CA1081076A - Process for refining tobacco - Google Patents
Process for refining tobaccoInfo
- Publication number
- CA1081076A CA1081076A CA299,837A CA299837A CA1081076A CA 1081076 A CA1081076 A CA 1081076A CA 299837 A CA299837 A CA 299837A CA 1081076 A CA1081076 A CA 1081076A
- Authority
- CA
- Canada
- Prior art keywords
- tobacco
- microorganisms
- nitrates
- culture
- nitrites
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- A—HUMAN NECESSITIES
- A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
- A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
- A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
- A24B15/18—Treatment of tobacco products or tobacco substitutes
- A24B15/20—Biochemical treatment
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Manufacture Of Tobacco Products (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
ABSTRACT
A process for finishing tobacco by which the nitrates and nitrites, contained in tobacco, are reduced by means of a pure culture of microorganisms, which has been brought and is kept in its exponential growth phase under anaerobic conditions, and whose effect upon the tobacco is stopped immediately when a sufficient reduction is reached.
A process for finishing tobacco by which the nitrates and nitrites, contained in tobacco, are reduced by means of a pure culture of microorganisms, which has been brought and is kept in its exponential growth phase under anaerobic conditions, and whose effect upon the tobacco is stopped immediately when a sufficient reduction is reached.
Description
10~L076 DE SCRIPTION
_ _ 1 The invention concerns a process for the finishing of tobacco through the reduction of nitrates and nitrites con-tained in tobacco.
Many tobaccos, Burley, for instance, contain nitrates and/or nitrites which can produce poisonous nitro~en oxides during smoking. There are known fermentation processes in which these nitrogen salts are reduced by ~ay of enzymes, however, only to a very small extent, ~nd only as a side-effect of other enzymatic conversions~
It is the task of the invention to reduce nitrates and nitrites to a wholesome residue level in order to reduce the share of nitrogen oxides in smoke as far as possible. The reduction of the nitrates and nitrites should take place as selectively as practicable, moreover, with the greatest possible avoidance of any conversions of the tobacco components accompany-ing nitrates and nitrites.
The invention specifies that a culture of micro-organisms requiring oxygen but capable of living by denitrating anaerobiously, which are brought to their exponential gro~th phase under anaerobic conditions, are made, with a nutritive solution for this culture, not containing an essential oxygen source available to the microorsanisms of this culture, under anaerobic conditions favorable to the microorganisms of this culture, to react on the nitrates, nitrites and other tobacco components accompanying these, until the nitrates and nitrites are reduced 3 _ .
. , ' . ! , , . ~ ~ ' ' , . ' ' : :
1 to the desired residue level, and that immediately then, at the latest after 24 hours, the produced effect of the microorganisms is stopped. Under these conditions the microorganisms will remain in their exponential growth phase as long as the necessary oxygen requirements can be filled from the nitrates and nitrites.
The microorganisms used are forced duriny the r~-action to cover their oxygen requirements from ~he to be reduced nitra~es and nitrites, whereby the latter are reduced to the level o~ nitrogen or ammonia respectively. Nitrogen can escape harmlessly, and nitrogen present in the form of ammonium salts, amino acids, and amines does not ~orm nitrogen oxides during smoking.
Since the added culture is already in its exponen-tial growth phase her microorganisms have a lead of about 8 hours over other undesired microorganisms, which are still in their latent phase. Such undesired microorganisms cannot recover this lead so far within the reaction period, limited to maximally 24 hours, that they could develop a noteworthy effect. This safe-guards that the effect promoted by the invention will be selective.
Un~ermented, air-dried tobaccos frequently have a nitrate content o~ about 50 g per kg dry weight. Nitrate quan-tities up to 80 g per kg dry weight were measured in extreme cases. The desired residue level will depend on the further use of the tobacco. For types of tobacco which constitute an essen-... ..
1(~810~6 1 tial part of the tobacco mixture ready for smoking, the desired residue level at its highest for the anions nitrate and nitrite rests at 5 g per kg dry weight; for types of tobacco constituting only smaller parts of the mixture ready for smoking, the desired residue level may be slightly higher. Generally the desired residue level rests within the range of 3 to 20 %, preferably at 5 %, of the original content of the to be finished tobacco, relative to the total weight of the anions nitrate and nitriteO
It is possible to avour the selective effect of the invention further by making the culture of microorganisms react so intensively that the nitrates and nitrites are reduced to a minimal residue level within 12 to 24 hours~ preferably within 20 hours, and that the produced effect oP the microorganisms is stopped immediately as soon as the desired residue level has been reached.
The minimal residue level, that iS9 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 to be finished tobacco, and amounts to from 0.01 to 0.1 % of the orginal content of the to be finished tobacco, in each instance relative to the total weight of the anions nitrate an(l nitrite. With such an intensive application the desired residue level can be reached after a few hours so that the effect can be stopped before ~he microorganisms of the culture have used up their approximately 8-hour lead or shortly therea~ter.
: .
. . .
~08~ 6 1 The ePfect of the microorganism culture can be in-tensified by bringing the anaerobic conditions for these micro-organisms to an optimum in regard to temperature, humidity, pH-level, and nutrient supply, and by making the culture react on the S nitrates and nitrites from the beginning in a high concentrationO
The optimal conditions can be ~ound 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 efPect 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~ Por instance, those from the genus Aerobacter, Pseudomonas, Micrococcus or Echerichia, or they may be Pungi such as from the genus Rhodutorula.
Preferable are bacteria belonging to the normal 2C microPlora of tobacco leaves because these ha~e a particularly rapid denitrifying effect and do not alter the tobacco in an un-desirable way.
The appropriate further development of the inven-tion specifies that a pure culture oP bacteria is used, which is obtained by inoculatin~ a watery smear of nitrate-containing . .. , ...... . . .... . .. ~ . ~ ..
, ' ~' ' ~,, - , . .
1081~6 1 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 35 C during 15 to 25 hours by shaking under far~reaching oxygen exclusion, and which is then used as active inoculation 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.
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 o this typ strain are available at the Deutsche Sammlung von Mikroorganismen, Gottingen, Grisebachstrasse 8, under the DSM-Registration-Number DSM 30053.
For parts o the tobacco, as for instance the stems, in which nitrates and nitrites are only with difficulty accessible to microorganisms acting from the outside, a further 37~
development o the invention is recommended, which specifies that nitrates and nitrites and other water-soluble components are removed with water from the to be finished tobaccoJ that the thus obtained tobacco-extract solution is isolated, inoculated with the microorganism culture, and, .
mixed with the nutritive solutionS is kept under greatly throttled air access for 12 to 24 hours, preferably 16 hours, undex anaerobic conditions avorable to these m cToorganisms, and that immediately afterwards the e~ect of the microorganisms is stopped by drawing off the actlve slush from the obtained nitrate~poor tobacco-extract solution, and that the solution components contained in the nitrate-poor tobacco-ex~ract solution are added to water washed tobacco.
However, when, for instance, the parts of the leaf between the ~tems, the so~called strips, or leaves with less pronounced stems, are to fie finished, or when it is not of importance to reduce the nitrate content also in the depth o~ the stems, the process can be made simpler by making an active suspension from the microorganism culture and the nutritive solution, by bringing the to be finished tobacco to a moisture level between 10 and 3Q %~ preerably 20 % and spraying it with the active suspension until it has reached a moisture level between 40 to 60 %, preferably 50 %, and by 20. ~hen keeping it under greatly ~hrottled air access for 12 to 24 hours, p~eferably 24 hours, under anaerobic conditions favorable to the micro- ;
organisms, and by i~mediately afterwards stopping the effect of the microorganisms by drying the tobacco .8-B
~. . . .
~8~ 6 1 to a moisture level between 10 to 30 %, pre~erably 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 s~elled into a suspension, and it is done preferably by grinding the to be ~inished -tobacco, swelling it with ~ater, and keeping it, mixed with the micro-organism culture and the nutritive solution, under greatly throttled air access during 12 to 24 hours, preferably 24 hours, under anaerobic conditions favorable to the microorganisms, and by immediately a~terwards 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 %, pre~erably 15 %. ~;
~5 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.
_ g _ . -- . . . -- , .
~a~81076 1 Characteristics of Enterobacter aerogenes are as follows:
Motile rods, 0.3 - 1.5 ~m;
Gram Development of gas at 37 C
Glycerin +
Inositol Andonitol +
Voses-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 proce-dural descriptions.
, .:
.. . . .
, .
.
- .' ': ~ ~
~0l~ 6 1 Example 1 .
Obtaining the Yure Culture:
20 g (grams) D-Glucose; 8.6 g Peptone;
6-4 g NaCl; 3.5 g KN03; 4.5 g KH2PO~ and 23.5 g Na2HP04 2H20 are dissolved in 1 l (liter) of water. The thus obtained nutri-tive solution is divided in 5 equal parts I through V of 200 ml (milliliter) each; each portion is placed into an Erlenmeyer flaslc holding 500 ml and the flask is closed with a porous stopper.
Then all oP 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 ~or 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 contalns the pure culture of microorganisms o~ 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 from the beginnung with other Burley-tobacco leaves. The microorganisms of this pure culture are at that point in time and until 2 hours - 11 - .
~ . , : . - . . , .
108~37'6 after~ards in their exponential growth phase~
Finishing of 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 1250 ml warm water of 70C~
This ~emo~es nitrates and nitrites contained in the stems together with other watex-soluble components. The thus obtained stem-extract solution is dra~n off from the stemsJ placed in a 2 l-Erlenmeyer flask, closed with a porous stopper, cooled to 30C, 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 30C so long, which is 16 hours, until the anions nitrate and nitrite are reduced to a content of 0 1 g per 1 The thus obtained nitrate-poor stem-extract solution is immediately centrifuged whereby the microorganisms are deposited and are drawn o~f as active slush~
The centrifuged substance from the nitrate-poor stem-extract solution, which still contains the remaining tobacco components removed ~rom the stems, is re~added to the predried, washed stems, which are then d~ied to a moisture level of 20 %. In this way all the tobacco components which had been removed beore ~ith the nitrates and nitrites from the stems are ~12~
- - . .
'7~i :
returned to the stems, so that the stems essentially contain their original components, except for nitrates and nitTiteS, which have been reduced to ~ ~-1/6 o~ their original conten~
Example 2 As example 1, wîth the single diference, that the centrifuged substance ~rom the nitrate-poor stem-extract solution is not re-added to those stems from which the extract had been obtained but to otheT stems which had been treated in the samt way~
Example 3 :
Finishing of TobaccQ Leaves:
1 kg Maryland-tobacco whose nitrates and nitrites are to be reduced is de~s~emmed, This yields 250 g stems and 750 g strips. The 250 g stems a~e treated as described in example l, The miCTOOTganiSmS of the thus obtained 150 ml active slush are still in their exponential growth phase ~hen 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 NaCl, 6~75 g KH2P04 and 35.25 g Na2P04 2H20 immediately into 1500 ml wateT.
Thus a suspension of active micToorganisms is obtained, which is then immediately sprayed in an even distribution onto the 750 g strips of a noisture content of 20 ~. The sprayed strips will then have a moisture ~ ;
content of 5Q % and are stored at 30C for 24 hours without air access.
During this time the microorganisms reducs the nitrates and nitrites con-tained in the strips to l/20 o their original content. The strips ~ith their thus reduced nitrate and 1 nitrite content are dried to a 20 % moisture level ~or their further processing. This inactivates any microorganisms still re~laining on the strips. The strips are now ready for fur~her processing.
~ E~
The pure culture obtained as in example 1.
Finishing o~ Tobacco ~eaves:
250 g Burley-tobacco leaves are washed in 1250 ml warm water of 70 C. The resulting tobacco-extract solution is treated further like the stem-extract solution of example 1, and the thus ulti-mately obtained nitrate-poor tobacco-extract solution is centri-fugally separated ~rom the active slush and re-added to the to-bacco leaves.
~ ' 1 kg tobacco waste in ~rhich nitrates and nitrites are to be re-duced is ground to a grain size of maximally 150 ~m fine. 150 ml active slush, obtained according to example 1 and still in their exponential growth phase, are lcept in their growth phase by placing the active slush with 30 g D-Glucose, 12.9 g Peptone, 9.6 g NaCl, 675 g KH2P04 (sic) and 35.25 g Na2P04 . 2H20 immedia~ely into 1500 ml water. This yields a suspension o~
active microorganisms which is stirred into the po1l~dered tobaccoO
The resulting pulp is kept ~or 24 hours at 30 C in a 3 l-Erlen-meyer ~lask, ~hich has been closed with a porous stopper. During that time the microorganisms reduce-nitrates and nitrites, . ~ .
. - .
.
~ : . ..
.
. . ..
7~i 1 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 spreafl 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 rege~erated tobacco, which are ready for further processing.
ExamPle 6 Like example 1 ~rith the single diference that the inoculated stem-extract solution is incubated on a shaker at 30 C for 8 hours instead of 16-hours, so that the anions nitrate and nitrite are reduced to a lesser extent than they are according to example 1.
:
The obtained nitrate-poor stem-extract solution is then further treated as in example 1 and re-added to the s~ems, pre-dried and washed, as in example 1, which are then dried to a mo~sture content of 20 %0 The thus treated stems contain one third of their original qontent of nitrate and nitrite.
Exarnple 7 Like example 3 with the single difference that the sprayed strips whose moisture content isl 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 fourth of their original content. The strips are 24 then further treated as described in example 3.
,~.
g~76 1 Exam~le 8 -~inishing of Tobacco Stems:
As example 1 ~rith the single difference that the inoculation will be carried out with a pure culture o bacteria Enterobactér aerogenes ATCC 13048.
Exam~le 9 As example ~ with the single difference that the centrifugalized substance 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 ~hich had been treated in the same way.
Example 10 ~inishing of 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 3.
The strips are treated as described in example 3 under using the active slush obtained by trea-ting the stems.
, . ' ,''. ' ;
.
; . .
, ~ 6 1 Exam~le 11 The pure culture obtained as in example 8 Finishing of Tobacco Leaves~
250 g Burley-tobacco leaves are washed in 1250 ml warm ~rater of 70 C. The resultin~ tobacco-extract solution is treated further like the stem-extract solution of example 8, and the thus ulti-mately obtained nitrate-poor tobacco-extract solution is centri-fugally separated from the active slush and re-added to the to-bacco leaves.
1G Example 12 .. . . ~ _ As example 5 with the single difference that an active slush is used, ~/hich t~as obtained r as in example 8.
i ' -Examp1e 13 ,.
Like.example 8 with the single diference that the inoculated stem-extract solution is;incubated on a shaker at 30 C for 8 hours instead of 16 hours, so that the anions nitrate and nitrite are reduced to a lesser extent than they are according to example 8.
The obtained nitrate-poor stem-extract solution is then further treated as in example 8 and re-added to the stems, pre-dried and ~/ashed, as in example 8, which are then dried to a moisture content of 20 %l The thus treated stems contain one third of their original content of nitrate and nitrite.
.; .
, ,. : .
.
-~0~ 76 1 Example 14 Like example 10 with the single difference that the sprayed strips whose moisture content is 50 % are stored only 8 hours in-stead of 24 hours at 30 C without air access. During this time the miCrQorganisms reduc~ ~ the nitrates and nitrites contained in the strips to one fourth of their original content. The strips are then further treated as describcd in example 10.
. .
: . ., . :
:.
, ~: .
,~
_ _ 1 The invention concerns a process for the finishing of tobacco through the reduction of nitrates and nitrites con-tained in tobacco.
Many tobaccos, Burley, for instance, contain nitrates and/or nitrites which can produce poisonous nitro~en oxides during smoking. There are known fermentation processes in which these nitrogen salts are reduced by ~ay of enzymes, however, only to a very small extent, ~nd only as a side-effect of other enzymatic conversions~
It is the task of the invention to reduce nitrates and nitrites to a wholesome residue level in order to reduce the share of nitrogen oxides in smoke as far as possible. The reduction of the nitrates and nitrites should take place as selectively as practicable, moreover, with the greatest possible avoidance of any conversions of the tobacco components accompany-ing nitrates and nitrites.
The invention specifies that a culture of micro-organisms requiring oxygen but capable of living by denitrating anaerobiously, which are brought to their exponential gro~th phase under anaerobic conditions, are made, with a nutritive solution for this culture, not containing an essential oxygen source available to the microorsanisms of this culture, under anaerobic conditions favorable to the microorganisms of this culture, to react on the nitrates, nitrites and other tobacco components accompanying these, until the nitrates and nitrites are reduced 3 _ .
. , ' . ! , , . ~ ~ ' ' , . ' ' : :
1 to the desired residue level, and that immediately then, at the latest after 24 hours, the produced effect of the microorganisms is stopped. Under these conditions the microorganisms will remain in their exponential growth phase as long as the necessary oxygen requirements can be filled from the nitrates and nitrites.
The microorganisms used are forced duriny the r~-action to cover their oxygen requirements from ~he to be reduced nitra~es and nitrites, whereby the latter are reduced to the level o~ nitrogen or ammonia respectively. Nitrogen can escape harmlessly, and nitrogen present in the form of ammonium salts, amino acids, and amines does not ~orm nitrogen oxides during smoking.
Since the added culture is already in its exponen-tial growth phase her microorganisms have a lead of about 8 hours over other undesired microorganisms, which are still in their latent phase. Such undesired microorganisms cannot recover this lead so far within the reaction period, limited to maximally 24 hours, that they could develop a noteworthy effect. This safe-guards that the effect promoted by the invention will be selective.
Un~ermented, air-dried tobaccos frequently have a nitrate content o~ about 50 g per kg dry weight. Nitrate quan-tities up to 80 g per kg dry weight were measured in extreme cases. The desired residue level will depend on the further use of the tobacco. For types of tobacco which constitute an essen-... ..
1(~810~6 1 tial part of the tobacco mixture ready for smoking, the desired residue level at its highest for the anions nitrate and nitrite rests at 5 g per kg dry weight; for types of tobacco constituting only smaller parts of the mixture ready for smoking, the desired residue level may be slightly higher. Generally the desired residue level rests within the range of 3 to 20 %, preferably at 5 %, of the original content of the to be finished tobacco, relative to the total weight of the anions nitrate and nitriteO
It is possible to avour the selective effect of the invention further by making the culture of microorganisms react so intensively that the nitrates and nitrites are reduced to a minimal residue level within 12 to 24 hours~ preferably within 20 hours, and that the produced effect oP the microorganisms is stopped immediately as soon as the desired residue level has been reached.
The minimal residue level, that iS9 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 to be finished tobacco, and amounts to from 0.01 to 0.1 % of the orginal content of the to be finished tobacco, in each instance relative to the total weight of the anions nitrate an(l nitrite. With such an intensive application the desired residue level can be reached after a few hours so that the effect can be stopped before ~he microorganisms of the culture have used up their approximately 8-hour lead or shortly therea~ter.
: .
. . .
~08~ 6 1 The ePfect of the microorganism culture can be in-tensified by bringing the anaerobic conditions for these micro-organisms to an optimum in regard to temperature, humidity, pH-level, and nutrient supply, and by making the culture react on the S nitrates and nitrites from the beginning in a high concentrationO
The optimal conditions can be ~ound 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 efPect 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~ Por instance, those from the genus Aerobacter, Pseudomonas, Micrococcus or Echerichia, or they may be Pungi such as from the genus Rhodutorula.
Preferable are bacteria belonging to the normal 2C microPlora of tobacco leaves because these ha~e a particularly rapid denitrifying effect and do not alter the tobacco in an un-desirable way.
The appropriate further development of the inven-tion specifies that a pure culture oP bacteria is used, which is obtained by inoculatin~ a watery smear of nitrate-containing . .. , ...... . . .... . .. ~ . ~ ..
, ' ~' ' ~,, - , . .
1081~6 1 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 35 C during 15 to 25 hours by shaking under far~reaching oxygen exclusion, and which is then used as active inoculation 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.
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 o this typ strain are available at the Deutsche Sammlung von Mikroorganismen, Gottingen, Grisebachstrasse 8, under the DSM-Registration-Number DSM 30053.
For parts o the tobacco, as for instance the stems, in which nitrates and nitrites are only with difficulty accessible to microorganisms acting from the outside, a further 37~
development o the invention is recommended, which specifies that nitrates and nitrites and other water-soluble components are removed with water from the to be finished tobaccoJ that the thus obtained tobacco-extract solution is isolated, inoculated with the microorganism culture, and, .
mixed with the nutritive solutionS is kept under greatly throttled air access for 12 to 24 hours, preferably 16 hours, undex anaerobic conditions avorable to these m cToorganisms, and that immediately afterwards the e~ect of the microorganisms is stopped by drawing off the actlve slush from the obtained nitrate~poor tobacco-extract solution, and that the solution components contained in the nitrate-poor tobacco-ex~ract solution are added to water washed tobacco.
However, when, for instance, the parts of the leaf between the ~tems, the so~called strips, or leaves with less pronounced stems, are to fie finished, or when it is not of importance to reduce the nitrate content also in the depth o~ the stems, the process can be made simpler by making an active suspension from the microorganism culture and the nutritive solution, by bringing the to be finished tobacco to a moisture level between 10 and 3Q %~ preerably 20 % and spraying it with the active suspension until it has reached a moisture level between 40 to 60 %, preferably 50 %, and by 20. ~hen keeping it under greatly ~hrottled air access for 12 to 24 hours, p~eferably 24 hours, under anaerobic conditions favorable to the micro- ;
organisms, and by i~mediately afterwards stopping the effect of the microorganisms by drying the tobacco .8-B
~. . . .
~8~ 6 1 to a moisture level between 10 to 30 %, pre~erably 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 s~elled into a suspension, and it is done preferably by grinding the to be ~inished -tobacco, swelling it with ~ater, and keeping it, mixed with the micro-organism culture and the nutritive solution, under greatly throttled air access during 12 to 24 hours, preferably 24 hours, under anaerobic conditions favorable to the microorganisms, and by immediately a~terwards 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 %, pre~erably 15 %. ~;
~5 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.
_ g _ . -- . . . -- , .
~a~81076 1 Characteristics of Enterobacter aerogenes are as follows:
Motile rods, 0.3 - 1.5 ~m;
Gram Development of gas at 37 C
Glycerin +
Inositol Andonitol +
Voses-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 proce-dural descriptions.
, .:
.. . . .
, .
.
- .' ': ~ ~
~0l~ 6 1 Example 1 .
Obtaining the Yure Culture:
20 g (grams) D-Glucose; 8.6 g Peptone;
6-4 g NaCl; 3.5 g KN03; 4.5 g KH2PO~ and 23.5 g Na2HP04 2H20 are dissolved in 1 l (liter) of water. The thus obtained nutri-tive solution is divided in 5 equal parts I through V of 200 ml (milliliter) each; each portion is placed into an Erlenmeyer flaslc holding 500 ml and the flask is closed with a porous stopper.
Then all oP 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 ~or 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 contalns the pure culture of microorganisms o~ 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 from the beginnung with other Burley-tobacco leaves. The microorganisms of this pure culture are at that point in time and until 2 hours - 11 - .
~ . , : . - . . , .
108~37'6 after~ards in their exponential growth phase~
Finishing of 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 1250 ml warm water of 70C~
This ~emo~es nitrates and nitrites contained in the stems together with other watex-soluble components. The thus obtained stem-extract solution is dra~n off from the stemsJ placed in a 2 l-Erlenmeyer flask, closed with a porous stopper, cooled to 30C, 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 30C so long, which is 16 hours, until the anions nitrate and nitrite are reduced to a content of 0 1 g per 1 The thus obtained nitrate-poor stem-extract solution is immediately centrifuged whereby the microorganisms are deposited and are drawn o~f as active slush~
The centrifuged substance from the nitrate-poor stem-extract solution, which still contains the remaining tobacco components removed ~rom the stems, is re~added to the predried, washed stems, which are then d~ied to a moisture level of 20 %. In this way all the tobacco components which had been removed beore ~ith the nitrates and nitrites from the stems are ~12~
- - . .
'7~i :
returned to the stems, so that the stems essentially contain their original components, except for nitrates and nitTiteS, which have been reduced to ~ ~-1/6 o~ their original conten~
Example 2 As example 1, wîth the single diference, that the centrifuged substance ~rom the nitrate-poor stem-extract solution is not re-added to those stems from which the extract had been obtained but to otheT stems which had been treated in the samt way~
Example 3 :
Finishing of TobaccQ Leaves:
1 kg Maryland-tobacco whose nitrates and nitrites are to be reduced is de~s~emmed, This yields 250 g stems and 750 g strips. The 250 g stems a~e treated as described in example l, The miCTOOTganiSmS of the thus obtained 150 ml active slush are still in their exponential growth phase ~hen 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 NaCl, 6~75 g KH2P04 and 35.25 g Na2P04 2H20 immediately into 1500 ml wateT.
Thus a suspension of active micToorganisms is obtained, which is then immediately sprayed in an even distribution onto the 750 g strips of a noisture content of 20 ~. The sprayed strips will then have a moisture ~ ;
content of 5Q % and are stored at 30C for 24 hours without air access.
During this time the microorganisms reducs the nitrates and nitrites con-tained in the strips to l/20 o their original content. The strips ~ith their thus reduced nitrate and 1 nitrite content are dried to a 20 % moisture level ~or their further processing. This inactivates any microorganisms still re~laining on the strips. The strips are now ready for fur~her processing.
~ E~
The pure culture obtained as in example 1.
Finishing o~ Tobacco ~eaves:
250 g Burley-tobacco leaves are washed in 1250 ml warm water of 70 C. The resulting tobacco-extract solution is treated further like the stem-extract solution of example 1, and the thus ulti-mately obtained nitrate-poor tobacco-extract solution is centri-fugally separated ~rom the active slush and re-added to the to-bacco leaves.
~ ' 1 kg tobacco waste in ~rhich nitrates and nitrites are to be re-duced is ground to a grain size of maximally 150 ~m fine. 150 ml active slush, obtained according to example 1 and still in their exponential growth phase, are lcept in their growth phase by placing the active slush with 30 g D-Glucose, 12.9 g Peptone, 9.6 g NaCl, 675 g KH2P04 (sic) and 35.25 g Na2P04 . 2H20 immedia~ely into 1500 ml water. This yields a suspension o~
active microorganisms which is stirred into the po1l~dered tobaccoO
The resulting pulp is kept ~or 24 hours at 30 C in a 3 l-Erlen-meyer ~lask, ~hich has been closed with a porous stopper. During that time the microorganisms reduce-nitrates and nitrites, . ~ .
. - .
.
~ : . ..
.
. . ..
7~i 1 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 spreafl 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 rege~erated tobacco, which are ready for further processing.
ExamPle 6 Like example 1 ~rith the single diference that the inoculated stem-extract solution is incubated on a shaker at 30 C for 8 hours instead of 16-hours, so that the anions nitrate and nitrite are reduced to a lesser extent than they are according to example 1.
:
The obtained nitrate-poor stem-extract solution is then further treated as in example 1 and re-added to the s~ems, pre-dried and washed, as in example 1, which are then dried to a mo~sture content of 20 %0 The thus treated stems contain one third of their original qontent of nitrate and nitrite.
Exarnple 7 Like example 3 with the single difference that the sprayed strips whose moisture content isl 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 fourth of their original content. The strips are 24 then further treated as described in example 3.
,~.
g~76 1 Exam~le 8 -~inishing of Tobacco Stems:
As example 1 ~rith the single difference that the inoculation will be carried out with a pure culture o bacteria Enterobactér aerogenes ATCC 13048.
Exam~le 9 As example ~ with the single difference that the centrifugalized substance 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 ~hich had been treated in the same way.
Example 10 ~inishing of 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 3.
The strips are treated as described in example 3 under using the active slush obtained by trea-ting the stems.
, . ' ,''. ' ;
.
; . .
, ~ 6 1 Exam~le 11 The pure culture obtained as in example 8 Finishing of Tobacco Leaves~
250 g Burley-tobacco leaves are washed in 1250 ml warm ~rater of 70 C. The resultin~ tobacco-extract solution is treated further like the stem-extract solution of example 8, and the thus ulti-mately obtained nitrate-poor tobacco-extract solution is centri-fugally separated from the active slush and re-added to the to-bacco leaves.
1G Example 12 .. . . ~ _ As example 5 with the single difference that an active slush is used, ~/hich t~as obtained r as in example 8.
i ' -Examp1e 13 ,.
Like.example 8 with the single diference that the inoculated stem-extract solution is;incubated on a shaker at 30 C for 8 hours instead of 16 hours, so that the anions nitrate and nitrite are reduced to a lesser extent than they are according to example 8.
The obtained nitrate-poor stem-extract solution is then further treated as in example 8 and re-added to the stems, pre-dried and ~/ashed, as in example 8, which are then dried to a moisture content of 20 %l The thus treated stems contain one third of their original content of nitrate and nitrite.
.; .
, ,. : .
.
-~0~ 76 1 Example 14 Like example 10 with the single difference that the sprayed strips whose moisture content is 50 % are stored only 8 hours in-stead of 24 hours at 30 C without air access. During this time the miCrQorganisms reduc~ ~ the nitrates and nitrites contained in the strips to one fourth of their original content. The strips are then further treated as describcd in example 10.
. .
: . ., . :
:.
, ~: .
,~
Claims (8)
1. A process for the finishing of tobacco through reduction of nitrates and nitrites, contained in tobacco, speci-fying that a culture of microorganisms, requiring oxygen but capable of living by denitrating anaerobiously, which has been.
brought to its exponential growth phase under anaerobic conditions, is made, with a nutritive solution for this culture, which does not contain an essential oxygen source available to this culture, to react under anaerobic conditions, favorable to the micro-organisms of this culture, on the nitrates, nitrites and other to-bacco components accompanying these until the nitrates and nitrites are reduced to the desired residue level, and that immediately then, at the latest after 24 hours, the produced effect of the microorganisms is stopped.
brought to its exponential growth phase under anaerobic conditions, is made, with a nutritive solution for this culture, which does not contain an essential oxygen source available to this culture, to react under anaerobic conditions, favorable to the micro-organisms of this culture, on the nitrates, nitrites and other to-bacco components accompanying these until the nitrates and nitrites are reduced to the desired residue level, and that immediately then, at the latest after 24 hours, the produced effect of the microorganisms is stopped.
2. A process in accordance with Claim 1 specifying that the culture of microorganisms is made to react so intensively that the nitrates and nitrites are reduced to a minimal residue level within 12 to 24 hours, and that the produced effect of the microorganisms is stopped immediately as soon as the desired residue level has been reached.
3. A process in accordance with Claim 1 or 2 specifying that 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 con-tains the amount of oxygen required for incubation predominantly in the form of nitrates, which is bufferd to between pH 6.6 and pH 7.5, and which is incubated at 25 to 35°C (degrees Celcius) during 15 to 25 hours by shaking under far-reaching oxygen exclusion, and which is then used as active inoculation 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 or 2 specifying that a pure culture of bacteria ATCC 13 048 Enterobacter aerogenes is used.
5. A process in accordance with Claim 1 specifying that nitrates and nitrites and other water-soluble components are removed with water from the to be finished tobacco, that the thus obtained tobacco-extract solution is isolated, inoculated with the microorganism culture, and, mixed with the nutritive solution, is kept under greatly throttled air access for 12 to 24 hours under anaerobic conditions favorable to these microorganisms, and that immediately afterwards the effect of the microorganisms is stopped by draw-ing off the active slush from the obtained nitrate-poor tobacco-extract solution, and that the solution components contained in the nitrate-poor tobacco-extract solution are added to water washed tobacco.
6. A process in accordance with either Claim 1 or Claim 5 specifying that an active suspension is formed from the microorganism culture and the nutritive solution, that the to be finished tobacco 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 greatly throttled air access for 12 to 24 hours under anaerobic conditions favorable to the microorganisms, and that the effect of the microorganisms is then stopped immediately by drying the tobacco to a moisture level between 10 and 30 %.
7. A process in accordance with Claim 5 specifying that the to be finished tobacco leaves are de-stemmed and the stems are separated from the strips, and that nitrates and nitrites are removed from the stems and re-duced by the microorganisms in the obtained tobacco-extract solution, and that the thus obtained active slush is prepared into an active suspension with the nutritive solution, which is sprayed onto the strips for the reduction of their nitrates and nitrites.
8. A process in accordance with Claim 5 specifying that the to be finished tobacco is ground and made into a slush with water, and, mixed with the microorganism culture and the nutritive solution, is kept during 12 to 24 hours under anaerobic conditions favorable to the microorganisms, and that immediately afterwards the effect of the microorganisms is stopped by forming the suspension into or onto sheets and drying them to a moisture level between 10 and 30 %.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| LU77272A LU77272A1 (en) | 1977-05-06 | 1977-05-06 | |
| LU77872A LU77872A1 (en) | 1977-07-29 | 1977-07-29 | PROCESS FOR REFINING TOBACCO |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1081076A true CA1081076A (en) | 1980-07-08 |
Family
ID=26640228
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA299,837A Expired CA1081076A (en) | 1977-05-06 | 1978-03-28 | Process for refining tobacco |
Country Status (7)
| Country | Link |
|---|---|
| AU (1) | AU520607B2 (en) |
| CA (1) | CA1081076A (en) |
| CH (1) | CH633169A5 (en) |
| DE (1) | DE2811690C3 (en) |
| FR (1) | FR2389341B1 (en) |
| GB (1) | GB1585023A (en) |
| NL (1) | NL188782C (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4651759A (en) * | 1983-04-12 | 1987-03-24 | Philip Morris Incorporated | Start-up process for the thermophilic denitrification of tobacco |
| US4685478A (en) * | 1981-10-01 | 1987-08-11 | Philip Morris Incorporated | Thermophilic denitrification of tobacco |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| LU79039A1 (en) * | 1978-02-09 | 1979-09-06 | Tabac Fab Reunies Sa | PROCESS FOR REFINING TOBACCO |
| DE2816427C2 (en) | 1977-05-06 | 1982-09-16 | Fabriques de Tabac Réunies S.A., 2003 Neuchâtel | Process for refining tobacco |
| EP0005082B1 (en) * | 1978-04-25 | 1982-05-12 | Philip Morris Incorporated | Microbial nitrate removal from tobacco materials by dissimilatory denitrification |
| US4556073A (en) * | 1978-06-15 | 1985-12-03 | Brown & Williamson Tobacco Corporation | Process for reduction of nitrate content of tobacco by microbial treatment |
| LU81611A1 (en) * | 1979-08-20 | 1981-03-24 | Tabac Fab Reunies Sa | METHOD FOR OBTAINING A NITRATE-FREE SOLUTION FROM A NITRATE-CONTAINING PRODUCT SOLUTION |
| DE3100715A1 (en) * | 1981-01-13 | 1982-07-22 | Fabriques de Tabac Réunies S.A., 2003 Neuchâtel | METHOD FOR PREPARING TOBACCO AND TOBACCO, PREPARED BY THIS PROCESS |
| CN113475744B (en) * | 2021-06-21 | 2022-05-24 | 河南中烟工业有限责任公司 | Method for preparing tobacco extract by using micrococcus |
| CN113475746B (en) * | 2021-06-21 | 2022-09-23 | 河南中烟工业有限责任公司 | Secondary extraction method of tobacco extract residues |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB121598A (en) * | 1917-12-15 | 1919-10-16 | Knud Erslev | Process for the Improvement of Tobacco. |
| FR821729A (en) * | 1936-05-19 | 1937-12-11 | Process for improving tobacco by fermentation | |
| DE669550C (en) * | 1936-05-20 | 1938-12-29 | Johannes Moser Dr | Process for fermentation of tobacco |
| US3747608A (en) * | 1971-06-18 | 1973-07-24 | Brown & Williamson Tobacco | Microbial digestion of tobacco materials |
| JPS4940960B2 (en) * | 1971-10-01 | 1974-11-06 | ||
| US4038993A (en) * | 1975-11-17 | 1977-08-02 | Brown & Williamson Tobacco Corporation | Process for reduction of nicotine content of tobacco by microbial treatment |
-
1978
- 1978-03-17 DE DE19782811690 patent/DE2811690C3/en not_active Expired
- 1978-03-23 GB GB1172978A patent/GB1585023A/en not_active Expired
- 1978-03-28 CA CA299,837A patent/CA1081076A/en not_active Expired
- 1978-03-30 CH CH342678A patent/CH633169A5/en not_active IP Right Cessation
- 1978-04-10 AU AU34921/78A patent/AU520607B2/en not_active Expired
- 1978-04-17 NL NL7804047A patent/NL188782C/en not_active IP Right Cessation
- 1978-05-02 FR FR7813771A patent/FR2389341B1/fr not_active Expired
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4685478A (en) * | 1981-10-01 | 1987-08-11 | Philip Morris Incorporated | Thermophilic denitrification of tobacco |
| US4651759A (en) * | 1983-04-12 | 1987-03-24 | Philip Morris Incorporated | Start-up process for the thermophilic denitrification of tobacco |
Also Published As
| Publication number | Publication date |
|---|---|
| CH633169A5 (en) | 1982-11-30 |
| DE2811690B2 (en) | 1981-07-30 |
| NL7804047A (en) | 1978-11-08 |
| FR2389341B1 (en) | 1982-09-24 |
| AU520607B2 (en) | 1982-02-11 |
| DE2811690A1 (en) | 1978-11-23 |
| NL188782B (en) | 1992-05-06 |
| AU3492178A (en) | 1979-10-18 |
| DE2811690C3 (en) | 1982-05-06 |
| FR2389341A1 (en) | 1978-12-01 |
| GB1585023A (en) | 1981-02-18 |
| NL188782C (en) | 1992-10-01 |
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