Note: Descriptions are shown in the official language in which they were submitted.
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BACKGROUND OY TEIE INVENTION(A) Field of the Invention
The present invention relates to a process for
reducing the nitrate content of tcbacco materLals by treating
the tobacco with cultures of microorganisms. More
specifically, the invention relates to a process for treating
tobacco materials to reduce the nitrate content thereof,
which, when incorporated into a tc,bacco smoking product, yield
smoke with reduced nitrogen oxides and hydrogen cyanide
deliveries without loss of desirable flavour and taste
properties or other smoking qualilies.
~B) Prior Art
For various reasons, it is often desirable to reduce the
nitrate content of tobacco. For example, in recent years, low
delivery cigarettes have gained substantial consumer
acceptance and numerous techniques have become available for
reducing smoke deliveries.
In the removal or reduction of the nitrate content, the
most common methods have included the use of chemical agents
in selective nitrate and ion removal from ~obacco ~xtracts by
ion retardation (U.S. 3,847,164) and ion exchange
(U.S. 3,616,801) techniques. ~lowever, there is no treatment
known which enables reduction of t:he nitrate content of
tobacco which includes the use of microorganisms.
SUMMARY OF THE INVENTION
-
It is an object of the present invention to provide a
process for reducing the nitrate content of tobacco. It is
another object of this invention t:o provide a process for the
preparation of an aqueous medium containing a microorganism
which may be used for the degradat:ion of the nitrate content
of tobacco. Other objects and ad~antages of this invention
will become apparent to those skilled in the art upon
consideration of the accompanying disclosure.
The present invention resides in the recognition that
certain microorganisms in an aqueous solution, when coming in
contact with tobacco, degrade the nitrate content of the
tobacco. It has been found that tobacco material treated with
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a pure culture of specific microorganisms degrade nitrates in
tobacco materials. In so doing; a tobacco material is
produced that, when placed in a blended cigarette, contributes
to decreasing deliveries of nitrogen oxides and hydrogen
cyanide. A preferred culture includes Micrococcus
denitrificans, (paracoccus denitrificans Am. Type Culture
Coll~ction Accession No. 17741) as described in Bergeys Manual
of Determinative Bacteriology, Edited by R. E. Buchanan and
N. E. Gibbons, pp. 43~-439, 8th E:dition. I~owever, it is
realized that other cultures may also be used, such as:
Micrococcus halodenitrificans, Alcaligenes faecalis, Bacillus
licheniformis, Bacillus stearothernlopllilus, ~rwinia
carotovora, Pseudomonas aeruginosa, Pseudomonas chlororaphis,
Pseudomonas fluorescens, Pseudomonas stutzeri, Thiobacillus
denitrificans. Also, nitrate-containing compounds may also be
used in combination with the microorganisms, such as,
potassium nitrate, sodium nitrate, ammonium nitrate, and the
like.
~sing the culture of the pL esent invention, it is
practical to treat burley or flue-cured lamina or stem and
remove the nitrates therein or to make a water extract of
either material and remove the nitrates and then reapply the
treated extract to the original tobacco materials. 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 for removing nitrate. It also avoids the loss of
other desirable tobacco components encountered in water
extraction and discard. This process is also useable in
reconstituted tobacco production systems wherein the tobacco
is extracted and the extract is a~ded back in subsequent
process steps, since this enzyme (microbial) system functions
efficiently in a liquid system, In the process, the nitrate
is broken down and converted to gaseous nitrogen, which is
released to the atmosphere.
It has been found that the nitrate-containing compound
in the aqueous medium must be at least 0.1 percent by weight
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in the medium and preferably in tl~e range of about 1 percent.
Fven though higher percentages of nitrate-containing compounds
may be used, increasing the nitrate-containing compound in
excess of I percent by weight doe; not appreciably assist in
the degrading capabilities of the microorganisms.
DESCRIPTION OF THE PRE!~ERRED EMBODI~ NTS
According to the present in7ention, one preferred merhod
for reducing the nitrate content of tobacco is to prepare an
aqueous medium containing microorganisms which will degrade
nitrates.
In the preparation of 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. In one proposed
method, 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 abo~lt 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 solution are
placed at a slant to provide a slanted surface in an autoclave
for at least 15 minutes at at lea;t 15 psig and at 121C. The
sterilized medium is then placed in a refrigerator for later
use.
A second solution is prepared which includes a
nitrate-containing substance therein which is to be treatecl by
the culture grown in the first medium. One such second
solution may be a nutrient broth containing nitratcs 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 broth concentration and achieve a useable culture.
This solution is also sterilized Eor 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 steri~ization.
114V876
5 .
Another example of a second solution may be a tobacco
extract broth containing nitrates. The tobacco extract brc>th
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 cookinq
the mixture in an autoclave for about 3n 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 extract may be used if desired. The mixture
is dispensed into flasks that are cotton-plugged and
sterilized for at least 15 minutes at 15 psig or yreater and
121C or greater for subsequent culture propagation. Prior to
use for culture growth, the pH is adjusted with appropriate
acid or base to about 7.2. The microorganism, preferably
Micrococcus denitrificans, is incubated on the nutrient agar
slants for from three to five days at 5 to 37C. The
resultant growth is then used to ~noculate the pH adjusted
tobacco extract broth, the inoculum being removed from the
slants by washing the slant surface with a predetermined
amount of distilled water. The inoculated tobacco extract
broth is then subjected to agitation for generally about 2
hours at 5 to 37C to promote growth of the culture.
The resultant inoculum is then ready for use in the
treatment of tobacco materials to reduce the nitrate content
therein.
In the treatment of tobacco materials (solids), the pH
of the tobacco is adjusted with a base and water mixture to
about 7.0 to 7.2. The culture is then applied along with
additional water and the tobacco ,o treated is usually placed
in plastic bags where nitrate degradation occurs.
A more comprehensive underslanding of the invention can
be obtained by considering the following examples. However,
it should be understood that the examples are not intended to
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be unduly li~itative of the inven~ion.
Example_l
The following example demonstrates the procedure that
was followed in the preparation of inoculum.
(a) Nutrient agar + l 0~ potassium nitrate.
Commercially prepared Nutrient Agar (dehydrated form)
from-Difco Laboratories was added to distilled water in the
ratio of 23 grams per liter. The 23 grams of Nutrient Agar
contained 3 grams of beef extract; 5 grams of peptone and 1
grams of agar. To ~his solution was added 1% of potassium
nitrate by weight to volume of water. The resulting solution
had a final p~ of 6.8.
This medium was then sterilized as tubed slants in an
autoclave for 15 minutes at 15 psig and 121, cooled, and
refrigerated for later use to gro~- cultures.
~ b) Nutrient Broth.
A solution of Nutrient Brotll media was prepared by
adding dehydrated Nutrient Broth from Difco l,aboratories at a
rate 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 Eor
lS minutes at 15 psig and 121C for later use in culture
growth.
(c) Flue-Cured/Burley Stem Tobacco Extr_ct Broth.
A flue-cured/burley stem tohacco extract broth was
prepared by adding 100 grams of f]ue-cured/burley stem to
1,000 ml of water and cooking in an autoclave for 40 minutes
at 15 psig and 121C. The resultant liquor extract was
removed and the liquid volume was adjusted to its original
amount with distilled water. The liquor was then mixed with
yeast extract ~YE) at a rate of 0.5% by wei~ht of yeast
extract per volume of liquor and the mixture dispensed into
flasks that were cotton-plugged and sterilized at lS psig for
15 minutes at 121C for subsequent culture propagation.
(d) Broth Inoculation.
The microorganism, Micrococ(us denitrificans (American
Type Culture Collection Accession Number 177~1), is incubated
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114~876
on the Nutrient Agar slants for from three to five days at
30C. Liquid media, for example, Nutrient Broth or
flue-cured/burley stem tobacco extract broth are inoculated at
a 2% ~v/v) rate with a sterile water wash of culture from
slants. The pH of the broth prior to inoculation is adjusted
with hydrochloric acid or sodium hydroxide to about 7.2 to
7.5; The flasks are then subjected to rotary agitation for
approximately 24 hours at 30C and 160 rpm.
Exampl~ 2
This example demonstrates the nitrate degradation that
occurs in flue-cured/burley stem extract and flue-cured stem
extract.
Micrococcus denitrificans (Am. Type Culture Collection
Accession Number 17741) was grown in flue-cured/burley stern
extract (+0.5% YE) prepared as described in Example 1 and in
flue-cured stem extract prepared as follows:
Fifteen pounds of flue-cured stem was extracted in 240
pounds of water at 90C for 30 minutes. The extract was
centrifugally separated, collecte~ and yeast extract added at
a 0.5% (wt/v) rate. The mixture was sterilized for 15 minutes
at 15 psig and 121C.
Both media were inoculated, after pH adjustment, with
washings from 4 day slants, at 10~ (v/v) rate and incubated at
160 rpm (rotary) and 30C for 24 hours in Erlenmeyer flasks
(250 ml/500 ml flask).
Results are shown in the following table:
Growth N03
MediumTi~~e~hrs) pH (~g/ml)
Flue-cured/burley Stem Extract
+0.5% Yeast Extract 0 ~7.50 2,500
16 8.10 59
8.29 57
24 -- 55
Flue-cured Stem Extract
~0.5% Yeast Extract 0 -7.50 716
16 -- 699
24 7.96 66
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It can be seen from the above data that the nitrate is
substantially degraded in both ex~:racts.
Example 3
This example demonstates the effects of aeration on
culture mass during nitrate degradation using the
microorganism Micrococcus denitri-icans.
-
A culture of Micrococcus denitrificans ~ATCC 17741) ~Jas
_
grown on Nutrient Agar + 1% KNO3 slants and then grown influe-cured/burley stem extract broth + 0.5% yeast extract in
shake flasks as described in Example 1.
This culture was split into two equal parts and used as
inoculum for two separate fermentors cf the same broth + 0.5%
yeast extract.
Growth parameters for the culture in each fermentor
were:
Parameters Ferment~r A Fermentor B
Medium Flue-cured/burley Flue-cured/burley
stem extract + stem extract +
0.5% YE 0.5% YE
20 Volume (liters) 8 8
Agitation (rpm)300 300
Aeration (cc/min.) 2,000 0 (none)
Temp. (C~ 30 30
pH Set/Control7.8 7.8
Inoculum Rate (% v/v) 5 5
(From Flasks)
Control Acid (2N) HCL HCL
Control Base (2N~ NaOH NaOH
The results of growth under these conditions are shown below.
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Fermentor A Fermentor B
(Aerat~d) (Unaerated)
Time
of CellsNO3 Cells NO~
_Sample (X106/ml) pH (~g/ml) (X106/ml) pH (~g/ml)
Before Inoc.* 0 6.78 2,ii30 0 6.99 2,900
Inoculum 9,500 8.10 0 9,500 8.10 0
0 hrs. after
Inoc. 370 7.91 2,690 90 7.84 2,5~0
10 16 hrs. after
Inoc. 7,100 7.82 560 4,500 7.85 350
17 hrs. after
Inoc. -- 7.83 0 -- 7.92 34
18 hrs. after
Inoc. 8,600 7.82 0 3,700 7.95 34
~9 hrs. after
Inoc. -- 7.81 0 -- 7.95 34
21 hrs. after
Inoc. 10,400 7.81 0 3,400 7.97 33
20 22.5 hrs. after
Inoc. 9,900 7.75 0 3,800 7.94 33
*Inoculation
These cultures were then used to treat burley tobacco
lamina with the following results.
Aerated Inoculum Unaerated Inoculum
From Fermentor A _rorn Fermentor B
Wet Tobacco NO3 Wet Tobacco NO3
Treatment Time (Hrs.) pll (%) pH (~)
Inoculated Tobacco
0 7.11 .. 27 7.52 3.~11
24 8.27 1~.57 8.13 1.:30
(2)
Uninoculated Control
0 7.17 3.14 7.20 2~85
24 7.60 :~.32 7.49 2.90
( 1 )
All Treatmens were: 90 gm dly weight burley lamina
20 ml lrl NaOH
116 ml ll2O
134 ml ~noculum
30C in plastic bags with restricted
air ava-~lability
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(2)
All controls were: 90 gm dry weight burley lamina
20 ml lN NaOH
250 ml ~2
No Inoculum
30C in plastic bags with restricted
air availability
It can be seen that the aerated c~lture produced the greatest
cel~ mass and degraded the leaf tobacco nitrate best.
However, the unaerated culture also produced a Iarge amount of
degradation of the leaf tobacco nitrate, Tobacco treated with
cultures grown under either set oE conditions is acceptable
for use in tobacco products.
Exampl,~ 4
This example demonstrates nitrate degradation of an
inoculated tobacco.
Five pounds of burley tobacco were treated with an
aerated culture of Micrococcus denitrificans (ATCC 17741)
grown for 22 hours as described in Example 3. The treated
tobacco was bulked at 30C in a plastic bag using the
following materials:
Inoculum (ml - 2880
Tobacco Weight (gm) - 2270
IN NaOH (ml) - 449.5
Tap Water (ml) - 2572
The results of this treatment were:
Wet Tobacco
Treatment Time (hrs.) _ pH NO~ (%)
Inoculated Tobacco
(2270 gm tob. wt.)
0 6.98 2.95
18 7.44 1.82
21 7.58 1.44
Uninoculated Control
(90 gm tob. wt.~
0 7.14 2.78
21 7.24 3.21
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It can be seen that the nitrate content of the treated tobacco
was reduced from 2.95% to 1.44~ (a 51~ reduction) while the
nitrate content of the control sanple did not decrease.
Example 5
This example demonstrates the reduction of nitrogen
oxides (NOx) and hydrogen cyanide (~ICN) in smoke from a
tobacco product when using a tobacco which has been subjecled
to nitrate degradation by the microorganism _icrococcus
denitrificans.
Nine hundred eight grams of burley tobacco lamina was
mixed with 2,864 ml of Micrococcu~s denitrificans grown in
flue-cured/burley stem extract as described in Example 1. ~o
additional water was added and no pH adjustment was made prior
to inoculation. The tobacco was t:horoughly mixed and placed
into a plastic bag and incubated at 30C for 24 hours.
Treatment Nitrate Moisture
Time (hrs.) (%) pH (%)
0 2.13 7.25 -75
18 1.57 -- --
24 0.91 7.95 ~75
In this tobacco treatment the liquid inoculum served three
purposes:
(1) Initial tobacco pH adjustment (pH at ~5.8
starting).
(2) Tobacco moisture elevat:ion (target 75~).
(3) Supply culture to degrade nitrate.
After microbial treatment, I:he burley tobaccos were
mixed with other standard blend components where the total
blend nitrate content was 1.16~ compared to 1.69~ for the
untreated control blend.
The separate blends were made into cigarettes and smoked
on a constant vacuum smoking machlne. The results were:
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114~876
Per Puff Deliveries
~lend NOx HCN
SampleNitrate (~ g) (~g) Puff No.
Untreated Control 1.69 40 13.5 7.2
Treated 1.16 33 11.8 7.3
It can be seen that the nitrogen oxides in smoke are
significantly reduced (17.5%) in t.he sample containing the
treated tobacco. Also, the hydrogen cyanide delivery is
reduced (12.6%) in the sample cont.aining treated tobacco. All
other delivery components remainecl virtually unchanged.
Example 6
This example demonstrates the reduction of nitrogen
oxides (NOx) and hydrogen cyanide (HCN) in a tobacco product
when using a tobacco which has been subjected to nitrate
degradation by the microorganism Micrococcus denitrificans.
Micrococcus denitrificans ~ATCC No. 17741) was grown as
described in Example 3 (Fermentor "A" conditions) and used to
treat burley tobacco for 24 hours in closed p.lastic bags at:
30C. The nitrate in the growth ~ledium was depleted at 17
hours.
The following amounts of ma~.erials were used:
Inoculum (ml) - 1716
lN NaOH (ml) - 270
Water (ml) - 1555
Tobacco (gm) - 1362
Tobacco treatment results were:
Treatment Wet Tobacco NO3 Moisture
Time (hrs) pH (~) (%)
-
~ Inoculated Tobacco
,__
0 7.21 2.51 75.7
21 7.70 1.33 73.9
24 -- 1.33 --
Air Dried 8.28 1.41 --
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After treatment, the burley lamina was blended with
other tobacco components and made into cigare~tes and smok~d
on a constant vacuum smoking machine. A control product,
without treated lamina, but incorporating untreated burley
lamina, was also machine smoked. The results were:
Per Puff Deliveries
Blend NOc EICN
Sample Nitrate (%)_(u'~ Puff No.
Control 1.70 6l 33 7.04
Experimental 1.36 4'3 30 7.08
It can be seen from the above data that nitrogen oxides were
significantly reduced (19.7%) in the product containing the
trcated tobacco. Also, the hydrogen cyanide delivery was
reduced (9.1~) in the product conl:aining treated tobacco.
~xample 7
This example demonstrates the procedure of extracting
tobacco lamina with water to remove nitrate, treating the
extract with M rococcus denitrificans (ATCC No. 17741) to
remove the nitrate therefrom, then adding the modified extract
back to the original tobacco.
A tobacco extract was prepared by mixing 100 gms of
burley lamina with one liter of water and allowing it to st:and
at room conditions for two hours. At this point, the extrlct
was collected by decanting the li(~uid and pressing the tobacco
to remove additional liquid. The tobacco was spread to dry in
room air while the extract (~700 Inl ) was subjected to
microbial treatment.
A mature culture of Micrococcus denitrificans was grown
in flue-cured/burley stem extract medium, prepared as
described in Exan~ple 1, and added to the tobacco eKtract,
prepared as described in the previous paragraph, at a 10~
(v/v) rate. Prior to culture addition, the extract pH was
raised to 7.0 + 0.1. 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:
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Micrococcus denitrificans T~eatment
of Burley Lamina Extrc~ct NO~(ug/ml)
_
Burley lamina extract 1872
Mature Micrococcus denitrificans
culture 64
Extract after treatment (l& hours) 66
The data indicate that nitrate was almost completely degraded -
(_9~) by the treatment.
After 18 hours incubation, the treated extract was added
back to the originally extracted tobacco in three stages
because of the large volume of treated extract. This was done
by adding a portion, mixing thoroughly and air drying, prior
to the next addition. The following chemical changes resulted
from this procedure:
Tobacco ~nalysis NO~
Burley lamina before extraction 1.96
~urley lamina after extraction 0.72
Burley lamina after treated extract added
back 0-44
Data show that 77% of the nitrate was removed by the
Micrococcus denitrificans treatment.
The tobaccos resulting from this operation were useahle
in manufacturing type operations.
In certain reconstituted tobacco manufacturing
processes, the step of extracting the tobacco solubles is an
integral part of the overall processing. lf preferred, the
resultant extracted tobacco could be processed by paper-making
techniques into base sheet to which the extract, from whicll
nitrate has been removed by microbial treatment, could then be
added back in the normal manner.
Examp~e B
.
This example demonstrates some differences in the final
) product which can be obtained by ~sing ultrafiltration
; equipment in conjunction with tobacco extraction, extract
treatment and extract addback as described in Example 7.
Tobacco used in this work was from the same source as that
used in Example 7.
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A burley lamina extract was prepared as in Example 7.
The extraet was then filtered witl a 0.2 micron pore siæe
filter in an Amicon ultrafiltraticn device (model TCF10) prior
to inoculating the filtered extra~t with Mierococeus
denitrifieans (ATCC No. 17741) and treating it as describecl in
Example 7. Following treatment, the extraet ~as again
filtered (0.2 mieron pore size filter) before addback
proeedures were started. The matl-rials retained on the filter
during the first filtration and the permeate from the second
filtration were added back to the extracted tobacco.
The materials retained by the filter during the seeond
filtration were not added back to the tobacco. The following
ehemieal ehanges oeeurred in the extraet:
Chemieal ehanges aeross ultrafilt~ation and Mierocoeeus
denitrifieans Treatment of Burley Tobaeec)
NO3
(~g/ml)
Burley lamina extract 1872
Mature Microcoecus denitrificans culture 64
20 Extract after filtration 2028
Extract after Micrococcus denitrilicans
treatment 646
The following ehemieal ehanges were measured in the tobacec~
across extraetion and treatment:
Tobaeeo Analysis
Burley Lamina NO~(%)
Before extraction 1.96
After extraction 0.72
After treated extract added back 0.85
These results show that nitrate i, removed from the extract by
Micrococcus denitrificans, but as opposed to Example 7, no
further removal from the extraetecl tobaeco oceurs during
addbaek proeedures. In this examole, the microbial cells clo
not eontaet the tobaeco, whereas in Example 7, the eells do
eontaet the tobaeeo during addbaek; and produce further
ehemieal ehanges.
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The tobaccos resulting from this operation were useable
in manufacturing type operations.
Other filters (with other pore sizes) can be used in the
first filtration step (in Examples 7 and 8) to keep many of
the larger extracted molecules from being exposed to potential
microbial action. If used, the r~sulting extract would be
less modified and a less modified tobacco would result.
Exampl~ 9
This example demonstrates t~e ability of Micrococcus
denitrificans (ATCC Accession No. 19367) to degrade nitrates
in tobacco.
Micrococcus denitrificans (ATCC Accession No. 19367) was
grown in flùe-cured/burley stem e~tract broth (+0.5~ YE)
prepared as in Example 1. Control and experimental culture
broths were pH adjusted to ~7.2 prior to use with 2.4 ml of lN
NaOH/flask. All flas~s were incubated at 30C and 160 rpm for
24 hours. Those flasks used for cell growth were inoculated
at 2~ (v/v) rate with Micrococcus denitrificans (ATCC No.
19367~.
The accompanying table illustrates the nitrate
degradation by this culture.
SET I
Time (hrs) p~lNOl(ug/ml)
Control Broth (Uninoculated)
0 7.28 2,251
6 7.~9 2,281
24 7.]8 2,025
Experimental Broth
(Inoculated~
0 7.19 1,975
6 7.~0 2,031
24 8.l8 51
,
Micrococcus denitrificans was grown in the same broth as sllown
above and chemical analyses were performed at a different
intermediate time interval with the following results:
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SET 2
Time (hrs.)_ pll_ NO~(~g/ml)
Control Broth (Uninoculated)
0 7.~8 2,251
6 7.13 2,281
24 7.~8 2,025
Experimental Broth
(Inoculatedj
0 7.22 2,226
18 7.92 1,605
24 8.17 0
The experimental cultures from Sets 1 and 2 were used to treat
burley lamina for 24 hours at 30C in plastic bags as follows:
Materials for Treatment
!Tobacco(gm) IN NH4O.~(ml) Water(ml) Inoculum(ml)
Treated 50 10.3 69.5 70.2
ControI 50 10.3 139.7 0
Treatment
Time p~l NO~(%)
20 Set 1
Treated 0 hrs. 7.3~ 2.91
24 hrs. 7.28 1.07
Control 0 hrs. 7.48 2.87
24 hrs. 7.03 2.58
Set 2
Treated 0 hrs. 7.49 3.10
24 hrs. 7.43 1.86
Control 0 hrs. 7.43 3.32
24 hrs. 7.04 3.61
It can be seen that Micrococcus denitrificans (ATCC No. 19:367)
degraded up to 63% of the nitrate in burley lamina while the
control tobacco showed little decrease in nitrate.
Example 10
This example demonstrates the effectiveness of
Micrococcus denitrificans (ATCC Accession No. 17741) in
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removing nitrate from an extract c,f a reconstituted tobacco
mixture.
A water extract was prepare(3 as follows:
150 g of reconstituted tobacco was pulped in one liter
of water for about one minute in .~ Waring blender. The
mixture was held at room temperature for 10 minutes after
which the liquid was centrifugally separated and brought back
to original volume for sterilization at 121C and 15 psig for
15 minutes. Yeast extract (YE) was added at 0.5% (wt/v) rate
prior to sterilization. Flue-cured/burley stem extract (with
0.5~ yeast extract added and prepared as in Example 1) was
used for standard extract. Broth pH was adjusted prior to
inoculating the standard ("control") extract and the
experimental extract with Micrococcus denitrificans.
The following results were obtained:
STANDARD EXTRACT
Growth Time (hrs.) NOl (~g~ml) pH
o 1,896 7.37
24 () 8.07
EXPERIMENTAL EXTRACT
.
Growth Time (hrs.) NO~ (~g/ml) pH
_
0 2,220 7.31
24 2,256 6.95
48 227 7.95
It can be seen that this data illustrates that the culture can
effectively degrade the nitrate of an extract of reconstituted
tobaccos.
Example 11
This example demonstrates the effects of aerobic and
anaerobic tobacco treatments.
Micrococcus denitrificans (ATCC No. 17741) was grown in
flue-cured/burley stem extract broth with 0.5% yeast extract
added for 24 hours in a New Brunswick Scientific Fermentor (MF
214) under the following conditiorls:
,
.:
; :
,
-
114V8~76
- 19
Paramet~rs
Agitation (rpm) 300
Aeration (cc/min.) 0
Medium Flue-cured/burley Stem Extract ~0.5% YE
Medium Vol. (liters) 8
Temperature (C) 30
pH starting (uncontrolled) 7.8
Inoc. Rate (%) 5
Inoc. Age (hrs) 24
Antifoam (Dow Chemical) P-1200
The culture at initiation and at 24 hours was characterizec3
by:
Time NO3
(hrs.) (~g/ml) pH
e 0 2169 7.74
24 52 8.20
At 24 hours, the culture was used to treat burley tobacco
under aerobic and anaerobic condit:ions with the following
results:
Aerobic Txeatments
-
Time Hrs.
0 24
pH _(%)NO~ pH (%~
Control 7.20 3.39 7.41 3.27
Treatment 7.59 3.39 7.92 1.81
Anaerobic Treatments
Control 6.93 3.39 7.03 3 79
Treatment 7.49 :3.39 7.65 1.61
All tobaccos were at _75~ moisture content and were
stored at 30C for 24 hours in plastic bags. Anaerobic
treatments were conducted in BBL (Baltimore Biological
Laboratories) "Gaspak" anaerobic system jars using BBL
catalyst to tie up atmospheric oxygen.
.- : ~. :
,. . :
- 20 -
It is seen from the above data that the present
invention can be carried out under anaerobic conditions and
under conditions when availabilit~ of oxygen i~ not
controlled.
Example 12
_
This example demonstates the effect of treating tobacco
with cells as well as supernatant liquor from the cell growth.
Micrococcus denitrificans (ATCC No. 17741) was grown in
flasks of flue-cured/burley stem extract broth, with 0.5%
(wt/vol) yeast extract added, pre~)ared 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.
-`' `:1
~, ~ .
' '
.
`~ 11408~76
- 21 -
TAnl.F A
Cultllre
l Split
Tobacco Centr]fuge (10,000 rpm for 15
Treatment~ minutes using Type GSA
/ \ head in Sorvall RC2-B
/ \ centrifuge)
Cell Pellet
Resuspend to Supernatant
Original Volume
in Sterile
Water ' ~
Split
/ Tobacco Split
Treatment
Mix/~uspend Millipore Inoculate
(0.22u) Fresh Flue-
Filtration cured/Burley
/ \ I Extract
~ Tobacco
Inoculate Treatment Tobacco
Fresh Flue- Treatment ~ I
cured/Burley Incu ate
Extract
Incubate
. . : ::
-
-, ~ : ' ' ':: ,
114~876
- 22 -
The following resulted from the operations shown in
Table A.
TABLE 1: CULT~RE PREPARATION
=UE-CURED/BURLEY EXTRACT BROTH WITH 0.5% YE
NO3
Time (hrs.~ (~g/ml) pH
Control 01618 7.13
(Uninoculated) 241550 7.0~1
Inoculated 01575 7.20
2436 8.02
Resuspended cells 0 8.1~
Supernatant 34 8.15
Filtered Supernatant 36 8.26
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 160 rpm. Extract was
prepared as in Example 1. The following was obtained:
TABLE 2
NO3
Ti e (hrs.) ( g/ml) pH
Suspended cells 0 1530 7.00
24 0 8.11
Filtered supernatant 0 1576 7.11
24 1464 6.99
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.
control sample was pH adjusted and water treated withou~
inoculum.
~14V876
- 23 -
TABLE 3- MATERIALS ADDED FOR TOB.~CCO TREATMENTS
Sterile IN NaOH
Tobacco Treated by: Distilled Water(ml) Base(ml) Inoculum(r
.
Control (none) 140.2 9.8 none
Original Culture 96.1 9.8 44.1
Resuspended cells 96.1 9.8 44.1
Supernatant 96.1 9.8 44.1
Filtered Supernatant 96.1 9.8 44.1
I
The following results were obtained from these tobacco
treatments (Table 4).
TABLE 4: TOBACCO TREATMENTS
-
Time (hrs) ~ %) pH
Control (no inoculum) 0 4.57 6.97
24 4.65 7.09
Original Culture 0 4.41 7.18
24 2.86 7.59
Resuspended cells0 4.52 7.01
24 0.94 7.65
Supernatant 0 4.45 7.27
24 4.38 7.13
Filtered Supernatant 0 4.41 7.07
24 4.48 7.15
It can be seen from the above data that the supernatant
liquor in which the culture is grown does not provide
sufficient culture for degradation of nitrates in tobacco.
