CH633169A5 - Method of upgrading tobacco - Google Patents

Description

The invention relates to a process for refining tobacco by breaking down nitrates and nitrites contained in tobacco.

Many tobacco products, such as Burley, contain nitrates and / or nitrites that can produce toxic nitrogen oxides when smoked. Fermentation processes are known in which these nitrogen salts are broken down enzymatically, but only to a very small extent and only as a side effect to other enzymatic reactions.

The object of the invention is to break down nitrates and nitrites to an acceptable residual content in order to reduce the proportion of nitrogen oxides in the smoke as much as possible. The breakdown of the nitrates and nitrites should take place as selectively as possible, that is to say as far as possible avoiding conversions of the tobacco components accompanying the nitrates and nitrites.

The invention is characterized in that a culture of microorganisms which require oxygen but are viable anaerobically denitrifying and which have been put into their exponential growth phase under anaerobic living conditions, with a nutrient solution for this culture which is not accessible to the microorganisms of this culture , essential oxygen source, under favorable anaerobic living conditions for the microorganisms of this culture, the nitrates, nitrites and other tobacco constituents accompanying them are brought into contact until the nitrates and nitrites are broken down to the desired residual content and then immediately, at the latest after 24 hours, the effect of the microorganisms is stopped. The microorganisms remain in their exponential growth phase under these conditions, as long as the required oxygen demand can be met from the nitrates and nitrites.

When applied, the microorganisms used are forced to cover their oxygen requirements from the nitrates and nitrites to be added, which reduces them to the level of nitrogen or ammonia. Nitrogen can escape without damage and in the form of ammonium salts, amino acids and

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Amines nitrogen does not form nitrogen oxides when fumigated

Since the added culture is already in its exponential growth phase, its microorganisms have a lead of about 8 hours over other undesirable microorganisms that are still in their latent phase. Such undesirable microorganisms cannot achieve this advantage within the exposure time, which is limited to a maximum of 24 hours, to such an extent that they can have a noticeable effect. In this way it can be ensured that the effect sought with the invention is selective.

Unfermented, air-dried tobacco often has a nitrate content of around 50 g per kg dry weight. In extreme cases, amounts of nitrate up to 80 g per kg dry weight were measured. The desired residual content depends on the reuse of the tobacco. For tobacco varieties that make up a significant part of the ready-to-smoke tobacco mixture, the desired residual content is a maximum of 5 g of the anions nitrate and nitrite per kg dry weight; in the case of tobacco varieties that only make up a small proportion of the ready-to-smoke mixtures, the desired residual content may be somewhat higher. In general, the desired residual content is in the range from 3 to 20%, preferably up to 5%, of the original content of the tobacco to be refined, based on the total weight of the anions nitrate and nitrite.

The selective effect of the invention can be further favored by bringing the culture of the microorganisms into so intense an effect that the nitrates and nitrites are broken down to a minimal residual content within 12 to 24 hours, preferably within 20 hours, and that caused action of the microorganisms is stopped immediately as soon as the desired residual content is reached.

The minimum residual content, that is the residual content that can be achieved with the invention without unusual measures, depends on the quality of the tobacco to be refined and it is 0.01 to 0.1% of the original content of the tobacco to be refined, in each case related on the total weight of the anions nitrate and nitrite. With such an intensive application, the desired residual content is reached after only a few hours, so that the action can be stopped before the microorganisms in the culture have used up their approximately 8-hour lead or shortly afterwards.

The impact of the microorganism culture can be intensified by optimizing the anaerobic living conditions for these microorganisms in terms of temperature, humidity, pH environment and nutrient supply and by bringing the culture into action from the start in high concentration on the nitrates and nitrites. The optimal conditions can be found by trying. The degradation of the nitrates and nitrites to the desired or minimum residual content can be analytically verified.

The exposure can be stopped by not maintaining the living conditions of the microorganisms, for example by sharply lowering the temperature, drying them, or by removing the microorganisms, for example by filtering them off as active sludge, if the exposure takes place in a liquid medium.

The microorganisms used can be bacteria, for example those of the genus Aerobacter, Pseudomonas, Micrococcus or Echerichia, but they can also be fungi, for example those of the genus Rhodutorula.

Bacteria belonging to the normal micro-fiora of tobacco leaves are preferred because they have a particularly fast denitrifying effect and do not undesirably change the tobacco.

A corresponding development of the invention is characterized in that a pure culture of bacteria is used, which is obtained by inoculating an aqueous swab of nitrate-containing leaves or decayed leaves of a nutrient solution which contains the oxygen content required for the incubation in the form of nitrates , to pH 6.6 to pH 7.5, preferably pH 7.4, is buffered and incubated at 25 to 35 ° C., preferably 30 ° C., for 15 to 25 hours, preferably 20 hours, by shaking with a substantial absence of oxygen and then used as active inoculum to inoculate a fresh such nutrient solution, with which the incubation is then repeated, and so on until the pure culture is formed.

The smear is preferably made from tobacco leaves. But you can also get a usable smear if you use forest soil that has arisen from mouldered leaves or contains mouldered leaves.

A pure culture is obtained by this process, the microorganisms of which are in an active, ie exponential, growth phase. This is either used immediately or kept and deactivated for this purpose and then reactivated before further use.

The invention can also be used advantageously in connection with a pure culture of the Enterobacter aerogenes bacterium, preferably from the standard strain ATCC 13 048. Pure cultures of this standard strain are available from the German Collection of Microorganisms, Göttingen, Grisebachstrasse 8, under the DSM image no. DSM 30 053 available.

In the case of tobacco parts in which, for example in the case of ribs, the nitrates and nitrites are difficult to access for microorganisms acting from outside, a further development of the invention is recommended, which is characterized in that nitrates and nitrites and other water-soluble constituents are mixed with water from the to be refined to refine tobacco, that the tobacco extract solution thus obtained is inoculated in isolation with the microorganism culture and mixed with the nutrient solution under strong restricted air access for 12 to 24 hours, preferably 16 hours, under favorable anaerobic living conditions for these microorganisms and that the action then immediately takes place the microorganisms is broken off by withdrawing the active sludge from the obtained low-nitrate tobacco extract solution and that the solution components contained in the low-nitrate tobacco extract solution are added with water from washed tobacco.

On the other hand, if, for example, the leaf parts between the ribs, the so-called strips, or leaves with ribs that are not as strong, are to be refined, or if it is not important to reduce the nitrate content in the depths of the ribs, then it is easier to proceed by an active suspension is formed from the microorganism culture and nutrient solution, the tobacco to be refined is brought to 10 to 30%, preferably 20%, moisture content and then sprayed with the active suspension until it has reached a moisture content of 40 to 60%, preferably 50% , and then stored under strongly restricted air access for 12 to 24 hours, preferably 24 hours, under favorable anaerobic living conditions for the microorganisms and that the action of the microorganisms is then stopped immediately and by drying the tobacco to 10 to 30%, preferably 20%, Moisture content.

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In many cases, for example in the reprocessing of tobacco waste, the tobacco is pulverized, slurried and formed into a film with binders, which is then solidified by drying. In such a case, it is advantageous to allow the microorganisms to act while the tobacco is slurried to the suspension, and this is preferably done by grinding the tobacco to be refined and slurrying with water and adding the microorganism culture and nutrient solution with greatly restricted air access for 12 to 12 hours 24 hours, preferably 24 hours, is kept under favorable anaerobic living conditions for the microorganisms and the effect of the microorganisms is then immediately stopped by shaping the suspension into or on foils and drying them to a moisture content of 10 to 30%, preferably 15% becomes.

The microorganism culture is preferably used as a pure culture, the degree of purity having to be sufficient to avoid significant side effects. The pure culture of tobacco leaves is preferably obtained.

The microorganism culture can be stored frozen in liquid nitrogen and is then thawed and reactivated for reuse. For ongoing use, it can be kept in an active state in a biostat from which portions are constantly being taken.

Properties of Enterobacter aerogenes:

Motile rods, 0.3-1.5 µm;

Gram - gas evolution at 37 ° C

Glycerin -f

Inositol +

Andonitol +

Voges-Proskauer +

Methyl red -

Phenylamine deaminase -

Urease -

Catalase +

Ornithine decarboxylase 4-

Lysine decarboxylase 4-

Hydrolysis of Aesculin + growth in the presence of KCN +

on malonate as the only carbon source +

The invention will now be explained in more detail with the aid of a few exemplary embodiments.

Example 1 Obtaining the Pure Culture:

20 g (grams) of D-glucose; 8.6 g peptone; 6.4 g NaCl; 3.5 g KN03; 4.5 g KH2P04 and 23.5 g Na2HP04-2H20 are dissolved in 1 liter (liter) of water. The nutrient solution obtained in this way is divided into 5 equal portions I to V of 200 ml (milliliters) each, each portion is placed in a 500 ml Erlenmeyer flask which is closed with a porous stopper. Then the whole is sterilized and stored at 20 ° C.

100 g of dry Burley tobacco leaves are washed with 500 ml of water under sterile conditions. 1 ml of the washing suspension formed is drawn off sterile and added to portion I of the nutrient solution. This portion I is incubated on a shaker at 30 ° C. for 16 hours. Then I ml of this portion is removed under sterile conditions and inoculated into Portion II of the nutrient solution, which is then treated like Portion I and from which 1 ml is inoculated into Portion III after incubation, and so on up to and including Portion V.

After the V portion has been incubated for 16 hours, it contains the pure culture of the microorganisms of a Pseu-domonas strain which covers its oxygen requirement by breaking down nitrates and nitrites. If this is not the case, everything is discarded and the extraction is repeated from the beginning with other Burley tobacco leaves. The microorganisms of this pure culture are in their exponential growth phase at this time and until 2 hours later.

Refinement of tobacco ribs:

1 kg of Maryland tobacco, in which the nitrates and nitrites are to be reduced, is stripped. That makes 250 g ribs and 750 g strips.

The 250 g ribs are washed with 1250 ml of 70 ° C warm water. Nitrate and nitrite present in the fins are extracted together with other water-soluble components. The rib extract solution obtained in this way is drawn off from the ribs, placed in a 21-Erlen-meyer flask which is sealed with a porous stopper, cooled to 30 ° C., mixed with 12.5 g of D-glucose and with 10 Inoculated ml of the pure culture mentioned above, namely within the period in which the microorganisms of this pure culture are still in their exponential growth phase.

The inoculated rib extract solution is incubated at 30 ° C. on a shaker for 16 hours until the anions nitrate and nitrite are broken down to a content of 0.1 g per liter.

The resulting low-nitrate rib extract solution is then immediately centrifuged, whereby the microorganisms settle, which are then removed as active sludge.

The centrifugate from the low-nitrate rib extract solution, which still contains the remaining tobacco components loosened from the ribs, is added to the pre-dried, washed-out ribs, which are then dried to a moisture content of 20%. The tobacco components previously released from the ribs with the nitrates and nitrites then return to the ribs, so that the ribs then essentially again contain their original components, with the exception of the nitrates and nitrites, which are reduced to 1/6 of the original content.

Example 2

As in Example 1, with the only difference that the centrifugate of the low-nitrate rib extract solution is not added back to the ribs from which the extract was obtained, but to other ribs which were treated in the same way.

Example 3 Refinement of tobacco leaves:

1 kg of Maryland tobacco, in which the nitrates and nitrites are to be reduced, is stripped. That makes 250 g ribs and 750 g strips. The 250 g ribs are treated as indicated in Example 1. When the active sludge is removed, the microorganisms of the 150 ml active sludge obtained are still in their exponential growth phase and are kept in this phase by this active sludge immediately together with 30 g D-glucose, 12.9 g peptone, 9.6 g NaCI, 6.75 g KH2P04 and 35.25 g Na2P04 • 2H20 in 1500 ml water. This gives a suspension of active microorganisms, which is then immediately sprayed in a uniform distribution onto the 750 g strips of 20% moisture content. The sprayed strips then have a moisture content of 50% and are stored for 24 hours at 30 ° C under air.

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finally stored. The microorganisms in the strips break down nitrates and nitrites down to V20 of the original content. The strips, which are reduced in their nitrate and nitrite content, are then dried to 20% moisture content for further processing. This inactivates the microorganisms remaining on the strips. The strips can now be processed further.

Example 4

Obtaining the pure culture as in Example 1.

Refining tobacco leaves:

250 g of Burley tobacco leaves are washed with 1250 ml of 70 ° C warm water. The resulting tobacco extract solution is further treated like the rib extract solution according to Example 1 and the low-nitrate tobacco extract solution finally obtained is centrifuged off from the active sludge and added to the tobacco leaves again.

Example 5

1 kg of tobacco waste, in which the nitrates and nitrites are to be reduced, is reduced to a grain size of at most 150 (finely ground. 150 ml of active sludge, which are obtained according to Example 1 and are still in their exponential growth phase, are in this held by immediately adding the active sludge together with 30 g D-glucose, 12.9 g peptone, 9.6 g NaCI, 675 g KH2P04 and 35.25 g Na2P04 • 2H20 in 1500 ml water Suspension of active microorganisms, into which the tobacco powder is then stirred in. The resulting slurry is stored for 24 hours at 30 ° C. in a 31-Erlenmeyer flask, which is closed with a porous stopper, in which case the microorganisms build nitrates and Nitrites down to Y10 of the original content .. Immediately afterwards, 150 g of carboxymethylcellulose are stirred into the slurry and the slurry is spread into a 3 mm thick layer and dried to 15% moisture content If the action of the microorganisms is ended and the porridge solidifies into films of regenerated tobacco, which can be processed further.

Example 6

Like example 1 with the only difference that the inoculated rib extract solution is incubated at 30 ° C on a shaker instead of 16 hours for only 8 hours, so that the anions nitrate and nitrite are degraded to a lesser extent than according to example 1.

The obtained low-nitrate rib extract solution is then further treated as in Example 1 and added to the ribs which have been pre-dried and washed out as in Example 1 and which are then dried to a moisture content of 20%. The ribs treated in this way contain a third of the original content of nitrate and nitrite.

Example 7

As example 3 with the only difference that the sprayed strips, whose moisture content is 50%,

instead of 24 hours, only 8 hours at 30 C with the exclusion of air. The microorganisms build in the nitrates and nitrites contained in the strips except for one

Quarter of the original salary. The strips are then treated as in Example 3.

Example 8

Refinement of tobacco ribs as in Example 1 with the only difference that the inoculation takes place with a pure culture of the bacterium Enterobacter aerogenes ATCC 13 048.

Example 9

Like Example 8, with the only difference that the centrifugate of the low-nitrate rib extract solution is not added back to the ribs from which the extract was obtained, but to other ribs that were treated in the same way.

Example 10 Refinement of tobacco leaves:

1 kg of Maryland tobacco, in which the nitrates and nitrites are to be reduced, is stripped. That makes 250 g ribs and 750 g strips. The 250 g ribs are treated as indicated in Example 8. The strips are treated using the active sludge obtained in the treatment of the ribs, as described in Example 3.

Example 11

Obtaining the pure culture as in Example 8. Refinement of tobacco leaves:

250 g of Burley tobacco leaves are washed with 1250 ml of 70 ° C water. The resulting tobacco extract solution is further treated like the rib extract solution according to Example 8 and the low-nitrate tobacco extract solution finally obtained is centrifuged off from the active sludge and added to the tobacco leaves again.

Example 12

As example 5 with the only difference that active sludge, which was obtained according to example 8, is used.

Example 13

Like example 8, with the only difference that the inoculated rib extract solution is incubated at 30 ° C. on a shaker instead of 16 hours for only 8 hours, so that the anions nitrate and nitrite are degraded to a lesser extent than according to example 8.

The low-nitrate rib extract solution obtained is then further treated as in Example 8 and added to the ribs which have been pre-dried and washed out as in Example 8 and which are then dried to a moisture content of 20%. The ribs treated in this way contain a third of the original content of nitrate and nitrite.

Example 14

Like example 10 with the only difference that the sprayed strips, whose moisture content is 50%, are stored for only 8 hours at 30 ° C. with the exclusion of air instead of 24 hours. The microorganisms break down the nitrates and nitrites contained in the strips to a quarter of their original content. The strips are then treated as in Example 10.

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Claims (8)

633 169 PATENT CLAIMS 1. A process for refining tobacco by breaking down the nitrates and nitrites contained in the tobacco, characterized in that a culture of microorganisms which require oxygen but are anaerobically denitrifying and which are in their exponential growth phase under anaerobic living conditions, with a nutrient solution for this culture, which has no essential oxygen source accessible to the microorganisms of this culture, is exposed to the nitrates, nitrites and other tobacco constituents accompanying them under favorable anaerobic living conditions for the microorganisms of this culture, until the nitrates and nitrites are broken down to the desired residual content and that the action of the microorganisms caused is then stopped immediately, at the latest after 24 hours. 2. The method according to claim 1, characterized in that the culture of the microorganisms is so intensely brought into action that the nitrates and nitrites are broken down to a minimal residual content within 12 to 24 hours and that the action of the microorganisms caused is stopped immediately as soon as the desired residual salary is reached. 3. The method according to claim 1 or 2, characterized in that a pure culture of bacteria is used, which is obtained by inoculating an aqueous smear of nitrate-containing leaves or decayed leaves of a nutrient solution, the oxygen content required for the incubation predominantly in the form of Contains nitrates, is buffered to pH 6.6 to pH 7.5 and is incubated at 25 to 35 ° C (degrees Celsius) for 15 to 25 hours by shaking with a substantial lack of oxygen and then used as active inoculum for inoculating a fresh such nutrient solution with which the incubation is then repeated and so on until the pure culture is formed. 4. The method according to claim 1 or 2, characterized in that a pure culture of the bacterium Enterobacter aerogenes from the strain ATCC 13 048 is used. 5. The method according to claim 2, 3 or 4, characterized in that nitrates and nitrites and other water-soluble constituents are dissolved out of the tobacco to be refined with water, that the tobacco extract solution thus obtained is inoculated in isolation with the microorganism culture and mixed with the nutrient solution with greatly restricted Air access is kept for 12 to 24 hours under favorable anaero-5 ben living conditions for these microorganisms and that the action of the microorganisms is then immediately stopped by removing the active sludge from the obtained low-nitrate tobacco extract solution and that the solution components contained in the low-nitrate tobacco extract-io solution Water from washed tobacco can be added. 6. The method according to claim 5, characterized in that an active suspension is formed from the microorganism culture and nutrient solution, that the tais bak to be refined is brought to 10 to 30% moisture content and then sprayed with the active suspension until it has a moisture content of 40 to 60% has been reached and is then stored under strongly restricted air access for 12 to 24 hours under favorable anaerobic living conditions for the micro-20 organisms and that the action of the microorganisms is then stopped immediately by drying the tobacco to 10 to 30% moisture content. 7. The method according to claim 5 and 6, characterized in that the tobacco leaves to be refined are stripped. 25 the ribs and the ribs are separated and that nitrates and nitrites of the ribs are extracted and broken down by the microorganisms in the tobacco extract solution obtained, and that the active sludge obtained is prepared with nutrient solution to form an active suspension which is used to break down the nitrates and nitrites of the strips are sprayed onto them. 8. The method according to claim 2, 3 or 4, characterized in that the tobacco to be refined is ground and slurried with water and with the micro 35 organism culture and nutrient solution are added to the microorganisms under strongly restricted air access for 12 to 24 hours under favorable anaerobic living conditions and that the action of the microorganisms is then immediately stopped 40 - by shaping the suspension into or on foils and to a moisture content of 10 to 30% is dried.