Note: Descriptions are shown in the official language in which they were submitted.
CA 02214036 1997-08-27
I WO93/1267S PCT/CA92/00566
:
TOBACCO TREATMENT
TECHNICAL FIELD
This is invention relates to the treatment of
tobacco material to reduce its protein content.
BACKGROUND ART
Investigators have found that tobacco quality is
improved by reducing its protein content. Although it is
relatively easy to remove protein from uncured tobacco
leaf, there are disadvantages to removing protein before
curing. The major problem is that protein broken down
during curing can form flavour compounds that are
important contributors to the organoleptic properties of
the smoke. Another disadvantage is that efficient
extraction of green leaf usually necessitates tobacco
structural changes which make it difficult to produce
shredded tobacco suitable for use as a cigarette filler.
Partial removal of protein from cured tobacco can be
accomplished by extraction with water, with the
efficiency of the extraction improving as the particle
size is reduced. However, for shredded tobacco of the
size normally used for cigarette manufacture, most of the
protein cannot be extracted by water alone.
United States Patent no. 4,407,307 describes the
removal of protein from tobacco strips in an aqueous
solution of a proteolytic enzyme whereby insoluble
proteins are decomposed into soluble fragments. The
extract is separated from the tobacco and inoculated with
a yeast culture, which, as it grows, removes the soluble
protein fragments in the extract by metabolic
assimilation. After removal of the yeast, the protein-
free extract is concentrated and added back to the
tobacco strips.
United States Patent no. 4,887,618 describes a
process in which tobacco is first extracted with water.
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The tobacco residue remaining after extraction is
separated from the solution, mixed with water and treated
with a proteolytic enzyme. The protein-reduced tobacco
is separated from the enzyme solution, rinsed and dried.
The water extract is concentrated and added back to the
protein reduced tobacco whereby water soluble flavour
tobacco components and the nicotine is retained in the
final product.
The processes of the above described United States
Patents rely on protease enzymes alone to remove protein
from tobacco material. Our own investigations have found
that enzymes which efficiently remove protein from
tobacco are expensive, while those enzymes which are
available in commercial quantities at a reasonable price,
are much less efficient for protein removal. United
States Patent no. 4,716,911 also recognizes this
disadvantage and proposes using either an alkali or a
combination of a protease and a non-protease depolymerase
to effect protein removal. However, we have found that
alkaline solutions may have a deleterious effect on the
physical structure of the tobacco, and the use of a
protease combined with a depolymerase may not be an
economical approach to protein removal. Therefore, it is
desirable to provide a technique for protein removal from
tobacco that does not cause a physical degradation of the
tobacco structure and is economical and efficient.
It is also desirable to provide an efficient and
cost effective process for removal of solubilized
polypeptides (which include proteins) from an aqueous
extract of tobacco, before the extract is added to solid
tobacco material. In United States Patent no. 4,407,307,
protein fragment in an aqueous extract was assimilated by
yeast. United States Patent no. 4,941,484 describes the
use of ultrafiltration to remove high molecular weight
compounds, (including proteins) from an aqueous extract
of tobacco before the extract is added to protein-reduced
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tobacco. The former method is unduly complicated by the
requirement to ferment the aqueous extract in the
presence of yeast. The ultrafiltration process requires
the use of special apparatus and may not be useful for
the removal of polypeptides outside the cut-off values of
the ultrafiltration membrane employed in the procedure.
It is known to treat an aqueous extract of tobacco
with a solid adsorbent to remove polyphenols, for
example, as described in United States Patent no.
3,561,451. Such adsorbents include alumina and poly~;de
which are not useful for removal of solubilized protein
or polypeptides from the aqueous extract. Heretofore,
there were no adsorbents known to be useful for removal
of the polypeptides found in a tobacco extract in
commercial batch processing.
DISCLOSURE OF THE lNV~NlION
This invention provides methods which involve the
extraction of tobacco material with a surfactant.
Tobacco material includes tobacco solids and any form of
solid tobacco, including cured tobacco. The surfactant
may be used alone or in combination with a proteolytic
enzyme. ln the latter instance it is possible to use
less surfactant and protein extraction is more efficient
than with enzyme treatment alone or with surfactant
treatment alone. The tobacco material may be first
extracted with an aqueous solvent or with a proteolytic
enzyme before extracting with a surfactant.
This invention also provides methods that involve
the use of hydroxyapatite and fuller's earth minerals
such as bentonite as insoluble adsorbents for removal of
polypeptides, including proteins, from aqueous extracts
of tobacco. Bentonite is a particularly effective
adsorbent because of its low cost and effectiveness in
small quantities. This is surprising since bentonite is
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w093/l267~ PCT/CA92/00566
known to be useful for adsorbing proteins in acidic
beverages such as wine but is now shown effective for
removal ,f proteins from more basic tobacco extracts.
Furthermore, it is known that bentonite will adsorb
nicotine, which may not be desirable in a tobacco
treatment. Surprisingly, bentonite may be used to
selectively adsorb polypeptides rather than nicotine.
Bentonite is also effective for removal of pigment
compounds from an aqueous extract of tobacco which is
advantageous because such compounds tend to darken
tobacco material when the extract is applied to the
material, particularly when the extract has been heated
to facilitate its concentration.
Accordingly this invention provides a method for
reducing the protein content of tobacco material which
includes extracting the tobacco material with a
surfactant or with a surfactant and a proteolytic enzyme.
This invention also provides the preceding method wherein
the tobacco material has been previously extracted with
an aqueous solvent to produce an aqueous extract or has
been previously extracted with a proteolytic enzyme.
This invention also provides a method for removing
polypeptides from an aqueous extract of tobacco material
which includes combining the extract with an insoluble
adsorbent selected from the group comprising hydroxy-
apatite and a fuller's earth mineral and, separating the
extract from the adsorbent.
This invention also provides tobacco material and
tobacco extracts produced according to the above
described methods, including an aqueous extract of
tobacco material having a reduced pigment and polypeptide
content.
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ln one aspect of this invention, tobacco material is
extracted directly with an aqueous solution of a
surfactant or a mixture of a surfactant with a
proteolytic enzyme, or alternatively, the tobacco
material is extracted sequentially with a proteolytic
enzyme and a surfactant, preferably with extraction by
the enzyme occurring first. The extract is separated
from the tobacco residue and treated in various ways to
remove surfactant, protein and/or protein fragments. The
treated extract is concentrated and added back to the
protein reduced tobacco material.
In another aspect of this invention, tobacco
material is first extracted with an aqueous solvent.
This method is preferred since it is easier to ensure
complete removal of surfactant and enzyme from the final
tobacco product. The initial aqueous extract is
separated from the insoluble tobacco residue and retained
for subsequent reconstitution. The aqueous extract may
be treated to remove solubilized polypeptides as
described below. The tobacco residue is resuspended in
an aqueous solution of a surfactant or a mixture of
surfactant and proteolytic enzyme. Alternatively,
sequential treatment with the enzyme and surfactant as
described above may be carried out. After further
protein has been solubilized, the latter solutions are
separated from the tobacco residue and discarded. The
extracted tobacco residue is rinsed and dried. The
aqueous extract from the initial extraction is sprayed
back onto the tobacco residue to make a smokable
cigarette filler. Preferably, the aqueous extract is
concentrated before applying to the tobacco residue.
The various tobacco extracts described above may
optionally be treated to remove soluble materials to
further enhance tobacco quality. For example, we have
found that the extract can be treated with
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polyvinylpolypyrrolidone (PVPP) as an insoluble adsorbent
for effective removal of polyphenols from the solution.
The extracts may be treated with hydroxyapatite or a
fuller's earth mineral such as bentonite or attapulgite
to remove solubilized polypeptides, and in the case of
bentonite treatment, to also remove pigment compounds.
In each case, the extract may be combined with the
adsorbent by simply suspending the adsorbent in the
solution and then removing the adsorbent by conventional
means such as filtration or centrifugation. Other
methods of combining the extracts or solutions with an
insoluble adsorbent are well known and may be used in the
method of this invention. For example, the adsorbent may
be enclosed in a column or other suitable container and
the extract is allowed to flow through the column or
container to permit adsorption to occur.
MODES FOR CARRYING OUT THE lNv~NlION
In one embodiment of this invention, strip, cut or
ground tobacco, and preferably cut tobacco, is extracted
at 35-65~C in an aqueous solution of a surfactant or a
mixture of surfactant and proteolytic enzyme. The
solvent, which is usually water, but can also contain
alcohols such as ethanol or methanol, is added to the
tobacco material in the ratio of between 10:1 and 30:1 by
weight. Preferably, the concentration of surfactant in
the solvent is 0.1% - 5% w/v.
The surfactant may be selected from the group
including the sodium alkylsulfonates, sodium
alkylsulfates, the sodium or potassium salts of
carboxylic acids, sodium alkylarylsulfonates and sodium
alkylsulfosuccinates. For these surfactants, the most
effective have a chain length of between 8 and 12 carbon
atoms. Particularly effective surfactants are sodium
dodecylsulfate, sodium dodecylbenzenesulfonate and sodium
dioctylsulfosuccinate (Aerosol OT-).
* Trademark
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-- 7
Cationic and non-ionic surfactants may be used but
these have been found to be less effective than the
anionic surfactants.
The proteolytic enzyme, if used, is preferably
chosen from the group comprising the bacterial and fungal
enzymes. Of most interest for the purpose of this
invention are the enzymes used commercially in the food
and detergent industries which are available at low cost.
Thus, Savinase , Neutrase~, Enzobake or Alcalase
available from Novo Inc. have been found to be effective
for protein removal from tobacco. The proteolytic
enzymes are preferably added to the solution in a
concentration range of 0.1%-5% wtw of the tobacco
material.
The suspension of tobacco material in the solution
of surfactant or surfactant and proteolytic enzyme is
stirred gently for 1-18 hours. The extracted tobacco
residue is separated from solubilized tobacco components
by filtration or centrifugation. Up to about 65% of the
initial tobacco weight may be solubilized during this
extraction step. The tobacco components that go into
solution are nicotine, sugars, polypeptides, ~;no acids,
pectins, polyphenols, flavours, inorganic salts, etc.
Alternatively, the tobacco material may be
extracted, as described above, sequentially with
solutions of surfactant and a proteolytic enzyme. In
some cases, sequential treatment, particularly with
enzyme treatment preceding surfactant treatment, provides
a greater reduction of tobacco protein.
The extract may be treated in a number of ways to
remove surfactant and polypeptides, or other components,
before the extract is added back in concentrated form to
the extracted tobacco.
*Trademark
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The surfactant may be removed by using either of the
following treatments or preferably both in sequence. The
solution is cooled to below the Krafft temperature of the
surfactant at which temperature, up to 50-70% of the
surfactant precipitates. Cooling the solution to 4~C is
effective. Re~i ni ng surfactant is precipitated using an
inorganic calcium or magnesium salt. The precipitated
surfactant and/or its insoluble calcium or magnesium
salts may be removed from the solution by filtration or
centrifugation.
Polypeptides may be removed from the solution using
an insoluble adsorbent such as hydroxyapatite, or one of
the fuller's earth minerals such as attapulgite or
bentonite. Larger amounts of adsorbent remove greater
amounts of protein. When hydroxyapatite is added in a
quantity of about 16-25% of the initial tobacco weight
(the weight of the tobacco used to provide the extract)
up to about 50~ of the dissolved protein is removed.
When about 10% of the initial tobacco weight of
attapulgite (Attagel 40 ; Engelhard) is used, all or a
large proportion of the dissolved protein is removed.
When bentonite is added to the tobacco extract in a
quantity that is about 3-4% of the weight of the tobacco
extracted, a large proportion of the protein nitrogen is
removed from solution. Some nicotine is also adsorbed
from solution, but this loss is minimal at the
concentrations of bentonite required to remove most of
the polypeptides. The quantity of bentonite may be
reduced if the bentonite is slurried in a small quantity
of water before adding it to the tobacco extract. Pre-
m; xi ng with water swells th- oentonite, which forms a
flocculent suspension when added to the tobacco extract.
Bentonite treatment is also effective in removing pigment
compounds found in a tobacco extract.
*Trademark
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It appears that a tobacco extract is an effective
buffer against bentonite's tendency to make a solution
more alkaline. Although it is generally unnecessary in
the methods of this invention to adjust the p~ of the
tobacco extract, the efficiency of adsorption by
bentonite may be increased by reducing the pH of the
extract. Flue-cured tobacco extracts typically have a pH
in the range 5-6. As the pH is lowered by adding an
acid, smaller quantities of bentonite may be required for
polypeptide and pigment removal. The optimum pH is about
3. The pH may be adjusted by addition of any suitable
acid such as hydrochloric.
At this stage, other components of the extract may
also be selectively removed. For example PVPP may be
used as an insoluble adsorbent using the same methods as
for absorbtion of polypeptides. PVPP in an amount
representing 5-10% of the initial tobacco weight removes
up to about 50-90% of the polyphenols in solution.
Preferably the extract is concentrated to a solids
concentration of between 20-50% by weight.
Concentrations of between 20-30% are most efficiently
achieved using reverse osmosis, using procedures known in
the art such as that disclosed in United States Patent
no.3,847,163. However, other methods of concentration,
particularly those which preserve the flavour and other
components of the extract are known and may be used.
The extracted tobacco, if in the cut or strip form,
may be dried by a variety of known methods. Also, a
rotary dryer with steel combs attached to the inside wall
of the drum, to prevent balling of the wet tobacco, may
be used to dry the tobacco.
The concentrated extract may be sprayed onto the
tobacco residue, during or after drying. This results in
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-- 10 --
a tobacco which is very similar in physical form and
appearance and smoking properties to the original
material, but with substantially reduced levels of
protein. When sufficient bentonite is used as an
adsorbent, the consequent removal of pigment compounds
results in a product that is not overly darkened by the
addition of the concentrated extract.
If the original tobacco is in the ground form, the
final product may be cast into a sheet, which, when
shredded, can form all or part of a cigarette filler.
In another embodiment of the invention, the tobacco
is first extracted with an aqueous solvent consisting
either of water or a mixture of water with an alcohol
(for example, methanol or ethanol). The ratio of solvent
to tobacco is preferably about 20:1 by weight but can be
as low as 12:1. The extraction time may be between
fifteen minutes to one hour, at a temperature between
15-60~C. The preferred conditions are 1/2 hour at 25~C.
The extraction step results in some of the polypeptides
and most of the sugars, nicotine, amino acids,
polyphenols, etc. being removed from the tobacco into
solution. The aqueous extract may be separated from the
tobacco by filtration or centrifugation.
Polypeptides, polyphenols, and pigment compounds
etc. may be removed from this extract by the methods
described in the first embodiment. The extract may be
concentrated by reverse osmosis or by other known
methods.
The extracted tobacco residue is subjected to a
further extraction step to remove protein. An aqueous
solution of a surfactant such as described in the first
embodiment, at a concentration in the range 0.01-5% (w/v)
is added to the wet or dried tobacco residue in the ratio
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-- 11 --
of 20:1 to 30:1 (solution: dry tobacco weight).
Alternatively, a proteolytic enzyme such as described in
the first embodiment, may be added to the surfactant
solution in a concentration range of 0.1-5%. If
surfactant alone is used, the tobacco slurry is agitated
gently for 1-18 hours at 24-65~C. For a mixture of
surfactant and enzyme, the same time may be allowed for
the extraction but a narrower temperature range such as
30-40~C should be used to avoid denaturing the enzyme.
Sequential treatment with enzyme and surfactant may be
carried out.
Following extraction, the tobacco residue may be
separated from the solution by filtration or
centrifugation and the residue rinsed thoroughly with
water. The tobacco residue may then be dried and the
concentrated extract sprayed back onto the tobacco
residue, as described in the first embodiment.
EXAMPLE I
Two hundred and fifty grams (250 g) of a single
grade of flue-cured tobacco, cut at 35 cpi, was extracted
with 5 litres of water cont~in;ng 100 g of sodium
dodecylsulphate (SDS). The extraction was carried out
for 18 hours at 60-70~C with gentle stirring. The tobacco
residue was separated from the solution by filtration and
dried using a small rotary drier. After correction for
moisture content, it was calculated that 66% of the
tobacco weight was in the solute. The initial nitrogen
content of the tobacco, as determined by the Kjeldahl
method, was 1.82% (on a dry weight basis) while the
extracted tobacco had a nitrogen content of 0.94% (on a
dry wei~ght basis). Thus 82% of the nitrogen in the
tobacco was solubilized.
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- 12 -
The extract was cooled to 4~C and the precipitated
SDS collected by filtration. This resulted in recovery
of 68% of the SDS. The rem~in;ng SDS was precipitated by
adding 6g of CaCl2 to the solution. The precipitate was
removed by filtration.
Fifty grams (50 g) of hydroxyapatite (Calcium
phosphate tribasic; Mallinckrodt)was added to the
solution, stirred for 1/2 hour, and removed by
filtration. The protein content of the solution was
measured before and after treatment by the BioRad method.
Hydroxyapatite reduced protein content by about 50%.
The extract was allowed to evaporate at 25~C until it
was sufficiently concentrated to spray back onto the
extracted tobacco residue.
EXAMPLE 2
Five hundred grams (500 g) of a single grade of
flue-cured tobacco, cut at 35 cpi. was extracted with 10
litres of water for 18 hours at 60-70~C.
The tobacco was separated from the solution by
filtration, and thoroughly rinsed with warm water, dried
to 13~ moisture in a rotary drier. The dried, water
extracted tobacco residue was divided into 20 g portions
and each portion was re-extracted at 60-70~C for 18 hours
in 600 ml of a solution containing 0-15 g of sodium
dodecylbenzenesulfonate (SDBS). The surfactant treated
tobacco residue was filtered, thoroughly rinsed with
water and dried. The dried residues were analyzed for
nitrogen using the Kjeldahl method. The results for
Kjeldahl nitrogen of the extracted tobacco at different
surfactant concentrations are given in Table I.
*Trademark
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W093/l2675 PCT/CA92/00566
Table I
SDBS Kjeldahl
concentration Nitrogen
(g/l~ %
~ ~ 2.03
0.83 2.03
2.5 1.93
5.0 1.87
10.0 1.67
15.0 1.74
20.0 1.60
25.0 1.33
EXAMPLE 3
Ten gram (10 g) portions of dried, water extracted
tobacco residue such as was procured in example 2 were
dispersed in a solution cont~;n;ng 300 ml of water, 0.25
g of Savinase (NOVO Industri, Denmark) with an activity
of 6.0 KNPU/g and various amounts of sodium
dodecylbenzenesulfonate. The slurries were gently
stirred for 18 hours at room temperature. The tobacco
residues were filtered from the slurry, thoroughly rinsed
with water and dried again in a rotary dryer. The
results for Kjeldahl nitrogen determinations on the
tobacco residues are given in table II.
Table II
SDBS Savinase Kjeldahl
Nitrogen
(q) (g) %
0 0 2.57
0 0~25 1.79
6.0 0 1.81
0.75 0.25 1.90
1.50 0.25 1.62
3.00 0.25 1.26
4.50 0.25 1.17
6.00 0.25 1.29
7.50 0.25 1.30
9.00 0.25 1.35
*Trademark
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~093/12675 PCT/CA92/00566
- 14 -
EXAMPLE 4
300 g of flue-cured shredded tobacco was extracted
with 6 litres of water for l hour at 30~C. The tobacco
extract was separated from the tobacco material by
centrifugation and divided into 200 ml aliquots, which
were treated with various quantities of either
hydroxyapatite (Mallinckrodt) or bentonite (Fisher;
Purified Grade). The adsorbents were added as dry
powders to the extracts and the resulting suspensions
were shaken for 15 minutes. The extracts were filtered
and protein nitrogen determined by the Bio Rad' method.
Kjeldahl nitrogen, nicotine and total sugars were
determined for freeze dried samples of the extract. The
results are given in Table III. The presence of pigment
compounds in the extract was noticeably reduced when the
amount of bentonite used was equivalent to 4%, or more,
of the weight of the tobacco used to provide the extract.
CA 02214036 1997-08-27
WO 93t1267~ 15 PCr/CA92/00566
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CA 02214036 1997-08-27
- 16 -
EXAMPLE 5
10 g samples of a Virginia lamina tobacco blend were
mixed with 300 ml of solutions containing 50 mg of type
XXIII protease enzyme (Sigma No. P4032) and/or various
amounts of SDBS. The tobacco material was left in
contact with the solution for 4 hours at room temperature
and then rinsed and dried. When the solutions were added
sequentially, the tobacco was rin~ed between treatments.
Tables IV and V give details of the treatments and
Rjeldahl nitrogen results. Sequential treatment with
this enzyme, particularly when enzyme treatment preceded
surfactant treatment, resulted in a significantly
reduced nitrogen as compared with simultaneous addition
of the reagents.
Table IV
% N
Unextracted tobacco 2.20
Water extracted tobacco2.03
SDBS only (6.0 g) 1.66
Enzyme only (50 mg) 1.30
Table V
% N
SDBS (lst) Enzyme (lst)
SDBS + Enzyme Added Together Enzyme (2nd) SDBS (2nd)
1.5g 50mg 1.27 0.76 0.49
3.3g 50mg 1.40 0.90 0.48
4.5g 50mg 1.46 0.84 0.57
6.0g 50mg 1.46 0.97 0.68
Various changes and modifications may be made in
practising this invention without departing from the
spirit and scope thereof.
