CA2889054C - Treatment of tobacco material - Google Patents
Treatment of tobacco material Download PDFInfo
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- CA2889054C CA2889054C CA2889054A CA2889054A CA2889054C CA 2889054 C CA2889054 C CA 2889054C CA 2889054 A CA2889054 A CA 2889054A CA 2889054 A CA2889054 A CA 2889054A CA 2889054 C CA2889054 C CA 2889054C
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- tobacco material
- enzyme
- protease
- tobacco
- operating conditions
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- 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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- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Manufacture Of Tobacco Products (AREA)
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Abstract
A method is provided for treating a tobacco material. The method comprises treating tobacco material with a combination of protease enzymes with different optimal operating conditions and adjusting the operating conditions during the enzyme incubation period. Also provided is a tobacco material which has been treated by such a method or a derivative thereof, and a smoking article which comprises a tobacco material treated by such a method.
Description
Treatment of Tobacco Material Field The present invention relates to a method for the treatment of tobacco material.
Background In some circumstances, it may be desirable to reduce the content of certain constituents from tobacco material before incorporating the tobacco material into a smoking article such as a cigarette.
Summary According to a first aspect, there is provided a method for treating a tobacco material, which comprises using a combination of protease enzymes with different optimal operating conditions and adjusting the operating conditions during the enzyme /5 incubation period.
In some embodiments, the method results in a reduction in the protein content of the treated tobacco material compared to the protein content of the untreated tobacco material.
In some embodiments, adjusting the operating conditions comprises adjusting the pH.
In some embodiments, adjusting the pH comprises decreasing the pH, optionally by the addition of an acid. In some embodiments, adjusting the pH comprises increasing the pH, optionally by the addition of an alkali.
In some embodiments, adjusting the operating conditions comprises adjusting the temperature. In some embodiments, adjusting the temperature comprises adjusting from a lower temperature to a higher temperature. In some embodiments, adjusting the temperature comprises a adjusting from a higher temperature to a lower temperature.
In some embodiments, the combination of enzymes comprises enzymes with two different optimal operating conditions. In some embodiments, the combination of enzymes comprises enzymes with three different optimal operating conditions.
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Background In some circumstances, it may be desirable to reduce the content of certain constituents from tobacco material before incorporating the tobacco material into a smoking article such as a cigarette.
Summary According to a first aspect, there is provided a method for treating a tobacco material, which comprises using a combination of protease enzymes with different optimal operating conditions and adjusting the operating conditions during the enzyme /5 incubation period.
In some embodiments, the method results in a reduction in the protein content of the treated tobacco material compared to the protein content of the untreated tobacco material.
In some embodiments, adjusting the operating conditions comprises adjusting the pH.
In some embodiments, adjusting the pH comprises decreasing the pH, optionally by the addition of an acid. In some embodiments, adjusting the pH comprises increasing the pH, optionally by the addition of an alkali.
In some embodiments, adjusting the operating conditions comprises adjusting the temperature. In some embodiments, adjusting the temperature comprises adjusting from a lower temperature to a higher temperature. In some embodiments, adjusting the temperature comprises a adjusting from a higher temperature to a lower temperature.
In some embodiments, the combination of enzymes comprises enzymes with two different optimal operating conditions. In some embodiments, the combination of enzymes comprises enzymes with three different optimal operating conditions.
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- 2 -In some embodiments, the protein content of the treated tobacco material is reduced by at least 50%, at least 60%, at least 70%, at least 80%, and/or at least 90%, compared to the protein content of the untreated tobacco material.
According to a second aspect, there is provided a tobacco material which has been treated by a method according to the first aspect, or a derivative thereof.
According to a third aspect, there is provided a smoking article comprising a tobacco material according to the second aspect.
According to a fourth aspect, there is provided the use of a combination of protease enzymes with different optimal operating conditions for removing one or more proteins from a tobacco material by adjusting the operating conditions during the enzyme incubation period.
According to another aspect, there is provided a method for treating a tobacco material, wherein the method comprises incubating the tobacco material for a period of time with a combination of protease enzymes with different optimal operating conditions and adjusting the operating conditions during the enzyme incubation period from the optimal temperature and/or pH of one protease enzyme to the optimal temperature and/or pH of another protease enzyme.
According to another aspect, there is provided use of a combination of protease enzymes with different optimal operating conditions for removing one or more proteins from a tobacco material by incubating the tobacco material with the protease enzymes for a period of time and adjusting the operating conditions during the enzyme incubation period from the optimal temperature and/or pH of one protease enzyme to the optimal temperature and/or pH of another protease enzyme.
Brief Description of the Drawings Embodiments of the invention are described, by way of example only, with reference to the accompanying drawings, in which:
- 2a -Figure 1 is a flow diagram illustrating a method of treating tobacco material in accordance with an embodiment of the present invention.
Figure 2 is a flow diagram illustrating a method of treating tobacco material in accordance with an embodiment of the present invention.
Figure 3 is a table of the results of enzymatic treatment of unwashed tobacco with two proteases with different optimal operating conditions with a shift in pH value and/or temperature during enzyme incubation.
Figure 4 is a table of the results of enzymatic treatment of washed tobacco with two proteases with different optimal operating conditions with a shift in pH value and/or temperature during enzyme incubation.
Figure 5 is a table of the results of enzymatic treatment with a protease with no shift in pH
value or temperature during enzyme incubation.
Figure 6 is a graph of the results of enzymatic treatment of washed tobacco with two proteases with different optimal operating conditions with a shift in pH value from pH
10 to 4 during enzyme incubation.
According to a second aspect, there is provided a tobacco material which has been treated by a method according to the first aspect, or a derivative thereof.
According to a third aspect, there is provided a smoking article comprising a tobacco material according to the second aspect.
According to a fourth aspect, there is provided the use of a combination of protease enzymes with different optimal operating conditions for removing one or more proteins from a tobacco material by adjusting the operating conditions during the enzyme incubation period.
According to another aspect, there is provided a method for treating a tobacco material, wherein the method comprises incubating the tobacco material for a period of time with a combination of protease enzymes with different optimal operating conditions and adjusting the operating conditions during the enzyme incubation period from the optimal temperature and/or pH of one protease enzyme to the optimal temperature and/or pH of another protease enzyme.
According to another aspect, there is provided use of a combination of protease enzymes with different optimal operating conditions for removing one or more proteins from a tobacco material by incubating the tobacco material with the protease enzymes for a period of time and adjusting the operating conditions during the enzyme incubation period from the optimal temperature and/or pH of one protease enzyme to the optimal temperature and/or pH of another protease enzyme.
Brief Description of the Drawings Embodiments of the invention are described, by way of example only, with reference to the accompanying drawings, in which:
- 2a -Figure 1 is a flow diagram illustrating a method of treating tobacco material in accordance with an embodiment of the present invention.
Figure 2 is a flow diagram illustrating a method of treating tobacco material in accordance with an embodiment of the present invention.
Figure 3 is a table of the results of enzymatic treatment of unwashed tobacco with two proteases with different optimal operating conditions with a shift in pH value and/or temperature during enzyme incubation.
Figure 4 is a table of the results of enzymatic treatment of washed tobacco with two proteases with different optimal operating conditions with a shift in pH value and/or temperature during enzyme incubation.
Figure 5 is a table of the results of enzymatic treatment with a protease with no shift in pH
value or temperature during enzyme incubation.
Figure 6 is a graph of the results of enzymatic treatment of washed tobacco with two proteases with different optimal operating conditions with a shift in pH value from pH
10 to 4 during enzyme incubation.
3 PCT/GB2013/053100 Figure 7 is a graph of the results of enzymatic treatment of washed tobacco with two proteases with different optimal operating conditions with a shift in pH value from pH
4 to 10 during enzyme incubation.
Figure 8 is a schematic side view of a smoking article including treated tobacco material according to embodiments of the invention.
Detailed Description The present invention relates to a method for treating a tobacco material, for removing proteins from tobacco material and/or reducing the protein content of tobacco io material.
Treating tobacco material may be used to remove one or more other chemical substances which are desirable to remove. Proteins in tobacco material are often found to be, and/or to be precursors of, such substances.
Alternatively or in addition, it may be desirable to reduce the protein content of tobacco material to improve the quality of the tobacco material.
Methods attempting to remove proteins have been proposed, although they have tended to be expensive, lengthy and/or detrimental to the physical structure of the tobacco material, and/or not reduce the protein content to a desired level.
The method of the present invention removes proteins from tobacco material.
Exemplary proteins that may be removed from the tobacco material include RuBisCO.
The method of the present invention results in a reduction in the protein content of tobacco material compared to the protein content of the untreated tobacco material.
Figure 1 is a flow diagram illustrating a method of treating tobacco material in accordance with an embodiment. Tobacco material 1 undergoes protease enzyme treatment 10. The solid and liquid components of the resulting suspension are separated 20, resulting in a tobacco material with reduced protein content 30 and a liquid component 31.
As used herein, the term "tobacco material" includes any part, such as for example the leaves or stems of any member of the genus Nicotiana and reconstituted materials thereof. The tobacco material for use in the present invention is preferably from the species Nicotiana tabacum.
Any type of tobacco can be used in the present invention. Examples of tobacco which can be used include but are not limited to Virginia, Burley, Maryland, Oriental and Rustica tobaccos.
The tobacco material may be treated in any suitable way before being treated according to the method of the invention.
As used herein, the terms "incubation period" and "enzyme incubation period"
refer to the period of time when the tobacco material is incubated with one or more protease enzyme(s).
/5 As used herein, the term "optimal operating conditions" refers to the reaction conditions under which the enzyme has optimal activity and/or activity sufficient for the reaction to proceed at a reasonable and/or desirable rate. The optimal operating conditions may comprise the pH and/or temperature ranges at which a protease enzyme has optimal activity and/or activity sufficient for the reaction to proceed at a reasonable and/or desirable rate.
In some embodiments, the method of the present invention relates to a method for reducing the protein content of cured tobacco material. The tobacco material may be cured using any suitable method of curing before being treated according to the method of the invention.
The tobacco material may be any suitable form of tobacco. The tobacco material may comprise lamina, sheet, reconstituted, ground and/or milled tobacco material.
When the tobacco material comprises lamina tobacco material, the lamina may be in the form of strip, cut or whole leaf tobacco. In some embodiments, the tobacco material is in the form of cut rag or whole leaf. In some embodiments, the tobacco material is cured cut rag and/or cured whole leaf tobacco.
In some embodiments, the tobacco material may be washed prior to protease enzyme treatment.
Figure 8 is a schematic side view of a smoking article including treated tobacco material according to embodiments of the invention.
Detailed Description The present invention relates to a method for treating a tobacco material, for removing proteins from tobacco material and/or reducing the protein content of tobacco io material.
Treating tobacco material may be used to remove one or more other chemical substances which are desirable to remove. Proteins in tobacco material are often found to be, and/or to be precursors of, such substances.
Alternatively or in addition, it may be desirable to reduce the protein content of tobacco material to improve the quality of the tobacco material.
Methods attempting to remove proteins have been proposed, although they have tended to be expensive, lengthy and/or detrimental to the physical structure of the tobacco material, and/or not reduce the protein content to a desired level.
The method of the present invention removes proteins from tobacco material.
Exemplary proteins that may be removed from the tobacco material include RuBisCO.
The method of the present invention results in a reduction in the protein content of tobacco material compared to the protein content of the untreated tobacco material.
Figure 1 is a flow diagram illustrating a method of treating tobacco material in accordance with an embodiment. Tobacco material 1 undergoes protease enzyme treatment 10. The solid and liquid components of the resulting suspension are separated 20, resulting in a tobacco material with reduced protein content 30 and a liquid component 31.
As used herein, the term "tobacco material" includes any part, such as for example the leaves or stems of any member of the genus Nicotiana and reconstituted materials thereof. The tobacco material for use in the present invention is preferably from the species Nicotiana tabacum.
Any type of tobacco can be used in the present invention. Examples of tobacco which can be used include but are not limited to Virginia, Burley, Maryland, Oriental and Rustica tobaccos.
The tobacco material may be treated in any suitable way before being treated according to the method of the invention.
As used herein, the terms "incubation period" and "enzyme incubation period"
refer to the period of time when the tobacco material is incubated with one or more protease enzyme(s).
/5 As used herein, the term "optimal operating conditions" refers to the reaction conditions under which the enzyme has optimal activity and/or activity sufficient for the reaction to proceed at a reasonable and/or desirable rate. The optimal operating conditions may comprise the pH and/or temperature ranges at which a protease enzyme has optimal activity and/or activity sufficient for the reaction to proceed at a reasonable and/or desirable rate.
In some embodiments, the method of the present invention relates to a method for reducing the protein content of cured tobacco material. The tobacco material may be cured using any suitable method of curing before being treated according to the method of the invention.
The tobacco material may be any suitable form of tobacco. The tobacco material may comprise lamina, sheet, reconstituted, ground and/or milled tobacco material.
When the tobacco material comprises lamina tobacco material, the lamina may be in the form of strip, cut or whole leaf tobacco. In some embodiments, the tobacco material is in the form of cut rag or whole leaf. In some embodiments, the tobacco material is cured cut rag and/or cured whole leaf tobacco.
In some embodiments, the tobacco material may be washed prior to protease enzyme treatment.
- 5 -Any suitable liquid medium may be used to wash the tobacco material. An exemplary washing medium is an aqueous solution.
In some embodiments, the tobacco material may be washed with water. The tobacco material may be washed with municipal water. Alternatively or in addition, the tobacco material may be washed with purified water. As used herein, "purified water"
relates to water treated to remove contaminants and/or impurities. In some embodiments, the purified water is deionised water.
/o Washing the tobacco material may remove proteins from the tobacco material. For example, when the tobacco material is washed with water, water-soluble proteins may be removed from the tobacco material.
Washing the tobacco material may confer the advantage that the tobacco material /5 already has a reduced protein content before protease enzyme treatment.
Thus, the reduction of the protein content using protease enzymes may be more efficient and/or more effective using washed tobacco material than unwashed tobacco material.
Alternatively or in addition, the enzyme incubation time required to remove a desired 20 level of proteins may be shorter and/or the enzyme concentration required to remove a desired level of proteins may be less with the inclusion of a washing step prior to enzyme treatment.
The tobacco material may be washed by placing in a suitable washing medium for up to 25 10 minutes, up to 15 minutes, up to 20 minutes, up to 25 minutes or up to 30 minutes.
The amount of washing medium may be sufficient to remove any proteins that are soluble in the washing medium from the tobacco material. The person skilled in the art will be aware that the extraction efficiency may be optimised by altering the ratio of the 30 washing medium to tobacco material.
The temperature of the tobacco material and washing medium may be higher than ambient temperature, as this may assist in the extraction process. The temperature of the tobacco material and washing medium may be up to 20 C, up to 30 C, up to 40 C, 35 up to 5o C, up to 60 C, up to 70 C or up to 80 C. Suitable temperatures of the tobacco material and washing medium will be known to the person skilled in the art.
In some embodiments, the tobacco material may be washed with water. The tobacco material may be washed with municipal water. Alternatively or in addition, the tobacco material may be washed with purified water. As used herein, "purified water"
relates to water treated to remove contaminants and/or impurities. In some embodiments, the purified water is deionised water.
/o Washing the tobacco material may remove proteins from the tobacco material. For example, when the tobacco material is washed with water, water-soluble proteins may be removed from the tobacco material.
Washing the tobacco material may confer the advantage that the tobacco material /5 already has a reduced protein content before protease enzyme treatment.
Thus, the reduction of the protein content using protease enzymes may be more efficient and/or more effective using washed tobacco material than unwashed tobacco material.
Alternatively or in addition, the enzyme incubation time required to remove a desired 20 level of proteins may be shorter and/or the enzyme concentration required to remove a desired level of proteins may be less with the inclusion of a washing step prior to enzyme treatment.
The tobacco material may be washed by placing in a suitable washing medium for up to 25 10 minutes, up to 15 minutes, up to 20 minutes, up to 25 minutes or up to 30 minutes.
The amount of washing medium may be sufficient to remove any proteins that are soluble in the washing medium from the tobacco material. The person skilled in the art will be aware that the extraction efficiency may be optimised by altering the ratio of the 30 washing medium to tobacco material.
The temperature of the tobacco material and washing medium may be higher than ambient temperature, as this may assist in the extraction process. The temperature of the tobacco material and washing medium may be up to 20 C, up to 30 C, up to 40 C, 35 up to 5o C, up to 60 C, up to 70 C or up to 80 C. Suitable temperatures of the tobacco material and washing medium will be known to the person skilled in the art.
- 6 -In some embodiments, the temperature of the tobacco material and washing medium is 50 C and the tobacco material is washed for 25 minutes.
The tobacco material and washing medium may be agitated during washing, for example by stirring and/or shaking.
Following washing, the tobacco material may be separated from the washing medium.
Suitable means for separating the tobacco material from the washing medium will be io known to the person skilled in the art, and may comprise filtration and/or centrifugation.
Following contact with the tobacco material, the washing medium may comprise components that are desirable to retain in tobacco material. For example, the washing /5 medium may comprise tobacco proteins that are soluble in the washing medium. Such proteins may confer particular organoleptic properties, such as tobacco flavour.
Alternatively or in addition, the washing medium may comprise nicotine, sugars and/or other components that may be desirable to retain in tobacco material.
Therefore, in some embodiments, following separation from the tobacco material the washing 20 medium is reapplied to tobacco material, for example, to tobacco material that has undergone protease enzyme treatment.
The method of the present invention reduces the protein content of the tobacco material by using protease enzyme activity.
Protease enzymes degrade proteins into smaller sized peptides and amino acids.
Therefore, in the method of the present invention, protease enzyme activity degrades the proteins in the tobacco material, which are released from the tobacco material, resulting in the removal of proteins from the tobacco material and/or the reduction in protein content of the tobacco material.
Enzyme treatment is considered to be one of the most effective methods of reducing the protein content of tobacco material. Furthermore, the use of enzymes to reduce the protein content of the tobacco material is advantageous as they only catalyse certain transformations while leaving other material intact, and/or the use of enzymes avoids the use of hazardous chemicals. In contrast, alternative methods of protein reduction
The tobacco material and washing medium may be agitated during washing, for example by stirring and/or shaking.
Following washing, the tobacco material may be separated from the washing medium.
Suitable means for separating the tobacco material from the washing medium will be io known to the person skilled in the art, and may comprise filtration and/or centrifugation.
Following contact with the tobacco material, the washing medium may comprise components that are desirable to retain in tobacco material. For example, the washing /5 medium may comprise tobacco proteins that are soluble in the washing medium. Such proteins may confer particular organoleptic properties, such as tobacco flavour.
Alternatively or in addition, the washing medium may comprise nicotine, sugars and/or other components that may be desirable to retain in tobacco material.
Therefore, in some embodiments, following separation from the tobacco material the washing 20 medium is reapplied to tobacco material, for example, to tobacco material that has undergone protease enzyme treatment.
The method of the present invention reduces the protein content of the tobacco material by using protease enzyme activity.
Protease enzymes degrade proteins into smaller sized peptides and amino acids.
Therefore, in the method of the present invention, protease enzyme activity degrades the proteins in the tobacco material, which are released from the tobacco material, resulting in the removal of proteins from the tobacco material and/or the reduction in protein content of the tobacco material.
Enzyme treatment is considered to be one of the most effective methods of reducing the protein content of tobacco material. Furthermore, the use of enzymes to reduce the protein content of the tobacco material is advantageous as they only catalyse certain transformations while leaving other material intact, and/or the use of enzymes avoids the use of hazardous chemicals. In contrast, alternative methods of protein reduction
- 7 -may damage the physical structure of the tobacco material, for example by requiring conditions that damage the tobacco material.
The protease enzyme to be used in the present invention may be any type of protease enzyme. The protease enzyme to be used in the present invention may be classified by the International Union of Biochemistry and Molecular Biology as EC 3.4 (i.e.
enzymes classified as peptidases).
Protease enzymes that are used commercially in the food and detergent industries that io are available at low cost may be suitable for the method of the present invention.
The protease enzyme may be obtained from any suitable source. For example, the protease enzyme may be of plant, animal, fungal, bacterial and/or viral origin.
Alternatively or in addition, the protease enzyme may be synthesised.
In some embodiments in which the protease enzyme has a bacterial origin, the protease may be obtained from the Bacillus genus. Exemplary protease enzymes obtained from the Bacillus genus that are commercially available include Savinase , Esperase , Neutrase and Alcalase . In some embodiments, the protease is obtained from the species Bacillus licheniformis. Other suitable bacterial protease enzymes will be known to the person skilled in the art.
In some embodiments in which the protease enzyme has a fungal origin, the protease may be obtained from the Aspergillus genus. In some embodiments, the protease is obtained from the species Aspergillus oryzae. An exemplary protease enzyme obtained from Aspergillus oryzae that is commercially available is Enzobake . In some embodiments, the protease is obtained from the species Aspergillus saitoi.
Other suitable fungal protease enzymes will be known to the person skilled in the art.
The protease enzyme may be an acidic enzyme. As used herein, the term "acidic enzyme" refers to an enzyme whose optimal activity comprises activity at acidic pH. An exemplary acidic protease enzyme is protease obtained from Aspergillus oryzae, which has an optimal activity in the pH range 3.0 to 7.0, optionally in the pH range 3.0 to 5.0 or at a pH of about 7Ø
The protease enzyme to be used in the present invention may be any type of protease enzyme. The protease enzyme to be used in the present invention may be classified by the International Union of Biochemistry and Molecular Biology as EC 3.4 (i.e.
enzymes classified as peptidases).
Protease enzymes that are used commercially in the food and detergent industries that io are available at low cost may be suitable for the method of the present invention.
The protease enzyme may be obtained from any suitable source. For example, the protease enzyme may be of plant, animal, fungal, bacterial and/or viral origin.
Alternatively or in addition, the protease enzyme may be synthesised.
In some embodiments in which the protease enzyme has a bacterial origin, the protease may be obtained from the Bacillus genus. Exemplary protease enzymes obtained from the Bacillus genus that are commercially available include Savinase , Esperase , Neutrase and Alcalase . In some embodiments, the protease is obtained from the species Bacillus licheniformis. Other suitable bacterial protease enzymes will be known to the person skilled in the art.
In some embodiments in which the protease enzyme has a fungal origin, the protease may be obtained from the Aspergillus genus. In some embodiments, the protease is obtained from the species Aspergillus oryzae. An exemplary protease enzyme obtained from Aspergillus oryzae that is commercially available is Enzobake . In some embodiments, the protease is obtained from the species Aspergillus saitoi.
Other suitable fungal protease enzymes will be known to the person skilled in the art.
The protease enzyme may be an acidic enzyme. As used herein, the term "acidic enzyme" refers to an enzyme whose optimal activity comprises activity at acidic pH. An exemplary acidic protease enzyme is protease obtained from Aspergillus oryzae, which has an optimal activity in the pH range 3.0 to 7.0, optionally in the pH range 3.0 to 5.0 or at a pH of about 7Ø
- 8 -The protease enzyme may be an alkaline enzyme. As used herein, the term "alkaline enzyme" refers to an enzyme whose optimal activity comprises activity at alkaline pH.
Exemplary alkaline protease enzymes include protease obtained from Bacillus licheniformis, which has an optimal activity in the pH range 3.0 to 11.5, and Savinase , which has an optimal activity in the pH range 7.0 to 11Ø
Alternatively or in addition, the protease enzyme may be an enzyme whose optimal activity is at neutral pH.
/o The protease enzyme treatment of the tobacco material may be carried out in solution.
In some embodiments, the protease enzyme treatment of the tobacco material is carried out in aqueous solution.
The solution in which the protease enzyme treatment of the tobacco material is carried /5 out may be selected according to the particular protease enzyme used.
Suitable solutions will be known to the person skilled in the art.
In some embodiments, protease enzyme is added to the selected solution after the addition of the tobacco material. In other words, in some embodiments, protease 20 enzyme is added to the tobacco material in the selected solution.
The conditions under which the enzyme is added may be selected to avoid loss of enzyme activity.
25 In some embodiments, the pH of the solution is adjusted prior to the addition of the enzyme to the selected solution. The pH may correspond to a pH at which the protease enzyme has optimal activity.
In some embodiments, the pH of the tobacco material in the selected solution 30 corresponds to a pH at which the protease enzyme has optimal activity, and therefore the pH of the tobacco material in the selected solution may not need to be adjusted.
When water is added to tobacco material, it may exhibit acidic or neutral characteristics. Accordingly, in some embodiments in which the tobacco material is 35 treated with acidic protease enzyme and the selected solution is water, such as
Exemplary alkaline protease enzymes include protease obtained from Bacillus licheniformis, which has an optimal activity in the pH range 3.0 to 11.5, and Savinase , which has an optimal activity in the pH range 7.0 to 11Ø
Alternatively or in addition, the protease enzyme may be an enzyme whose optimal activity is at neutral pH.
/o The protease enzyme treatment of the tobacco material may be carried out in solution.
In some embodiments, the protease enzyme treatment of the tobacco material is carried out in aqueous solution.
The solution in which the protease enzyme treatment of the tobacco material is carried /5 out may be selected according to the particular protease enzyme used.
Suitable solutions will be known to the person skilled in the art.
In some embodiments, protease enzyme is added to the selected solution after the addition of the tobacco material. In other words, in some embodiments, protease 20 enzyme is added to the tobacco material in the selected solution.
The conditions under which the enzyme is added may be selected to avoid loss of enzyme activity.
25 In some embodiments, the pH of the solution is adjusted prior to the addition of the enzyme to the selected solution. The pH may correspond to a pH at which the protease enzyme has optimal activity.
In some embodiments, the pH of the tobacco material in the selected solution 30 corresponds to a pH at which the protease enzyme has optimal activity, and therefore the pH of the tobacco material in the selected solution may not need to be adjusted.
When water is added to tobacco material, it may exhibit acidic or neutral characteristics. Accordingly, in some embodiments in which the tobacco material is 35 treated with acidic protease enzyme and the selected solution is water, such as
- 9 -deionised water, the tobacco material in the selected solution may be acidic, and the pH
may not require further adjustment.
In some embodiments in which the pH of the tobacco material in the selected solution does not correspond to a pH at which the protease enzyme has optimal activity, the pH
may be adjusted to the desired pH.
The skilled person will be aware of suitable methods of adjusting the pH. In some embodiments, the pH is adjusted by the addition of an acid such as citric acid and/or /o an inorganic acid, or by the addition of an alkali such as sodium hydroxide.
In some embodiments, a buffer may be added to maintain the pH of the solution.
Suitable buffers will be known to the person skilled in the art.
/5 In some embodiments, the temperature of the solution is adjusted prior to the addition of the enzyme to the selected solution. The temperature of the solution may be adjusted to a temperature at which the protease enzyme has optimal activity.
In some embodiments, the temperature of the selected solution corresponds to a 20 temperature at which the protease enzyme has optimal activity, and therefore the temperature of the selected solution may not need to be adjusted.
In some embodiments in which the temperature of the selected solution does not correspond to a temperature at which the protease enzyme has optimal activity, the 25 temperature may be adjusted to the desired temperature.
In some embodiments, the temperature of the selected solution is between about and 75 C. In some embodiments, the temperature of the selected solution is up to about 60 C, to minimise any negative effects on enzyme activity. In some embodiments in 30 which the tobacco material is treated with protease enzyme obtained from Aspergillus oryzae, the temperature of the selected solution may be about 30 C. In some embodiments in which the tobacco material is treated with Savinase , the temperature of the selected solution may be about 40 C.
35 Combining the tobacco material and protease enzyme may produce a tobacco/enzyme mixture.
may not require further adjustment.
In some embodiments in which the pH of the tobacco material in the selected solution does not correspond to a pH at which the protease enzyme has optimal activity, the pH
may be adjusted to the desired pH.
The skilled person will be aware of suitable methods of adjusting the pH. In some embodiments, the pH is adjusted by the addition of an acid such as citric acid and/or /o an inorganic acid, or by the addition of an alkali such as sodium hydroxide.
In some embodiments, a buffer may be added to maintain the pH of the solution.
Suitable buffers will be known to the person skilled in the art.
/5 In some embodiments, the temperature of the solution is adjusted prior to the addition of the enzyme to the selected solution. The temperature of the solution may be adjusted to a temperature at which the protease enzyme has optimal activity.
In some embodiments, the temperature of the selected solution corresponds to a 20 temperature at which the protease enzyme has optimal activity, and therefore the temperature of the selected solution may not need to be adjusted.
In some embodiments in which the temperature of the selected solution does not correspond to a temperature at which the protease enzyme has optimal activity, the 25 temperature may be adjusted to the desired temperature.
In some embodiments, the temperature of the selected solution is between about and 75 C. In some embodiments, the temperature of the selected solution is up to about 60 C, to minimise any negative effects on enzyme activity. In some embodiments in 30 which the tobacco material is treated with protease enzyme obtained from Aspergillus oryzae, the temperature of the selected solution may be about 30 C. In some embodiments in which the tobacco material is treated with Savinase , the temperature of the selected solution may be about 40 C.
35 Combining the tobacco material and protease enzyme may produce a tobacco/enzyme mixture.
- 10 -In some embodiments, the protease enzyme treatment comprises treating tobacco material with one type of protease enzyme. In some alternative embodiments, the protease enzyme treatment comprises treating tobacco material with a combination of different protease enzymes. The combination of protease enzymes may comprise at least two enzymes with different optimal operating conditions. Alternatively or in addition, the combination of protease enzymes may comprise at least three enzymes with different optimal operating conditions.
In some embodiments in which a combination of protease enzymes with different optimal operating conditions is used, the operating conditions may be adjusted or shifted during the enzyme incubation period. Adjusting the operating conditions may comprise adjusting the pH and/or the temperature.
/5 Treating the tobacco material with a combination of protease enzymes with different optimal operating conditions, which are added at the same time to the tobacco material, confers the advantage that just one enzyme addition step is required during the method of reducing the protein content of the tobacco material, which may be suitable for carrying out the method on an industrial or commercial scale.
In some embodiments in which a combination of protease enzymes with different optimal operating conditions is used, the protease enzymes may differ in terms of their optimal pH. The pH of the tobacco/enzyme mixture may therefore be adjusted during the enzyme incubation period, from the optimal pH of one protease enzyme to the optimal pH of another protease enzyme present in the tobacco/enzyme mixture.
The pH may be adjusted by decreasing or increasing the pH of the tobacco/enzyme mixture.
In some embodiments in which a combination of alkaline and acidic protease enzymes is used, the pH of the tobacco/enzyme mixture may be adjusted from alkaline to acidic pH or from acidic to alkaline pH. In other words, the pH may be adjusted from above 7.0 to below 7.0 or from below 7.0 to above 7Ø
In some embodiments in which a combination of alkaline enzymes and enzymes with an optimal activity at neutral pH is used, the pH of the tobacco/enzyme mixture may be
In some embodiments in which a combination of protease enzymes with different optimal operating conditions is used, the operating conditions may be adjusted or shifted during the enzyme incubation period. Adjusting the operating conditions may comprise adjusting the pH and/or the temperature.
/5 Treating the tobacco material with a combination of protease enzymes with different optimal operating conditions, which are added at the same time to the tobacco material, confers the advantage that just one enzyme addition step is required during the method of reducing the protein content of the tobacco material, which may be suitable for carrying out the method on an industrial or commercial scale.
In some embodiments in which a combination of protease enzymes with different optimal operating conditions is used, the protease enzymes may differ in terms of their optimal pH. The pH of the tobacco/enzyme mixture may therefore be adjusted during the enzyme incubation period, from the optimal pH of one protease enzyme to the optimal pH of another protease enzyme present in the tobacco/enzyme mixture.
The pH may be adjusted by decreasing or increasing the pH of the tobacco/enzyme mixture.
In some embodiments in which a combination of alkaline and acidic protease enzymes is used, the pH of the tobacco/enzyme mixture may be adjusted from alkaline to acidic pH or from acidic to alkaline pH. In other words, the pH may be adjusted from above 7.0 to below 7.0 or from below 7.0 to above 7Ø
In some embodiments in which a combination of alkaline enzymes and enzymes with an optimal activity at neutral pH is used, the pH of the tobacco/enzyme mixture may be
- 11 -adjusted from alkaline to neutral pH or from neutral to alkaline pH. In other words, the pH may be adjusted from above 7.0 to about 7.0 or from about 7.0 to above 7Ø
In some embodiments in which a combination of acidic enzymes and enzymes with an optimal activity at neutral pH is used, the pH of the tobacco/enzyme mixture may be adjusted from acidic to neutral pH or from neutral to acidic pH. In other words, the pH
may be adjusted from below about 7.0 to 7.0 or from about 7.0 to below 7Ø
In some embodiments in which a combination of alkaline, acidic enzymes and enzymes with an optimal activity at neutral pH is used, the pH of the tobacco/enzyme mixture may be adjusted from the pH for the optimal activity of the first enzyme, to the pH for the optimal activity of the second enzyme, to the pH for the optimal activity of the third enzyme.
/5 The change in pH may take place during the course of the reaction. In other words, the pH change may not be caused by the active adjustment of the pH by the operator.
Alternatively or in addition, the change in pH may be caused by the active adjustment of the pH.
To decrease the pH during the enzyme incubation period, an acid may be added.
Suitable acids will be known to the person skilled in the aft. In some embodiments, the pH is decreased by the addition of citric acid. Alternatively or in addition, the pH is decreased by bubbling CO, through the tobacco/enzyme mixture.
To increase the pH during the enzyme incubation period, an alkali may be added.
Suitable alkalis will be known to the person skilled in the art. In some embodiments, the pH is increased by the addition of sodium hydroxide.
In some embodiments of the method in which a combination of protease enzymes with different optimal operating conditions is used, the protease enzymes may differ in terms of their optimal temperature. The temperature of the tobacco/enzyme mixture may therefore be adjusted during the enzyme incubation period, from the optimal temperature of one protease enzyme to the optimal temperature of another protease enzyme present in the tobacco/enzyme mixture.
In some embodiments in which a combination of acidic enzymes and enzymes with an optimal activity at neutral pH is used, the pH of the tobacco/enzyme mixture may be adjusted from acidic to neutral pH or from neutral to acidic pH. In other words, the pH
may be adjusted from below about 7.0 to 7.0 or from about 7.0 to below 7Ø
In some embodiments in which a combination of alkaline, acidic enzymes and enzymes with an optimal activity at neutral pH is used, the pH of the tobacco/enzyme mixture may be adjusted from the pH for the optimal activity of the first enzyme, to the pH for the optimal activity of the second enzyme, to the pH for the optimal activity of the third enzyme.
/5 The change in pH may take place during the course of the reaction. In other words, the pH change may not be caused by the active adjustment of the pH by the operator.
Alternatively or in addition, the change in pH may be caused by the active adjustment of the pH.
To decrease the pH during the enzyme incubation period, an acid may be added.
Suitable acids will be known to the person skilled in the aft. In some embodiments, the pH is decreased by the addition of citric acid. Alternatively or in addition, the pH is decreased by bubbling CO, through the tobacco/enzyme mixture.
To increase the pH during the enzyme incubation period, an alkali may be added.
Suitable alkalis will be known to the person skilled in the art. In some embodiments, the pH is increased by the addition of sodium hydroxide.
In some embodiments of the method in which a combination of protease enzymes with different optimal operating conditions is used, the protease enzymes may differ in terms of their optimal temperature. The temperature of the tobacco/enzyme mixture may therefore be adjusted during the enzyme incubation period, from the optimal temperature of one protease enzyme to the optimal temperature of another protease enzyme present in the tobacco/enzyme mixture.
- 12 -The temperature may be adjusted by decreasing or increasing the temperature of the tobacco/enzyme mixture.
In some embodiments of the method in which a combination of protease enzymes with different optimal operating conditions is used, the protease enzymes may differ in terms of their optimal pH and their optimal temperature. The pH and temperature of the tobacco/enzyme mixture may therefore be adjusted during the enzyme incubation period, from the optimal pH and temperature of one protease enzyme to the optimal pH and temperature of another protease enzyme present in the tobacco/enzyme io mixture.
Using a combination of enzymes with different optimal operating conditions and adjusting the operating conditions may make the enzyme treatment step more effective and/or more efficient at reducing the protein content of the tobacco material.
/5 Alternatively or in addition, using a combination of enzymes with different optimal operating conditions and adjusting the operating conditions may minimise the effects on the physical properties of the tobacco material, such as damage to the structure of the tobacco material.
20 The tobacco material may be incubated with protease enzyme for a period of time sufficient to reduce the protein content of the tobacco material to a desired level.
In embodiments in which a combination of protease enzymes with different optimal operating conditions is used and the operating conditions are adjusted during the 25 incubation period, the tobacco material may be incubated with protease enzyme for up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, or up to 5 hours. In some embodiments in which a combination of protease enzymes with different optimal operating conditions is used, the incubation period is up to 1 hour.
30 In embodiments in which a combination of protease enzymes with different optimal operating conditions is used, the operating conditions may be adjusted after a proportion of the incubation period has passed. The operating conditions may be adjusted after about 25%, after about 50%, or after about 75% of the incubation period has passed. For example, in embodiments in which the incubation period is 1 hour and 35 the combination of protease enzymes comprises enzymes with two different optimal operating conditions, the operating conditions may be adjusted after about 50%
of the
In some embodiments of the method in which a combination of protease enzymes with different optimal operating conditions is used, the protease enzymes may differ in terms of their optimal pH and their optimal temperature. The pH and temperature of the tobacco/enzyme mixture may therefore be adjusted during the enzyme incubation period, from the optimal pH and temperature of one protease enzyme to the optimal pH and temperature of another protease enzyme present in the tobacco/enzyme io mixture.
Using a combination of enzymes with different optimal operating conditions and adjusting the operating conditions may make the enzyme treatment step more effective and/or more efficient at reducing the protein content of the tobacco material.
/5 Alternatively or in addition, using a combination of enzymes with different optimal operating conditions and adjusting the operating conditions may minimise the effects on the physical properties of the tobacco material, such as damage to the structure of the tobacco material.
20 The tobacco material may be incubated with protease enzyme for a period of time sufficient to reduce the protein content of the tobacco material to a desired level.
In embodiments in which a combination of protease enzymes with different optimal operating conditions is used and the operating conditions are adjusted during the 25 incubation period, the tobacco material may be incubated with protease enzyme for up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, or up to 5 hours. In some embodiments in which a combination of protease enzymes with different optimal operating conditions is used, the incubation period is up to 1 hour.
30 In embodiments in which a combination of protease enzymes with different optimal operating conditions is used, the operating conditions may be adjusted after a proportion of the incubation period has passed. The operating conditions may be adjusted after about 25%, after about 50%, or after about 75% of the incubation period has passed. For example, in embodiments in which the incubation period is 1 hour and 35 the combination of protease enzymes comprises enzymes with two different optimal operating conditions, the operating conditions may be adjusted after about 50%
of the
- 13 -incubation period has passed, i.e. after about 30 minutes. In embodiments in which the incubation period is 1 hour and the combination of protease enzymes comprises enzymes with three different optimal operating conditions, the operating conditions may firstly be adjusted after about 33% of the incubation period has passed, i.e. after about 20 minutes, and secondly after about 66% of the incubation period has passed, i.e. after about 40 minutes.
The tobacco material/enzyme mixture may be agitated, for example by stirring, rocking and/or shaking, during the enzyme incubation period. The tobacco material/enzyme io mixture may be agitated to provide adequate mixing without subjecting the tobacco fibres to mechanical stress that would break down the structure of the tobacco material.
The protease enzyme concentration in the tobacco/enzyme mixture may be sufficient to reduce the protein content to a desired level within the desired incubation time. At the /5 same time, the amount of protease enzyme used during the method may be sufficiently low so that the method is cost-effective.
The amount of protease enzyme and solution added to the tobacco material may be determined according to the desired enzyme concentration.
The concentration of the protease enzyme may be between about 0.01 g/1 and about 5.0 g/1 and/or between about 0.1 g/1 and about 2.0 g/l. In some embodiments, the concentration of the protease enzyme is about 1.0 g/l. In some embodiments, the concentration of the protease enzyme is about 1.8 g/l. To clarify, as used herein, the concentration of the protease enzyme does not equate to enzyme activity.
In some embodiments, the tobacco material is incubated for 1 hour with two protease enzymes with different optimal operating conditions, both at a concentration of 1.0 g/l.
The enzymatic treatment of the tobacco material results in the decomposition of protein fragments. Many of the resulting protein fragments are solubilised and/or dispersed in the liquid component of the tobacco/enzyme mixture, and are therefore easily separated from the tobacco material.
Following incubation of the tobacco material with the protease enzyme, the insoluble tobacco material may be separated from the liquid component of the tobacco/enzyme
The tobacco material/enzyme mixture may be agitated, for example by stirring, rocking and/or shaking, during the enzyme incubation period. The tobacco material/enzyme io mixture may be agitated to provide adequate mixing without subjecting the tobacco fibres to mechanical stress that would break down the structure of the tobacco material.
The protease enzyme concentration in the tobacco/enzyme mixture may be sufficient to reduce the protein content to a desired level within the desired incubation time. At the /5 same time, the amount of protease enzyme used during the method may be sufficiently low so that the method is cost-effective.
The amount of protease enzyme and solution added to the tobacco material may be determined according to the desired enzyme concentration.
The concentration of the protease enzyme may be between about 0.01 g/1 and about 5.0 g/1 and/or between about 0.1 g/1 and about 2.0 g/l. In some embodiments, the concentration of the protease enzyme is about 1.0 g/l. In some embodiments, the concentration of the protease enzyme is about 1.8 g/l. To clarify, as used herein, the concentration of the protease enzyme does not equate to enzyme activity.
In some embodiments, the tobacco material is incubated for 1 hour with two protease enzymes with different optimal operating conditions, both at a concentration of 1.0 g/l.
The enzymatic treatment of the tobacco material results in the decomposition of protein fragments. Many of the resulting protein fragments are solubilised and/or dispersed in the liquid component of the tobacco/enzyme mixture, and are therefore easily separated from the tobacco material.
Following incubation of the tobacco material with the protease enzyme, the insoluble tobacco material may be separated from the liquid component of the tobacco/enzyme
- 14 -mixture. The tobacco material and the liquid component may be separated with any suitable apparatus. In some embodiments, the tobacco material and the liquid component are separated using filtration. Suitable alternative methods of separating the tobacco material and liquid component will be known to the person skilled in the are, and may include centrifugation and/or using a press, sieve and/or belt filter.
In some embodiments, the tobacco material and the liquid component are separated using one or more filtration step(s). For example, the tobacco/enzyme mixture may be passed through a coarse filter and/or a fine filter. Suitable filters will be known to the io person skilled in the aft.
Figure 2 is a flow diagram illustrating a method of treating tobacco material in accordance with an embodiment. Whole leaf tobacco material 2 undergoes an optional washing step 3. The tobacco material is then added to the selected solution 4 and the pH and/or temperature of the tobacco material in solution is optionally adjusted 5 prior to the addition of protease enzyme. The tobacco material then undergoes protease enzyme treatment 10. The tobacco material and the liquid component of the resulting tobacco/enzyme mixture are separated 20, resulting in a tobacco material with reduced protein content 30 and a liquid component 31.
Following separation from the tobacco material, the liquid component may be discarded. Alternatively, the liquid component may be retained. For example, the liquid component may be retained to recover the solution, which may be reused.
Following separation from the liquid component of the tobacco/enzyme mixture, the tobacco material may be washed to remove any protein fragments and/or enzymes.
The tobacco material may be washed with any suitable medium. In some embodiments, the washing medium is heated. In some embodiments, the washing medium is salt water.
The tobacco material may be treated to deactivate any enzyme remaining in the tobacco fibre. Suitable deactivation methods will be known to the person skilled in the art. In some embodiments, the tobacco material may be heat-treated to deactivate any enzyme remaining in the tobacco fibre.
The treatment of the tobacco material according to a method which comprises using a combination of protease enzymes with different optimal operating conditions and
In some embodiments, the tobacco material and the liquid component are separated using one or more filtration step(s). For example, the tobacco/enzyme mixture may be passed through a coarse filter and/or a fine filter. Suitable filters will be known to the io person skilled in the aft.
Figure 2 is a flow diagram illustrating a method of treating tobacco material in accordance with an embodiment. Whole leaf tobacco material 2 undergoes an optional washing step 3. The tobacco material is then added to the selected solution 4 and the pH and/or temperature of the tobacco material in solution is optionally adjusted 5 prior to the addition of protease enzyme. The tobacco material then undergoes protease enzyme treatment 10. The tobacco material and the liquid component of the resulting tobacco/enzyme mixture are separated 20, resulting in a tobacco material with reduced protein content 30 and a liquid component 31.
Following separation from the tobacco material, the liquid component may be discarded. Alternatively, the liquid component may be retained. For example, the liquid component may be retained to recover the solution, which may be reused.
Following separation from the liquid component of the tobacco/enzyme mixture, the tobacco material may be washed to remove any protein fragments and/or enzymes.
The tobacco material may be washed with any suitable medium. In some embodiments, the washing medium is heated. In some embodiments, the washing medium is salt water.
The tobacco material may be treated to deactivate any enzyme remaining in the tobacco fibre. Suitable deactivation methods will be known to the person skilled in the art. In some embodiments, the tobacco material may be heat-treated to deactivate any enzyme remaining in the tobacco fibre.
The treatment of the tobacco material according to a method which comprises using a combination of protease enzymes with different optimal operating conditions and
- 15 -adjusting the operating conditions during the enzyme incubation period may result in a reduction in the protein content of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or at least 95%, based upon the protein content of the untreated tobacco material. In some embodiments, a reduction in protein content of at least 50% and/or at least 75% is achieved.
Alternatively or in addition, the treatment of the tobacco material according to a method which comprises using a combination of protease enzymes with different optimal operating conditions and adjusting the operating conditions during the enzyme io incubation period may result in the removal of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or at least 95% of the proteins from the tobacco material.
In some embodiments, the treatment of the tobacco material according to a method /5 which comprises using a combination of protease enzymes with different optimal operating conditions and adjusting the operating conditions during the enzyme incubation period results in the extraction of protein in an amount of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or at least 95%, based upon the protein content of the untreated tobacco material.
The method of the invention may comprise one or more further treatment steps.
Suitable additional treatment steps include, but are not limited to: treating the tobacco material with one or more suitable non-ionic liquids, such as water; treating the tobacco material with one or more additional enzymes, which may be enzymes which catalyse the modification of polyphenols, such as phenol-oxidising enzymes;
treating the tobacco material with one or more suitable surfactants, such as sodium dodecylsulfate (SDS), in any suitable solvent; treating the tobacco material with one or more suitable adsorbent materials, such as polyvinyl polypyrrolidone (PVPP), hydroxylapatite, bentonite, activated carbon or attapulgite, in any suitable solvent if appropriate; and treating the tobacco material with one or more suitable non-aqueous liquids, such as ionic liquids.
Alternatively or in addition, the tobacco material treated according to the method of the present invention may be subsequently subjected to further extraction processes.
Alternatively or in addition, the treatment of the tobacco material according to a method which comprises using a combination of protease enzymes with different optimal operating conditions and adjusting the operating conditions during the enzyme io incubation period may result in the removal of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or at least 95% of the proteins from the tobacco material.
In some embodiments, the treatment of the tobacco material according to a method /5 which comprises using a combination of protease enzymes with different optimal operating conditions and adjusting the operating conditions during the enzyme incubation period results in the extraction of protein in an amount of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or at least 95%, based upon the protein content of the untreated tobacco material.
The method of the invention may comprise one or more further treatment steps.
Suitable additional treatment steps include, but are not limited to: treating the tobacco material with one or more suitable non-ionic liquids, such as water; treating the tobacco material with one or more additional enzymes, which may be enzymes which catalyse the modification of polyphenols, such as phenol-oxidising enzymes;
treating the tobacco material with one or more suitable surfactants, such as sodium dodecylsulfate (SDS), in any suitable solvent; treating the tobacco material with one or more suitable adsorbent materials, such as polyvinyl polypyrrolidone (PVPP), hydroxylapatite, bentonite, activated carbon or attapulgite, in any suitable solvent if appropriate; and treating the tobacco material with one or more suitable non-aqueous liquids, such as ionic liquids.
Alternatively or in addition, the tobacco material treated according to the method of the present invention may be subsequently subjected to further extraction processes.
- 16 -Following treatment according to the method of the present invention, the tobacco material with reduced protein content may be incorporated into a smoking article.
As used herein, the term "smoking article" includes smokeable products such as cigarettes, cigars and cigarillos whether based on tobacco, tobacco derivatives, expanded tobacco, reconstituted tobacco or tobacco substitutes and also heat-not-burn products. The smoking article may be provided with a filter for the gaseous flow drawn by the smoker.
/o Following treatment according to the method of the present invention, the tobacco material may be further modified in any suitable way before being incorporated into a smoking article. For example, the tobacco material may be dried, certain chemical substances may be added to the tobacco material, such as flavourants where local regulations permit, and/or the tobacco material may be cut and/or shredded before /5 being incorporated into a smoking article using any suitable method of incorporation.
As used herein, the terms "flavour" and "flavourant" refer to materials which, where local regulations permit, may be used to create a desired taste or aroma in a product for adult consumers. They may include extracts (e.g., licorice, hydrangea, Japanese white 20 bark magnolia leaf, chamomile, fenugreek, clove, menthol, Japanese mint, aniseed, cinnamon, herb, wintergreen, cherry, berry, peach, apple, Drambuie, bourbon, scotch, whiskey, spearmint, peppermint, lavender, cardamon, celery, cascarilla, nutmeg, sandalwood, bergamot, geranium, honey essence, rose oil, vanilla, lemon oil, orange oil, cassia, caraway, cognac, jasmine, ylang-ylang, sage, fennel, piment, ginger, anise, 25 coriander, coffee, or a mint oil from any species of the genus Mentha), flavour enhancers, bitterness receptor site blockers, sensorial receptor site activators or stimulators, sugars and/or sugar substitutes (e.g., sucralose, acesulfame potassium, aspartame, saccharine, cyclamates, lactose, sucrose, glucose, fructose, sorbitol, or mannitol), and other additives such as charcoal, chlorophyll, minerals, botanicals, or 30 breath freshening agents. They may be imitation, synthetic or natural ingredients or blends thereof. They may be in any suitable form, for example, oil, liquid, or powder.
Referring to Figure 8, for purpose of illustration and not limitation, a smoking article 101 according to an exemplary embodiment of the invention comprises a filter 102 and 35 a cylindrical rod of smokeable material 1433, such as tobacco treated in accordance with the invention described herein, aligned with the filter 102 such that one end of the
As used herein, the term "smoking article" includes smokeable products such as cigarettes, cigars and cigarillos whether based on tobacco, tobacco derivatives, expanded tobacco, reconstituted tobacco or tobacco substitutes and also heat-not-burn products. The smoking article may be provided with a filter for the gaseous flow drawn by the smoker.
/o Following treatment according to the method of the present invention, the tobacco material may be further modified in any suitable way before being incorporated into a smoking article. For example, the tobacco material may be dried, certain chemical substances may be added to the tobacco material, such as flavourants where local regulations permit, and/or the tobacco material may be cut and/or shredded before /5 being incorporated into a smoking article using any suitable method of incorporation.
As used herein, the terms "flavour" and "flavourant" refer to materials which, where local regulations permit, may be used to create a desired taste or aroma in a product for adult consumers. They may include extracts (e.g., licorice, hydrangea, Japanese white 20 bark magnolia leaf, chamomile, fenugreek, clove, menthol, Japanese mint, aniseed, cinnamon, herb, wintergreen, cherry, berry, peach, apple, Drambuie, bourbon, scotch, whiskey, spearmint, peppermint, lavender, cardamon, celery, cascarilla, nutmeg, sandalwood, bergamot, geranium, honey essence, rose oil, vanilla, lemon oil, orange oil, cassia, caraway, cognac, jasmine, ylang-ylang, sage, fennel, piment, ginger, anise, 25 coriander, coffee, or a mint oil from any species of the genus Mentha), flavour enhancers, bitterness receptor site blockers, sensorial receptor site activators or stimulators, sugars and/or sugar substitutes (e.g., sucralose, acesulfame potassium, aspartame, saccharine, cyclamates, lactose, sucrose, glucose, fructose, sorbitol, or mannitol), and other additives such as charcoal, chlorophyll, minerals, botanicals, or 30 breath freshening agents. They may be imitation, synthetic or natural ingredients or blends thereof. They may be in any suitable form, for example, oil, liquid, or powder.
Referring to Figure 8, for purpose of illustration and not limitation, a smoking article 101 according to an exemplary embodiment of the invention comprises a filter 102 and 35 a cylindrical rod of smokeable material 1433, such as tobacco treated in accordance with the invention described herein, aligned with the filter 102 such that one end of the
- 17 -smokeable material rod 103 abuts the end of the filter 102. The filter 102 is wrapped in a plug wrap (not shown) and the smokeable material rod 103 is joined to the filter 102 by tipping paper (not shown) in a conventional manner.
In some embodiments, the methods described herein may comprise one or more further steps to modify the tobacco material in any suitable way. For example, the tobacco material may be modified to provide it with one or more characteristics desirable for a tobacco material. For example, where the treated tobacco material is to be incorporated into a smoking article such as a cigarette, the tobacco material may be io treated in order to modify the flavour it generates upon combustion, and/or may be treated in order to remove one or more of its chemical substances.
Experimental Work A series of experiments were carried out in order to investigate the removal of proteins /5 from tobacco material by using a combination of protease enzymes with different optimal operating conditions and adjusting the operating conditions during the enzyme incubation period. The disclosed experimental work is not intended to limit the scope of the invention.
20 Example 1 ¨ Unwashed tobacco material treatment with two proteases with different optimal operating conditions Treatment of Tobacco Material Whole tobacco leaves were dried to constant weight at 45 C in a drying chamber and were then crushed, to enable tobacco treatment on a laboratory scale. 25 g of the 25 crushed dried tobacco was placed in 500 ml of deionised water or buffer, selected according to the enzyme combination used (the enzymes were obtained from Sigma Aldrich ). Referring to Figure 3, tobacco treated with enzyme groups 2 and 4 was placed in deionised water, the alkaline buffer 0.1 M sodium phosphate pH 8.0 was used for tobacco treatment starting with an alkaline pH and the acidic buffer 0.1 M
sodium 30 acetate pH 4.0 was used for tobacco treatment starting with an acidic pH. Following the addition of two enzymes with different optimal operating conditions, each at a concentration of 1.0 g/l, the resulting suspension of tobacco material and enzymes was incubated in an incubation shaker at 120 revolutions per minute (rpm) for 1 hour. The temperature of the incubation shaker was set according to the optimal working 35 temperature of the enzymes, and in some cases the temperature was changed following 30 minutes of incubation. The pH value was set according to the optimal working pH of
In some embodiments, the methods described herein may comprise one or more further steps to modify the tobacco material in any suitable way. For example, the tobacco material may be modified to provide it with one or more characteristics desirable for a tobacco material. For example, where the treated tobacco material is to be incorporated into a smoking article such as a cigarette, the tobacco material may be io treated in order to modify the flavour it generates upon combustion, and/or may be treated in order to remove one or more of its chemical substances.
Experimental Work A series of experiments were carried out in order to investigate the removal of proteins /5 from tobacco material by using a combination of protease enzymes with different optimal operating conditions and adjusting the operating conditions during the enzyme incubation period. The disclosed experimental work is not intended to limit the scope of the invention.
20 Example 1 ¨ Unwashed tobacco material treatment with two proteases with different optimal operating conditions Treatment of Tobacco Material Whole tobacco leaves were dried to constant weight at 45 C in a drying chamber and were then crushed, to enable tobacco treatment on a laboratory scale. 25 g of the 25 crushed dried tobacco was placed in 500 ml of deionised water or buffer, selected according to the enzyme combination used (the enzymes were obtained from Sigma Aldrich ). Referring to Figure 3, tobacco treated with enzyme groups 2 and 4 was placed in deionised water, the alkaline buffer 0.1 M sodium phosphate pH 8.0 was used for tobacco treatment starting with an alkaline pH and the acidic buffer 0.1 M
sodium 30 acetate pH 4.0 was used for tobacco treatment starting with an acidic pH. Following the addition of two enzymes with different optimal operating conditions, each at a concentration of 1.0 g/l, the resulting suspension of tobacco material and enzymes was incubated in an incubation shaker at 120 revolutions per minute (rpm) for 1 hour. The temperature of the incubation shaker was set according to the optimal working 35 temperature of the enzymes, and in some cases the temperature was changed following 30 minutes of incubation. The pH value was set according to the optimal working pH of
- 18 -the enzymes, and was monitored during incubation with a pH meter, and in some cases the pH was changed following 30 minutes of incubation by the addition of 40%
acetic acid (to make the pH more acidic) or by the addition of 2 M sodium hydroxide solution (to make the pH more alkaline). The pH of the tobacco treated with enzyme groups 2 or 4 decreased during the course of the reaction and was not adjusted by the addition of an acid. Following incubation with the enzymes, the tobacco material was separated from the liquid component by two filtration steps, the first filtration step was carried out using coarse paper filter, and the filtrate from the paper filter was filtered with a 0.45 vtrn hydrophilic PVDF membrane syringe filter (Rotilabo , diameter 25 mm, Carl Roth, Germany).
The parameters used for the treatment of the tobacco material are outlined in Figure 3.
Analysis of Protein Removal The resulting liquid component of the samples prepared according to Figure 3 was analysed for the nitrogen content. Total nitrogen was measured with a TOC-N
Analyzer (Shimadzu) and/or a total nitrogen test kit (Spectroquant Nitrogen total photometric test, DMP 10 ¨ 150 mg/1, Merck).
The nitrogen content of the liquid component was corrected for the nitrogen present that was contributed by nicotine and enzyme proteins, taking the approximate proportion of nitrogen in nicotine to be 17% by weight, and the approximate proportion of nitrogen in protein to be 16% by weight.
Nicotine quantification was carried out using HPLC with a Phenomenex-Gemini column C18.3 lam, 150 x 3 mm, isocratic at 30 C, flow 0.6 ml/minute, injection volume 5 [11. Samples of the separated liquid component were diluted with acetonitrile-water 5o%, Na203, pH 9.8. The UV absorption was measured at 277 nm with an external calibration of the nicotine standard.
The results of the analyses are shown in Figure 3. The abbreviations in the table are as follows:
TN: total nitrogen TN-PN: nitrogen contributed by enzyme protein NN: nitrogen contributed by nicotine
acetic acid (to make the pH more acidic) or by the addition of 2 M sodium hydroxide solution (to make the pH more alkaline). The pH of the tobacco treated with enzyme groups 2 or 4 decreased during the course of the reaction and was not adjusted by the addition of an acid. Following incubation with the enzymes, the tobacco material was separated from the liquid component by two filtration steps, the first filtration step was carried out using coarse paper filter, and the filtrate from the paper filter was filtered with a 0.45 vtrn hydrophilic PVDF membrane syringe filter (Rotilabo , diameter 25 mm, Carl Roth, Germany).
The parameters used for the treatment of the tobacco material are outlined in Figure 3.
Analysis of Protein Removal The resulting liquid component of the samples prepared according to Figure 3 was analysed for the nitrogen content. Total nitrogen was measured with a TOC-N
Analyzer (Shimadzu) and/or a total nitrogen test kit (Spectroquant Nitrogen total photometric test, DMP 10 ¨ 150 mg/1, Merck).
The nitrogen content of the liquid component was corrected for the nitrogen present that was contributed by nicotine and enzyme proteins, taking the approximate proportion of nitrogen in nicotine to be 17% by weight, and the approximate proportion of nitrogen in protein to be 16% by weight.
Nicotine quantification was carried out using HPLC with a Phenomenex-Gemini column C18.3 lam, 150 x 3 mm, isocratic at 30 C, flow 0.6 ml/minute, injection volume 5 [11. Samples of the separated liquid component were diluted with acetonitrile-water 5o%, Na203, pH 9.8. The UV absorption was measured at 277 nm with an external calibration of the nicotine standard.
The results of the analyses are shown in Figure 3. The abbreviations in the table are as follows:
TN: total nitrogen TN-PN: nitrogen contributed by enzyme protein NN: nitrogen contributed by nicotine
- 19 -TN final: total nitrogen in the liquid component corrected for the nitrogen contributed by enzyme proteins and nicotine TN final (%): the group with the best nitrogen removal performance was set as l00%
and the remaining groups were compared to this result Example 2 - Washed tobacco material treatment with two proteases with different optimal operating conditions Treatment of Tobacco Material Whole tobacco leaves were treated according to Example 1, with the exception that the /o tobacco leaves were washed and dried prior to crushing. The tobacco leaves were washed with deionised water for 25 minutes at 50 C, stirring by hand every 5 minutes.
The aqueous solution was pressed out of the leaves by hand and the tobacco leaves were then dried to constant weight at 45 C in a drying chamber before undergoing enzyme treatment as described in Example 1.
The parameters used for the treatment of the tobacco material are outlined in Figure 4.
As for Example 1, tobacco treated with enzyme groups 2 and 4 was placed in deionised water.
Analysis of Protein Removal The resulting liquid component of the samples prepared according to Figure 4 was analysed for the nitrogen content and corrected for the nitrogen present that was contributed by nicotine and enzyme proteins according to the methods described in Example 1.
The results of the analyses are shown in Figure 4.
For the tobacco material prepared according to Example 2 (washed tobacco, Figure 4), the total nitrogen present in the liquid component was highest (i.e.
demonstrating a greater protein removal from the tobacco material) for groups 1 and 4. Group 4 was treated by a decrease in pH during the enzyme incubation period, and group 1 underwent a shift in temperature from 30 C to 40 C. An increase in pH during the enzyme incubation period was also seen to be effective for group 5.
A decrease in pH during the enzyme incubation period was seen to be effective for protein removal from unwashed tobacco material prepared according to Example 1
and the remaining groups were compared to this result Example 2 - Washed tobacco material treatment with two proteases with different optimal operating conditions Treatment of Tobacco Material Whole tobacco leaves were treated according to Example 1, with the exception that the /o tobacco leaves were washed and dried prior to crushing. The tobacco leaves were washed with deionised water for 25 minutes at 50 C, stirring by hand every 5 minutes.
The aqueous solution was pressed out of the leaves by hand and the tobacco leaves were then dried to constant weight at 45 C in a drying chamber before undergoing enzyme treatment as described in Example 1.
The parameters used for the treatment of the tobacco material are outlined in Figure 4.
As for Example 1, tobacco treated with enzyme groups 2 and 4 was placed in deionised water.
Analysis of Protein Removal The resulting liquid component of the samples prepared according to Figure 4 was analysed for the nitrogen content and corrected for the nitrogen present that was contributed by nicotine and enzyme proteins according to the methods described in Example 1.
The results of the analyses are shown in Figure 4.
For the tobacco material prepared according to Example 2 (washed tobacco, Figure 4), the total nitrogen present in the liquid component was highest (i.e.
demonstrating a greater protein removal from the tobacco material) for groups 1 and 4. Group 4 was treated by a decrease in pH during the enzyme incubation period, and group 1 underwent a shift in temperature from 30 C to 40 C. An increase in pH during the enzyme incubation period was also seen to be effective for group 5.
A decrease in pH during the enzyme incubation period was seen to be effective for protein removal from unwashed tobacco material prepared according to Example 1
- 20 -(unwashed tobacco, Figure 3), as seen for groups 2 and 3. An increase in pH
during the enzyme incubation period, as carried out on group 5, was also effective at removing protein from unwashed tobacco material.
Example 3 ¨ Unwashed and washed tobacco material treatment with one enzyme type under constant operating conditions Treatment of Tobacco Material Whole tobacco leaves were treated according to Examples 1 or 2, with the exception that the tobacco material was treated with a single enzyme type, i.e. with the same io optimal operating conditions. The pH and temperature were kept constant during the enzyme incubation period.
The parameters used for the treatment of the tobacco material are outlined in Figure 5.
Analysis of Protein Removal The resulting liquid component of the samples prepared according to Figure 5 was analysed for the nitrogen content and corrected for the nitrogen present that was contributed by nicotine and enzyme proteins according to the methods described in Example 1.
The results of the analyses are shown in Figure 5.
For the tobacco material treated with a single enzyme type and with constant pH and temperature during the enzyme incubation period, the total nitrogen present in the liquid component was highest (i.e. demonstrating a greater protein removal from the tobacco material) when the tobacco was treated with Bacillus species enzyme at a concentration of to g/1 at a pH of 7.5 and a temperature of 40 C. However, the level of protein removal was still lower than the level of protein removal achieved when the tobacco material is treated according to Examples 1 or 2, demonstrating that treating the tobacco material with a combination of enzymes with different optimal operating conditions and shifting the operating conditions during the enzyme incubation period can be more effective than treating the tobacco material with a single type of enzyme and/or not shifting the operating conditions during enzyme incubation.
Example 4 - Washed tobacco material treatment with two proteases with different optimal operating conditions
during the enzyme incubation period, as carried out on group 5, was also effective at removing protein from unwashed tobacco material.
Example 3 ¨ Unwashed and washed tobacco material treatment with one enzyme type under constant operating conditions Treatment of Tobacco Material Whole tobacco leaves were treated according to Examples 1 or 2, with the exception that the tobacco material was treated with a single enzyme type, i.e. with the same io optimal operating conditions. The pH and temperature were kept constant during the enzyme incubation period.
The parameters used for the treatment of the tobacco material are outlined in Figure 5.
Analysis of Protein Removal The resulting liquid component of the samples prepared according to Figure 5 was analysed for the nitrogen content and corrected for the nitrogen present that was contributed by nicotine and enzyme proteins according to the methods described in Example 1.
The results of the analyses are shown in Figure 5.
For the tobacco material treated with a single enzyme type and with constant pH and temperature during the enzyme incubation period, the total nitrogen present in the liquid component was highest (i.e. demonstrating a greater protein removal from the tobacco material) when the tobacco was treated with Bacillus species enzyme at a concentration of to g/1 at a pH of 7.5 and a temperature of 40 C. However, the level of protein removal was still lower than the level of protein removal achieved when the tobacco material is treated according to Examples 1 or 2, demonstrating that treating the tobacco material with a combination of enzymes with different optimal operating conditions and shifting the operating conditions during the enzyme incubation period can be more effective than treating the tobacco material with a single type of enzyme and/or not shifting the operating conditions during enzyme incubation.
Example 4 - Washed tobacco material treatment with two proteases with different optimal operating conditions
- 21 -Treatment of Tobacco Material Whole tobacco leaves were treated according Examples 1 or 2, with the exception that the pH was shifted by about one pH unit every 10 minutes during the enzyme incubation period. To decrease the pH, a 30% acetic acid solution was used. To increase the pH, a 1 M sodium hydroxide solution was used.
The protease enzymes used were Savinase (protease from Bacillus sp.) and protease from A. oryzae, both at a concentration of to g/l. The total enzyme incubation time was 1 hour and the temperature was maintained at 35 C during the enzyme incubation period.
lo Analysis of Protein Removal The resulting liquid component of the samples was analysed for the nitrogen content according to the methods described in Example 1, with the exception that the liquid component was not corrected for the nitrogen contributed by nicotine and enzyme /5 proteins.
The results of the analyses are shown in Figures 6 and 7.
Figures 6 and 7 illustrate that as the total nitrogen content of the liquid component 20 increases (and therefore the protein removal from the tobacco material increases) as the enzyme incubation time increases and the pH is shifted. There is a more continuous increase in the nitrogen content of the liquid component when the pH is increased from 4 to 10 (shown in Figure 7) than when the pH is decreased from 10 to 4 (shown in Figure 6). When the pH was shifted from pH 10 to 4, the highest nitrogen removal was 25 seen after 40 minutes of incubation with the enzymes, when the pH was between about 7 and 5.
In order to address various issues and advance the aft, the entirety of this disclosure shows by way of illustration various embodiments in which the claimed invention(s) 30 may be practiced and provide for superior tobacco treatment, tobacco material, and products incorporating tobacco material. The advantages and features of the disclosure are of a representative sample of embodiments only, and are not exhaustive and/or exclusive. They are presented only to assist in understanding and teach the claimed features. It is to be understood that advantages, embodiments, examples, functions, 35 features, structures, and/or other aspects of the disclosure are not to be considered limitations on the disclosure as defined by the claims or limitations on equivalents to
The protease enzymes used were Savinase (protease from Bacillus sp.) and protease from A. oryzae, both at a concentration of to g/l. The total enzyme incubation time was 1 hour and the temperature was maintained at 35 C during the enzyme incubation period.
lo Analysis of Protein Removal The resulting liquid component of the samples was analysed for the nitrogen content according to the methods described in Example 1, with the exception that the liquid component was not corrected for the nitrogen contributed by nicotine and enzyme /5 proteins.
The results of the analyses are shown in Figures 6 and 7.
Figures 6 and 7 illustrate that as the total nitrogen content of the liquid component 20 increases (and therefore the protein removal from the tobacco material increases) as the enzyme incubation time increases and the pH is shifted. There is a more continuous increase in the nitrogen content of the liquid component when the pH is increased from 4 to 10 (shown in Figure 7) than when the pH is decreased from 10 to 4 (shown in Figure 6). When the pH was shifted from pH 10 to 4, the highest nitrogen removal was 25 seen after 40 minutes of incubation with the enzymes, when the pH was between about 7 and 5.
In order to address various issues and advance the aft, the entirety of this disclosure shows by way of illustration various embodiments in which the claimed invention(s) 30 may be practiced and provide for superior tobacco treatment, tobacco material, and products incorporating tobacco material. The advantages and features of the disclosure are of a representative sample of embodiments only, and are not exhaustive and/or exclusive. They are presented only to assist in understanding and teach the claimed features. It is to be understood that advantages, embodiments, examples, functions, 35 features, structures, and/or other aspects of the disclosure are not to be considered limitations on the disclosure as defined by the claims or limitations on equivalents to
- 22 -the claims, and that other embodiments may be utilised and modifications may be made without departing from the scope and/or spirit of the disclosure. Various embodiments may suitably comprise, consist of, or consist essentially of, various combinations of the disclosed elements, components, features, parts, steps, means, etc.
In addition, the disclosure includes other inventions not presently claimed, but which may be claimed in future.
In addition, the disclosure includes other inventions not presently claimed, but which may be claimed in future.
Claims (14)
1. A method for treating a tobacco material, wherein the method comprises incubating the tobacco material for a period of time with a combination of protease enzymes with different optimal operating conditions and adjusting the operating conditions during the enzyme incubation period from the optimal temperature and/or pH of one protease enzyme to the optimal temperature and/or pH of another protease enzyme.
2. A method according to claim 1, wherein the method results in a reduction in the protein content of the treated tobacco material compared to the protein content of the tobacco material prior to being treated.
3. A method according to claims 1 or 2, wherein adjusting the operating conditions comprises adjusting the pH.
4. A method according to claim 3, wherein adjusting the pH comprises decreasing the pH.
5. A method according to claim 4, wherein the pH is decreased by the addition of an acid.
6. A method according to claim 3, wherein adjusting the pH comprises increasing the pH.
7. A method according to claim 6, wherein the pH is increased by the addition of an alkali.
8. A method according to any one of claims 1 to 7, wherein adjusting the operating conditions comprises adjusting the temperature.
9. A method according to claim 8, wherein adjusting the temperature comprises adjusting from a lower temperature to a higher temperature.
10. A method according to claim 8, wherein adjusting the temperature comprises adjusting from a higher temperature to a lower temperature.
11. A method according to any one of claims 1 to 10, wherein the combination of protease enzymes comprises two enzymes each having different optimal operating conditions.
12. A method according to any one of claims 1 to 11, wherein the combination of protease enzymes comprises three enzymes each having different optimal operating conditions.
13. A method according to any one of claims 1 to 12, wherein the protein content of the treated tobacco material is reduced by at least 50%, at least 60%, at least 70%, at least 80%, and/or at least 90%, compared to the protein content of the tobacco material prior to being treated.
14. Use of a combination of protease enzymes with different optimal operating conditions for removing one or more proteins from a tobacco material by incubating the tobacco material with the protease enzymes for a period of time and adjusting the operating conditions during the enzyme incubation period from the optimal temperature and/or pH of one protease enzyme to the optimal temperature and/or pH of another protease enzyme.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB1221193.4 | 2012-11-26 | ||
| GBGB1221193.4A GB201221193D0 (en) | 2012-11-26 | 2012-11-26 | Treatment of tobacco material |
| PCT/GB2013/053100 WO2014080223A1 (en) | 2012-11-26 | 2013-11-25 | Treatment of tobacco material |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2889054A1 CA2889054A1 (en) | 2014-05-30 |
| CA2889054C true CA2889054C (en) | 2017-03-07 |
Family
ID=47560636
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2889054A Expired - Fee Related CA2889054C (en) | 2012-11-26 | 2013-11-25 | Treatment of tobacco material |
Country Status (8)
| Country | Link |
|---|---|
| US (1) | US20150296869A1 (en) |
| EP (1) | EP2922421A1 (en) |
| JP (1) | JP6067874B2 (en) |
| BR (1) | BR112015012064A2 (en) |
| CA (1) | CA2889054C (en) |
| GB (1) | GB201221193D0 (en) |
| RU (1) | RU2015125060A (en) |
| WO (1) | WO2014080223A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105266188A (en) * | 2015-11-25 | 2016-01-27 | 红云红河烟草(集团)有限责任公司 | Method for increasing permeability of feed liquid in tobacco flakes by applying complex enzyme preparation |
| CN105686067A (en) * | 2016-03-11 | 2016-06-22 | 李贵生 | Rosemary type cigarette |
| CN109554306B (en) * | 2018-11-14 | 2022-04-19 | 河南中烟工业有限责任公司 | Compound preparation of protease and protein degrading bacteria and application of compound preparation in cigars |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4887618A (en) * | 1988-05-19 | 1989-12-19 | R. J. Reynolds Tobacco Company | Tobacco processing |
| US4941484A (en) * | 1989-05-30 | 1990-07-17 | R. J. Reynolds Tobacco Company | Tobacco processing |
| US5343879A (en) * | 1991-06-21 | 1994-09-06 | R. J. Reynolds Tobacco Company | Tobacco treatment process |
| GB0810850D0 (en) * | 2008-06-13 | 2008-07-23 | British American Tobacco Co | Tobacco treatment |
| IN2012DN02364A (en) * | 2009-10-12 | 2015-08-21 | Du Pont |
-
2012
- 2012-11-26 GB GBGB1221193.4A patent/GB201221193D0/en not_active Ceased
-
2013
- 2013-11-25 RU RU2015125060A patent/RU2015125060A/en not_active Application Discontinuation
- 2013-11-25 EP EP13799348.1A patent/EP2922421A1/en not_active Withdrawn
- 2013-11-25 CA CA2889054A patent/CA2889054C/en not_active Expired - Fee Related
- 2013-11-25 US US14/647,081 patent/US20150296869A1/en not_active Abandoned
- 2013-11-25 WO PCT/GB2013/053100 patent/WO2014080223A1/en active Application Filing
- 2013-11-25 BR BR112015012064A patent/BR112015012064A2/en not_active IP Right Cessation
- 2013-11-25 JP JP2015543525A patent/JP6067874B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| RU2015125060A (en) | 2017-01-10 |
| US20150296869A1 (en) | 2015-10-22 |
| JP2015535428A (en) | 2015-12-14 |
| EP2922421A1 (en) | 2015-09-30 |
| GB201221193D0 (en) | 2013-01-09 |
| CA2889054A1 (en) | 2014-05-30 |
| WO2014080223A1 (en) | 2014-05-30 |
| JP6067874B2 (en) | 2017-01-25 |
| BR112015012064A2 (en) | 2017-07-11 |
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