GB1585023A

GB1585023A - Process for the treating of tobacco

Info

Publication number: GB1585023A
Application number: GB1172978A
Authority: GB
Original assignee: Fabriques de Tabac Reunies SA
Current assignee: Philip Morris Products SA
Priority date: 1977-05-06
Filing date: 1978-03-23
Publication date: 1981-02-18
Also published as: DE2811690B2; DE2811690A1; NL188782B; AU520607B2; FR2389341B1; CA1081076A; FR2389341A1; NL7804047A; NL188782C; AU3492178A; DE2811690C3; CH633169A5

Abstract

In a method of upgrading tobacco, the nitrates and nitrites in the tobacco are reduced by a pure culture of micro-organisms which, under anaerobic conditions, are advanced into and kept in their phase of exponential growth, their action on the tobacco being discontinued immediately sufficient reduction has taken place.

Description

(54) A PROCESS FOR THE TREATING OF TOBACCO (71). We, FABRIQUES DE TEBAC REUNIES S.A., a Swiss corporate body of Quai Jeanrenaud 3, CH-2003 Neuchatel, Switzerland do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: The invention concerns a process for the treating of tobacco through the reduction of nitrates and nitrites contained in tobacco.

Many tobaccos, Burley, for instance, contain nitrates and/or nitrites which can produce irritant nitrogen oxides during smoking. There are known fermentation processes in which these nitrogen salts are reduced by way of enzymes; however, such reduction is only to a very small extent, and only as a side-effect of other enzymatic conversions.

It is an object of the invention to reduce nitrates and nitrites to a wholesome residue level in order to reduce the share of nitrogen oxides in smoke. The reduction of the nitrates and nitrites should take place as selectively as practicable and, moreover, with the maximum avoidance of any conversions of the tobacco components accompanying nitrates and nitrites.

According to the present invention, there is provided a process of treating tobacco which comprises anaerobically culturing a nitrate and/or nitrite - reducing micro organism in the presence of at least part of the nitrate and/or nitrite content of the tobacco as an essential oxygen source for the micro-organism, the micro-organism being at an exponential growth phase when initially contacted with the said content and the reduction being immediately terminated when the desired reduced nitrate and/or nitrite content is reached and at most after 24 hours. Under the said conditions the micro organism will remain in its exponential growth phase as long as the necessary oxygen requirements can be filled from the nitrates and nitrites. Preferably, the micro-organism has been brought to its exponential growth phase under anaerobic conditions.

The micro-organisms used are forced during the reaction to cover their oxygen requirements from the nitrate and/or nitrites content, whereby the latter are reduced to the level of nitrogen or ammonia respectively. Nitrogen can escape harmlessly, and nitrogen present in the form of ammonium salts, amino acids, and amines does not form nitrogen oxides during smoking.

Since the adding culture is already in its exponential growth phase the micro-organisms have a lead of about 8 hours over other undesired micro-organisms, which are still in their latent phase. Such undesired micro-organisms cannot recover this lead within the reaction period, limited to maximally 24 hours, to produce a significant effect. This ensures 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 measured in extreme cases. The desired residue level will depend on the further use of the tobacco. For types of tobacco which constitute an essential tial part of the tobacco mixture ready for smoking, the desired maximum residue level for the nitrate and nitrite anions is 5 g per kg dry weight; for types of tobacco constituting only smaller parts of the mixture ready for smoking, the desired maximum residue level may be slightly higher. Generally, the desired residue level is 25% or below, usually within the range of 3 to 20%, preferably 5%, of the original content of the tobacco to be treated relative to the total weight of nitrate and nitrite anions.

It is possible to favour the selective effect of the invention further by making the culture of microorganisms react so intensively that the nitrates and nitrites would be reduced to a minimal residue level within 12 to 24 hours, preferably within 20 hours, and that the effect of the microorganisms is stopped immediately the desired residue level has been reached.

The minimal residue level, that is, the residue level which can be obtained with the help of the invention without resorting to any exceptional measures, depends on the quality of the tobacco to be treated, and amounts to from 0.01 to 0.1% of the original content of the tobacco to be treated, in each instance relative to the total weight of nitrate and nitrite anions. With such an intensive application the desired residue level can be reached after a few hours so that the effect can be stopped before the microorganisms of the culture have used up their approximately 8-hour lead or shortly thereafter.

The effect of the microorganism culture can be intensified by bringing the anaerobic conditions for these microorganisms to an optimum in regard to temperature, humidity, pH-level, and nutrient supply, and by making the culture react on the nitrates and nitrites from the beginning in a high concentration. The optimal conditions can be found through testing. That the reduction of nitrates and nitrites to the desired or minimal residue level, respectively, has taken place can be ascertained analytically.

The effect can be stopped by not maintaining living conditions for the microorganisms, for instance, by greatly lowering the temperature, by drying, and also by removing the microorganisms, as for instance, through filtering as active slush when the reaction takes place in liquid surroundings.

The microorganisms used can be bacteria, for instance, those from the genus Aerobacter, Pseudomonas, Micrococcus or Echerichia, or they may be fungi such as from the genus Rhodutorula.

Preferable are bacteria belonging to the normal microflora of tobacco leaves because these have a particularly rapid denitrifying effect and do not alter the tobacco in an undesirable way.

In a preferred embodiment of the invention, a pure culture of bacteria is used, which culture is obtained by inoculating a watery smear of nitrate-containing leaves or decayed leaves into a nutritive solution, which contains the amount of oxygen required for incubation predominantly in the form of nitrates, is buffered to between pH 6.6 and pH 7.5, and which is incubated at 25 to 350C during 15 to 25 hours by shaking under substantially complete exclusion of oxygen, and which is then used as active inoculation material for the inoculation of another fresh nutritive solution which the incubation is repeated, and so forth until the pure culture has been formed. Preferably the smear is made from tobacco leaves, but a useable smear can also be obtained from forest soil made up of decayed leaves or containing decayed leaves.

In this process a pure culture is obtained whose microorganisms are in their active, that is, their exponential growth phase. This culture is either immediately applied to further use or it is preserved and inactivated for this purpose and reactivated before later use.

The invention is advantageous applicable also in connection with a pure culture of bacteria Enterobacter Aerogenes preferably of typ strain ATCC 13 048. Pure cultures of this typ strain are available at the Deutsche Sammlung von Mikroorganismen, Göttingen, Grisebachstrasse 8, under the DSM-Registration-Number DSM 30053.

For parts of the tobacco, as for instance the stems, in which nitrates and nitrites are only accessible with difficulty to microorganisms acting from outside, a further embodiment of the invention is recommended, in which nitrates and nitrites and other water-soluble components are extracted with water from the tobacco to be treated, the thus obtained tobacco-extract solution is isolated, inoculated with the microorganism culture, and, mixed with the nutritive solution, kept under substantially complete exclusion of air for 12 to 24 hours, preferably 16 hours, under anaerobic conditions favorable to the microorganisms, and immediately afterwards the effect of the microorganisms is stopped by drawing off the active slush from the obtained nitrate-poor tobacco-extract solution, and the solution components contained in the nitrate-poor tobacco-extract solution are added to waterwashed tobacco.

However, when, for instance, the parts of the leaf between the stems, the so-called strips, or leaves with less pronounced stems, are to be treated, or when it is not of importance to reduce the nitrate content in the depth of the stems, the process can be made simpler by making an active suspension from the microorganism culture and a nutritive solution, bringing the tobacco to be treated to a moisture level between 10 and 30%, preferably 20% and spraying it with the active suspension until it has reached a moisture level between 40 to 60%, preferably 50%, and then keeping it under substantially complete exclusion of air for 12 to 24 hours, preferably 24 hours, under anaerobic conditions favorable to the microorganisms, and immediately afterwards stopping the effect of the microorganisms by drying the tobacco to a moisture level between 10 to 30%, preferably 20%.

In many cases, for instance in the reconstituting of tobacco waste, the tobacco is pulverised, made into slush, and, with the help of adhesives, rolled into a sheet which is hardened through drying. In such a case the microorganisms can be applied advantageously while the tobacco is swelled into a suspension, and it is done preferably by grinding the tobacco to be treated, swelling it with water, and keeping it, mixed with the microorganism culture and the nutritive solution, under substantially complete exclusion of air during 12 to 24 hours, preferably 24 hours, under anaerobic conditions favorable to the microorganisms, and by immediately afterwards stopping the effect of the microorganisms by forming the suspension into or onto sheets and drying them to a moisture level between 10 and 30%, preferably 15%.

The microorganism culture is preferably used as a pure culture, whereby the degree of purity must be sufficient to prevent substantial side-effects. Preferably the pure culture is obtained from tobacco leaves.

The microorganism culture can be preserved frozen in liquid nitrogen and is thawed and reactivated before later use. For continuing use it can be kept in an active state in a biostat, from which the continually required portions can be removed.

Characteristics of Enterobacter aerogenes are as follows: Motile rods, 0.3 - 1.5 Fm; Gram Development of gas at 370C Glycerin + Inositol + Andonitol + Voges-Proskauer + Methylred Phenylamindesaminase Urease Catalase + Ornithindecarboxylase + Lysindecarboxylase + Hydrolyse of Aesculin + Growth in presence of KCN + upon Malonat as the only source of carbon + The invention will now be exemplified further by several procedural descriptions.

EXAMPLE 1 Obtaining the Pure Culture: 20 g (grams) D-Glucose; 8.6 g Peptone; 6.4 g NaCI; 3.5 g KNO3; 4.5 g KH2PO4 and 23.5 g Na2HPO4 2H2O are dissolved in 11 (liter) of water. The thus obtained nutritive solution is divided in 5 equal parts I to V of 200 ml (milliliter) each; each portion is placed into an Erlenmeyer flask holding 500 ml and the flask is closed with a porous stopper. Then all of it is sterilized and kept at 20"C.

100 g dry Burley-tobacco leaves are washed in 500 ml water under sterile conditions. 1 ml of the resulting wash-suspension is drawn off under sterile conditions and added to portion I of the nutritive solution. This portion I is incubated on a shaker for 16 hours at 30"C. Then 1 ml of this portion is removed under sterile conditions and inoculated into portion II of the nutritive solution, which is then treated like portion I, and from which, after completed incubation, 1 ml is inoculated into portion III, and so forth until portion V inclusive.

After portion V has been incubated for 16 hours, it contains the pure culture of microorganisms of a genus Pseudomonas, which covers its oxygen requirements through the reduction of nitrates and nitrites. If this is not the result, everything is thrown away and the process is repeated with other Burley-tobacco leaves. The microorganisms of this pure culture are at that point in time and until 2 hours afterwards in their exponential growth phase.

Treating tobacco stems: 1 kg Maryland-tobacco whose nitrates and nitrites are to be reduced is de-stemmed. This yields 250 g stems and 750 g strips.

The 250 g stems are washed with 250 ml warm water of 70"C. This removes nitrates and nitrites contained in the stems together with other water-soluble components. The thus obtained stem-extract solution is drawn off from the stems, placed in a 2 1-Erlenmeyer flask, closed with a porous stopper, cooled to 300C, mixed with 12.5 g D-Glucose as well as inoculated with 10 ml of the above mentioned pure culture, which has to be done within the time period during which the microorganisms of this pure culture are still in their exponential growth phase.

The inoculated stem-extract solution is incubated on a shaker at 30 C until the anions nitrate and nitrite are reduced to a content of 0.1 g per 1 (16 hours).

The thus obtained nitrate-poor stem-extract solution is immediately centrifuged whereby the micro-organisms are deposited and are drawn off as active slush.

The centrifuged liquid from the nitrate-poor stem-extract solution, which still contains the remaining tobacco components removed from the stems, is re-added to the pre-dried, washed stems, which are then dried to a moisture level of 20%. In this way all the tobacco components which has been removed with the nitrates and nitrites from the stems are returned to the stems, so that the stems essentially contain their original components, except for nitrates and nitrites, which have been reduced to 1/6 of their original content.

EXAMPLE 2 Example 1 is repeated with the single difference that the centrifuged liquid from the nitrate-poor stem-extract solution is not re-added to those stems from which the extract had been obtained but to other stems which had been treated in the same way.

EXAMPLE 3 Treating Tobacco Leaves: 1 kg Maryland-tobacco whose nitrates and nitrites are to be reduced is de-stemmed. This yields 250 g stems and 750 g strips. The 250 g stems are treated as described in Example 1.

The microorganisms of the thus obtained 150 ml active slush are still in their exponential growth phase when the active slush is drawn off and are kept in their growth phase by putting the active slush with 30 g D-Glucose, 12.9 g Peptone, 9.6 g Nail, 6.75 g KH2PO4 and 35.25 g Na2PO4 2H2O immediately into 1500 ml water. Thus a suspension of active microorganisms is obtained, which is then immediately sprayed in an even distribution onto the 750 g strips of moisture content 20%. The sprayed strips will then have a moisture content of 50% and are stored at 30"C for 24 hours without air access. During this time the microorganisms reduce the nitrates and nitrites contained in the strips to 1/20 of their original content.The strips with their thus reduced nitrate and nitrite content are dried to a 20% moisture level for their further processing. This inactivates any microorganisms still remaining on the strips. The strips are now ready for further processing.

EXAMPLE 4 Pure culture is obtained as in Example 1.

Treating Tobacco Leaves: 250 g Burley-tobacco leaves are washed in 1250 ml warm water of 70"C. The resulting tobacco-extract solution is treated in the same manner as the stem-extract solution of Example 1, and the thus ultimately obtained nitrate-poor tobacco-extract solution is centrifugally separated from the active slush and re-added to the tobacco leaves.

EXAMPLE 5 1 kg tobacco waste in which nitrates and nitrites are to be reduced is ground to a grain size of maximally 150 Fm fine. 150 ml active slush, obtained according to Example 1 and still in the exponential growth phase, are kept in their growth phase by placing the active slush with 30 g D-Glucose, 12.9 g Peptone, 9.6 g NaCI, 675 g KH2PO4 (sic) and 35.25 g Na2PO4 2H2O immediately into 1500 ml water. This yields a suspension of active microorganisms which is stirred into the powdered tobacco. The resulting pulp is kept for 24 hours at 300C in a 3 1-Erlenmeyer flask, which has been closed with a porous stopper. During that time the microorganisms reduce nitrates and nitrites, contained in the tobacco powder, to 1/10 of the original content.Immediately afterwards 150 g carboxymethylcellulose are stirred into the pulp, and the pulp is spread out in a layer of 3 mm thickness and dried to a 15% moisture level. This terminates the effect of the microorganisms and hardens the pulp into sheets of regenerated tobacco, which are ready for further processing.

EXAMPLE 6 Example 1 is repeated with the single difference that the inoculated stem-extract solution is incubated on a shaker at 300C for 8 hours instead of 16 hours, so that the nitrate and nitrite anions are reduced to a lesser extent than in Example 1.

The obtained nitrate-poor stem-extract solution is then further treated as in Example 1 and re-added to the stems, pre-dried and washed, as in Example 1, which are then dried to a moisture content of 20%. The thus treated stems contain one third of their original content of nitrate and nitrite.

EXAMPLE 7 Example 3 is repeated with the single difference that the sprayed strips whose moisture content is 50% are stored only 8 hours instead of 24 hours at 30"C without air access.

During this time the microorganisms reduce the nitrate and nitrite contained in the strips to one quarter of their original content. The strips are then further treated as described in Example 3.

EXAMPLE 8 Treating Tobacco Stems: Example 1 is repeated with the single difference that the inoculation is will be carried out with a pure culture of bacteria Enterobacter aerogenes ATCC 13048.

EXAMPLE 9 Example 8 is repeated with the single difference that the centrifuged liquid from the nitrate-poor stem-extract solution is not re-added to those stems from which the extract had been obtained but to other stems which had been treated in the same way.

EXAMPLE 10 Treating Tobacco Leaves: 1 kg Maryland-tobacco whose nitrates and nitrites are to be reduced is de-stemmed. This yields 250 g stems and 750 g strips. The 250 g stems are treated as described in Example 8.

The strips are treated as described in Example 3 using the active slush obtained by treating the stems.

EXAMPLE 11 Pure culture is obtained as in Example 8.

Treating Tobacco Leaves: 250 g Burley-tobacco leaves are washed in 1250 ml warm water of 70"C. The resulting tobacco-extract solution is treated in the manner of the stem-extract solution of Example 8, and the thus ultimately obtained nitrate-poor tobacco-extract solution is centrifugally separated from the active slush and re-added to the tobacco leaves.

EXAMPLE 12 Example 5 is repeated with the single difference that an active slush obtained as in Example 8 is used.

EXAMPLE 13 Example 8 is repeated with the single difference that the inoculated stem-extract solution is incubated on a shaker at 30"C for 8 hours instead of 16 hours, so that the nitrate and nitrite anions are reduced to a lesser extent than in Example 8.

The obtained nitrate-poor stem-extract solution is then treated as in Example 8 and re-added to the stems, pre-dried and washed as in Example 8, which are then dried to a moisture content of 20%. The thus treated stems contain one third of their original content of nitrate and nitrite.

EXAMPLE 14 Example 10 is repeated with the single difference that the sprayed strips whose moisture content is 50% are stored only 8 hours instead of 24 hours at 30"C without air access.

During this time the microorganisms reduce the nitrates and nitrites contained in the strips to one quarter of their original content. The strips are then further treated as described in Example 10.

WHAT WE CLAIM IS 1. A process of treating tobacco which comprises anaerobically culturing a nitrate and/or nitrite-reducing micro-organism in the presence of at least part of the nitrate and/or nitrate content of the tobacco as an essential oxygen source for the micro-organism, the micro-organism being at an exponential growth phase when initially contacted with the said content and the reduction being immediately terminated when the desired reduced nitrate and/or nitrite content is reached and at most after 24 hours.

2. A process as claimed in Claim 1 wherein the micro-organism has been brought to its exponential growth phase under anaerobic conditions.

3. A process as claimed in Claim 1 or Claim 2 wherein the culture of micro-organisms is made to react so intensively that the nitrates and nitrites would be reduced to a minimal residue level (as hereinbefore defined) within 12 to 24 hours.

4. A process as claimed in Claim 3 wherein said minimal residue level would be reached within 20 hours.

5. A process as claimed in Claim 4 wherein the said desired reduced nitrate and/or nitrite content is said minimal residue level.

6. A process as claimed in any one of the preceding Claims wherein the said desired reduced nitrate and/or nitrite content is 25% or below of the original nitrate and/or nitrite content in the tobacco to be treated.

7. A process as claimed in Claim 6 wherein said reduced content is within the range 3% to 20% of said original content.

8. A process as claimed in Claim 7 wherein said reduced content is 5% of said original content.

9. A process as claimed in any one of the preceding Claims wherein a pure culture of bacteria Is used, which is obtained by inoculating a watery smear of nitrate-containing leaves or decayed leaves into a nutritive solution, which contains the amount of oxygen 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 during 15 to 25 hours by shaking under substantially complete exclusion of oxygen, and which is then used as active inoculation material for the inoculation of another fresh nutritive solution with which the incubation is repeated, and said inoculation and incubation is repeated until the pure culture has been formed.

10. A process as claimed in any one of Claims 1 to 8 wherein the micro-organism is a bacteria of the genus Aerobacter, Pseudomonas, Micrococcus or Echerichia or a fungi of the genus Rhodutorula.

11. A process as claimed in Claim 10 wherein the micro-organism is a pure culture of bacteria ATCC 13 048 Enterobacter aerogenes.

12. A process as claimed in any one of the preceding Claims wherein nitrates and nitrites and other rgater-soluble components are extracted with water from the tobacco to be treated, the thus obtained tobacco-extract solution cultured with the micro-organism and the resultant low-nitrate and/or nitrate tobacco extract is added to water washed tobacco.

13. A process as claimed in Claim 12 wherein the said tobacco-extract solution is isolated, inoculated with the micro organism culture, and, mixed with the nutritive solution, kept under substantially complete exclusion of air for 12 to 24 hours under anaerobic conditions favourable to the micro-organisms, and immediately afterwards the effect of the micro organisms is stopped by drawing off the active slush from the obtained nitrate-poor tobacco-extract solution, and the solution components contained in the nitrate-poor tobacco-extract solution are added to water washed tobacco.

14. A process as claimed in any one of Claims 1 to 11 wherein an active suspension is formed from micro organism culture and a nutritive solution, the tobacco to be treated is brought to a moisture content of 10 to 30% and then sprayed with the active suspension until it has reached a moisture level between 40 and 60% and is then kept under substantially complete exclusion of air for 12 to 24 hours under anaerobic conditions favourable to the micro organisms, and that the effect of the micro organisms is then stopped immediately by drying the tobacco to a moisture level between 10 and 30%.

15. A process as claimed in Claim 13 and Claim 14 wherein tobacco leaves to be treated are de-stemmed and the stems are separated from the remaining leaf strips, nitrates and nitrites are removed from the stems by the process of Claim 13 and the active slush obtained thereby is prepared into an active suspension with a nutritive solution, which is sprayed

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. EXAMPLE 14 Example 10 is repeated with the single difference that the sprayed strips whose moisture content is 50% are stored only 8 hours instead of 24 hours at 30"C without air access. During this time the microorganisms reduce the nitrates and nitrites contained in the strips to one quarter of their original content. The strips are then further treated as described in Example 10. WHAT WE CLAIM IS

1. A process of treating tobacco which comprises anaerobically culturing a nitrate and/or nitrite-reducing micro-organism in the presence of at least part of the nitrate and/or nitrate content of the tobacco as an essential oxygen source for the micro-organism, the micro-organism being at an exponential growth phase when initially contacted with the said content and the reduction being immediately terminated when the desired reduced nitrate and/or nitrite content is reached and at most after 24 hours.

onto the strips for the reduction of their nitrate and nitrite content by the process of Claim 14.

16. A process in accordance with and as claimed in any one of Claims 1 to 11 wherein the tobacco to be treated is ground and made into a slush with water, and, mixed with the micro organism culture and a nutritive solution, kept during 12 to 24 hours under anaerobic conditions favourable to the micro organisms, and 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 between 10 and 30%.

17. A process as claimed in any one of Claims 1 to 8, and 12 to 16 wherein the micro-organism naturally occurs in tobacco.

18. A process of treating tobacco to reduce the nitrate and/or nitrite content thereof substantially as hereinbefore described.

19. Tobacco whenever treated by a process as claimed in any one of the preceding Claims.