EP0056268A1 - Process for the preparation of tobacco - Google Patents

Abstract

For the denitration of tobacco, the soluble nitrates are extracted from the tobacco and broken down in the extract by metabolic assimilation. The proteins of the resulting biomass are hydrolyzed and aroma substances are obtained from the amino acids obtained by Maillard's reaction, which are then added to the tobacco.

Description

The invention relates to a process for the preparation of tobacco, in which soluble tobacco constituents are extracted with water and from the separated solution are eliminated by metabolic assimilation caused by microorganisms and subsequent separation of the biomass low molecular weight nitrogen compounds and the solution constituents remaining in the residual solution are added to an extracted tobacco .

In such a known method, nitrates, nitrites and ammonium compounds are extracted from the tobacco because in some cases these lead to substances which are undesirable for human consumption, for example nitrogen oxides, in tobacco smoke.

Flavor is also required for the preparation of tobacco, which are aroma-forming substances that form a taste characteristic of tobacco when smoking tobacco. Such flavor is obtained, for example, from amino acids that are subjected to the so-called Maillard reaction. The quality of the flavor obtained depends on the amino acids used.

The object of the invention is to provide the tobacco with a pleasant tobacco taste using tobacco components as far as possible.

The invention is characterized in that the proteins of the separated biomass are hydrolytically broken down into amino acids and that tryptophan is thereby destroyed to less than 0.01 Percent based on the weight of the amino acid mixture obtained and that the amino acid mixture obtained is then isolated with reducing sugar and converted with heat (Maillard reaction) into flavor (flavoring agent), which is then metered in. Tobacco is added.

The invention surprisingly leads to excellent flavoring and, apart from relatively cheap sugar, does not require any essential additional materials, because the yeast required as a starting product for the production of the amino acids is obtained in sufficient quantities when the tobacco is denitrated.

The flavor obtained can be used to prepare any tobacco, particularly of poor quality, for flavoring. However, it is advantageously used for tobacco which has been prepared by the denitration described at the outset and has therefore inevitably also lost flavor components which can then be compensated for by the flavoring. Flavoring according to the invention is also particularly advantageous in the case of reconstituted tobacco which has suffered such losses as a result of the pretreatment of the reconstitution.

A further development is characterized in that microorganisms, which can cover their nitrogen requirement through nitrate and nitrite degradation, are separated on the
Solution are prepared, which solution is enriched with a carbon source in a concentration of 16.5 ^± 10 assimilable carbon atoms per nitrate molecule and other nutrients required apart from nitrogen and with assimilation with 0.5 to 2.5 1. 1 ^-1 min ^-1 aerated and kept in the pH range from 3.5 to 6 and in the temperature range from ₂₅ ⁰ C to 37 ° C, and that the assimilation is carried out until the nitrate content to a maximum content of 10 ppm is reduced. When the nitrates are reduced, the nitrites and ammonium salts are inevitably reduced at the same time.

Preferably, the ventilation is carried out with 1.4 to 1.6 1.1 .min and preferably the pH to 5.5 ^± 0.3 and the temperature to 30 ° C ± 3 ⁰ C is maintained. Many yeasts are suitable in connection with the invention, but assimilation is preferably carried out by using a yeast from the group consisting of the Candida utilis NCYC 707, Candida berthetii CBS 5452, Candida utilis NCYC 321 and Candida utilis DSM 70167.

Not only water-soluble nitrates and nitrites lead to undesirable components in the smoke, this can also be caused by the nitrogen content of proteins or protein subunits. This is justified by a further development of the invention, which is characterized in that the insoluble protein components and protein subunits of the tobacco to be processed are first broken down into soluble protein fragments by enzymatic treatment and then these soluble protein fragments are extracted together with the other soluble tobacco components with water and then in the separated solution are subjected to metabolic assimilation. As a result, the nitrogen portions of the proteins are subjected to the same assimilation as the soluble nitrates and the like. Enzymes suitable for the enzymatic treatment are given in Table ₁ .

• Bei einer Temperatur von 50 - ₁₃0°C, vorzugsweise 90°C, während einer Zeitdauer von 2 - 300 Stunden, vorzugsweise 110 Stunden,
• bei einem Hefefeststoffgehalt von 5 - 50 Prozent (Gewichtsprozent), vorzugsweise 20 Prozent,
• bei einer Säurekonzentration von 0,5 - 45 N (normal), vorzugsweise bei Salzsäure 6 N und bei Phosphorsäure 45 N,
• bei einem Druck von 1 - 3 Atmosphären, vorzugsweise ₁ Atmosphäre und unter dauerndem Umrühren.

The proteins in the resulting biomass are hydrolyzed to obtain the amino acid mixture. This can be done by isolating the proteins from the biomass beforehand or by leaving them in the biomass. The hydrolysis is expediently carried out under the following conditions:

• At a temperature of _50-13 0 ° C, preferably 90 ° C, for a time from 2 to 300 hours, preferably 110 hours,
• with a yeast solids content of 5-50 percent (percent by weight), preferably 20 percent,
• at an acid concentration of 0.5 - 45 N (normal), preferably 6 N for hydrochloric acid and 45 N for phosphoric acid,
• at a pressure of 1-3 atmospheres, preferably ₁ atmosphere and with constant stirring.

_M can, in order to get from the amino acid mixture obtained to the flavor, first of all free the mixture obtained from the hydrolysis of the solid components. But this is not absolutely necessary. These solid components - the insoluble cell residues of the biomass - can also be left and subjected to the Maillard reaction. In such a case, however, the solution obtained in the hydrolysis will be concentrated in order to achieve the necessary concentration for the Maillard reaction. However, a method is preferred in which the amino acids are isolated before being subjected to the Maillard reaction. This is advantageously done by isolating the amino acid mixture contained in the solution by adsorption chromatography and then evaporating it to dryness.

• bei einem pH-Wert von 3 - ₁2, vorzugsweise 6 - 7,
• bei Angleichen des pH-Wertes durch Ammoniumhydroxyd oder Kaliumhydroxyd oder Phosphorsäure,
• bei einer Behandlungszeit von 1 - 200 Stunden, vorzugsweise 70 - 140 Stunden,
• bei einer Temperatur von 20 - 180 °C, vorzugsweise 90 - 140 °C,
• bei einem molaren Verhältnis von Aminosäuren zu zugesetztem reduzierenden Zucker von 4 : 1 bis 1: 4, vorzugsweise ₁ : 1 bis 2 : 1,
• in wässriger Lösung bei einem Feststoffgehalt von 20 bis 70 Gewichtsprozent, vorzugsweise 45 - 55 Gewichtsprozent,
• bei einem Druck von 1 - 5 Atmosphären, vorzugsweise 1 Atmosphäre und unter ständigem Rühren.

The Maillard reaction is conveniently carried out under the following conditions:

• at a pH of 3 - ₁ 2, preferably 6 - 7,
• when adjusting the pH value with ammonium hydroxide or potassium hydroxide or phosphoric acid,
• with a treatment time of 1 - 200 hours, preferably 70 - 140 hours,
• at a temperature of 20-180 ° C, preferably 90-140 ° C,
• with a molar ratio of amino acids to added reducing sugar of 4: 1 to 1: 4, preferably ₁ : 1 to 2: 1,
• in aqueous solution with a solids content of 20 to 70 percent by weight, preferably 45 to 55 percent by weight,
• at a pressure of 1-5 atmospheres, preferably 1 atmosphere and with constant stirring.

The reaction mixture resulting from the Maillard reaction can be used directly as a flavor and applied to the tobacco, for example by spraying. However, the essential components for the flavor can also be extracted from the reaction mixture, for example by shaking against dichloromethane or against isobutyl alcohol. The substances essential for the flavor can be isolated from the reaction product of the Maillard reaction by fractional distillation.

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

example 1 a) Deproteinization and denitration of tobacco

3.75 g (gram) of the enzyme Protease EC 3.4.24.4 with an enzyme activity of 1.0 enzyme unit per mg (milligram) were dissolved in 10 1 (liter) of water. One unit of enzyme is so that hydrolyzed activity casein at pH 7.5 and 37 ⁰ C (degrees Celsius), that the amount per minute of 1 Mykromol tyrosine is released. 1 kg (kg) of tobacco mixture (American blend) to be prepared in the form of so-called strips (stripped leaves) were slurried into this 10 1 enzyme solution. This slurry was allowed to stand for 6 hours at 37 ⁰ C and occasionally stirred. Then the aqueous phase was separated from the strips and then the strips were washed twice with 2.5 l of water at 80 ° C. and then pressed out. The aqueous phase, the wash water and the liquid obtained during the squeezing out were combined to form the solution. There was a total of 12 1 solution.

The pretreated tobacco, that is the pressed strips, was dried in flowing warm air to a residual moisture content of ₁ 8% (percent) and stored. The tobacco mixture to be prepared, the solution and the pretreated tobacco were analyzed analytically. This resulted in analytical values as in

TABLE 3 specified.

TABLE 3 shows that 58 dry weight percent of the proteins present in the tobacco mixture to be processed has been broken down and the breakdown products have been converted into the solution.

b) Metabolic assimilation

The following additions were entered into the 12 1 solution:

The solution prepared in this way was sterilized in an autoclave at 105 ° C. under pressure and then relieved of pressure and cooled to 30 ° _C. and transferred to a 20 l fermenter. The 30 ° C, prepared solution was inoculated with 600 ml (milliliter) of a culture of Candida utilis NCYC 707 which is in its exponential growth phase. The inoculated solution was left in the fermenter for 8 hours with aeration and stirring. The pH was first stabilized to pH 5.5 with KOH (potassium hydroxide) and later with citric acid. The proteins, amino acids, nitrates and nitrites were broken down by metabolic assimilation. After the 8 hours, the biomass was centrifuged off. This gave 2.25 l of biomass with a solids content of 8.5 _%, corresponding to ₁₉₀ g of anhydrous biomass.

The supernatant obtained by centrifugation - the so-called residual solution - contained the tobacco alkaloids in the originally present concentration, but beyond that only traces of soluble nitrogen compounds. The total volume of the residual solution of 9.75 l was reduced to 2 l and added to the existing tobacco by spraying. This tobacco was then dried. Table 2 shows analyzes of cigarettes made from such tobacco.

c) Obtaining amino acids

The biomass obtained in accordance with paragraph b) was dried. 1 kg of biomass obtained in this way was mixed with 3.5 l of HC1 (6N) and boiled under reflux for 140 hours. The resulting hydrolyzate was centrifuged at 15,000 xg for 20 minutes and the supernatant evaporated to a quarter of its original volume. The distillate contained the HC1 and could be used for further hydrolysis.

The same volume of an ion exchanger (300 g of Amberlite IR-120, Fluka) was added to the residue, 0.6 l of HC1 (0.1N) was added and the whole was shaken for 2 hours at room temperature. The ion exchanger was then separated from the liquid phase by filtration and washed with 250 ml of deionized water. The filtrate was discarded.

The ion exchanger in turn was mixed with 0.8 1 (NH ₄ ) OH (7N) and shaken for 2 hours at room temperature. The ion exchanger was filtered off again and washed with 150 ml of deionized water. The amino acids were in the filtrate, which was evaporated to dryness. In this way, '300 g of a mixture of amino acids could be obtained from the 1 kg of biomass. The composition of this mixture is shown in Table 4.

d) Obtaining the flavor

₁ 00 g in accordance with paragraph c) amino acid mixture obtained, were added 55 g of glucose and 188 g of deionized water. The pH was adjusted to 9.5 with a 25% ammonium hydroxide solution. The reaction mixture was then refluxed for 70 hours. The resulting brown product solution was filtered. The filtrate was sprayed on reconstituted tobacco in amounts of 20%.

Example 2 a) Denitration of tobacco

₁ kg of a tobacco mixture consisting of Burley ribs was washed with 14 l of water for 1 hour at 80 ° C. and then pressed out. The squeezed out wash water gave a total of 12 l of solution and contained 1140 ppm of nitrate nitrogen.

b) Metabolic assimilation. Like example 1.

• c) Obtaining amino acids. Like example 1.
• d) Obtaining the flavor. Like example 1.

Example 3

• a) Denitration of tobacco. Like example 2.
• b) Metabolic assimilation. Like example 2.
• c) Obtaining amino acids

The biomass obtained according to paragraph b) was dried. 1 kg of biomass obtained in this way was mixed with 5 1 H ₃ P0 ₄ (85%) and boiled under reflux for 140 hours. The resulting hydrolyzate was filtered through a frit and 300 g of Amberlite IR-120 and 0.6 l of HC1 (0.1N) were added to the filtrate. This mixture was shaken for ₁ 0 hours at room temperature. The Amberlite was then separated from the liquid phase by filtration and washed with 360 ml of deionized water. The filtrate was discarded.

The ion exchanger in turn was mixed with 0.8 1 (NH ₄ ) OH (7N) and shaken for 2 hours at room temperature. The ion exchanger was filtered off again and washed with ₁₅ 0m1 deionized water. The amino acids were in the filtrate, which was evaporated to dryness. In this way, 320 g of a mixture of amino acids could be obtained from the ₁ kg of biomass. The composition of this mixture is shown in Table 4.

d) Obtaining the flavor. Like example 1. Example 4

• a) Denitration of tobacco. Like example 2.
• b) Metabolic assimilation. Like example 2
• c) Obtaining amino acids

The biomass obtained in accordance with paragraph b) was dried to a moisture content of 60%. This gave 500 g of moist yeast. 2.5 kg of yeast thus obtained were mixed with 6 1 H ₃ P0 ₄ (85%) and hydrolyzed as indicated in paragraph c) Example 4. 300 g of amino acid mixture were obtained, the composition of which is given in Table 4.

c) Obtaining the flavor. Like example 1. Example 5

As in Example 4, with the only difference that when amino acids are obtained, 1.2 kg of yeast with a moisture content of 70% are hydrolyzed and 120 g of amino acid mixture are obtained therefrom.

Example 6

As Example 3 with the difference that 5 1 KOH (6N) are used instead of H ₃ P0 ₄ in the extraction of the amino acids. The pH of this solution was adjusted to 1.1 with HC1 before the ion exchanger was added. 280 g of an amino acid mixture could be obtained, the composition of which can be seen from Table 4.

Example 7

• a) Deproteinization and denitration of tobacco. Like example 1.
• b) Metabolic assimilation. Like example ₁ .
• c) Obtaining amino acids. Like example 1.
• d) Obtaining the flavor.

332 g of an amino acid mixture obtained according to paragraph c) were mixed with 252 g xylose and 510 ml dionized water. The pH was adjusted to 7.0 with a 25% ammonium hydroxide solution. The reaction mixture was then kept for ₁ 40 Held under reflux at 90 ° C. The resulting brown product liquid was filtered. The filtrate was sprayed on reconstituted tobacco in amounts of up to 20%.

Example 8

• a) Deproteinization and denitration of tobacco. Like example ₁ .
• b) Metabolic assimilation. Like example 1.
• c) Obtaining amino acids. Like example 1.
• d) Obtaining the flavor.

219 g of an amino acid mixture obtained in accordance with paragraph c) were mixed with 120 g of glucose and 435 ml of deionized water. The pH was adjusted to 6.8 with a 25% ammonium hydroxide solution. Then the reaction mixture for 24 hours at ₁ 05 ⁰ C was maintained under pressure. The resulting brown product solution was filtered. The filtrate was sprayed on bad Burley tobacco in amounts of 20%.

Example 9

• a) Denitration of tobacco. Like example 2.
• b) Metabolic assimilation. Like example 2.
• c) Obtaining amino acids. Like example 2.
• d) Obtaining the flavor

180 g of an amino acid mixture obtained according to paragraph c) were mixed with 76 g glucose and 400 ml deionized water. The reaction mixture was then boiled under reflux at 90 ° C. for 120 hours. The resulting brown product solution was filtered. The filtrate was sprayed on extracted tobacco strips in amounts of up to 40%.

Example 10

• a) _D enitration of tobacco. Like example 2.
• b) Metabolic assimilation. Like example 2.
• c) Obtaining amino acids. As example 3.
• d) Obtaining the flavor

217 g of an amino acid mixture obtained in accordance with paragraph c) were mixed with 91 g of glucose and 480 ml of deionized water. The reaction mixture was then kept under pressure at 110 ° C. for _{1 to} 20 hours. The resulting brown product solution was filtered. The filtrate was sprayed on extracted tobacco fins in amounts of up to 80%.

Example 11

• a) Denitration of tobacco. Like example 2.
• b) Metabolic assimilation. Like example 2.
• c) Obtaining amino acids. As example 4.
• d) Obtaining the flavor as in Example 10. However, a temperature of 180 ° C. was used.

Example 12

• a) Deproteinization and denitration of tobacco. Like example 1.
• b) Metabolic assimilation. Like example 2.
• c) Obtaining amino acids. As example 5.
• d) Obtaining the flavor.

As in Example 8. However, the pH was adjusted to 11.5.

Example 13

• a) Deproteinization and denitration of tobacco. Like example 1.
• b) Metabolic assimilation. Like example 2.
• c) Obtaining amino acids. As example 6.
• d) Obtaining the flavor.

As in Example 8. However, the reaction was only carried out for 1 hour and the filtrate was added in amounts of 15% reconstituted tobacco.

Example 14

As in example 9. However, 350 g of glucose were used.

Example 15

As in Example 9. The reaction was carried out at 50 ° C for 200 hours.

• Anstelle des im Beispiel 1 genannten Enzyms kann ein anderes Enzym aus Tabelle 1 eingesetzt werden.
• Anstelle der Glucose kann eine equimolare Menge Fructose, Galactose oder dergleichen eingesetzt werden.
• Anstelle der Candida utilis NCYC 707 kann eine der nachfolgenden Candidas eingesetzt werden: Candida berthetii CBS 5452, Candida utilis NCYC 32₁ und Candida utilis DSM 70167.

The examples can be modified as follows:

• Instead of the enzyme mentioned in Example 1, another enzyme from Table 1 can be used.
• Instead of glucose, an equimolar amount of fructose, galactose or the like can be used.
• Instead of the Candida utilis NCYC 707, one of the following Candidas can be used: Candida berthetii CBS 5452, Candida utilis NCYC 32 ₁ and Candida utilis DSM 70167.

• NCYC National Collection of Yeast Cultures, Brewing Industry Research Foundation;
• CBS Centraal Bureau voor Schimmelcultures;
• DSM Deutsche Sammlung von Mikroorganismen.
  
  
  
  

The strains mentioned are available at the depository locations indicated by the abbreviations in the form required for patent disclosure under the number given. The abbreviations mean:

• NCYC National Collection of Yeast Cultures, Brewing Industry Research Foundation;
• CBS Centraal Bureau voor Schimmelcultures;
• DSM German collection of microorganisms.
  
  
  
  

Claims (10)

1. A process for the preparation of tobacco, in which soluble tobacco components are extracted with water and from the separated solution by metabolic assimilation caused by microorganisms and subsequent separation of the biomass low molecular weight nitrogen compounds are eliminated and the solution components remaining in the residual solution are added to an extracted tobacco characterized in that the proteins of the separated biomass are hydrolytically broken down into amino acids and that tryptophan is thereby destroyed to less than 0.01 percent based on the weight of the amino acid mixture obtained and that the amino acid mixture obtained is then isolated with reducing sugar with heating (Maillard reaction ) is converted into flavor (aroma generator), which is then added to tobacco in doses. 2. The method according to claim 1, characterized in that microorganisms that can cover their nitrogen requirement through nitrate and nitrite degradation are prepared on the separated solution, which solution with a carbon source in a concentration of 16.5 ± 10 assimilable carbon atoms each a nitrate molecule and other nutrients required apart from nitrogen is enriched and with assimilation with ₀ , ₅ to ₂ , 5 1. 1 ^-1 . min ^-1 aerated and kept in the pH range from 3.5 to 6 and in the temperature range from 25 ° C to 37 ⁰ C, and that the assimilation is carried out until the nitrate content is reduced to a maximum content of 10 ppm . 3. The method according to claim 1 or 2, characterized in that the assimilation is carried out by using a yeast from the group consisting of the Candida utilis NCYC 707, Candida berthetii CBS 5452, Candida utilis NCYC 321 and Candida utilis DSM 70167 and that the ventilation with 1.4 to 1.6 1. 1 ^-1 . min ^-1 and that the pH is kept at 5.5 ± 0.3 and the temperature at 30 ° C ± 3 ° C. 4. The method according to any one of the preceding claims, characterized in that first the insoluble protein constituents and protein subunits of the tobacco to be processed are broken down into soluble protein fragments by enzymatic treatment, and then these soluble protein fragments are extracted together with the remaining soluble tobacco constituents with water and then in the separated solution are subjected to metabolic assimilation. ₁₅ . Method according to one of the preceding claims, characterized in that the hydrolysis of the proteins of the biomass takes place under conditions as follows: At a temperature of 50 - 130 ° C for a period of 2 - 300 hours with a yeast solids content of 5 - 50 percent (weight percent) at an acid concentration of 0.5 - 45 N (normal) at a pressure of 1-3 atmospheres and with constant stirring. 6. The method according to claim 5, characterized in that the insoluble residual constituents of the biomass are separated from the hydrolyzed solution before the solution is further processed. 7. The method according to claim 6, characterized in that the amino acid mixture contained in the solution is isolated by adsorption chromatography and then evaporated to dryness. 8. The method according to any one of the preceding claims, characterized in that the amino acid mixture obtained in the Maillard reaction is converted into flavor under the following conditions: At a pH of 3 - 12, when adjusting the pH value with ammonium hydroxide or potassium hydroxide or phosphoric acid, with a treatment time of 1 - 200 hours, at a temperature of 20 - 180 ° C, with a molar ratio of amino acids to the added reducing sugar from 4: 1 to 1: 4, in aqueous solution, at a solids content of 20 to 70 percent by weight, at a pressure of 1 - 5 atmospheres and with constant stirring. 9. The method according to claim 8, characterized in that the reaction mixture generated with the Maillard reaction is sprayed as a flavor on tobacco in an amount of 0.05 to 0.8 milliliters per gram of tobacco material. 10. The method according to any one of the preceding claims, characterized in that reconstituted tobacco is treated with the flavor obtained.