CONTROL OF SPORES ON TOBACCO

Abstract: In a first method of spore control, tobacco or tobacco extract liquor is treated with D-alanine to prevent the germination of Bacillus spores. Existing vegetative Bacillus cells are killed by heat treatment at a temperature of at least 60°C. In a second method of spore control, tobacco or tobacco extract liquor is treated with L-alanine to initiate germination of the Bacillus spores to vegetative Bacillus cells, which are then killed by heat treatment.

Description: This invention relates to a method of controlling the microflora present on tobacco. At present, microbiological activity on tobacco or in the aqueous extracts of tobacco made during the production of so-called "reconstituted tobacco" has been effected by treating the tobacco or extract with an agent which kills microorganisms. One such agent is propyl-4-hydroxybenzoate (propylparaben), another is sorbate. However, it has been observed that, whilst the microorganism population achieved initially after treatment with such agents is low, this initial level is not always maintained over a long period. It has now been surprisingly observed that the microflora population on tobacco, and hence in tobacco extracts, are principally of the Bacillus family. Bacillus life cycle involves sporulation, dormancy, spore germination, outgrowth and vegetative growth. On tobacco, most of the Bacillus are present in the dormant spore form. Spores are inactive with no detectable metabolic activity. They are resistant to harsh environmental factors such as heat, dessication and chemicals such as sorbate and propylparaben. They germinate in a suitable environment, to give vegetative cells, having proliferation capacity and metabolic activity. It is the products of cell metabolism which produce the characteristic indications of microbiological activity in tobacco. Tobacco flavouring for smoking products , J.C. Leffingwell et al , 1972, p.15, discloses the use of DL-alanine and L-alanine as tobacco flavourants; both are said to impart a "sweet" flavour to the tobacco smoke taste and aroma. Chemical Abstracts , vol.90, 1979, p.248, no. 36161a, discloses the inhibition of L-alanine initiated germination of Bacillus subtilis spores by, among other compounds, D-alanine. According to the present invention there is provided a method of controlling the level of Bacillus spores on tobacco or in tobacco extract liquor comprising treating the tobacco or tobacco extract liquor with L-alanine to cause at least a portion of Bacillus spores to germinate, and then. subjecting the tobacco or tobacco extract liquor to heat to kill the germinated cells. When L-alanine is applied to whole tobacco, it can conveniently be incorporated into the casing normally applied to tobacco. When L-alanine is applied to an aqueous extract for use in the production of "reconstituted tobacco", it is incorporated into the aqueous phase either before or after extraction of the tobacco. It may be the case that the temperature to which whole tobacco is subjected during drying or to which tobacco extracts are subjected during the extraction process is sufficiently high to kill the existing vegetative Bacillus cells without further heat treatment. It may be the case that the temperature to which whole tobacco is subjected during drying or to which tobacco extracts are subjected during the extraction process is sufficiently high to kill the vegetative Bacillus cells without further heat treatment. The invention will be further described in the following example. In the example, the spore and vegetative cell populations were measured using a plate count method, in which diluted extracts are spread onto petri dishes. In order to measure spore populations, the extracts were first heated for 12 minutes at 70°C, to kill any vegetative cells present. Example Tobacco was extracted with a 5.10⁻³ M aqueous solution of L-alanine (weight ratio 1:10) at 70°C for 20 minutes, with gentle stirring. The extract was slowly cooled to 40°C for 20 minutes, and then rapidly heated to 80°C for 10 minutes. A control extract, containing no L-alanine, was prepared in an identical way. Samples of the extracts were incubated at 37°C for 24 hours. Measurement of spore and vegetative cell counts (obtained by counting total mesophilic bacteria present and substracting the spore count) were as follows. After extraction After cooling After heating Extract Control pores 4.5X10⁶ per gram veg.cell 1X10³ per gram spores 4X10⁶ per gram veg.cell 0.5X10⁶ per gram spores 3.9X10⁶ per gram veg.cell 2X10³ per gram Trial with L-alanine 5 mM 4.5x10⁶ per gram 1X10³ per gram 1X10⁵ per gram 4.4X10⁶ per gram 5X10⁴ per gram 1X10³ per gram The levels in the untreated tobacco were: Bacillus spores: 4.5.10⁷ per gram Vegetative : 4.7.10⁷ per gram From the table it can be seen that the levels of both spores and vegetative cells present in the newly extracted liquors are the same for the control and the treated extract. However, after the cooling stage, it is apparent that most of the spores in the treated extract have germinated to vegetative cells. These are killed in the heat treatment stage, resulting in a much lower bacterial population in the treated extract than in the control sample. It has been found that particularly good results are achieved if the L-alanine concentration is 2.10⁻²M, or higher. The tobacco is extracted with aqueous L-alanine preferably at a temperature between 70°C and 80°C and is then incubated, preferably at a temperature between 30°C and 45°C. It is believed that germination is activated during extraction, by the 'heat shock' imparted to the spores by the hot aqueous L-alanine solution, and that the spores lose their heat resistance during incubation. A relatively short incubation, of perhaps 5 minutes, may well be sufficient to allow heat treatment at about 60°C to kill all or most of the Bacillus cells present on the tobacco.

Claims: A method of controlling the level of Bacillus spores on tobacco or in tobacco extract liquor comprising treating the tobacco or tobacco extract liquor with L-alanine to cause at least a portion of Bacillus spores to germinate, and then subjecting the tobacco or tobacco extract liquor to heat to kill the germinated cells. A method according to claim 1 in which tobacco is extracted with an aqueous solution of L-alanine to form an aqueous extract. A method according to claim 2, in which the concentration of L-alanine in the solution is at least 5.10⁻³M. A method according to claim 2 or 3 in which the concentration of L-alanine in the solution is at least 2.10⁻²M. A method according to claim 2,3 or 4, in which the extraction takes places at a first temperature, the resulting extract is held at second, lower, temperature to allow germination of the Bacillus spores and is then heated to a third temperature to kill the germinated cells. A method according to any of claims 2 to 5 in which the aqueous solution of L-alanine is at between 70°C and 80°C during the extraction step. A method according to any of claims 2 to 6 in which the extract is cooled to between 30°C and 45°C. A method according to any preceding claim in which the tobacco or tobacco extract liquor is subjected to a temperature of at least 60°C to kill the germinated cells. A method according to any preceding claim, in which the tobacco or tobacco extract liquor is subjected to a temperature of no more than 80°C to kill the germinated cells.

Inventors: HOFER, MICHEL GEORGES
Applicants: FABRIQUES DE TABAC REUNIES S.A.
Application year: 1989
Main classification: A24B15
IPC codes: A24B15, A01N37