 17% by weight, and the approximate
proportion
of nitrogen in protein to be 16% by weight.
Nicotine quantification was carried out using HPLC with a Phenomenex-Gemini
column C18.3 lam, 150 x 3 mm, isocratic at 30 C, flow 0.6 ml/minute, injection
volume
5 [11. Samples of the separated liquid component were diluted with
acetonitrile-water
5o%, Na203, pH 9.8. The UV absorption was measured at 277 nm with an external
calibration of the nicotine standard.
The results of the analyses are shown in Figure 3. The abbreviations in the
table are as
follows:
TN: total nitrogen
TN-PN: nitrogen contributed by enzyme protein
NN: nitrogen contributed by nicotine
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TN final: total nitrogen in the liquid component corrected for the nitrogen
contributed
by enzyme proteins and nicotine
TN final (%): the group with the best nitrogen removal performance was set as
l00%
and the remaining groups were compared to this result
Example 2 - Washed tobacco material treatment with two proteases with
different
optimal operating conditions
Treatment of Tobacco Material
Whole tobacco leaves were treated according to Example 1, with the exception
that the
/o tobacco leaves were washed and dried prior to crushing. The tobacco
leaves were
washed with deionised water for 25 minutes at 50 C, stirring by hand every 5
minutes.
The aqueous solution was pressed out of the leaves by hand and the tobacco
leaves were
then dried to constant weight at 45 C in a drying chamber before undergoing
enzyme
treatment as described in Example 1.
The parameters used for the treatment of the tobacco material are outlined in
Figure 4.
As for Example 1, tobacco treated with enzyme groups 2 and 4 was placed in
deionised
water.
Analysis of Protein Removal
The resulting liquid component of the samples prepared according to Figure 4
was
analysed for the nitrogen content and corrected for the nitrogen present that
was
contributed by nicotine and enzyme proteins according to the methods described
in
Example 1.
The results of the analyses are shown in Figure 4.
For the tobacco material prepared according to Example 2 (washed tobacco,
Figure 4),
the total nitrogen present in the liquid component was highest (i.e.
demonstrating a
greater protein removal from the tobacco material) for groups 1 and 4. Group 4
was
treated by a decrease in pH during the enzyme incubation period, and group 1
underwent a shift in temperature from 30 C to 40 C. An increase in pH during
the
enzyme incubation period was also seen to be effective for group 5.
A decrease in pH during the enzyme incubation period was seen to be effective
for
protein removal from unwashed tobacco material prepared according to Example 1
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(unwashed tobacco, Figure 3), as seen for groups 2 and 3. An increase in pH
during the
enzyme incubation period, as carried out on group 5, was also effective at
removing
protein from unwashed tobacco material.
Example 3 ¨ Unwashed and washed tobacco material treatment with one enzyme
type
under constant operating conditions
Treatment of Tobacco Material
Whole tobacco leaves were treated according to Examples 1 or 2, with the
exception
that the tobacco material was treated with a single enzyme type, i.e. with the
same
io optimal operating conditions. The pH and temperature were kept constant
during the
enzyme incubation period.
The parameters used for the treatment of the tobacco material are outlined in
Figure 5.
Analysis of Protein Removal
The resulting liquid component of the samples prepared according to Figure 5
was
analysed for the nitrogen content and corrected for the nitrogen present that
was
contributed by nicotine and enzyme proteins according to the methods described
in
Example 1.
The results of the analyses are shown in Figure 5.
For the tobacco material treated with a single enzyme type and with constant
pH and
temperature during the enzyme incubation period, the total nitrogen present in
the
liquid component was highest (i.e. demonstrating a greater protein removal
from the
tobacco material) when the tobacco was treated with Bacillus species enzyme at
a
concentration of to g/1 at a pH of 7.5 and a temperature of 40 C. However, the
level of
protein removal was still lower than the level of protein removal achieved
when the
tobacco material is treated according to Examples 1 or 2, demonstrating that
treating
the tobacco material with a combination of enzymes with different optimal
operating
conditions and shifting the operating conditions during the enzyme incubation
period
can be more effective than treating the tobacco material with a single type of
enzyme
and/or not shifting the operating conditions during enzyme incubation.
Example 4 - Washed tobacco material treatment with two proteases with
different
optimal operating conditions
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Treatment of Tobacco Material
Whole tobacco leaves were treated according Examples 1 or 2, with the
exception that
the pH was shifted by about one pH unit every 10 minutes during the enzyme
incubation period. To decrease the pH, a 30% acetic acid solution was used. To
increase
the pH, a 1 M sodium hydroxide solution was used.
The protease enzymes used were Savinase (protease from Bacillus sp.) and
protease
from A. oryzae, both at a concentration of to g/l. The total enzyme incubation
time
was 1 hour and the temperature was maintained at 35 C during the enzyme
incubation
period.
lo
Analysis of Protein Removal
The resulting liquid component of the samples was analysed for the nitrogen
content
according to the methods described in Example 1, with the exception that the
liquid
component was not corrected for the nitrogen contributed by nicotine and
enzyme
/5 proteins.
The results of the analyses are shown in Figures 6 and 7.
Figures 6 and 7 illustrate that as the total nitrogen content of the liquid
component
20 increases (and therefore the protein removal from the tobacco material
increases) as
the enzyme incubation time increases and the pH is shifted. There is a more
continuous
increase in the nitrogen content of the liquid component when the pH is
increased from
4 to 10 (shown in Figure 7) than when the pH is decreased from 10 to 4 (shown
in
Figure 6). When the pH was shifted from pH 10 to 4, the highest nitrogen
removal was
25 seen after 40 minutes of incubation with the enzymes, when the pH was
between about
7 and 5.
In order to address various issues and advance the aft, the entirety of this
disclosure
shows by way of illustration various embodiments in which the claimed
invention(s)
30 may be practiced and provide for superior tobacco treatment, tobacco
material, and
products incorporating tobacco material. The advantages and features of the
disclosure
are of a representative sample of embodiments only, and are not exhaustive
and/or
exclusive. They are presented only to assist in understanding and teach the
claimed
features. It is to be understood that advantages, embodiments, examples,
functions,
35 features, structures, and/or other aspects of the disclosure are not to
be considered
limitations on the disclosure as defined by the claims or limitations on
equivalents to
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the claims, and that other embodiments may be utilised and modifications may
be
made without departing from the scope and/or spirit of the disclosure. Various
embodiments may suitably comprise, consist of, or consist essentially of,
various
combinations of the disclosed elements, components, features, parts, steps,
means, etc.
In addition, the disclosure includes other inventions not presently claimed,
but which
may be claimed in future.
