CA1140876A - Process for reduction of nitrate content of tobacco by microbial treatment - Google Patents

Abstract

TITLE

PROCESS FOR REDUCTION OF NITRATE CONTENT OF
TOBACCO BY MICROBIAL TREATMENT

INVENTOR

Vernon Louis GEISS, Lawrence Edmond GRAVELY, and Charles Fred GREGORY

ABSTRACT

A process for the reduction of the nitrate content of tobacco materials by microbial treatment is disclosed wherein tobacco materials are subjected, under controlled conditions, to the action of a microorganism effective to degrade nitrates through a biochemical reaction. Tobacco materials treated in accordance with this process, when incorporated into a tobacco smoking product, produce a mild smoke having reduced nitrogen oxides and hydrogen cyanide deliveries without loss of desirable flavor, taste or other smoking properties.

Description

` 1141~876 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 X~

. , : .
:;
' 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 -` ~ 114~876 _ 4 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.

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 .
'.

i . .

;

114~876 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 ;.
..~ `1 .

`
. ............................................... .

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 Flue-cured Stem Extract ~0.5% Yeast Extract 0 -7.50 716 24 7.96 66 ., , ` ' : ` ' ~

--114~876 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.

.~,.

:

~Q876 g .
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 .. . . .

: ' :
' ' : ' ~ ~' ' , ., ,', ' , -, --~ 114~8~

(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 `' ;~'~

, .. . . .

. ' ~

~ .
'' ' : . ,, . . : .

)876 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:

' , , , ~ .~
- ~ :

. - .

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 --., ' I\``' I
, .
:
- ' ' 8~76 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:

, ", .

-114~876 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.
,:

;~ :. ~ ; , ...... ...
~ . ; ' q3876 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) (~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.
;

.' ' , ~, ' ' ~"

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:

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 ., . ~
' ' ~. .

.
. .

114~3876 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 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.

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.

.- : ~. :

,. . :

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 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 The following resulted from the operations shown in Table A.
TABLE 1: CULT~RE PREPARATION
=UE-CURED/BURLEY EXTRACT BROTH WITH 0.5% YE

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:

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 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.

Claims (3)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for preparing a microbial containing medium for use in reducing the nitrate content of a substance to be treated comprising the stpes of:
(a) adding at least 5 percent by weight nutrient agar to water to form a first solution;
(b) adding from about 0.5 to 1.0 percent by weight of a nitrate containing compound to said first solution;
(c) sterilizing said first solution by subjecting said first solution to at least 15 psig at least 121°C for a period of at least 15 minutes;
(d) adding Micrococcus dentrificans to the first medium and allowing said first solution to incubate for from about 3 to 5 days at from about 5°C to about 37°C.

2. The process of claim 1 said nitrate containing compound being potassium nitrate.

3. The process of claim 2, said sterilizing of said first medium is accomplished within a test tube on a slant whereby a slanted surface is provided for growth.