CA1116971A

CA1116971A - Process for refining tobacco

Info

Publication number: CA1116971A
Application number: CA000319932A
Authority: CA
Inventors: Helmut Gaisch; Urs Nyffeler
Original assignee: Fabriques de Tabac Reunies SA
Current assignee: Philip Morris Products SA
Priority date: 1978-02-09
Filing date: 1979-01-19
Publication date: 1982-01-26
Also published as: GB2014031B; GB2014031A; DE2901310A1; CH640393A5; NL188733C; FR2416654B1; IT7919955A0; LU79039A1; NL188733B; IT1110105B; BE874050A; AU523558B2; NL7900761A; DE2901310C2; FR2416654A1; AU4347279A

Abstract

A B S T R A C T

A process for reducing the content of nitrate and/or nitrite salts contained in tobacco is disclosed whereby tobacco is treated, under controlled aerobic conditions, with microorganisms capable of degrading nitrates and/or nitrites to other nitrogen-containing compounds, such as proteins and amino acids.

Description

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 ' 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.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for finishing tobacco by reduction of nitrates and nitrites and other nitrogen containing compounds contained in tobacco, wherein a culture of microorganisms, requiring nitrogen but capable of living by denitrating aerobically, which has been brought to its exponential growth phase under aerobic conditions by adding a nutritive solution which does not contain a nitrogen source essential to this culture, is made to react under aerobic conditions, favorable to the microorganisms of this culture, on the nitrates, nitrites and other accompanying tobacco components until the nit-rates and nitrites are reduced to the desired residue level, and then immedi-ately, or at the latest after 24 hours, the operation of the microorganisms is stopped.

2. A process in accordance with Claim 1 wherein the culture of microorganisms is made to react so intensively that the nitrates and nitrites are reduced to a minimum residue level within 6 to 16 hours, and the produced effect of the microorganisms is stopped as soon as said minimum residue level has been reached.

3. A process in accordance with Claim 1 wherein a pure culture of bacteria is used, which is obtained by inoculating a watery smear of nit-rate-containing leaves or decayed leaves into a nutritive solution, which contains the amount of nitrogen required for incubation predominantly in the form of nitrates, which is buffered to between pH 6.6 and pH 7.5, and which is incubated at 25 to 35°C (degrees centigrade) for 6 to 16 hours by shaking under sterile, intensive aeration, and which is then used as active inocula-tion material for the inoculation of another fresh nutritive solution with which the incubation is repeated, and so forth until the pure culture has been formed.

4. A process in accordance with Claim 1 wherein a pure culture of bacteria ATCC 13 048 Enterobacter aerogenes is used.

5. A process in accordance with Claim 2 wherein a pure culture of bacteria ATCC 13 048 Enterobacter aerogenes is used.

6. A process in accordance with Claim 2 or 3 wherein nitrates and nitrites and other water-soluble components are removed with water from the tobacco to be finished, and the tobacco-extract solution so obtained is iso-lated, inoculated with the microorganism culture, and, mixed with the nutri-tive solution, is kept under sterile, intensive aeration for 6 to 16 hours under aerobic conditions favorable to these microorganisms, and immediately afterwards the effect of the microorganisms is stopped and the solution com-ponents contained in the nitrate-poor tobacco-extract solution are added to water from washed tobacco.

7. A process in accordance with Claim 2 or 3 wherein the tobacco to be finished is ground and made into a slush with water, and, mixed with the microorganism culture and the nutritive solution, is kept for 6 to 16 hours under sterile, intensive aeration and aerobic conditions favorable to the microorganisms, and that immediately afterwards the effect of the micro-organisms is stopped by forming the suspension into or onto sheets and drying them to a moisture level of from 10 to 30 %.

8. A process in accordance with Claim 4 or 5 wherein nitrates and nitrites and other water-soluble components are removed with water from the tobacco to be finished, and the tobacco-extract solution so obtained is iso-lated, inoculated with the microorganism culture, and, mixed with the nutri-tive solution, is kept under sterile, intensive aeration for 6 to 16 hours under aerobic conditions favorable to these microorganisms, and immediately afterwards the effect of the microorganisms is stopped and the solution com-ponents contained in the nitrate-poor tobacco-extract solution are added to water from washed tobacco.

9. A process in accordance with Claim 4 or 5 wherein the tobacco to be finished is ground and made into a slush with water, and, mixed with the microorganism culture and the nutritive solution, is kept for 6 to 16 hours under sterile, intensive aeration and aerobic conditions favorable to the microorganisms, and that immediately afterwards the effect of the microorgan-isms is stopped by forming the suspension into or onto sheets and drying them to a moisture level of from 10 to 30 %.