Note: Descriptions are shown in the official language in which they were submitted.
PROCESS FOR IMPROVING TOBACCO
The invention concerns a process for treating tobacco whereby
the nitrates and/or nitrites contained in tobacco are reduced.
Many tobaccos, Burley for example, contain salts of nitrates
and/or nitrites. There are known fermentation processes in which these
nitrogen salts are reduced by way of enzymes, however, only to a very small
extent, and only as a side-effect of other enzymatic conversions.
It is the purpose of this invention to reduce nitrates and/or
nitrites to a lower level in tobacco in order to improve the smoking qualities
thereof. The selective reduction of nitrates and/or nitrites is achieved
without adversely affecting other constituents present in tobacco.
According to the present invention there is provided a process
for finishing tobacco by reduction of nitrates and nitrites and other nitro-
gen containing compounds contained in tobacco, wherein a culture of micro-
organisms, requiring nitrogen but capable of living by denitrating aerobically,
which has been brought to its exponential growth phase under aerobic condi-
tions by adding a nutritive solution which does not contain a nitrogen source
essential to this culture, is made to react under aerobic conditions, favor-
able to the micro-organisms of this culture, on the nitrates, nitrites and
other accompanying tobacco components until the nitrates and nitrites are
reduced to the desired residue level, and then immediately, or at the latest
after 24 hours, the operation of the microorganisms is stopped.
Under these conditions, the microorganisms will remain in their
exponential growth phase as long as the necessary nitrogen requirements can
be derived from the nitrates and/or nitrites.
The microorganisms used herein utilize nitrogen from the nitrates
and/or nitrites, whereby the latter are degraded to other nitrogen-con~aining
compounds, such as amino acids and proteins. Both amino acids and proteins
are naturally occurring compounds in tobacco and are considered desirable in
that they are known to improve the flavor of tobacco smoke.
Since the added culture is already in its exponential growth phase,
the microorganisms have a lead of about 8 hours over other microorganisms
pTesent, which are still in their lag phase. Such microorganisms thus can-
not catch-up this lead within the reaction period, which is maximally 24
hours, and preferably about 16 hours, so that their effect is insignificant.
This insures that the effect promoted by the invention will be selective.
Unfermented, air-dried tobaccos frequently have a nitrate content
of about 50 g per kg dry weight. Nitrate quantities up to 80 g per kg dry
weight have been found in extreme cases. The desired level of nitrates will
depend on the ultimate use of the tobacco. For present purposes, however,
the desired level of nitrates should be within the range of 3 to 20%, and
preferably about 5% of the original content of the tobacco treated relative
to the total weight of the anions of nitrate and/or nitrite.
It is possible to influence the selective effect of the micro-
organisms further by allowing a highly concentrated culture of microorganisms
to react so that the nitrates and/or nitrites are reduced to a minimal level
within 6 to 16 hollrs. Thereafter, the produced effect of the microorganisms
is terminated immediately.
The minimal level, i.e., the level of nitrates and nitrites that
can be achieved by the process of the invention without resorting to any ex-
traordinary measures, depends on the quality of the tobacco, and amounts to
from 0.01 to 0.1% of the original content of the treated tobacco, in each
instance relative to the total weight of the anions of nitrate and/or nit-
rite. With such a concentrated application of microorganisms, the desired
level can be realized after a few hours so that the effect of the micro-
organisms can be ternlinated before the microorganisms o the culture have
used up their approximately 8-hour lead or shortly thereafter.
The effect of the microorganism culture can be intensified by con-
trolling the substantially aerobic conditions for the microorganisms to an
optimum with regard to temperature, humidity, pH level, nutrient supply, and
by using a highly concentrated culture for reducing the nitrates and/or nit-
rites. The optimal conditions will be described hereinafter. The degree ofreduction of nitrates and nitrites in tobacco may be ascertained analytical-
ly by known methods.
The effect of the microorganisms can be terminated by failure to
maintain growing conditiGns for the microorganisms; for example, by greatly
lowering or raising the temperature, by drying and also by removing the
microorganisms, as for example by filtration when the reaction is carried
out in a liquid medium.
The microorganisms useful for this invention may be those selected
from the genus Aerobacter, Pseudomonas, Micrococcus, or ~cherichia; or al-
ternatively, they may be fungi selected from the genus Rhodutorula orCandida. Microorganisms isolated from the normal microflora of tobacco
leaves are especially useful in that they have a particularly rapid dentrify-
ing effect and do not adversely alter the tobacco in an undesireable way.
One aspect of the invention provides a culture of microorganisms
obtained by inoculating a watery smear of nitrate-containing leaves or de-
cayed leaves into a nutrient solution. The solution contains a source of
nitrogen required for growth, and is predominantly in the form of nitrates.
The solution is buffered to a pH between about 6.6 and 7.5, and is then inc-
ubated aerobically at 25 to 35C for 6 to 16 hours with shaking and with in-
tensive aeration under sterile conditions. The degree of aeration varies
^
depending on the circumstances. Aeration in the range of about 150 to 400
ml/min is generally used during culture preparation while increased aeration
up to about 3000 ml/min is generally used when tr ating tobacco materials.
The thus prepared culture is then used as an active inoculum for the inocu-
lation of another fresh nutrient solution, which is incubated in a similar
manner. Transfers are repeated until a pure culture is obtained.
Preferably the smear is made from tobacco leaves. But a useable
smear can also be obtained from forest soil comprising decayed leaves or
containing decayed leaves therein. According to this method, a pure culture
may be obtained wherein the microorganisms are in their active, i.e., their
exponential growth phase. This culture is either used immediately or it is
inactivated and preserved for later use.
The invention may advantageously be practiced utilizing a pure
culture of Enterobacter aerogenes, preferably of type strain ATCC 13048.
Pure cultures of this type strain may be obtained from the American Type
Culture Collection, 12301 Park Lain Drive, Rockville, Maryland 20852, USA
and under the Collection No. DSM 30053 from the Deutsche Sammlung von
Mikroorganismen, Gottingen, CrisebachstraBe 8, Federal Republic of Germany.
This type strain belongs to prior art and is not object of this invention.
When, for example, the tobacco to be treated includes strip, leaf,
or stems, denitration is greatly facilitated if the tobacco is first ex-
tracted with water to remove the soluble nitrates and/or nitrites. There-
after, the aqueous extract is inocuiated with the microorganism culture, the
nutrient solution is added, and the mixture is incubated for 6 to 16 hours
under sterile aerobic conditions with substantial aeration oF about 1000 to
about 1500 ml/min of air. After an appropriate time period, the effect of
the microorganisms is stopped by removing the active microorganisms by fil-
tration, centrifugation, or the like. The treated extract solution from
which the nitrates and/or nitrites have been reduced is concentrated and re-
applied to the original tobacco material. In some instances, the entire
~r~
denitrated extract containing the microorganisms may be concentrated and
reapplied to tobacco materials, for example by spraying, and thereafter the
tobacco is dried for a period of time sufficient to deactivate all microbial
activity. The final moisture content of the tobacco should be in the range
of about 10 to about 30%.
In some instances, as for example in making reconstituted tobacco,
the tobacco materials are homogenized and made into a slurry. The slurry is
cast into a sheet, which is then dried. In this instance, the microor-
ganisms may be applied advantageously to the tobacco slurry. Preferably the
tobacco is ground and mixed with water. The microorganism culture and the
nutrient solution are added to the slurry, and the mixture is incubated for
6 to 16 hours under aerobic conditions. The effect of the microorganisms
is stopped by casting the slurry into or onto sheets and drying them to a
moisture content between about 10 and 30%.
The microorganism culture used is preferably a pure culture where-
by the degree of purity must be sufficient to prevent substantial side ef-
fects. The microorganism culture can be preserved by freezing in liquid
nitrogen and is thawed and reactivated before use. For immediate use, it
can be kept in an active state in a biostat from which the continually re-
quired portions can be removed.
Characteristics of Enterobacter aero~enes 13048 are as follows:
Motile rods 0.3 - 1.5 ~m
Gram
Development of gas at 37&
Glycerin
Inositol
Andonitol +
Voges-Proskauer
Methlred
Phenylamindesaminase
-- 5 --
'
97~ `
Urease
Catalase
Ornithindecarboxylase +
Lysindecarboxylase
Hydrolyse of Aesculin +
Growth:
In presence of KCN +
Upon Malonate as the only
source of carbon
.
The invention is exemplified by the descriptions hereinbelow.
Example 1
Pre ~ration of the Pure Culture
Twenty g of D-glucose, 6.4 g of NaCl, 3.5 g of KNO3; 4.5 g of
KH2P04, and 23.5 g of Na2HP04 2H20 were dissolved in 1 liter of water. The
thus obtained nutrient broth was divided into 5 equal aliquots of 200 ml
each. Each aliquot was placed in a 500 ml Erlenmeyer flask, and the flasks
were closed with a porous stopper in order to allow gasses formed during the
process to escape and to facilitate sterilization. The broths were steril-
ized and stored at ~0C.
One-hundred grams of dry Burley tobacco leaves were washed with
500 ml of water under sterile conditions. One ml of the resulting wash
suspension was drawn off under sterile conditions and added to aliquot I of
the nutrient broth. Aliquot I was incubated on a shaker for 16 hours at
30C with aeration of 200 ml/min of air. Then 1 ml of the incubated aliquot
was removed under sterile conditions and inoculated into aliquot 11 of the
nutrient solution, and the incubation was repeated. This serial transfer
procedure was repeated until the fifth aliquot was treated.
After aliquot V had been incubated for 16 hours, it contained a
pure culture of microorganisms of a genus Enterobacter that derives its
Dq~J~7~l
nitrogen requirements via the reduction of nitrates and/or nitrites. The
microorganisms of this pure culture are in their exponential growth phase
and remain so for approximately 8 hours.
Example 2
One kg of Maryland tobacco was processed to separate the stems
from the strip. This yielded 250 g of stems and 750 g of strips. The 250 g
of stems were washed with 1250 ml warm water at 70C. This removed nitrates
and nitrites contained in the stems together with o~her water-soluble com-
ponents. The aqueous stem extract solution was separated from the stems,
placed in a 2 liter Erlenmeyer flask, closed with a porous stopper, and
cooled to 30C. Then 12.5 g of D glucose and 10 ml of the culture prepared
in Example 1 were added to the flask. The microorganisms of the pure cul-
ture were still in their exponential growth phase.
The inoculated stem extract solution was incubated on a shaker at
30C for 16 hours and aerated under sterile conditions at 1300 ml/min of
air. The thus obtained denitrated stem extract solution was immediately
centrifugedl and the residual microorganisms were removed.
The centrifuged, denitrated stem extract was concentrated and re-
applied to the predried, washed stems, which were then dried to a moisture
level of 20%. In this manner, all of the soluble tobacco components that
had been removed previously with the nitrates and/or nitrites were returned
to the stems so that the stems contained essentially all of their original
components with the exception of the nitrates and/or nitrites.
Example 3
A tobacco stem extract was treated in a similar manner to Example
2. After separating the denitrated stem extract solution by centrifuging,
the extract was applied to a different type of washed tobacco stems.
Example 4
Using the same procedure as shown in Example 2, tobacco stems were
incubated for approximately 20 hours. At this point it was noted that
approximately 50% o the nicotine present in the extract had been reduced
and there were only trace amounts of nitrates and nitrites present.
Example 5
One kg of Maryland tobacco was destemmed and yielded 250 g of
stems and 750 g of strips. The 250 g of stems were treated according to
the proc~ss of Example 2.
The 750 grams of tobacco strip were dipped in 1250 ml water at
50C whereby the nitrates and nitrites were extracted from the surface re-
gions of the strip. The resulting extract was treated in a similar manner
to Example 2. The nitrates and nitrites were reduced to approximately 0.1
g per liter of extract. The denitrated extract was then centrifuged, con-
centrated, and reapplied by spraying to the previously extracted tobacco
strip.
Example 6
Two hundred fifty g of Burley tobacco leaves were washed in 1250
ml warm water at 50C. The resulting tobacco extract solution was treated
in a similar manner to Example 2. Thereafter, the denitrated tobacco ex-
tract solution was centrifuged to separate and recover the active micro-
organisms, and the denitrated extract was reapplied to the tobacco leaves.
Example 7
One kg of tobacco scraps was ground to a granular size no greater
than 150 ~m. One hundred fifty ml of a suspension of active microorganisms
obtained according to Example 1 and still in their exponential growth phase
was added to a broth containing 30 g D-glucose, 9.6 g NaCl, 6.75 g KH2P04,
and 35.25 g Na2PO4 2H20 in 1500 ml water. The mixture of active micro-
organisms and nutrients was stirred into the powdered tobacco, and the re-
sulting slurry was incubated for 24 hours at 30C in a 10-liter Erlenmeyer
flask equipped with a porous stopper. The mixture was aerated under sterile
conditions at 3000 ml/min of air. The microorganisms reduced the nitrates
and/or nitrites contained in the tobacco slurry to 1/10 of the original
~ - 8 -
t7~
content, Immediately afterwards, 150 g carboxymethylcellulose was stirred
into the slurry, and the slurry was cast in a layer of 3 mm thickness and
dried to a 15% moisture content. This terminated the effect of the micro-
organisms and solidified the slurry into sheets of reconstituted tobacco,
which were then ready for further processing.
Exarnple 8
The procedure of Example 7 was repeated in a similar manner except
that the period of incubation was extended in analogy to Example 4 until ap-
proximately 50% of the nicotine had also been degraded.
Example 9
Tobacco stems were extracted according to the method of Example 2,
and the extract was inoculated with a pure culture of Enterobacter aerogenes
ATCC 13048 prepared generally according to the procedure detailed in Example
1. Incubation conditions and reapplication of the denitrated extract were
identical to Example 2.
Example 10
The process of Example 9 was repeated under identical conditions
with the exception that the denitrated stem extract was reapplied to a dif-
ferent type of washed tobacco stems.
Example 11
Two hundred fifty g of burley tobacco leaves were wnshed in 1250
ml warm water at 50&. The resulting tobacco extract solution was inoculat-
ed with Enterobacter aerogenes 13048 as in Example 2. Following denitra-
tion, the tobacco extract solution was centrifuged to separate the micro-
organisms. 'rhereafter, the extract was reapplied to the tobacco leaves.
Example 12
Tobacco stems were extracted as in Example 2 and inoculated with
a pure culture of Enterobacter aerogenes 13048. The stem extract solution
was incubated on a shaker at 30C for 8 hours so that the anions of nitrate
and nitrite were reduced to a lesser extent than they were according to
Example 2.
The denitrated stem extract solution was centrifuged, concentrat-
ed, and reapplied to the stems, that had been predried and washed. The
stems were dried to a moisture content of 20%.
Example 13
Maryland tobacco leaves were extracted in a similar manner to
Example 6, inoculated with a culture of Enterobacter Aerogenes 13048, and
incubated for 8 hours at 30C. During this period the microorganisms re-
duced the nitrates and/or nitrites contained in the strips. The strips were
then further treated as described in Example 11.
- 10 -
