JP6067874B2 - Tobacco material processing - Google Patents

Description

  The present invention relates to a method for treating tobacco material.

  In some situations, it may be desirable to reduce the content of certain components of the tobacco material before making it a smoking article such as a cigarette.

  According to a first aspect, a method for treating tobacco material is provided, the method comprising the steps of using a combination of proteolytic enzymes having different optimal operating conditions and adjusting the operating conditions during enzyme incubation. .

  In certain embodiments, tobacco materials treated in this manner have a reduced protein content compared to untreated tobacco material.

  In some embodiments, adjusting the operating conditions includes adjusting the pH. In some embodiments, the pH adjustment includes adding an acid as needed to lower the pH. In some embodiments, pH adjustment includes adding alkali as needed to increase pH.

  In some embodiments, adjusting the operating conditions includes temperature adjustment. In some embodiments, the temperature adjustment includes adjustment to increase the low temperature. In some embodiments, the temperature adjustment includes adjustment to lower the high temperature.

  In one embodiment, the enzyme combination consists of enzymes with two different optimal operating conditions. In one embodiment, the enzyme combination consists of enzymes with three different optimal operating conditions.

  In certain 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 untreated tobacco material.

  In a second aspect, there is provided a tobacco material or derivative thereof treated by the method of the first aspect.

  In a third aspect, there is provided a smoking article comprising the tobacco material according to the second aspect.

  In a fourth embodiment, a combination of proteolytic enzymes with different optimal operating conditions is used to remove one or more proteins from tobacco material by adjusting the operating conditions during enzyme incubation.

Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings.
3 is a flowchart illustrating a method for treating tobacco material according to an embodiment of the present invention. 3 is a flowchart illustrating a method for treating tobacco material according to an embodiment of the present invention. FIG. 6 is a table showing the results of treating unwashed tobacco with two proteolytic enzymes having different optimal operating conditions with varying pH values and / or temperatures during enzyme incubation. FIG. 6 is a table showing the results of enzymatic treatment of washed tobacco with two proteolytic digestions with different optimum operating conditions with varying pH values and / or temperatures during enzyme incubation. It is a table | surface which shows the result of having processed with the proteolytic enzyme, without changing the pH value or temperature during enzyme incubation. FIG. 5 is a graph showing the results of treating washed tobacco with two proteolytic enzymes having different optimum operating conditions with pH values changed during enzyme incubation to pH 10-4. FIG. 6 is a graph showing the results of treating washed tobacco with two proteolytic enzymes having different optimum operating conditions with the pH value during enzyme incubation changed to pH 4-10. 1 is a schematic view of a smoking article comprising tobacco material treated according to an embodiment of the present invention. FIG.

  The present invention relates to a method of treating tobacco material to remove protein from tobacco material and / or reduce the protein content of tobacco material.

  The tobacco material may be processed to remove one or more other chemicals that it is desirable to remove. Proteins in tobacco materials are often found to be such substances and / or their precursors.

  Alternatively or in addition, it may be desirable to reduce the protein content of the tobacco material in order to improve the quality of the tobacco material.

  Methods have been proposed to attempt protein removal, but they tend to be costly, time consuming and / or adversely affect the physical structure of the tobacco material and / or reduce the protein content to the desired level It may not be done.

  The method of the present invention removes protein from tobacco material. Examples of proteins that are removed from tobacco material include RuBisCO. The method of the present invention reduces the protein content of tobacco material compared to untreated tobacco material.

  FIG. 1 is a flowchart illustrating a method for treating tobacco material according to one embodiment. The tobacco material 1 is subjected to proteolytic enzyme treatment 10. By separating 20 the solid component and the liquid component of the obtained suspension, the tobacco material 30 and the liquid component 31 having a low protein content are obtained.

  As used herein, the term “tobacco material” includes any part, for example, the leaves and stems of any species of the genus Tobacco, and their reconstituted materials. The tobacco material used in the present invention is preferably from the Nicotiana tabacum species.

  Any kind of tobacco is used in the present invention. Examples of tobacco that can be used include, but are not limited to, Virginia, Burley, Maryland, Oriental and Rustica tobacco.

  The tobacco material may be treated in any suitable manner before being treated with the method of the present invention.

  As used herein, the terms “in incubation” and “in enzyme incubation” refer to the period during which tobacco material is incubated with one or more proteolytic enzymes.

  As used herein, the term “optimal operating conditions” refers to reaction conditions at which the enzyme exhibits optimal activity, and / or reaction conditions that exhibit sufficient activity such that the reaction proceeds at an appropriate and / or desired rate. Optimal operating conditions include a range of pH and / or temperature at which the proteolytic enzyme exhibits optimal activity and / or sufficient activity such that the reaction proceeds at an appropriate and / or desired rate.

  In one embodiment, the method of the invention relates to a method for reducing the protein content of a preserved tobacco material. The tobacco material may be stored by any suitable storage processing method prior to processing by the method of the present invention.

  The tobacco material may be any suitable form of tobacco. The tobacco material may comprise a thin layer, sheet, reconstituted, crushed and / or ground tobacco material. If the tobacco material consists of a thin layer of tobacco material, the thin layer may be in the form of strips, chops, or whole leaf tobacco. In some embodiments, the tobacco material is in the form of cut rags or whole leaves. In some embodiments, the tobacco material is pre-saved chopped waste or whole leaf tobacco.

  In some embodiments, the tobacco material may be washed prior to proteolytic enzyme treatment.

  Any suitable liquid medium may be used for cleaning the tobacco material. A typical cleaning medium is an aqueous solution.

  In some embodiments, the tobacco material may be washed with water. The tobacco material may be washed with tap water. Alternatively or in addition, the tobacco material may be washed with purified water. As used herein, “purified water” corresponds to water that has been subjected to treatment for removing contaminants and / or impurities. In some embodiments, the purified water is deionized water.

  Washing the tobacco material removes protein from the tobacco material. For example, washing tobacco material with water removes water soluble proteins from the tobacco material.

  Cleaning the tobacco material has the advantage that the protein content of the tobacco material has already been reduced prior to proteolytic enzyme treatment. Accordingly, it is more efficient and / or more effective to use washed tobacco material than unwashed tobacco material to reduce protein content using proteolytic enzymes.

  Alternatively or in addition, if a washing step is included prior to the enzyme treatment, the enzyme incubation time required for the desired level of protein removal may be shorter and / or the enzyme required for the desired level of protein removal. The concentration may be lower.

  The tobacco material may be placed in a suitable cleaning medium and washed for up to 10 minutes, up to 15 minutes, up to 20 minutes, up to 25 minutes, or up to 30 minutes.

  The amount of the washing medium may be sufficient to remove any protein soluble in the washing medium from the tobacco material. One skilled in the art will recognize that extraction efficiency can be optimized by changing the ratio of cleaning medium to tobacco material.

  The temperature of the tobacco material and cleaning medium may be higher than the ambient temperature, which facilitates the extraction process. The temperature of the tobacco material and cleaning medium may be up to 20 ° C, up to 30 ° C, up to 40 ° C, up to 50 ° C, up to 60 ° C, up to 70 ° C, or up to 80 ° C. Suitable temperatures for the tobacco material and cleaning medium are well known to those skilled in the art.

  In one embodiment, the temperature of the tobacco material and the cleaning medium is 50 ° C. and the tobacco material is washed for 25 minutes.

  The tobacco material and cleaning medium may be agitated during cleaning, for example, by stirring and / or shaking.

  After washing, the tobacco material may be separated from the washing medium. Suitable means for separating tobacco material from the washing medium are well known to those skilled in the art and include filtration and / or centrifugation.

  After contact with the tobacco material, the cleaning medium may include components that are desired to be retained in the tobacco material. For example, the cleaning medium may include tobacco proteins that are soluble in the cleaning medium. Such proteins impart certain sensory characteristics such as tobacco flavor. Alternatively or additionally, the cleaning medium may include nicotine, sugar and / or other ingredients that are desirably retained in the tobacco material. Thus, in one embodiment, after separation from the tobacco material, the cleaning medium is reapplied to the tobacco material, eg, to tobacco material that has been treated with a proteolytic enzyme.

  The method of the present invention uses proteolytic enzyme activity to reduce the protein content of tobacco material.

  Proteolytic enzymes break down proteins into smaller sized peptides and amino acids. Accordingly, in the method of the present invention, the protein of the tobacco material is decomposed with proteolytic enzyme activity, and the decomposed product is released from the tobacco material, so that the protein is removed from the tobacco material and / or the protein of the tobacco material. The content is reduced.

  Enzymatic treatment is considered one of the most effective ways to reduce the protein content of tobacco material. In addition, the use of enzymes to reduce the protein content of tobacco materials is advantageous because the enzymes catalyze only specific transformations while leaving other materials intact, and the use of enzymes that are harmful to the enzyme. Because it is not necessary. On the other hand, other protein reduction methods require, for example, various conditions that damage the tobacco material, and may damage the physical structure of the tobacco material.

  The proteolytic enzyme used in the present invention may be any type of proteolytic enzyme. The proteolytic enzyme used in the present invention is classified into EC 3.4 (that is, an enzyme belonging to peptidase) by the International Union of Biochemistry and Molecular Biology.

  Proteolytic enzymes that are commercially used in the food and detergent industries that can be used at low cost are suitable for the method of the present invention.

  The proteolytic enzyme may be obtained from any source if suitable. For example, the proteolytic enzyme may be derived from plants, animals, fungi, bacteria and / or viruses. Alternatively or in addition, a proteolytic enzyme may be synthesized.

  In certain embodiments where the proteolytic enzyme is derived from bacteria, the proteolytic enzyme may be obtained from the genus Bacillus. Examples of commercially available Bacillus derived proteolytic enzymes include Savinase (registered trademark), Esperase (registered trademark), Neutrase (registered trademark) and Alcalase (registered trademark). In one embodiment, the proteolytic enzyme is obtained from a Bacillus licheniformis species. Other suitable bacterial proteolytic enzymes are well known to those skilled in the art.

  In certain embodiments where the proteolytic enzyme is derived from a fungus, the proteolytic enzyme may be obtained from Aspergillus. In one embodiment, the proteolytic enzyme is obtained from an Aspergillus oryzae species. An example of a commercially available proteolytic enzyme derived from Aspergillus niger is Enzobake (registered trademark). In one embodiment, the proteolytic enzyme is obtained from an Aspergillus saitoi species. Other suitable fungal derived proteolytic enzymes are well known to those skilled in the art.

  The proteolytic enzyme may be an acidic enzyme. As used herein, the term “acidic enzyme” refers to an enzyme that is optimally active at acidic pH. An example of an acidic proteolytic enzyme is one derived from Aspergillus niger, which is in the range of pH 3.0 to 7.0, optionally in the range of pH 3.0 to 5.0, or about pH 7.0. Shows the optimal activity.

  The proteolytic enzyme may be an alkaline enzyme. As used herein, the term “alkaline enzyme” refers to an enzyme that is optimally active at alkaline pH. Examples of alkaline proteolytic enzymes are those derived from Bacillus licheniformis (showing optimum activity in the range of pH 3.0 to 11.5) and Savinase (registered trademark) (optimum in the range of pH 7.0 to 11.0) Activity).

  Alternatively or in addition, the proteolytic enzyme may be an enzyme that exhibits optimal activity at neutral pH.

  The proteolytic enzyme treatment of tobacco material may be performed in solution. In certain embodiments, the proteolytic treatment of tobacco material is performed in an aqueous solution.

  The solution for subjecting the tobacco material to the proteolytic enzyme treatment may be selected according to the specific proteolytic enzyme used. Suitable solutions are well known to those skilled in the art.

  In some embodiments, the proteolytic enzyme is added after the tobacco material is added to the selected solution. In other words, in one embodiment, the proteolytic enzyme is added to the tobacco material in the selection solution.

  The conditions at the time of enzyme addition are selected so that there is no loss of enzyme activity.

  In some embodiments, the pH of the solution is adjusted prior to adding the enzyme to the selection solution. The pH corresponds to the pH at which the proteolytic enzyme exhibits optimal activity.

  In certain embodiments, it may not be necessary to adjust the pH of the tobacco material in the selection solution because the pH of the tobacco material in the selection solution corresponds to the pH at which the proteolytic enzyme exhibits optimal activity.

  When water is added to the tobacco material, it exhibits acidic or neutral properties. Thus, in some embodiments where the tobacco material is treated with an acidic proteolytic enzyme and the selection solution is water such as deionized water, the tobacco material in the selection solution is acidic and no further adjustment of the pH is required. There is also.

  In some embodiments where the pH of the tobacco material in the selective solution does not correspond to the pH at which the proteolytic enzyme exhibits optimal activity, the pH may be adjusted to the desired pH.

  Those skilled in the art know suitable methods for adjusting the pH. In some embodiments, the pH is adjusted by adding acids such as citric acid and / or inorganic acids or by adding alkali such as sodium hydroxide.

  In certain embodiments, a buffer may be added to maintain the pH of the solution. Suitable buffering agents are well known to those skilled in the art.

  In some embodiments, the temperature of the solution is adjusted prior to adding the enzyme to the selection solution. The temperature of the solution may be adjusted to a temperature at which the proteolytic enzyme exhibits optimum activity.

  In certain embodiments, it may not be necessary to adjust the temperature of the selection solution because the temperature of the selection solution corresponds to the temperature at which the proteolytic enzyme exhibits optimal activity.

  In some embodiments where the temperature of the selected solution does not correspond to the temperature at which the proteolytic enzyme exhibits optimal activity, the temperature may be adjusted to the desired temperature.

  In some embodiments, the temperature of the selection solution is between about 15 ° C and 75 ° C. In certain embodiments, the temperature of the selection solution is up to about 60 ° C. to minimize adverse effects on enzyme activity. In some embodiments where the tobacco material is treated with a proteolytic enzyme derived from Aspergillus oryzae, the temperature of the selective solution may be about 30 ° C. Depending on the embodiment in which the tobacco material is treated with Savinase®, the temperature of the selection solution may be about 40 ° C.

  The tobacco material and proteolytic enzyme may be combined to produce a tobacco / enzyme mixture.

  In certain embodiments, the proteolytic enzyme treatment comprises treating tobacco material with a type of proteolytic enzyme. In certain other embodiments, the proteolytic enzyme treatment comprises treating tobacco material in combination with different proteolytic enzymes. The combination of proteolytic enzymes may include at least two enzymes with different optimal operating conditions. Alternatively or in addition, the combination of proteolytic enzymes may include at least three enzymes with different optimal operating conditions.

  Depending on the embodiment in which proteolytic enzymes having different optimal operating conditions are used in combination, the operating conditions may be adjusted or changed during the enzyme incubation. Adjustment of operating conditions may include pH and / or temperature adjustment.

  Combining proteolytic enzymes with different optimal operating conditions and adding them simultaneously to the tobacco material to treat the tobacco material results in a single enzyme addition step required during the procedure to reduce the protein content of the tobacco material. And is suitable for carrying out the process on an industrial or commercial scale.

  Depending on the embodiment in which proteolytic enzymes having different optimum operating conditions are used in combination, the optimum pH of the proteolytic enzyme may be different. Thus, the pH of the tobacco / enzyme mixture may be adjusted during enzyme incubation from the optimum pH of one proteolytic enzyme in the tobacco / enzyme mixture to the optimum pH of another proteolytic enzyme.

  The pH may be adjusted by lowering or increasing the pH of the tobacco / enzyme mixture.

  In some embodiments using a combination of alkaline and acidic proteolytic enzymes, 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 more than 7.0 to less than 7.0, or from less than 7.0 to more than 7.0.

  Depending on the embodiment in which an alkaline enzyme and an enzyme that exhibits optimal activity at neutral pH are used in combination, the pH of the tobacco / enzyme mixture may be adjusted from alkaline to neutral pH or from neutral to alkaline pH. In other words, the pH may be adjusted from greater than 7.0 to about 7.0, or from about 7.0 to greater than 7.0.

  Depending on the embodiment using a combination of an acidic enzyme and an enzyme that exhibits optimal activity at neutral pH, 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 less than 7.0 to about 7.0, or from about 7.0 to less than 7.0.

  In some embodiments using a combination of alkaline enzyme, acidic enzyme and an enzyme that exhibits optimal activity at neutral pH, the pH of the tobacco / enzyme mixture is from the optimal activity pH of the first enzyme to the optimal activity pH of the second enzyme. In addition, the pH may be adjusted to the optimum activity of the third enzyme.

  Changes in pH may occur during the reaction. In other words, the pH may not be changed by the operator intentionally adjusting the pH. Instead of or in addition to natural changes, the pH may be changed by intentionally adjusting the pH.

  Acid may be added to lower the pH during enzyme incubation. Suitable acids are well known to those skilled in the art. In some embodiments, the pH is lowered by adding citric acid. Alternatively or in addition, the pH is lowered by blowing carbon dioxide into the tobacco / enzyme mixture.

  Alkaline may be added to increase the pH during enzyme incubation. Suitable alkalis are well known to those skilled in the art. In certain embodiments, the pH is increased by adding sodium hydroxide.

  Depending on the embodiment of the method in which proteolytic enzymes having different optimum operating conditions are used in combination, the optimal temperature of the proteolytic enzyme may be different. Thus, the temperature of the tobacco / enzyme mixture may be adjusted during enzyme incubation from the optimal temperature of one proteolytic enzyme in the tobacco / enzyme mixture to the optimal temperature of another proteolytic enzyme.

  The temperature may be adjusted by decreasing or increasing the temperature of the tobacco / enzyme mixture.

  Depending on the embodiment of the method using a combination of proteolytic enzymes having different optimal operating conditions, the optimal pH and temperature of the proteolytic enzyme may be different. Thus, the pH and temperature of the tobacco / enzyme mixture may be adjusted during enzyme incubation from the optimum pH and temperature of one proteolytic enzyme in the tobacco / enzyme mixture to the optimum pH and temperature of another proteolytic enzyme. .

  When enzymes having different optimum operating conditions are used in combination and the operating conditions are adjusted, the enzyme treatment step for reducing the protein content of the tobacco material can be performed more effectively and / or more efficiently. As an alternative to or in addition to this method, using a combination of enzymes with different optimal operating conditions and adjusting the operating conditions can affect the physical properties of the tobacco material, for example, the structure of the tobacco material Damage is minimized.

  The tobacco material may be incubated with a proteolytic enzyme for a time sufficient to reduce the protein content of the tobacco material to a desired level.

  In embodiments where proteolytic enzymes with different optimal operating conditions are used in combination and the operating conditions are adjusted during incubation, the tobacco material is up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, or 5 hours. May be incubated with proteolytic enzymes. In some embodiments using a combination of proteolytic enzymes with different optimal operating conditions, the incubation time is up to 1 hour.

  In embodiments where a combination of proteolytic enzymes having different optimal operating conditions is used, the operating conditions may be adjusted after a certain incubation time has elapsed. The operating conditions may be adjusted after about 25% of the incubation time has passed, after about 50% has passed, or after about 75% has passed. For example, in an embodiment where the incubation time is 1 hour and the combination of proteolytic enzymes consists of enzymes having two different optimal operating conditions, the operating conditions are after about 50% of the incubation time has passed, ie about 30 You may adjust after minutes. In embodiments where the incubation time is 1 hour and the combination of proteolytic enzymes consists of enzymes with three different optimal operating conditions, the operating conditions are after about 33% of the incubation time has elapsed, i.e. after about 20 minutes. The first adjustment may be made and after about 66% of the incubation time has elapsed, ie after about 40 minutes, the second adjustment may be made.

  The tobacco material / enzyme mixture may be agitated during the enzyme incubation, for example, by stirring, shaking and / or shaking. The tobacco material / enzyme mixture may be agitated to moderately mix without exposing the tobacco fibers to mechanical stresses that damage the structure of the tobacco material.

  The concentration of the proteolytic enzyme in the tobacco / enzyme mixture may be sufficient to reduce the protein content to the desired level within the desired incubation time. At the same time, the amount of proteolytic enzyme used during the process may be small enough, making the process more cost effective.

  The amount of proteolytic enzyme and solution added to the tobacco material may depend on the desired enzyme concentration.

  The concentration of the proteolytic enzyme may be between about 0.01 g / L and about 5.0 g / L and / or between about 0.1 g / L and about 2.0 g / L. In one embodiment, the concentration of proteolytic enzyme is about 1.0 g / L. In certain embodiments, the concentration of proteolytic enzyme is about 1.8 g / L. For clarity, as used herein, the concentration of proteolytic enzyme is not the same as the enzyme activity.

  In one embodiment, the tobacco material is incubated for 1 hour with two proteolytic enzymes at different optimal operating conditions (both at a concentration of 1.0 g / L).

  It is broken down into protein fragments by enzymatic treatment of tobacco material. Many of the resulting protein fragments are easily separated from tobacco material because they are solubilized and / or dispersed in the liquid component of the tobacco / enzyme mixture.

  After incubating the tobacco material with a proteolytic enzyme, the insoluble tobacco material may be separated from the liquid component of the tobacco / enzyme mixture. The tobacco material and liquid component may be separated using any suitable device. In some embodiments, the tobacco material and the liquid component are separated by filtration. Other suitable methods for separating tobacco material and liquid components are well known to those skilled in the art and may include the use of centrifuges and / or presses, sieves and / or belt filters.

  In some embodiments, the tobacco material and liquid component are separated using one or more filtration steps. For example, the tobacco / enzyme mixture may be passed through a coarse filter and / or a fine filter. Suitable filters are well known to those skilled in the art.

  FIG. 2 is a flowchart illustrating a method for treating tobacco material according to one embodiment. Whole leaf tobacco material 2 is passed through a washing step 3 as necessary. Next, the tobacco material is added 4 to the selection solution, and the pH and / or temperature of the tobacco material in the solution is adjusted 5 prior to the addition of the proteolytic enzyme as necessary. The tobacco material is then subjected to proteolytic enzyme treatment 10. Separating 20 the tobacco material and the liquid component of the resulting tobacco / enzyme mixture results in a tobacco material 30 and a liquid component 31 having a low protein content.

  After separation from the tobacco material, the liquid component may be discarded. Or you may leave a liquid component. For example, the solution may be recovered while leaving the liquid component and reused.

  After 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 cleaning medium is heated. In some embodiments, the cleaning medium is saline.

  The tobacco material may be treated to inactivate any enzyme remaining in the tobacco fiber. Suitable inactivation methods are well known to those skilled in the art. In certain embodiments, the tobacco material may be heat treated to inactivate any enzymes remaining in the tobacco fibers.

  When tobacco material is treated in a way that consists of combining proteolytic enzymes with different optimum operating conditions and adjusting the operating conditions during enzyme incubation, the protein content is the protein content of the untreated tobacco material Of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or at least 95 % Reduction. In some embodiments, the protein content is reduced by at least 50% and / or at least 75%.

  Alternatively or additionally, at least 5% when the tobacco material is treated in a manner comprising a combination of proteolytic enzymes having different optimum operating conditions and adjusting the operating conditions during enzyme incubation, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or at least 95% protein tobacco Can be removed from the material.

  In one embodiment, when tobacco material is treated in a manner comprising a combination of proteolytic enzymes having different optimum operating conditions and adjusting the operating conditions during enzyme incubation, it is contained in the untreated tobacco material. At least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or An amount of at least 95% protein is extracted.

  The method of the present invention may include one or more additional processing steps. Suitable additional processing steps include, but are not limited to, treating tobacco material with one or more suitable non-ionic liquids, eg, water; one or more additional enzymes (catalyzing modification of polyphenols) Treatment of tobacco material with, for example, phenol oxidase; treatment of tobacco material with one or more suitable surfactants such as sodium dodecyl sulfate (SDS) in any suitable solvent; If the tobacco material is treated with one or more suitable adsorbents, such as polyvinyl polypyrrolidone (PVPP), hydroxyapatite, bentonite, activated carbon, or attapulgite, in any suitable solvent; and one or more suitable Treatment of tobacco material with a non-aqueous liquid, such as an ionic liquid.

  Instead of or in addition to the treatment step, the tobacco material treated by the method of the present invention may be further subjected to an extraction step thereafter.

  After treatment with the method of the present invention, the low protein content tobacco material may be made into a smoking article.

  As used herein, the term “smoking article” refers to smokeable products such as cigarettes, cigars and cigarillos (which consist of any tobacco, tobacco derivative, expanded tobacco, regenerated tobacco or tobacco substitute). As well as heat-not-burn products. A smoke filter may be provided with a gas filter that is sucked by the smoker.

  After treatment with the method of the present invention, the tobacco material may be further modified in any suitable manner before making it a smoking article. For example, the tobacco material may be dried, certain chemicals, for example, savory substances in the range permitted by law, added to the tobacco material, and / or the tobacco material cut and / or shredded and any suitable You may make it a smoking article in a simple way.

  As used herein, the terms “flavor” and “flavor substance” refer to materials that can be used to the extent permitted by law to produce the desired taste and aroma in products for adult consumers. These include extracts (e.g., licorice, hydrangea, park leaves, chamomile, foliage, clove, menthol, mint, aniseed fruit, cinnamon, herbs, cherries, berries, peaches, apples, durum buoys, bourbon, scotch, Whiskey, spearmint, peppermint, lavender, cardamom, celery, cascarilla, nutmeg, sandalwood, bergamot, geranium, honey extract, rose oil, vanilla, lemon oil, orange oil, cassia, tiger fennel, cognac, jasmine, ylang ylang, sage, fennel , Allspice, ginger, anise, coriander, coffee, or mint oil from any species of the mint family), flavor enhancers, bitter taste receptor site inhibitors, sensory receptor site activators or stimulants, sugars and / or Or sugar bill Products (eg, sucralose, acesulfame potassium, aspartame, saccharin, cyclamate, lactose, sucrose, glucose, fructose, sorbitol or mannitol), as well as other additives (eg, charcoal, chlorophyll, minerals, plant components, or breath refreshment) Agent). These may be imitation, synthetic or natural ingredients, or a mixture thereof. These may be in any suitable form, for example oil, liquid or powder.

  Referring to FIG. 8, the smoking article 101 according to an exemplary embodiment of the present invention includes a filter 102 and a cylindrical smoking material rod 103, and the smoking material rod 103 is made of a smoking material. For example, it is made of tobacco processed according to the invention described in the present specification, and the smoking material rod 103 is linearly connected to the filter 102 so that one end of the smoking material rod 103 contacts the end of the filter 102. As is conventional, the filter 102 is wrapped with a plug wrapper (not shown), and the smoking material rod 103 is joined to the filter 102 with tipping paper (not shown).

  In certain embodiments, the methods described herein may include one or more additional steps of modifying the tobacco material in any suitable manner. For example, the tobacco material may be modified to impart one or more desirable properties to the tobacco material. For example, if the treated tobacco material is a smoking article, such as a cigarette, the tobacco material may be treated to change the flavor produced during combustion and / or one or more chemicals may be removed.

Experimental Work A series of experiments were performed using a combination of proteolytic enzymes with different optimal operating conditions to examine protein removal from tobacco material and adjusting the operating conditions during enzyme incubation. The experimental work disclosed is not intended to limit the scope of the invention.

Examples Example 1-Treatment of unwashed tobacco material with two proteolytic enzymes with different optimal operating conditions

Tobacco Material Processing Whole leaf tobacco was dried in a 45 ° C. drying chamber to a constant weight and then crushed so that tobacco could be processed on a laboratory scale. 25 g of the crushed dry tobacco was placed in 500 mL of deionized water or buffer (selected according to the enzyme combination used, the enzyme obtained from Sigma Aldrich®). Referring to FIG. 3, tobacco treated with Group 2 and Group 4 enzymes is placed in deionized water and an alkaline buffer (0.1 M sodium phosphate, pH 8.0) is used for tobacco treatment starting from alkaline pH. Used, acidic buffer (0.1 M sodium acetate, pH 4.0) was used for tobacco treatment starting from acidic pH. After adding two enzymes each having different optimum operating conditions at a concentration of 1.0 g / L, the resulting suspension of tobacco material and the enzyme was incubated for 1 hour on an incubation shaker at 120 rpm. Incubated. The temperature of the incubation shaker was set according to the optimum working temperature of the enzyme, and in some cases the temperature was changed after 30 minutes of incubation. The pH value is set according to the optimal working pH of the enzyme and is observed with a pH meter during the incubation, optionally after addition of 40% acetic acid (to make the pH more acidic) or 2M water after 30 minutes of incubation. Sodium oxide solution was added to change the pH (to make the pH more alkaline). No adjustment was made to the acid addition of the tobacco group treated with the Group 2 or Group 4 enzymes that had decreased in the course of the reaction. After incubation with the enzyme, the tobacco material is separated from the liquid components in two filtration steps, the first filtration step is performed with coarse filter paper, and the filtrate is filtered from the filter paper to a 0.45 μm hydrophilic PVDF membrane syringe filter ( Rotilabo (registered trademark), diameter 25 mm, manufactured by Carl Roth, Germany).

  The parameters used to process the tobacco material are outlined in FIG.

Analysis of protein removal The amount of nitrogen contained in the liquid component of the sample prepared and obtained according to FIG. 3 was analyzed. Total nitrogen was measured using a TOC-N analyzer (manufactured by Shimadzu Corporation) and / or a total nitrogen test kit (Spectroquant (registered trademark) total nitrogen photometric test, DMP 10 to 150 mg / L, manufactured by Merck).

  The nitrogen content of the liquid component was corrected for the nitrogen content of nicotine and enzyme protein, with the approximate proportion of nitrogen in nicotine being 17% by weight and the approximate proportion of nitrogen in protein being 16% by weight.

  Nicotine quantification was performed using HPLC with a Phenomenex-Gemini column C18.3 μm, 150 × 3 mm, isocratic at 30 ° C., a flow rate of 0.6 mL / min, and an injection volume of 5 μL. A sample of the separated liquid component was diluted with acetonitrile-water 50%, Na2O3, pH 9.8. UV absorption at 277 nm was measured with a nicotine external calibration standard.

The analysis results are shown in FIG. Abbreviations in the table are as follows.
TN: Total nitrogen TN-PN: Nitrogen by enzyme protein NN: Nitrogen by nicotine TN final: Total nitrogen TN final in liquid component corrected for nitrogen content by enzyme protein and nicotine (%): Group with the best nitrogen removal efficiency Compare the remaining groups with this result, assuming 100%

  Example 2 Treated Tobacco Material with Two Proteolytic Enzymes with Different Optimal Operating Conditions

Treatment of tobacco material Whole leaf tobacco was treated according to Example 1 except that it was washed and dried prior to crushing. The tobacco leaf was washed with deionized water at 50 ° C. for 25 minutes while stirring the tobacco leaf by hand every 5 minutes. After the aqueous solution was pushed out of the leaves by hand, the tobacco leaves were dried in a drying chamber at 45 ° C. until a constant weight and then enzymatically treated as described in Example 1.

  The parameters used to process the tobacco material are outlined in FIG. As in Example 1, tobacco treated with Group 2 and Group 4 enzymes was placed in deionized water.

Analysis of protein removal The amount of nitrogen prepared according to FIG. 4 and contained in the liquid component of the obtained sample was analyzed according to the method described in Example 1, and the nitrogen content by nicotine and enzyme protein was corrected.

  The analysis results are shown in FIG.

  The tobacco material prepared according to Example 2 (washed tobacco, FIG. 4) had the highest total nitrogen in the liquid components of Groups 1 and 4 (ie, more protein was removed from the tobacco source). Showing). Group 4 was treated with a reduced pH during enzyme incubation and Group 1 was changed in temperature from 30 ° C to 40 ° C. Increased pH during enzyme incubation was also seen in group 5.

  As seen in Groups 2 and 3, the reduction in pH during enzyme incubation was effective in removing protein from unwashed tobacco material prepared according to Example 1 (unwashed tobacco, FIG. 3). . Increased pH during enzyme incubation (as done in Group 5) also had an effect on protein removal from unwashed tobacco material.

  Example 3-Treatment of unwashed and washed tobacco material with one enzyme under certain operating conditions

Treatment of tobacco material Whole leaf tobacco was treated according to Example 1 or 2, except that the tobacco material was treated with one enzyme, ie under the same optimal operating conditions. The pH and temperature were kept constant during enzyme incubation.

  The parameters used to process the tobacco material are outlined in FIG.

Analysis of protein removal The amount of nitrogen prepared according to FIG. 5 and contained in the liquid component of the obtained sample was analyzed according to the method described in Example 1 to correct nitrogen content due to nicotine and enzyme protein.

  The analysis results are shown in FIG.

  For tobacco material treated with one enzyme and at a constant pH and temperature during enzyme incubation, when the tobacco material was treated with an enzyme of Bacillus species at a concentration of 1.0 g / L, pH 7.5, temperature 40 ° C. However, the total nitrogen in the liquid component was the highest (ie, more protein was removed from the tobacco source). However, the level of protein removal is even lower than the level of protein removal when processed according to Example 1 or 2, which treats tobacco material in combination with enzymes having different optimal operating conditions, and during enzyme incubation It is shown that changing the operating conditions is more effective than treating the tobacco material with only one enzyme and / or not changing the operating conditions during enzyme incubation.

  Example 4-Washed tobacco material is treated with two proteolytic enzymes with different optimal operating conditions

Treatment of tobacco material Whole leaf tobacco was treated according to Example 1 or 2, except that the pH was changed by about 1 pH unit every 10 minutes during the enzyme incubation. A 30% acetic acid solution was used to lower the pH. 1M sodium hydroxide solution was used to increase the pH.

  The proteolytic enzymes used were Savinase® (proteolytic enzyme derived from Bacillus species) and A. These were oryzae-derived proteolytic enzymes, both at a concentration of 1.0 g / L. The total enzyme incubation time was 1 hour, and the temperature during enzyme incubation was maintained at 35 ° C.

Analysis of protein removal The amount of nitrogen contained in the liquid component of the obtained sample was analyzed according to the method described in Example 1 except that the liquid component was not corrected for nitrogen by nicotine and enzyme protein.

  The analysis results are shown in FIG. 6 and FIG.

  FIGS. 6 and 7 show that as the total nitrogen content of the liquid component increases (which increases protein removal from the tobacco material), the enzyme incubation time increases and the pH changes. The nitrogen content of the liquid component increases more continuously when the pH is increased to 4-10 (shown in FIG. 7) than when the pH is reduced to 10-4 (shown in FIG. 6). Maximum nitrogen removal was seen after 40 minutes of enzyme incubation when the pH changed to pH 10-4, at which time the pH was between about 7-5.

  In order to address various issues and to advance the present technology, the entirety of the present disclosure shows various embodiments by way of example, in which the claimed invention is implemented and a superior tobacco treatment, tobacco material, And a product comprising tobacco material is provided. The advantages and features of the disclosure are merely representative of embodiments, and are not exhaustive and / or exclusive. These are merely presented to help understand and teach the claimed features. Of course, the advantages, embodiments, examples, functions, features, structures, and / or other aspects of the present disclosure are not intended to limit the present disclosure or its equivalents as defined in the claims, Other embodiments may be used or modified without departing from the scope and / or concept of the present disclosure. The various embodiments may suitably comprise, consist of, or consist essentially of various combinations of the disclosed elements, components, features, parts, steps, means, etc. . Further, the present disclosure includes other inventions that are not currently claimed but may be claimed in the future.

Claims (12)

A method of treating tobacco material, comprising combining a protease material having different optimal operating conditions with the tobacco material and simultaneously adding the operating conditions to the optimal temperature of one proteolytic enzyme and / or during enzyme incubation adjusting the pH to the optimum temperature and / or pH of another type of proteolytic enzyme.   2. The method of claim 1, wherein the protein content of the treated tobacco material is reduced compared to the untreated tobacco material.   The method according to claim 1 or 2, wherein the adjustment of the operating condition includes pH adjustment.   4. The method of claim 3, wherein the pH adjustment includes adding an acid as needed to lower the pH.   4. The method of claim 3, wherein the pH adjustment includes adding an alkali as necessary to increase the pH.   6. The method according to claim 1, wherein the adjustment of the operating condition includes a temperature adjustment.   The method of claim 6, wherein the temperature adjustment includes an adjustment to increase a low temperature.   The method of claim 6, wherein the temperature adjustment includes an adjustment to lower a high temperature.   The method according to any one of claims 1 to 8, wherein the combination of enzymes comprises enzymes having two different optimum operating conditions.   The method according to any one of claims 1 to 9, wherein the combination of enzymes comprises enzymes having three different optimum operating conditions.   Reducing the protein content of the treated tobacco material by at least 50%, at least 60%, at least 70%, at least 80%, and / or at least 90% as compared to untreated tobacco material. Item 11. The method according to any one of Items 1 to 10. Proteolytic enzymes with different optimal operating conditions are combined and added simultaneously to the tobacco material, and the operating conditions are changed from the optimal temperature and / or pH of one proteolytic enzyme during enzyme incubation to the optimal temperature and / or pH of another proteolytic enzyme. Or use of a combination of proteolytic enzymes with different optimal operating conditions to remove one or more proteins from tobacco material by adjusting to pH.

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