Tobacco extract treatment with insoluble adsorbent

This invention provides a method for reducing the protein content of tobacco material which includes extracting the tobacco material with a solution containing a surfactant. The tobacco material may be first extracted with an aqueous solvent to produce an aqueous extract before being treated with the solution containing a surfactant. This invention also provides a method for removing polypeptides from an aqueous extract of tobacco material which includes treating the extract with an insoluble adsorbent selected from the group comprising hydroxyapatite and a fuller's earth mineral such as bentonite. Treatment of the aqueous extract with bentonite will produce an extract having a reduced pigment and polypeptide content.

BACKGROUND OF THE INVENTION 
Several 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. Several inventors have found that proteolytic 
enzymes will break down tobacco protein into readily soluble fragments and 
that strip or cut tobacco can be treated by such enzymes. Thus Gaisch et 
al. (U.S. Pat. No. 4,407,307) described 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. Bernasek et 
al. (U.S. Pat. No. 4,887,618) describe a process in which tobacco is first 
extracted with water. 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. The advantage described by 
Bernasek et al. for this process is that the water soluble flavour 
components of tobacco and the nicotine can be retained in the final 
product. 
The above described processes 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. Poulose et al. (U.S. 
Pat. No. 4,716,911) has also realized this disadvantage and proposed using 
either an alkali or a combination of a protease and a non-protease 
depolymerase to effect protein removal in an overall processing scheme 
similar to that of Gaisch et al. However, we have found that alkaline 
solutions at the strengths quoted by Poulose et al. may have a deleterious 
effect on the physical structure of the tobacco. Moreover, the use of a 
protease combined with a depolymerase may not be an economical approach to 
protein removal. 
It is desirable to provide a technique for protein removal from tobacco 
material which does not cause a physical degradation of the tobacco 
structure and is economical and efficient. Tobacco material includes 
tobacco solids and any solid form of tobacco including cured tobacco. 
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 back to tobacco 
material. In the aforementioned patent of Gaisch et al., this was 
accomplished by assimilation of protein fragments by yeast. Clapp et al. 
(U.S. Pat. No. 4,941,484) describes the use of ultrafiltration to remove 
high molecular weight compounds (e.g. proteins) from an aqueous extract of 
tobacco before the extract is added back to protein-reduced tobacco. The 
process of Gaisch et al. is complicated by the requirement to ferment the 
aqueous extract in the presence of yeast. .The ultrafiltration process of 
Clapp et al. requires the use of ultrafiltration apparatus and may not be 
useful for the removal of proteins or polypeptides outside the cut-off 
values of the ultrafiltration membrane employed in the procedure. 
It is also known to treat aqueous extracts of tobacco with solid adsorbents 
which will remove polyphenols from the extract according to the patent of 
Jacin, et al. (U.S. Pat. No. 3,561,451). Such adsorbents include alumina 
and polyamide 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. 
SUMMARY OF THE INVENTION 
This invention provides methods which involve the extraction of tobacco 
material with surfactants either used alone or in combination with a 
proteolytic enzyme. In 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. 
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 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 
known to be useful for absorbing proteins in acidic beverages such as beer 
and wine but would not be expected to be effective for removal of proteins 
from more basic solutions such as a tobacco extract. Furthermore, it is 
also 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 
often advantageous because such compounds tend to darken tobacco material 
when the extract is applied to the material, particularly if the extract 
has been heated (for example, to facilitate concentration of the extract). 
Accordingly this invention provides a method for reducing the protein 
content of tobacco material which includes extracting the tobacco material 
with a solution containing a substance selected from the group comprising 
a surfactant and a surfactant combined with a proteolytic enzyme. This 
invention also provides the preceding method wherein the tobacco material 
to be extracted with the solution has been previously extracted with an 
aqueous solvent to produce an aqueous extract. 
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. 
In one aspect of this invention, the tobacco is extracted directly with an 
aqueous solution of a surfactant or a mixture of a surfactant with a 
proteolytic enzyme. 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. 
In another aspect of this invention, the tobacco is first extracted with an 
aqueous solvent. The extract is separated from the insoluble tobacco 
residue and retained for subsequent reconstitution. The extract may be 
treated to remove solubilized proteins (polypeptides) as described below. 
The tobacco residue is resuspended in an aqueous solution of a surfactant 
or a mixture of surfactant and proteolytic enzyme. After further protein 
has been solubilized in this mixture, the solution is discarded and the 
extracted tobacco residue is rinsed and dried. The aqueous extract from 
the initial extraction is preferably concentrated and sprayed back onto 
the tobacco to make a smokable cigarette filler. This embodiment is 
preferred since it is easier to ensure complete removal of surfactant and 
enzyme from the final tobacco product. 
The tobacco extracts described above can optionally be treated to remove 
soluble materials to further enhance tobacco quality. For example, we have 
found that the extract can be treated with 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 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. There are other ways of 
combining the extracts or solutions with an insoluble adsorbent that are 
well known and may be used in the method of this invention. For example, 
the adsorbent may be contained in a column or other suitable container and 
the extract is allowed to flow through the column or container to permit 
adsorption to occur. 
It will be apparent that the methods of this invention may be combined with 
known methods for treating tobacco to obtain the advantages of this 
invention.

DETAILED DESCRIPTION OF THE INVENTION 
In one embodiment of this invention, strip, cut or ground tobacco 11, and 
preferably cut tobacco, is extracted at 35.degree.-65.degree. C. in an 
aqueous solution 12 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. 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*). Cationic and non-ionic surfactants 
may be used but these have been found to be less effective than the 
anionic surfactants. The surfactant is added to the solvent in the 
concentration range 0.1%-5% w/v solution. The proteolytic enzyme, if used, 
is 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 lnc. have been found to be effective for protein removal from 
tobacco. The proteolytic enzymes are added to the solution in the 
concentration range 0.1%-5% w/w of the tobacco material. 
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The suspension of tobacco material in the solution of surfactant and 
proteolytic enzyme is stirred gently for 1-18 hours. The extracted tobacco 
13 is separated from the solubilized tobacco 14 components by filtration 
or centrifugation 15. 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, proteins and/or polypeptides and amino 
acids, pectins, polyphenols, flavours, inorganic salts, etc. 
The extract 14 may be treated in a number of ways to remove surfactant and 
polypeptides 17, or other components, before the extract is added back in 
concentrated form to the extracted tobacco. 
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.degree. C. is 
effective. Remaining 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. 
Protein (polypeptides) 17 may be removed from the solution 14 using an 
insoluble adsorbent 24 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 is used, all or a large proportion of the dissolved protein is 
removed. 
Bentonite is also an effective adsorbent for polypeptides. 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 protein. 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-mixing with water swells the 
bentonite, 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 which, if not removed, tend to darken 
the extract after concentration, particularly if the extract is heated. 
In the case of bentonite, it appears that a tobacco extract is an effective 
buffer against the adsorbent's tendency to make a solution more alkaline. 
Although it is generally unnecessary in the methods of this invention to 
adjust the pH 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 18 using 
the same methods as for absorption of polyphenol. 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 19 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 by Molyneux (U.S. Pat. No. 3,847,163). However, 
other methods of concentration, particularly those which preserve the 
flavour and other components of the extract are known and can be used. 
The extracted tobacco 13, if in the cut or strip form, may be dried 22 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 20 onto the tobacco, for example 
during or after drying. This results in a tobacco 21 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 11 is first extracted 
with an aqueous solvent 12 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 and one hour at a 
temperature between 15-60.degree. C. The preferred conditions are 1/2 hour 
at 25.degree. C. This extraction step results in some of the protein and 
most of the sugars, nicotine, amino acids, polyphenols, etc. being removed 
from the tobacco into solution. The aqueous extract may be separated 15 
from the tobacco by filtration or centrifugation. 
Polypeptides, polyphenol 18, and pigment compounds etc. can be removed from 
this extract 14 by the methods described in the first embodiment. The 
extract may be concentrated by reverse osmosis or by other known methods. 
The extracted tobacco is subjected to a further extraction step 23 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 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 the concentration range of 0.1-5%. If surfactant alone is 
used, the tobacco slurry is agitated gently for 1-18 hours at 
24.degree.-65.degree. 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.degree.-40.degree. C. should be used to avoid denaturing the 
enzyme. 
Following extraction, the tobacco may be separated from the solution by 
filtration or centrifugation and rinsed thoroughly with water. The tobacco 
residue may then be dried and the concentrated extract sprayed back onto 
the tobacco material, as described in the first embodiment. 
EXAMPLE 1 
Two hundred and fifty grams (250 g) of a single grade of flue-cured 
tobacco, cut at 35 cpi, was extracted with 5 liters of water containing 
100 g of sodium dodecylsulphate (SDS). The extraction was carried out for 
18 hours at 60.degree.-70.degree. C. with gentle stirring. The tobacco 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 weight basis). Thus 82% of the nitrogen in the tobacco was 
solubilized. 
The extract was cooled to 4.degree. C. and the precipitated SDS collected 
by filtration. This resulted in recovery of 68% of the SDS. The remaining 
SDS was precipitated by adding 6 g of CaCl.sub.2 to the solution. The 
precipitate was removed by filtration. 
Fifty grams (50 g) of hydroxyapatite 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%. 
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The extract was allowed to evaporate at 25.degree. C. until it was 
sufficiently concentrated to spray back onto the treated tobacco. 
EXAMPLE 2 
Five hundred grams (500 g) of a single grade of flue-cured tobacco, cut at 
35 cpi. was extracted with 10 liters of water for 18 hours at 
60.degree.-70.degree. C. 
The tobacco was separated from the solution by filtration and thoroughly 
rinsed with warm water. The water extracted tobacco residue was dried to 
13% moisture in a rotary drier. 
The water extracted tobacco residue was divided into 20 g portions and each 
was re-extracted at 60.degree.-70.degree. C. for 18 hours in 600 ml of a 
solution containing 0-15 g of sodium dodecylbenzenesulfonate. The 
surfactant treated tobacco 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. 
TABLE I 
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SDBS 
concentration Kjeldahl Nitrogen 
(g/l) % 
______________________________________ 
0.0 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 
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EXAMPLE 3 
Ten gram (10 g) portions of water extracted tobacco residue such as was 
procured in example 2 were dispersed in a solution containing 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 in a rotary dryer. The results for Kjeldahl nitrogen determinations 
on the tobacco residues are given in table II. 
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TABLE II 
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SDBS Savinase Kjeldahl Nitrogen 
(g) (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 
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EXAMPLE 4 
300 g of flue-cured shredded tobacco was extracted with 6 liters of water 
for 1 hour at 30.degree. C. The tobacco extract was separated from the 
tobacco by centrifugation and divided into 200 ml aliquots, which were 
treated with various quantities of either hydroxyapatite or bentonite. 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. 
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Various changes and modifications may be made in practicing this invention 
without departing from the spirit and scope thereof. 
TABLE III 
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Protein Kjeldahl 
Absorbent Concentration 
Nitrogen Nitrogen 
Nicotine 
Total Sugars 
Sugars (mg/ml) 
(as % Tob. wt.) 
(Control = 100) 
(%) (%) (%) 
__________________________________________________________________________ 
Hydroxyapatite 
0 (0) 100 2.29 4.21 36.7 
8 (16) 52 2.21 4.26 37.0 
24 (48) 57 2.17 4.26 37.2 
60 (120) 14 2.29 4.28 37.3 
Bentonite 
0 (0) 100 2.33 4.20 38.1 
0.5 (1) 12 2.35 4.17 
1.0 (2) 20 2.26 4.06 
1.5 (3) 16 2.33 3.95 
2.0 (4) 3 2.27 3.83 
2.5 (5) 1 2.21 3.53 
4.0 (8) 5 1.97 3.21 
5.0 (10) 3 1.83 2.92 39.5 
7.5 (15) 0 1.94 2.23 
10.0 (20) 0 1.61 1.62 
20.0 (40) 3 1.37 0.54 40.2 
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