Note: Descriptions are shown in the official language in which they were submitted.
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BACKGROUND OF THE INVENTION
_
(A) Field of the Invention
The present invention relates to a process o~ reducing
the nitrate and nicotine contents of tobacco by treating the
tobacco with a culture of a microorganism. More specifically,
the invention relates to a process for treating tobacco to
reduce the nitrate and nicotine contents thereoE, which, when
incorporated into a tobacco smoking product, yields smoke with
reduced nitrogen oxides, hydrogen cyanide and nicotine
deliveries without loss of desirable flavor and taste
properties or other smoking qualities.
tB) Prior Art
For various reasons, it is often desirable to reduce the
nitrate and nicotine contents of tobacco. For example, in
recent years, low nicotine content cigarettes have gained
substantial consumer acceptance. Also, demand has increased
for low delivery cigarettes and numerous techniques have
become available for reducing either the nitrate content or
the nicotine content of tobacco.
In the removal or reduction of the nitrate content, the
most common method has included the use of chemical agents in
selective nitrate removal from tobacco extracts by ion
retardation techniques. Reduction of nicotine content from
tobacco has been accomplished by both chemical means as well
as by microbial treatment. ~.S. 4,011,141; U.S. 4,037,609;
and U.S. 4,038,993 teach microbial treatment means for the
reduction of the nicotine content of tobacco. However, there
is no treatment known which enables slective simultaneous
reduction of both the nitrate and nicotine content of tobacco
in one treatment without reducing all flavor components,
particularly one including the use of microorganisms.
SUMMARY OE' THE INVENTION
It is an object of the present invention to provide a
process for reducing the nitrate and nicotine content of
tobacco. It is another object of this invention to provide
a process for the preparation of an aqueous medium containing
a microorganism which may be used for the degradation of
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'7:3L
nitrate and nicotine content of tohacco materials. Other
objects and advantages of this invention will become apparent
to those skilled in the ar-t upon consideration of the
accompanying disclosure.
The present invention resides in the recognition that
certain microor~anisms in an aqueous solution, when coming in
contact with tobacco, degrade the nitrate and nicotine content
of t'ne tobacco. It has been found that tobacco material
treated with a pure culture of a microorganism grown in a
nitrate-containing medium degrades both nitrate and alkaloids
(nicotine) in tobacco materials simultaneously. In so doing,
a tobacco material is produced that, when placed in a blended
cigarette, contributes to decreasing deliveries of nitrogen
oxides, hydrogen cyanide, and nicotine. The preerred culture
is Cellulomonas sp. and is fully described in U.S.A. Patent
4,038,993, and includes a preferred nitrate-containing
compound added to the growth media, potassium nitrate.
However, it is realized that other cultures may be used and
other ni~rate-containing compounds, such as sodium nitrate,
ammonium nitrate, and the like may also be used.
Using the culture o~ the present invention, it is
practical to treat tobacco lamina or stem and remove nitrate
and nicotine simultaneously or to make a water extract of
either material and remove nitrate and nicotine and -then
reapply treated ex-tract to the original tobacco materials or a
reconstituted tobacco. The capability of treating the extract
and then reapplying it to the original tobacco avoids the
solubles weight loss encountered when using water extraction
and discard as a vehicle Eor removing nitrate and nicotine.
It also avoids the loss of other desirable tobacco components
encountered in water extraction and discard. The process of
the present invention also oEfers potential for removing both
nitrate and nicotine in reconstituted tobacco production
systems, wherein the tobacco is extracted and the extract is
added back in subsequent process steps, since this enzyme
(microbial) system functions efficiently in a liquid system.
,~,
In the pro~ess, the nitrate is broken down and converted to
gaseous nitrogen, which is released to the atmosphere. I-t has
been found that the pH of the aqueous medium containing the
microorganism prior to the addition to the tobacco materials
must be maintained in the range o~ at least greater than 5.~
in order to profide a microorganism which will successEully
and simultaneously degrade nitrates and nicotine. The
preferred initial pH~of the aqueous medium is about 7 to 9~5O
It has also been found that the nitrate containing compound in
the aqueous medium must be at least about 0.1 percent by
weight in the medium and preferably about 1 percent. Even
though higher percentages of nitrate-containing materials may
be used, increasing the nitrate-containing compound in excess
of 1 percent by weight does not appreciably assist in the
degrading capabilities of the microorganisms, although higher
concentrations are usable and the organism will degrade
nitrate compounds at higher concentrations.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
According to the present invention, one preferred method
for simultaneously reducing the nitrate and nicotine content
of tobacco is to prepare an aqueous medium containing
microorganisms.
In the preparation o an aqueous medium, a nutrient agar
(first) solution is prepared by adding a commercially
available nutrient agar to distilled water, the amount of agar
generally being at least 5 grams per liter. To this is added
a nitrate-containing compound, preferably potassium nitrate,
which is at least 0.1 percent by weight of nitrate per volume
of water and is generally about 1 percent by weight of nitrate
per volume of water. This solution is then sterilized as
tubed slants; that is, test tubes containing the nutrient agar
are placed at a slant to provide a slanted surface, in an
autoclave for at least iteen minutes and at least 15 psig
and at least 121C. The sterilization medium is then placed
in a refrigerator for later use.
A second solution is then prepared which includes
nicotine and a nitrate containing substance therein which is
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to be treated by the culture grown in the sterilized medium.
One such second solution may be a nutrient broth containing
only nitrates therein which is prepared by dissolving a
commercially available nutrient broth in distilled water, the
amount of nutrient broth being from about 5 to 10 grams per
liter. However, it is realized that those skilled in the art
may vary the nutrient bro-th concentration and achieve a
useable culture. This solution is also sterilized for at
least 15 minutes at at least 15 psig and 121C or greater in
an autoclave. Potassium nitrate or other nitrate-containing
compounds may be added to this solution prior to the
sterilization.
Another example of a second solution may be a -tobacco
extract broth containing both nitrates and nicotine. The
tobacco extract broth is prepared by taking usually about 100
grams of tobacco material, such as, a flue cured burley stem
mixture and mixing this with about 1,000 milliliters of water
and then cooking the mixture in an autoclave for a least 30 to
60 minutes at at least 15 psig and 121C or greater. The
resultant liquid extract is then removed and the liquid volume
is adjusted to the original amount of the extract by adding
distilled water. The extract is then mixed with yeast
extract, the yeast extract being generally at least 0.3
percent by weight to volume of liquid. However, greater
amounts of yeast extrac-t may be usd if desired. The mixture
is dispensed into flasks that are co-tton-plugged and
sterilizecd for a least 15 minutes at 15 psig or greater and
121C or greater for subsequent culture propagation. Prior to
use for culture grownth, the pH is adjustecl with appropriate
acid or base to about 7.2.
The microorganism, preferably Cellulomonas sp., is
incubated on nutrient agar slants, including the
nitrate-containing compound, for 3 to 5 days at 20C to 40C.
The resultant grownth is then used to inoculate the ~obacco
extract broth, the inoculum being removed from the slants by
washing the slant surface with a predetermined amount of
sterile distilled water. Tne tobacco extract broth is then
, ,,'~,
subjected to agitation for generally about 24 hours a-t about
20C to 40C to promote growth of the microorganism which was
addéd. Lesser or greater growth periods, up to as long as
about 48 hours, are acceptable.
The resultant inoculum is -then ready for use in the
treatment of additional tobacco materials to reduce the
nitrate and nicotine content thereof.
A more comprehensive understanding of the invention can
be obtained by considering the following examples. However,
it should be understood that -the examples are not intended to
be unduly limitative of the invention.
Example 1
The following example demonstrates the procedure that
was followed in -the preparation if inoculum.
(a) Nutrient agar ~ 1.0% potassium nitrate.
Commercially prepared ~utrient Agar (dehydrated for~)
from Difco Laboratories was added to distilled water in -the
ratio of 23 grams per literO The 23 grams of nutrient agar
contained 3 grams of beef extract; 5 grams of peptone and, 15
grams of agar. To this solution was added 1~ of potassium
nitrate by weight to volume of water. The resulting solution
had a final pH of 6.8.
This medium was then sterilized as tubed slants in an
autoclave for 15 minutes at 15-psig and 121C and refrigerated
for later use to grow cultures.
(b) Nutrient Broth.
A solution of Nutrient Broth media, was prepared by
adding dehydrated Nutrient Broth from Dico Laboratories at a
ra-te of 8 grams per liter to distilled water. The Nutrient
Broth contained 5 grams of peptone and 3 grams of beef
extract. The resulting aqueous medium was then sterilized or
15 minutes at 15 psig and 121 C for later use in culture
growth.
(c) Flue-Cured/Burley Stem Tobacco Extract Broth.
A flue-cured/burley stem tobacco extract broth was
prepared by adding 100 grams of flue-cured/burley stem to 100
ml of water and cooked in an autoclave for 40 minutes at 15
,~
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psig and 121C. The resultant liquor extract was removed and
the liquid volume was adjus-ted to its original amount with
distilled water. The liquor was then mixed with yeast extract
at a rate of 0.5~ by weight of yeast extract per volume of
liquor and the mixture dispensed into flasks which were -then
cotton-plugged and sterilized for 15 minutes at 15 psig and
121C for culture propogation.
(d) Broth Inoculation.
The micrborganism, Cellulomonas sp., is incubated on the
nutrient agar slants for from 3 to 5 days at 30C. Liquid
media, for example, Nutrient Broth or flue-cured/burley stem
tobacco extract broth are inoculated with a sterile water wash
from slants at a 2% (v/v) rate. The pH of the broth prior to
inoculation is adjusted with hydrochloric acid or sodium
hydroxide to a pH of 7.2 to 7.5. The flasks are then
subjected to rotary agitation for approximately 24 hours at
30C and 220 rpm.
Example 2
This example demonstrates the nitrate and nicotine
degradation that occurs in burley stem extract at different p~l
levels.
A water extract of burley stem was prepared according to
the procedure described in Example l(c) and dispensed into 500
ml Erlenmeyer flasks at 250 ml/flask. These media were used
to determine nitrate and nicotine degradation capabilitles of
Cellulomonas sp. with the results shown below.
__
NO3 Alkaloid (Nicotine)
p~ (~g/ml) (mg/ml)
Burley Stem Extract Broth -
pH 7.2
0 hours 7.18 220 0.32
7 hours 7.08 80 0.04
25 hours 7.75 0 0.02
30 hours 8.15 0 0.02
,~",
5~Z7~
. ~
NO3 Alkaloid (Nicotine)
pH (~g/ml) (mg/ml)
Burley Stem Extract Broth -
pH 5.6
0 hours 5.60 295 0.41
7 hours 5.59 305 0 39
25 hours 5.65 265 0.39
30 hours 5.70 300 0.37
Burley Stem Extract Broth -
10 pH 4.8
0 hours 4.82 305 0.41
7 hours 4.85 310 0.42
25 hours ~.90 285 0.40
30 hours 4.80 300 0.40
It can be seen from the above data that Cellulomonas
sp. at pH of 7.2 degraded most of the nitrate and nicotine
available in the extract, whereas at a lower pH (5.6 and 4.8),
very little, if any, degradation occurred.
Example 3
This example demonstrates nitrate degradation in
materials other than tobacco.
Cellulomonas sp. was grown under ~he conditions
described below in a Nutrient Broth + 0.1~ KNO3 medium using a
New Brunswick Scientific Fermentor (MF21~). The inoculating
culture was prepared as in Example 1 using the nutrient agar
of Example l(a) and the nicotine-free nutrient broth of
Example l(b). Growth conditions were:
Agitation (rpm) - 300
Aeration (cc/min.) - 4,000
Medium - Nutrient Broth + 0.1% KNO3 (wt/v)
Medium Volume (L) - 8
Temperature (C) - 30
pH - 7.0
Inoc. Rate (v/v) - 5~
Inoc. Age (hrs.) - 20
Inoc. Medium - Nutrient Broth + 0.1~ KNO3
Antifoam - P-1200 (Dow Chemical Company)
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pH Control - 2N HCL
2N NaOH
The following changes in nitrate content occurred:
Growth Time NO3 Cell Count
(hrs.) (~g/ml) pH (Xl05)
-
Inoculum 138 7.70 4,100
1 hr. after inoc.126 6.90 53
2 hrs. after inoc.120 7.00 350
4 hrs. after inoc.114 7.20 1,600
6 hrs. after inoc.108 7.20 1,100
21 hrs. after inoc. 132 7.18 3,400
29 hrs. after inoc. 0 7.05 3,100
45 hrs. after inoc. 0 7.55 4,700
It can be~seen from the above data that nitrate was removed by
the Cellulomonas sp. culture prior to 29 hours at a pH of
7.0 7.2.
Example 4
This example demonstates the nitrate and nicotine
degradation that occurs in burley extract broth having a
relatively high nitrate concentration.
Cellulomonas ~.was grown in a New Brunswick Fermentor
(MF214) in burley extract broth prepared as in Fxample l(c)~
Conditions for growth were the same as in Example 3 except
that the growth medium was burley extract broth.
The following changes in nitrate and alkaloid content
occurred:
Alkaloid
Growth Tirne NO3 (Nicotine)
(hrs.) (~g/ml) (mg/ml) pH
Before Inoculation4,680 0.430 6.55
Inoculum 0 0.0~8 8.14
After Inoculation4,380 0.240 7.02
lhr. after inoc.4,500 0.202 6.90
2 hrs. after inoc.4,380 0.136 6~91
4 hrs. after inoc.4,200 0.036 7.18
: 6 hrs. after inoc.2,910 0.040 7.62
8 hrs. after inoc.2,040 0.038 7.57
, . ~ .
,~
.
.. . . . .
:
:,
Alkaloid
Growth Time NO3 (Nicotine)
(hrs.) (~g/ml) (mg/ml) pH
9 hrs. after inoc2,040 0.038 7.82
24 hrs. after inoc.1,350 0.040 7.20
26 hrs. after inoc.1,320 0.040 7.22
30 hrs. after inoc.1,380 0.036 7.21
48 hrs. after inoc.900 0.034 7.05
50 hrs. ater inoc.900 0.034 7.00
It can be seen from the above data that Cellulomonas sp.
degraded most of the nitrate and nico-tine available in the
extract
Example S
This example demonstrates different levels of a nitrate-
containing compound that may be used in the growing of a
microorganism for degrading nitrates.
Cellulomonas sp. was grown in a nicotine free nutrient
broth (N~) ~ 0.1~ KNO3 prepared as in Example l(b). The
culture was used to inoculate nutrient broth with varying
levels of KNO3 added on a wt/vol basis. The followiny changes
occurred during agitation oE these cultures at 30C and 160
rpm (rotary).
NO3 (~g/ml) pH
0 hrs.25 hrs. 0 hrs. 25 hrs.
Inoculated
335 155 6.97 8.17
500 240 7~00 7.g5
3,0002,370 6.95 8.05
4,9~04,560 6.92 8.15
Control - Uninoculated
~60400 6.99 7.19
It can be seen that Cellulomonas sp. degraded a portion of
the nitrate at all initial nitrate concentrations from 335
~g/ml to 3000 ~g/ml nitrate in Nutrient Broth and degraded a
small amount of the nitrate above 4,980 ~g/ml. The slight
change in "control" nitrate concentration is close to
analytical error. It was not due to microbial action since
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no culture was added to the control media.
E~ample 6
This example demonstrates the effect of aeration on the
cultures growth in tobacco extract.
Cellulomonas sp. was grown in a water extract o~ flue-
cured/burley stem, prepared as described in Example l(c),
under the following controlled conditions in a New Brunswick
Scientific Fermentor~(MF214):
Agitation (rpm) - 600
Aeration (cc~min.) - 8,000
pH - 7.3
TeMperature (C) - 30
Time (hrs.) - 22
Antifoam - P-1200 (Dow Chemical Company)
Inoc. Rate (v/v) - 5%
Medium (vol.) - 8L
Medium Type ~ Water extract of flue-cured/
burley stem. pH was controlled
using 2N HCL and 2N NaOH
Cell mass increase and chemical changes during growth were:
Cell Nitrate Alkaloid (Nicotine)
Time Count (X106/ml) pH (~g/ml) (mg/ml)
Before Inoculation 0 7.31 1,S34 0.32
Inoculum 5,000 8.17 0 0.02
After Inoculation 350 7.40 1,486 0.30
1 hr.after inoc. 490 7.40 1,448 0.27
3 hrs. aEter inoc. 640 7.41 1 r 491 0.20
5 hrs. aEter inoc. 1,220 7.35 l,449 0.08
22 hrs. after inoc.4,200 7.23 1,450 0.02
The above data indicate that under the conditions used,
specifically a high (8,000 cc/min) aeration rate, nitrate is
not degraded but alkaloids were degraded.
The culture grown in this fashion was used to treat
burley lamina as follows:
Tobacco Dry Wt. Culture NaOH (lN) Water
(lbs.) (ml) (ml) (ml)
3.8 2,436 379.5 2,2~9
~5~
Treatment was conducted in a plastic bag (non-aerated
environment) at 30C for 24 hours with the following results:
Treatment NO3 Alkaloids Moisture
Time (hrs.) (~ ) (..%.? .. ~ P~
0 3.54 1.42 7~.4 7.33
24 0.22 0.32 76.4 8.38
It can be seen that in a non-aerated environment, the
_ellulomonas sp. degraded both nitrate and nicotine. The
lowered nitrate and nicotine burley tobacco was blended with
other tobacco materials and compared to a control blend
containing untreated burley tobacco with results as shown
below:
Blend Chemical Properties
Alkaloids
NO3 (Nicotine)
~ ) pH
Control** 1~63 1.79 5.47
Experimental* 1.04 1.32 6.00
** Contained untreated burley
lamina
* Contained treated burley lamina
These blends were manufactured into cigarettes and
machine smoked with the following smoke delivery reductions in
nitrogen oxides, hydrogen cyanide and nicotine.
Per Puff Deliveries
NOx HCN Nicotine
(~g) (~9) (mg) Puffs
Control 54 28.4 0.13 7.3
Experimental33 22.8 0.11 7.2
The smoke data show: 38.8% reduction in nitrogen oxides
(NOx); 19.7% ~eduction in hydrogen cyanide and a 15.3%
reduction in nicotine.
Example 7
This example demonstrates the effect of aeration in the
culture growth wherein reduction aeration provides the
environment for nitrate degradation in liquid systems.
Cellulomonas _ . was grown in a water extract of flue-
.~ ;27~
cured/burley stem, prepared as described in Example ltc),under the following conditions in a New Brunswick Scientific
Fermentor (MF214):
Agitation (rpm) - 600 (lst 4 hrs.) 300 llast 20 hrs.)
Aeration (cc/min.) - 8,000 (lst 4 hrs. only)
none (last 20 hrs.)
pH - 7.0
Temperature (C) - 30
Time (hrs.) - 24
Antifoam - P-1200 (Dow Chemical Company)
Inoc. Rate (~) (v/v) - 5
Medium (vol.) -8L
Medium type - Water extract of flue-cured/
burley stem
pH was controlled using 2N HCL and 2N NaOH.
Cell mass increase and chemical changes during growth
were:
Alkaloid
Cell Nitrate (Nicotine)
Time Count (X106) pH(~/ml) (mg/ml)
Before Inoculation * 7.12 3,173 0.48
Inoculum 7,400 7.4050 0.05
After Inoculation155 7.27NoD. N.D.
1 hr. after inoc.430 7.25N.D. N.D.
2 hrs. after inoc. 410 7.17 N.D. N.D.
3 hrs. after inoc. 840 7.14 2,534 N.D.
4 hrs. after inoc. 1,040 7.02 1,171 0.06
6 hrs. after inoc. 1,490 7.08 50 N.D.
~ hrs. after inoc. 2,500 7.15 50 0.06
24 hrs. after inoc. 8,000 7.34 50 0.06
*Slight conta~nination
N.D. = No Analysis
The above data indicate that under the conditions used,
specifically an initial high aeration rate (4 hrs.), and then
no appreciable aeration (20 hrs.), both nitrate and alkaloids
were degraded. More specifically, it can be seen that the
nitrate degradation started very soon after the aeration was
discontinued.
`,~
The culture grown as described in this example was used
to treat a flue-cured/burley stem mixture for 27 hours by
applying inoculum at a rate of 2.4 mls./gram tobacco weight
and incubating the tobacco at 30C. The following chemical
changes typically occurred:
Treatment NO3 ~lkaloids
Time (hrs.) (~) (%)
0.0 2.8 0.34
6.5 2.8 No Data
1027.0 0.4 0.0~
The treated tobaccos were blended with other tobacco materials
and compared to a control blend, which contained untreated
stems, as shown below for two different inclusion levels of
treated materials:
Blend Chemical Properties
Alkaloids
Stem NO3 (Nicotine)
Sample Inclusion Leve (%) (%) pH
Control Normal 1.33 1.85 5.45
2.5x normal 1.67 1.47 5.48
Experimental* Mormal 0.85 1.79 5.77
2.5x normal 0.69 1.26 6.42
*Contained treated stem materials.
These blends were manufactured into cigarettes and machine
smoked with the following difEerences resulting between
control and experimental products:
Per Puff Deliveries
Stem MOx HCN Nicotine
Sam~ Incluslon Levels (~g) (~ (mg) PuEfs
30 Control Normal 44.4 24.4 0.13 8.8
2.5x normal 51.8 18.7 0.11 8.3
Experimental Normal 32.2 19.1 0.13 9.5
2.5x normal 20.7 7.4 0.09 10.0
The smoke delivery date shown: 27% and 60% reductions in
nitrogen oxides and 21.7% and 60.4% reductions in hydrogen
cyanide for normal and 2.5x normal inclusion rates oE treated
; stem material. The data also reflect a significant increase
.. ;
,;
:
in puff number where treated materials were incorporated into
the blend at 2.5x normal rate.
Example 8
This example demonstrates the procedure used for
extracting tobacco lamina with water to remove nltrate and
nicotine, treating the extract with Cellulomonas sp. to remove
the nitrate and nicotine, followed by adding the modified
extract back to the~original tobacco~
A tobacco extract was prepared by mixing lO0 gms. of
burley lamina with one liter of water and allowing it to stand
at room conditions for two hours. At this point, the extract
was collected by decanting the liquid and pressing the tobacco
to remove additional liquid. The tobacco was spread to dry in
room air while the extract (700 ml) was subjected to microbial
treatment as discussed hereinafter.
A mature culture of Cellulomonas sp. was grown in a
separate tobacco extract medium, prepared as described in
Example l~c) and added to the tobacco extract as described
above, at a lO~ (v/v) rate. Prior to adding the culture, the
extract p~I was raised to 7.0 + O.l. The culture was incubated
in the extract in an Erlenmeyer flask on a rotary shaker at
30C. The following chemical changes occurred across the 18
hour incubation time:
Cellulomonas sp. Treatment of Burle~ Lamina Extract
NO3 Alkaloid (Wicotine)
~Lml) (mg/ml)
Burley lamina extract l,8-12 1.47
Mature Cellulomonas sp. culture 0 0
Extract after treatment 66 0.09
It can be seen that nitrate and nicotine were almost
completely degraded (96.5~ and 93.9~, respectively) in view of
the treatment.
After 18 hours, the treated extract was added back to
the originally extracted tobacco in three stages because oE
the large amount of extract involved. This was done by adding
a portion, mixing thoroughly, and air drying prior to the next
",
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addition. The following chemical changes occurred during
these procedures:
Tobacco Analysis
NO~%)Alkaloid (Nicotine) (%)
Burley Lamina Before
Extraction 1.96 2.46
Burley Lamina After
Extraction 0.72 0.97
Burley Lamina After Treated
Extract Addbac~ 0.39 0
It can be seen that the nitrates and alkaloids (nicotine~ are
removed from t'ne extract and, therefore, are significantly
lowered in the tobacco to which treated extract is added
back. 80% of the nitrate and 100~ of the alkaloids were
removed by this method. Partof the nitrate and alkaloids are
removed from tobacco by the culture during drying following
addback.
The tobaccos resulting from this operation were usable
in manufacturing type operations.
Example 9
This example demonstrates some differences in the final
product which can be obtained by using ultrafiltration
equipment in conjunction with tobacco extraction~ extract
treatment and extract addback as described in Example 8.
Tobacco used in this example was t:he same as that used in
Example 8.
A burley lamina extract was prepared as in Example 8.
The extract was then filtered with a 0.2 mlcron pore size
filter in an Amicon ultrafiltration device (Model TC~10) prior
to inoculating the filtered extract with Cellulomonas sp. and
treating it as described in Example 8. Following treatment,
the extract was again fillered before addback procedures were
started. The materials retained on the filter during the
first filtration were also added back to the extracted
tobacco.
The materials retained b~ the filter during the second
/ filtration were not added back to the tobacco. The following
`1~ chemical changes occurred in the extract:
.,
.
,
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- 16 -
Chemical Changes Across Ultrafiltrati~n and Cellulomonas sp.
Treatement of surley Extract
NO3 Alkaloid (Nicotine)
(~g/ml) (mg/ml)
Burley Lamina Extract1,872 1.47
~ature Cellulomonas _ . Culture 0 0
Extract After Filtration2,0~8 1.~8
Extract After Cellulomonas sp.
Treatment 110 0.12
The following chemical c'nanges were measured in the extracted
tobacco across extraction and treatment:
Tobacco Analysis
Alkaloid
Burley Lamina NO~ (%) (Nicotine) (%)
Before Extraction 1.96 2.46
After Extraction 0.72 0.79
After Treated Extract Added Back 0.75 0.72
It can be seen that nitrates and alkaloids (nicotine) are
removed from the extract by Cellulomonas sp. but, as opposed
to Example 8, no further removal from the extracted tobacco
occurs during addback procedures. In this example, the
microbial culture never comes into contact with the tobacco,
whereas in Example 8, the culture does contact the tobacco
during addback.
j The tobaccos resulting from this operation were usable
in manufacturing type operations.
Example 10
This example demonstrates the effectiveness of
Cellulomonas _ . in removing nitrate and nicotine from
reconstituted tobacco materials.
A water exkract broth was prepared as follows:
150g of reconstituted tobacco was pulped in one liter of water
in a Waring blender for one minute. Following this pulping,
the mixture was held at room temperature for 10 minutes after
which the liquid was centrifuyally separated and brought back
to the original volume with distilled water for sterilization
at 121C and 15 psig for 15 minutes. Separate preparations
,. ~.
/
.
were made to which yeast extract (YE) was added at 0~5~
(wt/vol) rate prior to sterilization. Flue-cured/burley stem
extract (with 0.5% yeast extract) was prepared as in Example
l~c) and was used for "Control" extract. The broths' pH was
adjusted to 7.2 prior to inoculation with Cellulomonas sp.
The following results were obtained:
Control
~ Alkaloids
Growth Time (hrs.) NO3 (~g/~l) (Nicotine (mg/ml) pH
0 2,246 0~23 7.30
24 0 0 8.50
48 0 0 8.12
Experimental
Alkaloids
No~(~g/ml) (Nicotine (mg/ml) pH
Without Yeast Extract
0 1,859.0 1.12 7.34
20 24 1,641.0 0.88 7.46
48 39.0 0.08 8.08
With Yeast Extract
0 1,878.0 1.09 7.21
24 0.28 0.35 ~.04
48 0.14 0.06 8.17
It can be seen that the culture can effectively degrade
the nitrate and alkaloids (nicotine) of reconstituted tobacco
materials with or without the addition of yeast extract.
Example 11
This example demonstrates the effects of aerobic and
anaerobic tobacco treatments.
_llulomonas sp. was grown in flue-cured/burley extract
broth, prepared as described in Example l(c) but without yeast
extract added, for 25.5 hrs. in a New Brunswick Scientific
Fermentor (MF214) under the ~ollowing conditions:
Agitation (rpm) - 600 (lst 4 hrs.) 300(1ast 21.5hrs.)
Aeration (cc/min.) - 8,000 (lst 4 hrs.) O(last 2105 hrs.)
Medium - Flue-cured/burley extract broth
Medium Volume (L) - 8
i~
~5~7~
- 18 -
Temperature (C) - 30
pH - 7.0
Inoculum Rate (% v/v) - 5
Inoculum Age (hrs.) - 22
Antifoam - P-1200 (Dow Chemical)
Inoculum Agitation Rate (rpm) - 160
Inoculum Medium - Flue-cured/burley Inoculum for
extract broth MF214 Growth Cycle
Time (hrs. (~g/ml) (mg/ml) pH
Initial 3,565 2.84 7.15
2.5 0 0.24 7.06
At 25.5 hrs., the culture was used to treat flue-
cured/burley stem under aerobic and anaerobic conditions with
the following results-
Aerobic Treatments
Time (hrs.)
.
0 24
pH NO~(~) Alkaloids(%) NO~(~) Alkaloids(~)
6.48 2.75 0.17 0.12 0.10
20 Treated
7.53 2.75 0.17 0.13 0.09
Control 5.20 2.75 0.17 2.72 0.12
Anaerobic Treatments
Time (hrs.)
0 24
-
pH NO~(%) Alkaloids(%) NO~(%) Alkaloids(%)
6.82 2.75 0.17 0.12 0.09
Treated
7.22 2.75 0.17 0.15 0.09
Control 5.20 2.75 0.17 2.78 0.19
All treatments were at 75% moisture content and
conducted at 30C for 24 hours in plastic bags. Also,
anaerobic treatments were conducted in BBL (Baltimore
Biological Laboratories) "GASPAK " anaerobic system jars using
BBL catalyst for tying up atmospheric oxygen.
It is seen from the above data that the present
/
7~L
- 19 -
invention can be carried out under anaerobic conditions and
under conditions when availability of oxygen is not
controlled.
Example 12
This example demonstrates the effects of treating
tobacco with cells as well as supernatant liquor from the cell
growth.
Cellulomonas sp. was grown in Elasks of flue-cured/
burley stem extract broth, with 0.5% (wt/vol) yeast extract
added, prepared as in Example l(c).
Flask Inoculation and incubation were conducted as
described in Example l(d). At the end of the growth period,
the culture was processed as shown in Table A.
,:
- ~o -
TABLE A
Culture
I Split
Treatment Centrifuge (10~000 rpm for 15
A minutes using Type ~SA
/ \ head in Sorvall RC2-B
/ \ centrifuge)
Cell Pellet
Res~uspend to Supernatant
Original Volume
in Sterile
Water ` ~
Split
/ Tobacco Split
/ Treatment
Mix/Suspend Millipore Inoculate
/ \ (0.22u) Fresh Flue-
/ \ F'iltration cured/Burley
Extract
Tobacco
Inoculate Treatment Tobacco
Fresh Flue- Treatment ~ /
cured/Burley
Extract Incubate
Incubate
'~;
'
.
.
7~
- 21
The following resulted from the operation shown in Table
A.
TAsLE 1: CULTURE PREPARATION
NO3 Alkaloids
(~g/ml) (mg/ml) pH
Flue-cured/Burley
EX tract Broth with 0.5% YE
Control O~hrs. 1618 0.290 7~13
(uninoculated) 24 hrs 1550 0.290 7.04
Inoculated0 hrs. 1559 00280 7.11
24 hrs. 39 0.028 8.06
Resuspended Cells 0 0 8.32
Supernatant 36 0.026 8.16
Filtered Supernatant 40 0.026 8.27
Resuspended cells and filtered supernatant were used to
inoculate separate fresh flasks of flue-cured/burley extract
broth at 10 ml/flask (250 ml extract/500 ml flask) and
incubated at 30C for 24 hours at 220 rpm. Extract was
prepared as in Example l(c). The following was obtained:
20 TABLE 2
NO3 Alkaloids
Time (hrs) (~g/ml) (mg/ml) pH
Resuspended Cells 0 1482 0.27 7.02
24 0 0 8.15
Eiltered Supernatant
0 1522 0.27 7.21
24 1022 0.30 7,75
Resuspended cells, original culture, filtered
supernatant and unfiltered supernatant were all used
separately to treat 50 gm samples of flue-cured/burley stem at
about 75% moisture for 24 hours at 30C in plastic bags. A
control sample was pH adjusted and water treated without
inoculum. The following results were obtained:
~( ~
~5~
22 -
TABLE 3: TOBACCO TREATMENTS
Alkaloids
Time(hrs~) NO~(%) _Nicotine)(~) _pH
Control (no inoculum) 0 4.34 0.59 6.83
24 4.12 0.37 6.99
Original Culture0 4.48 0.56 7.22
24 0.61 0.05 8.54
Resuspended cells 0 4.33 0.56 7.03
24 2.82 0018 8.06
Supernatant O 4.65 0.56 7.25
2~ 4.51 0.~2 7.24
Filtered Supernatant 0 4.46 0.57 7~26
24 4.04 0.49 7.12
It can be seen from the above data that the supernatant
liquor when separated from the culture, does not provide the
capability for degradation of ni-trates and nicotine in
tobacco.
