CN105764359A

CN105764359A - Methods for reducing matrix-bound nicotine-derived nitrosamine ketone in tobacco plant material

Info

Publication number: CN105764359A
Application number: CN201480064294.2A
Authority: CN
Inventors: G·朗; J-P·沙勒; A·维亚尔诺兹
Original assignee: Philip Morris Products SA
Current assignee: Philip Morris Products SA
Priority date: 2013-12-18
Filing date: 2014-12-18
Publication date: 2016-07-13
Anticipated expiration: 2034-12-18
Also published as: KR20160094941A; CA2931761A1; US20160295907A1; MX2016007987A; JP2017500048A; BR112016012407B1; KR102329410B1; RU2016124655A; AP2016009294A0; CN105764359B; EP3082478A1; EP3082478B1; US20230028025A1; JP6596000B2; RU2671616C1; BR112016012407A2; WO2015091880A1; PH12016500819A1

Abstract

A method of reducing the amount of matrix-bound NNK in cured tobacco plant material is provided comprising reducing the amount of lignin in the cured tobacco plant material. A further method of reducing the formation of matrix-bound NNK during the curing of tobacco plant material is described comprising reducing the amount of lignin therein prior to curing.

Description

For the method reducing the Nicotine derivative nitrosamine ketone of binding matrix in Tobacco plant material

Technical field

The present invention relates generally to for reducing the method for the amount of Nicotine derivative nitrosamine ketone or 4-(Methylnitrosamino)-1-(3-pyridine radicals)-1-butanone (NNK) in Tobacco plant material.

Background technology

In the manufacture and processing procedure of tobacco product, produce the side-product of such as tobacco stem and scattered leaf.Tobacco stem and Nicotiana tabacum L. particle from manufacture process are not suitable for being directly used in manufacture tobacco product.Owing to described stem and particle account for a large portion quantity that raw material puts into, these stems and particle are further converted into product (such as reclaimed tobacco material (such as reconstituted tobacco sheet)) by the method that has been developed for, and then it can mix use with relatively large amount with acceptable processed tobacco leaf.Reconstituted tobacco can be manufactured in slurry or cast sheet technique, wherein the slurry of the tobacco stem ground and the other parts of tobacco leaf are ground, and mix with the solution being likely to containing different additive.Then spray to form thin film to gained tobacco pulp, dry, rolling and cutting into inch strips, described bar is added in implant.

Nitrosamine is the organic compound found in many consumer goodss (such as Nicotiana tabacum L., foods and cosmetics).Nitrosamine has caused strong scientific interest, because some compound in this classification it is verified that be carcinogenic in laboratory animal.It is reported, some drying Nicotiana tabacum L.s contain in smokeless tobacco, the main flume of medicated cigarette and side-stream smoke it appeared that tobacco specific nitrosamines.In Nicotiana tabacum L., produce at least 4 kinds of nitrosamine of perceptible amount.These are Nicotine derivative nitrosamine ketone or 4-(Methylnitrosamino)-1-(3-pyridine radicals)-1-butanone (NNK), N-nitrosonornicotine (NNN), N-nitrosoanatabine (NAT) and N-nitroso-group anabasine (NAB).It is not considered as in tobacco plant that tobacco specific nitrosamines is present in growth with notable quantity or fresh cutting Nicotiana tabacum L. (green Nicotiana tabacum L.), but is formed in Nicotiana tabacum L. drying technics process.Except forming tobacco specific nitrosamines in the drying technics process of greenery, tobacco specific nitrosamines can also be used for preparing technique (such as the technique for preparing reconstituted tobacco) the middle formation of aqueous tobacco pulp.

In the trial reducing tobacco specific nitrosamines, it has been proposed that the various process of the Nicotiana tabacum L. of tobacco plant or results, including radiation treatment, chemical treatment and extraction.MacKown et al. (J.Agric.FoodChem.36,1031-1035,1988) has been proposed for other method for reducing tobacco specific nitrosamines.These methods are directed to use with the process of sterilizing, microbial inhibitor, alkali (being used for increasing pH) or ascorbic acid (for reducing the accumulation of tobacco specific nitrosamines in the production process of reconstituted tobacco sheet).WO2012160133 describes the method for the level of the tobacco specific nitrosamines reduced in Nicotiana tabacum L. homogenate by increasing its pH, particularly when setting up, by the nitrite level raised, the Volatile Nitrosamines Levels raised.

A problem relevant with the level of the tobacco specific nitrosamines managed in reduction Nicotiana tabacum L. is, some nitrosamine (including NNK) in the Nicotiana tabacum L. dried exist with the form combined or substrate combines, it may be difficult to remove or extraction.The NNK that substrate combines can use 0.1NKOH solution to extract from through the burley implant of water washing.This alkali processes the NNK level (Keene, C.K., 1992, TheEffectofBaseDigestiononTSNAinExtractables-DepletedFil lers.LegacyTobaccoDocuments) that also can reduce in flue gas.But, described process is likely to introduce in Nicotiana tabacum L. compound relevant in toxicology, and is likely to significantly decrease the quality of Nicotiana tabacum L., and it has height controversial for tobacco business.

WO2010/021809 describes a kind of method of nitrogen compound for reducing in Nicotiana tabacum L. and lignin.Nitrogen compound is removed by solvent extraction；Remove lignin in a separate step.

Needing the effective and cost-effective method being used for reducing the NNK of binding matrix, the NNK of described binding matrix is formed in the drying process of Nicotiana tabacum L..Especially, it is especially desirable to the cost-effective and simple method of the level of the NNK of a kind of binding matrix for reducing in drying Nicotiana tabacum L., described method does not introduce poisonous or is likely to poisonous compound and will not reduce the quality of tobacco product.

Summary of the invention

The present invention is based at least partially on the NNK of the binding matrix surprising discovery jointly positioned with the lignin in tobacco plant.Especially, inventor has observed that, in the drying part of tobacco plant (such as in stem and middle arteries and veins), the NNK of binding matrix and lignin (the such as especially lignified tissue in vascular bundle (such as xylem), and the surrounding tissue of not such as cortex) common location or mainly common location or specifically jointly position.Therefore, removal lignin (such as, by by lignified tissue and non-wood separate tissue) can reduce the NNK of the binding matrix in vegetable material and the amount of metabolite thereof.The NNK of binding matrix can covalently or noncovalently be bonded to lignin.The cigarette of expection vegetable material release the NNK level with minimizing and the sense quality possibly improved.Present disclosure can be applicable to the NNK of accumulation binding matrix or has those vegetable materials of probability of NNK of accumulation binding matrix.Especially, present disclosure can be applicable to the low price vegetable material of the NNK including binding matrix for some tobacco process.Method described herein can carry out when not using any additive and is not therefore incorporated in vegetable material by the compound that extra toxicology is relevant.In the drying process of Tobacco plant material or drying after can remove lignin.Can at removal lignin before drying to prevent, reduce or to suppress the NNK of the binding matrix together with lignin jointly to position.

One general purpose of present disclosure is the amount reducing the NNK for the binding matrix in smoking or the Nicotiana tabacum L. that consumes by other means and metabolite thereof.Another general purpose is to reduce the carcinogenic potential of tobacco product (including medicated cigarette, cigar, chewing tobacco, Folium Nicotianae preparatum and the chewing gum containing Nicotiana tabacum L. and lozenge).Another general purpose another is the amount of NNK and the metabolite thereof reducing or reducing the binding matrix in Tobacco plant material and in tobacco product.Another general purpose is the amount of NNK and the metabolite thereof reducing the binding matrix in drying (such as partially or completely drying) Tobacco plant material.Another general purpose is to reduce the amount of NNK and metabolite thereof in aerosol (including cigarette).Another purpose of present disclosure is to reduce the amount of NNK or its metabolite in people, described people aspirates, consumes or otherwise take in some form of Nicotiana tabacum L., the tobacco product being suitable for people's consumption is provided, described tobacco product contains NNK or its metabolite of minimizing amount, thus reduces the carcinogenic potential of this product.

In one aspect, the amount of the NNK of a kind of binding matrix reduced in drying tobacco plant or in drying Tobacco plant material is provided, described method includes the amount by lignified tissue and non-wood separate tissue reduce lignin therein, preferably, chemical mode and/or tool mode is wherein adopted to reduce the amount of lignin.

In one embodiment, tobacco plant or Tobacco plant material are processed so that non-wood plant tissue expands.By based on expanding and the different densities (such as, buoyant density) of unexpansive plant tissue and/or its varying strength and/or its different size and/or its Different Weight are isolated the amount reducing lignified tissue.The plant tissue of expansion can be collected to carry out further Nicotiana tabacum L. process.

It has been observed that substantially reducing of NNK and the level of TSNA that is associated thereof can be obtained by dividing lignified tissue.The independent chemistry of nitrogen compound and/or lignin be extracted as need not.

In one embodiment, by removing vascular bundle or xylem or lignifying sclerenchyma from plant or vegetable material or its two or more combination reduces the amount of lignin.Lignin can be located in vascular bundle.Lignin can be specifically located in vascular bundle.Lignin can be specifically located in vascular bundle and non-surrounding tissue.Lignin can be located in xylem.Lignin can be specifically located in xylem.Lignin can be specifically located in xylem and non-surrounding tissue.Lignin can be located in sclerenchyma.Lignin can be specifically located in sclerenchyma.Lignin can be specifically located in sclerenchyma and non-surrounding tissue.The usual not outer layer of arteries and veins in plant of lignified tissue.

In one embodiment, according to the plant of the process of present disclosure or vegetable material include arteries and veins in plant or axis or plant haulm or its two or more combination, by arteries and veins in plant or axis or plant haulm or its two or more form substantially by arteries and veins in plant or axis or plant haulm or its two or more form.

In one embodiment, by reducing the amount of lignin from plant or vegetable material harvesting cortex (such as exodermis).

In one embodiment, said method comprising the steps of: (a) provides drying tobacco plant or drying Tobacco plant material；B () reduces the amount of the lignin in drying tobacco plant or drying Tobacco plant material by dividing Tobacco plant material；And (c) is wherein compared the drying tobacco plant measuring minimizing of the amount minimizing of the middle drying tobacco plant provided of step (a) or drying Tobacco plant material lignin and the NNK of binding matrix or drying Tobacco plant material.

In one embodiment, another step of NNK or its combination measuring free NNK or binding matrix is there is after step (a), and optionally, after step (b), wherein there is another step of the amount of NNK or its combination measuring free NNK or binding matrix.

In one embodiment, another step (d) that the level of level and the NNK of the binding matrix measured after step (b) that described method includes the NNK by least binding matrix measured after step (a) compares, the described amount of the NNK of the binding matrix in the described tobacco-containing material wherein obtained in step (b) is compared the described amount reducing the NNK indicating the binding matrix in described tobacco-containing material of the described tobacco-containing material provided in step (a) and is reduced.

On the other hand, it is provided that a kind of reduce the method for the formation of the NNK of binding matrix in the drying process of tobacco plant or Tobacco plant material, described method includes the amount of minimizing lignin therein before drying.

In one embodiment, said method comprising the steps of: (a) provides not drying tobacco plant or not drying Tobacco plant material；B () reduces the amount of the lignin in not drying tobacco plant or not drying Tobacco plant material before drying；The tobacco plant provided in (c) drying step (b) or Tobacco plant material；And (d) is wherein compared the drying tobacco plant of the wherein NNK minimizing measuring unbated comparison binding matrix of lignin element or drying Tobacco plant material.

In one embodiment, another step of NNK or its combination measuring free NNK or binding matrix is there is after step (a), and preferably, after step (b), wherein there is another step of the amount of NNK or its combination measuring free NNK or binding matrix, and preferably, after step (c), wherein there is another step of NNK or its combination measuring free NNK or binding matrix.

In one embodiment, another step (d) that the level of level and the NNK of the binding matrix measured after step (b) that described method includes the NNK by least binding matrix measured after step (a) compares after step (c) or step (d), the described amount of the NNK of the binding matrix in the described tobacco-containing material wherein obtained in step (b) or step (c) is compared the described amount reducing the NNK indicating the binding matrix in described tobacco-containing material of the described tobacco-containing material provided in step (a) and is reduced.

In yet another aspect, it is provided that obtained by method described herein or available Tobacco plant material.

In yet another aspect, provide the use of tobacco plant or Tobacco plant material, wherein the comparison Tobacco plant material of the Nicotiana tabacum L. that the amount of lignin therein compares the level of the minimizing for manufacturing the NNK with binding matrix reduces, and wherein the level of the NNK of binding matrix compares described comparison minimizing.

In yet another aspect, it is provided that for the method producing reconstituted tobacco, said method comprising the steps of: (a) carries out method described herein；B the tobacco-containing material obtained in step (a) is manufactured reconstituted tobacco by ()；And reconstituted tobacco is optionally incorporated in tobacco product by (c).

In yet another aspect, it is provided that obtained by method described herein or available reconstituted tobacco.

In yet another aspect, it is provided that for the method preparing the Nicotiana tabacum L. cutting implant as Nicotiana tabacum L., said method comprising the steps of: (a) carries out method described herein；And (b) rolling and cutting tobacco-containing material cut implant for use as Nicotiana tabacum L..

On the other hand, it is provided that the drying Tobacco plant material of the lignin of the minimizing level of the comparison Tobacco plant material reduced containing the described amount comparing wherein lignin, and wherein the described amount of the NNK of binding matrix is preferably about 3500ng/g or less.

In one embodiment, mean diameter is more than about 0.5 millimeter.

In one embodiment, the amount of free NNK less than approximately 330ng/g, optionally wherein NNN content less than approximately 1700ng/g, and optionally wherein nicotine content less than approximately 2610 μ g/g.

In one embodiment, drying Tobacco plant material includes plant cortex (such as outside plant cortex), is made up of plant cortex (such as outside plant cortex), is substantially made up of plant cortex (such as outside plant cortex).

In one embodiment, vascular bundle or xylem or lignifying sclerenchyma or its two or more combination be not substantially in drying Tobacco plant material.

In one embodiment, drying Tobacco plant material includes plant cortex (such as outside plant cortex), form by plant cortex (such as outside plant cortex), be substantially made up of plant cortex (outside such as plant cortex), and vascular bundle or xylem or lignifying sclerenchyma or its two or more combination are substantially not wherein.

In one embodiment, drying Tobacco plant material arteries and veins or axis or plant haulm or its two or more combination in plant obtains maybe can obtaining.

In one embodiment, mean diameter is more than about 0.5 millimeter.

In one embodiment, the amount of free NNK is less than approximately 330ng/g.

In one embodiment, NNN content is less than approximately 1700ng/g.

In one embodiment, nicotine content is less than approximately 2610 μ g/g.

On the other hand, a kind of tobacco product or reconstituted tobacco product are provided, it includes vegetable material as herein described or drying vegetable material, is made up of vegetable material as herein described or drying vegetable material, or is substantially made up of vegetable material as herein described or drying vegetable material.

On the other hand, it is provided that a kind of aerocolloidal method of generation, wherein the amount of NNK compares comparison aerosol minimizing, said method comprising the steps of: (a) provides drying tobacco plant or drying Tobacco plant material；B () reduces the amount of the lignin in drying tobacco plant or drying Tobacco plant material；C () is wherein compared the drying tobacco plant of the amount minimizing of the amount minimizing of the middle drying tobacco plant provided of step (a) or drying Tobacco plant material lignin and the NNK of binding matrix or drying Tobacco plant material；And (d) heats the drying tobacco plant from step (c) or drying Tobacco plant material to produce aerosol.

On the other hand, it is provided that a kind of aerocolloidal method of generation, wherein the amount of NNK compares comparison aerosol minimizing, said method comprising the steps of: (a) provides not drying tobacco plant or not drying Tobacco plant material；B () reduces the amount of the lignin in not drying tobacco plant or not drying Tobacco plant material before drying；The tobacco plant provided in (c) drying step (b) or Tobacco plant material；And (d) is wherein compared the drying tobacco plant of the wherein NNK minimizing measuring unbated comparison binding matrix of lignin element or drying Tobacco plant material；(e) the drying tobacco plant from step (d) or drying Tobacco plant material are heated to produce aerosol.

On the other hand, one is provided to produce aerocolloidal method, wherein the amount of NNK compares comparison aerosol minimizing, said method comprising the steps of: (a) provides tobacco product or reconstituted tobacco product, it includes being obtained by method described herein or available Tobacco plant material or drying vegetable material, by being obtained by method described herein or available Tobacco plant material or drying vegetable material forms or substantially by being obtained by method described herein or available Tobacco plant material or drying vegetable material form；And (b) heats tobacco product or reconstituted tobacco product to produce aerosol.

In yet another aspect, it is provided that obtained by method described herein or available aerosol.

On the other hand, it is provided that the drying Tobacco plant material being substantially made up of tobacco plant cortex, and wherein the NNK of binding matrix amount reduce, as described herein.

On the other hand, thering is provided Nicotiana tabacum L. that a kind of blending is wherein blended with the dissimilar Nicotiana tabacum L. of at least two method to form tobacco composition, described method comprises the steps: that (a) provides the first drying Tobacco plant material and reduce the amount of wherein lignin；B () measures total NNK of the first drying Tobacco plant material and/or the NNK content of binding matrix and selects the NNK content of wherein total NNK and/or binding matrix to compare the drying Tobacco plant material that the first drying Tobacco plant material provided in step (a) reduces；C () provides the total NNK of the first drying Tobacco plant material obtained in relatively step (b) and/or the NNK of binding matrix the second drying Tobacco plant material of the NNK content with higher total NNK and/or binding matrix, and optionally measure the NNK content of the total NNK in the second drying Tobacco plant material and/or binding matrix；D first and second drying Tobacco plant material from step (b) He (c) is blended together by (), and optionally measure the NNK content of total NNK and/or the binding matrix blended in Tobacco plant material；And (e) obtains described total NNK of wherein said blending Tobacco plant material and/or the NNK content of the binding matrix blending Tobacco plant material lower than the second drying Tobacco plant material provided in step (c), optionally, wherein step (a) and (b) carry out after step (c).

In yet another aspect, it is provided that obtained by method described herein or available smoking mixture vegetable material.

Each in embodiment discussed above is open as the embodiment of each in the aspect of the present invention.Predict the combination of one or more embodiment.

Accompanying drawing explanation

Fig. 1 is the chart of the distribution that arteries and veins in free NNK, the NNK of binding matrix and drying white rib or the lignifying (L) in drying white rib stem stalk and the lignin in non-wood (NL) tissue are described.

Fig. 2 is the cross-sectional view of display lignifying (L) and the moisture drying white rib stem stalk of non-wood (NL) tissue.Use phloroglucinol that lignified tissue is dyed redness.

Fig. 3 is for illustrating the chart of the distribution of the lignin in the lignifying (L) of arteries and veins in using NNK free after sodium nitrite solution (reaching 4 hours by rocking at room temperature 1.5mL (in water 10mg/mL)) nitrosation, the NNK of binding matrix and green white rib and non-wood (NL) tissue.

Fig. 4 is the chart of the NNK of the free NNK in portion's screening part of the lyophilizing burley axis that explanation is milled and binding matrix.

Fig. 5 is the chart associated between NNK and the lignin of the binding matrix in the screening part of the lyophilizing burley axis that display is milled.

Fig. 6 is shown in the green of TN90 in arteries and veins nitrosation and the chart of the concentration (μ g/g) of the NNK of binding matrix in sclerenchyma (S) and in the outer layer (NS) of middle arteries and veins after washing.Also show false oxidation nicotine (PON) (μ g/g) and nicotine (μ g/g).Also show the NNK level (ng/g) of binding matrix in the lignifying (CS) of the drying white rib stem sample of business and non-wood (CNS) part.Also show NNN (ng/g) and the level of nicotine (μ g/g).

Definition

Technical term used herein and expression are typically assigned the implication being usually applied to them in plant and molecular biological association area.The definition of all following terms is applicable to the entire content of present disclosure.

Word " includes " being not excluded for other element or step, and indefinite article " (a) " or " one (an) " are not excluded for multiple/multiple.

Each this attribute maybe this value is also accurately limited especially about the term " substantially " of attribute or value, " about ", " about " etc..

Term " plant " refers to be in any plant in any stage of its life cycle or growth or its any part and offspring thereof.In one embodiment, plant is tobacco plant, its plant referring to belong to Nicotiana.Preferred tobacco plant kind is described herein as.

" plant cell " refers to structure and the physiological unit of plant.Plant cell can take the protoplast without cell wall, separated unicellular or cultivate the form of cell, or the part as more high fabric unit, for instance but be not limited to plant tissue, plant organ or whole plant.In one embodiment, plant cell is tobacco plant cell.

Term " vegetable material " refers to the mixture of any part of plant or the different piece of plant or the mixture of different plant, and includes but not limited to Tissues of Tobacco, scattered leaf, green scattered leaf, stem, the dirt produced in the plant course of processing and the nascent strip of foil of tobacco leaf and combination thereof.In certain embodiments, vegetable material will include the mixture (in such as plant arteries and veins or axis or plant haulm) of plant part or the plant part containing lignin or its two or more combination, by the mixture (in such as plant arteries and veins or axis or plant haulm) of plant part or the plant part containing lignin or its two or more form or be substantially made up of the mixture (in such as plant arteries and veins or axis or plant haulm) of plant part or the plant part that contains lignin or its two or more combination.Tobacco plant material can have the form of processed tobacco section or block, the mixture in substantially natural thin slice or the drying and aging Nicotiana tabacum L. of stem form, tobacco extract or aforementioned substances, such as, the mixture of the drying and aging natural baccy sheet combination of tobacco pulp and pelletize that will extract.Vegetable material can be solid form, in liquid form, in semi-solid form, in ground form, in crushing form, in sieving form or in particular form etc. or additionally treated to reduce particle diameter.Described vegetable material can be the form of the homogenate having be carried out homogenization (include but not limited to cutting and mill or its combination).Homogenate can from whole plant or from the mixture of plant component (such as contain the mixture of the plant part of lignin, for instance, have be carried out the middle arteries and veins of homogenization or stem or stem stalk or its two or more combination) prepare.Described vegetable material can be the form of slurry, the suspension in aqueous solution or solvent including vegetable material or plant homogenate.Described slurry can be the vegetable material of 5% (w/v), 10% (w/v), 15% (w/v), 20% (w/v) or 25% (w/v) or higher mixture in aqueous solution or solvent.In one embodiment, vegetable material is the vegetable material including lignin (such as lignified tissue), being made up of lignin (such as lignified tissue) or be substantially made up of lignin (such as lignified tissue).In one embodiment, vegetable material is the vegetable material including vascular bundle, being made up of vascular bundle or be substantially made up of vascular bundle.In one embodiment, vegetable material is the vegetable material including xylem, being made up of xylem or be substantially made up of xylem.In one embodiment, vegetable material is the vegetable material including sclerenchyma, being made up of sclerenchyma or be substantially made up of sclerenchyma.In one embodiment, vegetable material include arteries and veins in plant or axis or plant haulm or its two or more combination, by arteries and veins in plant or axis or plant haulm or its two or more form substantially by arteries and veins in plant or axis or plant haulm or its two or more form.In one embodiment, plant cell is Tobacco plant material.

Term " tobacco product " includes goods and the smokeless tobacco product that smoking product maybe can aspirate.

Term " free NNK " refers to the NNK concentration of the NNK cubage from the extract by room temperature using the aqueous buffer described vegetable material of extraction to prepare.Ultra Performance Liquid Chromatography tandem mass spectrum (UPLC-MS/MS) can be used to determine the free NNK content in this extract.

Term " total nitrosamine (NNK) " refers to extracting the nitrosamine concentration calculated after mixture carries out method described herein (such as, by heating to about 130 DEG C of maintenances about 4 hours) and the aliquot of filtrated extract.UPLC-MS/MS can be used to determine the total NNK content in this extract.

Difference between term as used herein " NNK of combination " or " NNK of binding matrix " expression " total NNK " and " free NNK " concentration.

The amount of the lignin that term " content of lignin of minimizing " or " content of lignin of reduction " or the variation of its " non-wood " grammer refer to when comparing with the amount of the lignin in comparable check plant in plant measure quantitative minimizing.Can known by described technical field and include carat pine (Klason) lignin test (MethodinEnzymol., 161:87-101 (1988)), acetyl bromide test (WoodSci.Technol., photometry (the J.Chem.Ecol. of derivatization 22:271-2801988)) or based on use TGA, 28,2483-2501 (2002)) test be readily determined the quantitative minimizing of lignin.In non-woodization tissue, the amount of lignin compares the amount that comparable check plant reduces and can hold or partly remove lignin fully, in fact.In non-woodization tissue, if when reduced with the amount of wherein lignin comparable compare and compare time exist lignin amount measure quantitative minimizing, then can there is the detectable amount of lignin.In non-woodization tissue, detect any amount of lignin perhaps impossible.Non-woodization tissue can contain the gross dry weight of the lignin in plant or plant part less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%, lignin is removed from plant or plant part.

" check plant " or " check plant cell " refers to have the plant of the content of lignin without operation or amendment and/or NNK content or plant cell (such as natural or spontaneous plant or plant cell).Check plant material includes deriving from, derived from or can derived from the vegetable material of check plant or check plant cell or its combination.Check plant or check plant cell can be same kind of plant or plant cell, for instance, with and the plant of its plant compared or plant cell same species or plant cell.Check plant or check plant cell may correspond to wild-type plant or wild type plant cell.

Term " minimizing " or " reduction " or the variation of its grammer refer to the minimizing from about 10% to about 99%, or quality, quantity or at least the 10% of activity, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or at least 100% or more reduce.

Term " suppression " or its grammer variation refer to total amount, quantity or activity from about 98% to about 100% reduce or at least 98%, at least 99% but especially 100% minimizing.

Term " increase " or the variation of its grammer refer to total amount, quantity or activity from the increase of about 5% to about 99%, or at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or at least 100% or more increase.

Refer in the 20% of specified value or scope about the term " about " of given digital value or scope, value in 10% or in 5% or scope.

Term " at least partially " or the variation of its grammer include total amount, quantity or activity at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98% or at least about 99%.

Detailed description of the invention

In general, present disclosure can be applicable to any type of Tobacco plant material, and wherein NNK or its metabolite or its combination can be formed or formed.Suitably, NNK's is at least some of in combining form.At least some of and the lignin (such as lignified tissue) of the form of binding matrix positions jointly.It is well-known in the art and described herein for measuring free nitrosamine and substrate in conjunction with the method for nitrosamine.In brief, the aliquot of tobacco sample can be extracted, and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) can be used to analyze content of nitrosamines therein.Generally, the standard substance that one or more are corresponding with wanting quantitative nitrosamine are mixed in the aliquot of tobacco sample.From the sample concentration of extract calculating corresponding to " free NNK " concentration sample.Process by method described herein after extracting mixture (such as, by heating to about 130 DEG C of maintenances about 4 hours), measure nitrosamine concentration again by UPLC-MS/MS." total NNK " concentration sample can be calculated from these values." NNK that substrate combines " concentration is the difference between " total NNK " and " free NNK " concentration.

Nicotiana tabacum L. is carried out much research, especially with regard to the specific nitrosamine of Nicotiana tabacum L..The Nicotiana tabacum L. of fresh harvest is referred to as " green Nicotiana tabacum L. ", and not thinks and contain nitrosamine, but green Nicotiana tabacum L. is not suitable for human consumption.The method of drying green Nicotiana tabacum L. depends on the type of the Nicotiana tabacum L. of results.Such as, cigarette roasting (light color (bright)) Nicotiana tabacum L. in Virginia is usually flue baking, and burley and some dark strain are often dried.Compared with one or two moon air-dry, the flue baking of Nicotiana tabacum L. generally carries out within the period of five to seven days.Many great chemistry and Biochemical changes start in drying technics process, and continue the commitment that perfoliate-leaf dries.Nicotiana tabacum L. typically results in the formation of nitrosamine (including NNK) and a large amount of accumulation and the content of microorganisms increased from its yellow to brown color transition.The precise mechanism forming the specific nitrosamine of Nicotiana tabacum L. (including NNK) is failed to understand, it is believed that can be strengthened by microbial activity, relates to microorganism nitrate reductase in drying technics process in the generation of nitrite.

As described above, it has been found that the lignin in the NNK of substrate combination and tobacco plant or in Tobacco plant material positions jointly.Owing to the oxide compound of 4-hydroxyl phenyl propyl couples, lignin is the generic term of large quantities of aromatic polymer.These polymer major sedimentary are in the wall of secondary thick cell (such as fiber and tracheary element) so that it is hard and can not permeate.The mechanical stiffness of lignin makes these tissues strengthen, so that tracheary element can bear the negative pressure that evaporation produces when tissue does not disintegrate.Except providing mechanical strength, lignin has protective function.For example, the physical toughness of lignin and chemical durability can prevent herbivores from taking food.Wooden turning to is infected or injured frequency response, and it can provide physical barrier to block the infiltration of pathogen.The basic building block of lignin is hydroxycinnamoyl alcohols (or single lignose) 4-hydroxy-3-methoxycinnamic alcohol and sinapinic alcohol, has usual small amount of tonquinol.Single lignose is synthesized from phenylalanine by general benzenpropanoic acid and single specific approach of lignose.Phenylalanine is derived from the shikimic acid biosynthesis pathway in chromatoplast.Some enzyme (i.e. cytochrome P 450 enzymes cinnamic acid 4-hydroxylase (C4H), coumaric acid 3-hydroxylase (C3H) and ferulate 5-hydroxylase (F5H)) of Lignin biosynthesis approach be considered at the cytosol side place of endoplasmic reticulum be activity memebrane protein.Although having shown metabolic pathway effect between PAL (PAL) and C4H, but whether other path enzyme is also maintained as the unknown for a part for the metabolite compounds at endoplasmic reticulum place.The unit caused by single lignose when being incorporated to lignin polymers is referred to as guaiacyl (G), Syringa oblata Lindl. base (S) and hydrogen-oxygen phenylacetic acid (H) unit.

Lignin is typically found in (such as) plant in arteries and veins, axis or plant haulm.Therefore, the material used in present disclosure can include arteries and veins in plant or axis or plant haulm or its mixture or its two or more combination, and can through removing.Lignin is arranged in (such as) and can be found in the vascular bundle of tobacco plant of plant arteries and veins, axis, plant haulm etc..Vascular bundle is made up of multiple relatively hard cellulose components, and described cellulose component connects tissue tight by vegetable fibers and is fixed together.Being cortex around described vascular bundle, described cortex is formed or coated with the major part of component stem and feature thereof and the characteristic part close to the blade of Nicotiana tabacum L. by relatively spongiform vegetable tissue.Lignin is usually located in vascular bundle.Vascular bundle is a part for the induction system in vascular plant.Conveying itself betides in vascular tissue, and it exists in two forms, i.e. xylem and phloem.These tissue both of which are present in vascular bundle, and it additionally will include supporting and protection tissue.In these vascular tissues, in xylem, only find lignin.

The amount of lignin can reduce substantially in arteries and veins, axis or plant haulm etc. or its two or more mixture or combination in plant.Lignin can be removed substantially in arteries and veins, axis or plant haulm etc. or its two or more mixture or combination in plant.Suitably, vascular bundle or xylem or its be combined in plant arteries and veins, axis or plant haulm etc. or its two or more mixture or combination reduce substantially.Suitably, vascular bundle or xylem or its be combined in plant arteries and veins, axis or plant haulm etc. or its two or more mixture or combination removed substantially.Suitably, lignifying sclerenchyma can be removed substantially in arteries and veins, axis or plant haulm etc. or its two or more mixture or combination in plant.

Owing to lignin can easily separate with the other parts of plant or other plant tissue, therefore the NNK of binding matrix and lignin jointly position and have superiority.In one embodiment, by lignified tissue and non-wood separate tissue being reduced the amount of lignin.For example, exodermis can easily separate with the vascular bundle containing lignin, thus obtains the vegetable material with the levels of lignin of minimizing.Lignin containing minimizing level or be substantially free of the vegetable material of lignin and can be used for manufacturing tobacco-containing material or there is the tobacco product of NNK of binding matrix of minimizing level, as described herein.Optionally, discardable or use otherwise containing with the independent vegetable material of the NNK of the binding matrix lignin jointly positioned.

In one aspect, it is provided that the method for the amount of the NNK of a kind of binding matrix reducing or reducing in drying tobacco plant or in drying Tobacco plant material, described method includes the amount that reduces wherein lignin.According to described method, it is provided that a kind of drying tobacco plant or drying Tobacco plant material.The amount of the lignin in drying tobacco plant or drying Tobacco plant material reduces.Lignin is completely removed or part is removed.Obtaining drying tobacco plant or drying Tobacco plant material subsequently, wherein the amount of lignin reduces and the amount of NNK of binding matrix is compared the drying tobacco plant or drying Tobacco plant material that are initially provided of or compares comparison and also reduce.

Treat that the lignin removed wholly or in part can be located in vascular bundle.Treat that the lignin removed wholly or in part can be specifically located in vascular bundle.Treat that the lignin removed wholly or in part can be specifically located in vascular bundle and not surrounding tissue.Therefore, by reducing the amount of the NNK of the binding matrix in the fascicular amount drying tobacco plant of minimizing in drying tobacco plant or in drying Tobacco plant material or in drying Tobacco plant material.

Treat that the lignin removed wholly or in part can be located in xylem.Treat that the lignin removed wholly or in part can be specifically located in xylem.Treat that the lignin removed wholly or in part can be specifically located in xylem and not surrounding tissue.Therefore, by reducing the amount measuring the NNK reducing the binding matrix in drying tobacco plant or in drying Tobacco plant material of the xylem in drying tobacco plant or in drying Tobacco plant material.

Treat that the lignin removed wholly or in part can be located in lignifying sclerenchyma.Treat that the lignin removed wholly or in part can be specifically located in lignifying sclerenchyma.Treat that the lignin removed wholly or in part can be specifically located in lignifying sclerenchyma and not surrounding tissue (in such as the outer layer of arteries and veins).Therefore, by reducing the amount of the NNK of the binding matrix in the lignifying sclerenchymatous amount drying tobacco plant of minimizing in drying tobacco plant or in drying Tobacco plant material or in drying Tobacco plant material.

On the other hand, it is provided that a kind of minimizing, reduce, prevent or suppress the method for the formation of the NNK of binding matrix in the drying process of tobacco plant or Tobacco plant material, described method includes the amount that reduces lignin therein before drying.At least initial, tobacco plant or Tobacco plant material can be not drying or substantially not drying.Described method can be used for reducing, reduce, prevent or suppress the NNK common location with lignin, and it is likely to otherwise occur in drying process subsequently.According to present aspect, it is provided that not drying tobacco plant or not drying Tobacco plant material or substantially not drying tobacco plant or substantially not drying Tobacco plant material, and before drying or in drying process, reduce the amount of lignin therein.Lignin is completely removed or part is removed.Lignin can be located in vascular bundle.Lignin can be specifically located in vascular bundle.Lignin can be specifically located in vascular bundle and non-surrounding tissue.Therefore, vascular bundle is completely removed or part removal.Lignin can be located in xylem.Lignin can be specifically located in xylem.Lignin can be specifically located in xylem and non-surrounding tissue.Therefore, xylem is completely removed or part removal.Lignin can be located in lignifying sclerenchyma.Lignin can be specifically located in lignifying sclerenchyma.Lignin can be specifically located in lignifying sclerenchyma and be not surrounding tissue (in such as the outer layer of arteries and veins).Therefore, lignifying sclerenchyma is completely removed or part removal.Lignin is completely removed or part is removed.After drying tobacco plant or Tobacco plant material, use the well-known method in described field, the wherein amount of the NNK of binding matrix and the amount of lignin can be obtained and compare parent material or compare drying tobacco plant or the drying Tobacco plant material that comparison reduces.

Use the well-known various methods in described field can reduce the amount of the lignin in tobacco plant or in Tobacco plant material.In a method, batch stem etc. can soak or soak in fluid (such as water), and its cortex softens and expands or protuberance, makes lignin be in unswollen state simultaneously.Cortex manually (such as, with hands) can be removed and keeps, and discardable containing the vascular bundle of lignin or use elsewhere.Therefore, cortex (such as, exodermis) can separate with lignified tissue and keep, and abandons unexpanded lignified tissue.May then use that cortex is to carry out further Nicotiana tabacum L. process.Also disclosing plant cortex (such as, plant exodermis) or the plant cortex (such as, the plant exodermis of expansion) expanded, wherein the amount of the NNK of binding matrix is lower than detectable level.Therefore, non-wood plant tissue is inflatable with by lignified tissue and non-wood separate tissue.Suitably, non-wood is organized in lignified tissue property ground selected over or preferably expands.

By the mode of another example, suitable skinning machine separating lignin can be used.Skinning machine is the machine for peeling off bark, timber and plant haulm etc..

In other method, mechanical separation can be used.For example, the immersion of batch stem etc. is carried out after freeze drying.In another example, lyophilizing, mill and sieve after carry out the immersion of batch stem etc..Suitably, use for milling, the equipment of grinding etc. and technology, Tobacco plant material is milled into powder type.Suitably, mill or in the process ground, Tobacco plant material is the form of relatively dry at the equipment of use (such as hammer mill, cutterhead, gas control grinder etc.).

Various methods known in the art can be used to reduce the size of vegetable material to form granule or particle matter.Apart granule or particle matter can be passed through to obtain the part of the NNK of the binding matrix of lignin and the minimizing level with minimizing level.In an appropriate method, process vegetable material by compression, such as by using the compression of one or more object harder than pending vegetable material.In one embodiment, the compression of metal (such as metal ball) is used.Various method (such as vibration) can be used to realize compressing.For example, by with 300rpm vibrate 15 minutes use steel ball (2 steel balls, diameter 2cm) compress vegetable material.Can use vibrating sieving machine that granule or particle matter are separated into the part with different-grain diameter by size.Suitably, average grain is more than about 0.5 millimeter, more than about 0.85 millimeter or more than about 1 millimeter.These grades can have the lignin of minimizing level and the NNK of the binding matrix of minimizing level.

When vegetable material water content less than approximately 15 weight % to less than approximately 5 weight % time, it is possible to milled or ground.Can pulverizing Tobacco plant material.Fine ground tobacco-containing material is generally of the particle diameter of about 30-600 micron.

In one embodiment, described method includes expanding, for instance by contacting with liquid (such as, pass through water soaking), then lyophilizing, it will cause that vegetable material expands, and described vegetable material does not contain lignin or contains only low-level lignin.Lignified plant material will keep higher density, higher building reason intensity and less particle diameter compared with turgid plant tissue, consequently allow for passing through apart.In another embodiment, described method includes expansion, lyophilizing subsequently, mills (such as subsequently, compression by as described above), the classification (such as, by size classes) of gained fragment subsequently and there is the selection of fragment of NNK of the lignin of minimizing level and the binding matrix of minimizing level.Screening can be used for this purpose.In another embodiment, described method includes expanding, then mill or crush or its combination (such as, by compressing, as described above), then by the selection of size classes gained granule (such as using screening or density based and/or mechanical strength) and the granule of the NNK with the lignin of minimizing level and the binding matrix of minimizing level.Various sizes of part is also different at its free NNK content or its NNN content or its nicotine content or its two or more combination.

After removing lignin wholly or in part, vegetable material is optionally after further treatment in tobacco product.By the mode of example, this material is formed as water slurry body.Gained slurry can contain the gluey cortical granule of the major part being dispersed therein.The grinder (such as ball mill or colloid mill) that can use suitable type completes the conversion to water slurry body of the Nicotiana tabacum L. cortex.Cortex and processing further of water slurry body are described in herein.

In certain embodiments, obtained by method described herein or available vegetable material includes the following, is made up of the following or is substantially made up of above those: the tissue around vascular bundle or the tissue around xylem or the tissue around lignifying sclerenchyma or there is its two or more its two or more the combination combined or be created substantially absent of vascular bundle or xylem or lignified thickness portion tissue.In certain embodiments, vegetable material includes the following, is made up of the following or is substantially made up of above those: vascular bundle or xylem or the tissue around lignifying sclerenchyma or its two or more combination and substantially without vascular bundle or without xylem or organize without lignified thickness portion or its two or more combination.In certain embodiments, vegetable material includes the following, is made up of the following or is substantially made up of above those: vascular bundle or xylem or the tissue around lignifying sclerenchyma or its two or more combination and without vascular bundle or without xylem or organize without lignified thickness portion or its two or more combination.In certain embodiments, vegetable material includes outside Nicotiana tabacum L. cortex, is made up of outside Nicotiana tabacum L. cortex or is substantially made up of outside Nicotiana tabacum L. cortex.In certain embodiments, vegetable material includes the outer layer of arteries and veins in plant, is made up of the outer layer of arteries and veins in plant or is substantially made up of the outer layer of arteries and veins in plant.

Method described herein can include the NNK of NNK or the binding matrix measured and optionally relatively dissociate or one or more other step of the level of its combination.Method for measuring the NNK of free NNK and binding matrix is described in herein.In one embodiment, the NNK of free NNK or binding matrix or the amount of its combination are determined in drying tobacco plant or in drying Tobacco plant material.After the amount of the lignin in reducing drying tobacco plant or drying Tobacco plant material, can again measure the NNK of free NNK or binding matrix or the amount of its combination.At least the level of the NNK of binding matrix can make comparisons the level that determines whether to reduce the NNK of binding matrix with initial parent material.In this step, the level of the level of the NNK of previously measured at least binding matrix with the NNK measured after reducing the amount of lignin can be made comparisons.The amount of the NNK reducing instruction binding matrix of the amount of the NNK of the binding matrix compared in the tobacco-containing material that obtains after the amount of lignin reduces with the tobacco-containing material being initially provided of reduces.

Method described herein can include the NNK of NNK or the binding matrix measured and optionally relatively dissociate or one or more other step of the level of its combination.In one embodiment, the NNK of free NNK or binding matrix or the amount of its combination are measured in not drying tobacco plant or in not drying Tobacco plant material.Optionally, after reducing the amount of lignin in not drying tobacco plant or not drying Tobacco plant material before drying, the NNK of free NNK or binding matrix or the amount of its combination can again be measured.Described method can include one or more comparison step.By the mode of example, described method can include another step level of NNK of the initial at least binding matrix measured as described above compared with the level of the NNK measured afterwards, and the amount of the NNK wherein comparing the binding matrix reduced in instruction tobacco-containing material of the amount of the NNK of the binding matrix in tobacco-containing material with the tobacco-containing material being initially provided of reduces.

Can when described method starts and/or when described method terminates and/or described procedure is measured the NNK of free NNK or binding matrix or its combination.Can discontinuously or compartment of terrain measure NNK or its combination of free NNK or binding matrix.Described interval can be fixed interval or random interval.NNK or its NNK or its combination combining the free NNK of verification or binding matrix that can measure free NNK or binding matrix when described method terminates are present in aequum, concentration or scope.

Lignin can covalently or noncovalently combine to lignin.Describe and include covalently or noncovalently combining the complex of the lignin to NNK.Also disclose and include the plant cell of complex, plant tissue or plant or vegetable material.The method being also described the amount of the NNK of a kind of binding matrix reduced in drying tobacco plant or in drying Tobacco plant material, described method includes the amount reducing complex therein.

On the other hand, it is provided that the drying plant tissue of the lignin of the minimizing level of the check plant material reduced containing the described amount comparing wherein lignin, and wherein the described amount of the NNK of binding matrix be about 3500ng/g or less.The amount of the NNK of binding matrix can be about 3000ng/g or less, about 2500ng/g or less, about 2000ng/g or less, about 1500ng/g or less, about 1000ng/g or less or about 500ng/g or less.Suitably, the mean diameter of this drying plant tissue can more than about 0.5 millimeter, more than about 0.85 millimeter or more than about 1 millimeter.Suitably, the amount of the free NNK in this drying plant tissue can be about 330ng/g or less, about 300ng/g or less, about 250ng/g or less, about 200ng/g or less, about 150ng/g or less, about 100ng/g or less, about 50ng/g or less.Suitably, the amount of the NNN in this drying plant tissue can be about 1700ng/g or less, about 1500ng/g or less, about 1300ng/g or less, about 1100ng/g or less, about 1000ng/g or less or about 500ng/g or less.Suitably, the amount of the nicotine in this drying plant tissue can be about 2600 μ g or less, about 2300 μ g or less or about 2100 μ g or less.Suitable, the amount of the lignin in this drying plant tissue can be about the 6% of gross dry weight content of about 6.5% or plant tissue less, drying of the gross dry weight content of drying plant tissue, about the 5% of gross dry weight content of drying plant tissue, the gross dry weight content of about 4% or drying plant tissue of the gross dry weight content of drying plant tissue about 3% or less.

In one embodiment, there is provided containing the drying plant tissue comparing the wherein lignin of the minimizing level measuring unbated check plant tissue of lignin, and the amount of the wherein NNK of binding matrix is about 3500ng/g or less, and mean diameter is about 0.5mm or bigger.Suitably, the amount of free NNK is about 300ng/g or less.Suitably, the amount of NNN is about 1700ng/g or less.Suitably, about the 6.4% or less of the gross dry weight content that amount is drying plant tissue of the lignin in this drying plant tissue.Suitably, the amount of nicotine is about 2600 μ g or less.

In another embodiment, there is provided containing the drying plant tissue comparing the wherein lignin of the minimizing level measuring unbated check plant tissue of lignin, and the amount of the wherein NNK of binding matrix is about 1900ng/g or less, and mean diameter is about 0.85mm and about between 1mm.Suitably, the amount of free NNK is about 250ng/g or less.Suitably, the amount of NNN is about 1270ng/g or less.Suitably, about the 4.4% or less of the gross dry weight content that amount is drying plant tissue of the lignin in this drying plant tissue.Suitably, the amount of nicotine is about 2300 μ g or less.

In another embodiment, it is provided that the drying plant tissue of the lignin containing the minimizing level measuring unbated check plant tissue comparing wherein lignin, and the amount of the wherein NNK of binding matrix is about 1600ng/g or less, and mean diameter is more than about 1mm.Suitably, the amount of free NNK is about 200ng/g or less.Suitably, the amount of NNN is about 1100ng/g or less.Suitably, about the 3% or less of the gross dry weight content that amount is drying plant tissue of the lignin in this drying plant tissue.Suitably, the amount of nicotine is about 2100 μ g or less.

The tobacco plant or the Tobacco plant material that start with in method described herein can include the following, be made up of the following or be substantially made up of the following: not drying tobacco plant or not drying Tobacco plant material or drying tobacco plant or drying Tobacco plant material.The method of drying Nicotiana tabacum L. (such as Nicotiana tabacum L., particularly green Nicotiana tabacum L.) is well known to the skilled person, and includes, but is not limited to air-dry, baking, flue baking and sun solarization.The method of drying Nicotiana tabacum L. depends on the type of the Nicotiana tabacum L. of results.Such as, cigarette roasting (light color) Nicotiana tabacum L. in Virginia is usually flue baking, and burley usually dries with some dark strain, and pipe tobacco, chewing tobacco and Folium Nicotianae preparatum are usually baking.Derive from any kind of tobacco plant or Tobacco plant material although can use, but certain form of Nicotiana tabacum L. is preferred.Particularly preferred tobacco-containing material selects the group of free the following composition: Nicotiana tabacum L., Turkish tobacco, burley, Virginia tobacco, Maryland Nicotiana tabacum L., spice Nicotiana tabacum L. or its two or more any combination that flue toasts.The shape of tobacco-containing material usually not limits.It can be the form of filamentary tobacco material.Nicotiana tabacum L. homogenate (the Nicotiana tabacum L. homogenate such as, but not limited to drying) can use multiple method known in the art to prepare from tobacco-containing material, for instance, Nicotiana tabacum L. can be tear up, grind, pelletize, fine particles or powder type.In certain embodiments, some mechanical separation method owing to can be used for separating lignin is likely to need to grind and/or screening step, it is therefore desirable to do not originate in the tobacco-containing material of grinding or powder type.

nullTobacco-containing material that is that use or that obtain can include additive，Described additive includes, but is not limited to one or many person in following components and combination thereof: spice、Organic filler and inorganic filler are (such as，Particulate、Process particulate、Loose particulate、Maltodextrin、Dextrose、Calcium carbonate、Calcium phosphate、Corn starch、Lactose、Mannitol、Xylitol、Sorbitol、Pulverizing cellulose etc.)、Binding agent is (such as，Polyvidone、The binding agent of carmethose and other modified cellulosic type、Sodium alginate、Xanthan gum、Binding agent based on starch、Radix Acaciae senegalis、Lecithin etc.)、Coloring agent is (such as，Dyestuff and pigment，Including burnt sugar coloring and titanium dioxide etc.)、Wetting agent is (such as，Glycerol、Propylene glycol etc.)、Oral care additives、Preservative is (such as，Potassium sorbate etc.)、Syrup is (such as，Mel、High-fructose corn syrup etc.，As spice) and disintegrate auxiliary agent is (such as，Microcrystalline Cellulose、Cross-linked carboxymethyl cellulose sodium、Crospovidone、Primojel、Pregelatinized com starch etc.).Examples of such additives is well known by persons skilled in the art, and can exist with amount known in the art and form.

Nicotiana tabacum L. is formed as reconstituted tobacco.Therefore, in one embodiment, approach described herein may be used for preparing reconstituted tobacco, such as reconstituted tobacco (leaf) sheet.These sheets are the paper-like material can made with the Nicotiana tabacum L. particle of recirculation, tobacco stem and " grade Nicotiana tabacum L. ", and the tobacco particle being typically smaller than 30 order sizes that described " grade Nicotiana tabacum L. " was collected by any stage processed Nicotiana tabacum L. forms.Extract the soluble chemical material in tobacco by-products, the remaining baccy fiber deriving from extraction is processed into paper, then the material of the extraction in conc forms is applied again on paper to prepare reconstituted tobacco.Generally can form reconstituted tobacco in many ways.Such as, in one embodiment, it is possible to use band casting forms reconstituted tobacco.Generally adopt the slurry of pulverizing tobacco section and binding agent with casting, described slurry is coated on steel band, then dries.After dry, sheet blended with natural tobacco swath or tears up and be used in various tobacco product, including as cigarette filler.Some examples for producing the method for reconstituted tobacco describe in US3,353,541, US3,420,241, US3,386,449, US3,760,815 and US4,674,519.Reconstituted tobacco can also be formed by papermaking process.Describe at US3,428,053, US3,415 for some examples forming the method for reconstituted tobacco according to the method, 253, US3,561,451, US3,467,109, US3,483,874, US3,860,012, US3,847,164, US4,182,349, US5,715,844, US5,724,998 and US5,765,570 in.Such as, use the formation of reconstituted tobacco of paper technology to comprise the steps of mix with water by Nicotiana tabacum L., extract the composition of solubility, the composition of concentration solubility, refining Nicotiana tabacum L. from it, form net, the composition of the solubility that again applies to concentrate, dry and beat.Multiple components (such as taste or color treatments agent) can be put on net.

That obtained by method described herein or available Nicotiana tabacum L. can be formed tobacco sheet, such as reconstituted tobacco sheet.According to described embodiment, described method may comprise steps of: (a) obtains tobacco-containing material according to method described herein, such as Nicotiana tabacum L. homogenate；B () prepares the slurry of Nicotiana tabacum L. homogenate；The slurry of (c) casting Nicotiana tabacum L. homogenate；(d) slurry of dry Nicotiana tabacum L. homogenate is to form reconstituted tobacco sheet.According to another embodiment, described method may comprise steps of: (a) obtains tobacco-containing material (such as Nicotiana tabacum L. homogenate) according to method described herein and prepare tobacco pulp；The slurry of (b) casting Nicotiana tabacum L. homogenate；(c) slurry of dry Nicotiana tabacum L. homogenate is to form tobacco sheet.

Use any casting known in the art or papermaking process, it is possible to carry out the step of the slurry of casting Nicotiana tabacum L. homogenate.As an example, casting method describes at US5, and 724,998 and in US5,584,306；Papermaking process describes in US4,341,228, US5,584,306 and US6,216,706.Casting method generally include slurry is cast on continuous stainless steel band, the slurry of dry casting to be to form reconstituted tobacco sheet and to take off described.Papermaking process generally includes and is cast to mesh screen from head cartridge to be used for forming desired by aqueous slurry.Aqueous slurry can be separated into soluble part and fiber part.Water is discharged from fiber part, and with post processing and the dry sheet being thusly-formed.

Tobacco pulp may further include one or more binding agents, such as natural gum and pectin.As mentioned above, tobacco pulp for preparing reconstituted tobacco sheet may further include common additive, and described additive includes, but is not limited to one or more and these the combination in following components: wood cellulose fibers, aerosol former, sugar and spice and binding agent.The additive of above-mentioned list is well known by persons skilled in the art, and can be present in these aqueouss slurry with amount known in the art and form.

After preparation, it is possible to with intact leaves tobacco like mode cut reconstituted tobacco sheet described herein, to produce to be suitable for the tobacco filler of medicated cigarette and other tobacco product.Reconstituted tobacco sheet mechanical finger described herein can be torn (trash) further or peel off into a certain size the block being similar to natural baccy strip of foil, or cut at the diamond block that side is about 50-100mm.Reconstituted tobacco sheet block described herein can blend further with other Nicotiana tabacum L. (such as the Nicotiana tabacum L. etc. of the Nicotiana tabacum L. of flue baking, burley, Maryland Nicotiana tabacum L., Oriental tobacco, rare Nicotiana tabacum L., special Nicotiana tabacum L., expansion).Precise volume for manufacturing the every class Nicotiana tabacum L. in the tobacco composition of particular cigarette brand is different with brand.Referring to, such as, TobaccoEncyclopaedia, Voges 44-45 page (1984), Browne, TheDesignofCigarettes, 3rd edition, 43rd page (1990) and TobaccoProduction, ChemistryandTechnology, Davis et al. (volume) the 346th page (1999).Then whole mixture can be shredded into cutting implant and mix in tobacco product.

According on the other hand, it is provided that a kind of for blending the method wherein blending at least two distinct types of Nicotiana tabacum L. to form the Nicotiana tabacum L. of tobacco composition.Various tobacco compositions have the distinct methods for blending different tobacco type.By the mode of example, tobacco type can be burley, flue-cured tobacco, spice Nicotiana tabacum L., dull finish tobacco and reconstituted tobacco.Burley, flue-cured tobacco and spice Nicotiana tabacum L. are certain types of Nicotiana tabacum L., and dull finish tobacco is flue-cured tobacco and the pre-blended of spice Nicotiana tabacum L..According to the present invention, it is provided that first (planting type) drying Tobacco plant material, and reduce the amount of lignin therein.Any method as herein described can be used to reduce the amount of lignin.The NNK content that can measure total NNK of the first drying Tobacco plant material and/or the NNK content of binding matrix and optional wherein said total NNK and/or binding matrix compares the drying Tobacco plant material of the be initially provided of first drying Tobacco plant material minimizing with further use.Second drying Tobacco plant material of the NNK content compared with the NNK of total NNK of the first drying Tobacco plant material and/or binding matrix with higher total and/or binding matrix is provided subsequently.In some embodiments, it may be possible to the NNK content of the total and/or binding matrix of this material known, therefore the measurement of these values will do not needed.In other embodiments, total and/or binding matrix NNK content of this material is possibly unknown, is therefore measured by needs.Therefore, in the process, total and/or binding matrix the NNK content measured in the second drying Tobacco plant material is optional step.Well-known method in described field can be used to be blended together by first and second the drying Tobacco plant material obtained from these steps.Optionally, the NNK content of total and/or binding matrix in the Tobacco plant material of final blending can be measured.According to described method, the NNK content blending Tobacco plant material lower than the second drying Tobacco plant material of the wherein total and/or binding matrix of the final Tobacco plant material blended can be obtained.Advantageously, described method can be used for providing the mixture of tobacco-containing material, and the overall NNK content of wherein said mixture reduces.Substantially, the tobacco-containing material that wherein amount of lignin is reduced is for diluting or reducing the overall NNK content blended in tobacco-containing material.

That obtain according to disclosure or available tobacco-containing material may be also used in Nicotiana tabacum L. cutting implant neutralization and is used in the smoking product that the tobacco rod from cutting implant is formed.Routinely, the cutting implant tobacco product for smoking product is mainly formed from the sheet segment of tobacco leaf, and described sheet segment stem portion with leaf in the process of beating separates.After thin slice has removed and separated, remaining most of stem portion do not use.In order to increase the amount of commercial operable tobacco-containing material, it is possible to some tobacco stems are added back to together with thin slice in cutting implant.In order to improve taste and the combustion characteristic of the tobacco stem being used in cutting implant, first stem often carrying out one or more and processes operation, described operation can include operation described herein.Rolling step can be carried out on the tobacco stem of method being already subjected to present disclosure.Can by stem rolling to desired thickness, the average thickness of all 0.6mm to 0.8mm according to appointment.In processing subsequently and storage steps, it is possible to make stem be expanded to the final thickness of about 0.8mm to about 1.0mm.After rolling, by dry for stem and be transferred to tobacco leaf production factory, herein, they cut and add in Nicotiana tabacum L. cutting implant.In some cases, rolling step can be included alternatively as a part for the online production method of cutting implant.Generally, before rolling, the water content of tobacco stem is about 28% to about 34% Oven Volatiles, in order to prevent caulomic damage.If it is necessary to tobacco stem can be regulated before rolling, in order to by water content increase so far level.The known method of adjustment tobacco stem comprises makes described stem contact with the mixture of water, steam or water and steam.Including rolling step wherein online with in the method using dry stem, it is more of a specified duration that regulating step generally carries out, thereby increases and it is possible to needs soaking step, is soaked a few hours by stem in soaking step before rolling in water.A step rolling method rolling tobacco stem can be used, so that the thickness of stem is decreased to desired average thickness.After rolling, it is possible to stem is cut into the cutting width of 0.1mm to 0.2mm.Then optionally use known stem expansion technique to make the stem of cleaved rolling expand, then dry.When by pre-for stem rolling and dry, it is often necessary to regulate stem before being cut, in order to make the water content of tobacco stem increase back by 28% to 34% Oven Volatiles.This can increase the flexibility of tobacco stem, in order to the damage being limited in cutting process stem or fracture.Finally, the stem of cleaved rolling is combined with Nicotiana tabacum L. cutting thin slice and any other tobacco-containing material, in order to form the cutting implant of the tobacco stem of the cleaved rolling with at least 5 weight %.

Thus, in yet another aspect, it is provided that for the method preparing the Nicotiana tabacum L. cutting implant as Nicotiana tabacum L., described method comprises the steps: that (a) carries out method as described herein；(b) rolling and cutting tobacco-containing material cut implant for use as Nicotiana tabacum L..Also describing the method processing the tobacco-containing material (such as tobacco stem) being used in Nicotiana tabacum L. cutting implant, described method comprises the steps: that (a) carries out method as described herein；(b) rolling tobacco-containing material；The tobacco-containing material of (c) cutting pass；(d) the rolling stem of optionally dry cutting.Tobacco stem through rolling can be combined with reconstituted tobacoo so that described step carries out on the tobacco stem and thin slice of combination.Described cutting step can include the stem through rolling is cut the cutting width into about 0.3mm to 1.3mm.Stem is removed from tobacco leaf；Described stem is cut the average length into about 15mm to 80mm；With the thickness that described stem is rolled into 0.1mm to 0.5mm.Additionally providing the cutting producing to include rolling tobacco stem and fill raw method, institute's art method includes: use method described herein to process tobacco stem；Blend to produce to cut implant with by least one class in treated stem and reconstituted tobacoo, the Nicotiana tabacum L. of expansion or reconstituted tobacco.

Obtained by the method or available Nicotiana tabacum L. cutting implant can comprise the reconstituted tobacoo of at least 60 weight % and preferably at least 80 weight %, described reconstituted tobacoo has the average cutting width of 0.8mm to 1.1mm (suitably, about 0.9mm) and the average thickness of about 0.2mm.Nicotiana tabacum L. cutting implant can contain up to the reconstituted tobacoo of 95 weight %, and described reconstituted tobacoo has the average cutting width of about 0.8mm to 1.1mm, more suitably about 0.9mm and the average thickness of about 0.2mm.Therefore, the granule of reconstituted tobacoo in cutting implant have with the particulate species of tobacco stem like size.So, tobacco stem visually will be same as reconstituted tobacoo, even at de-luxe compartment containing under ratio.Furthermore it is possible to advantageously transport and the mixture of processing cigarette grass blade and thin slice effectively, without the notable sedimentation of stem.Suitably, the average cutting width of the tobacco stem of cleaved rolling is in the about 0.1mm of the average thickness of the reconstituted tobacoo in cutting implant, more suitably in about 0.05mm.Cutting implant can be mixed in multiple smoking product.For example, it is possible to cutting implant is used in the tobacco rod of combustible smoking product (such as cigarette with filter tip, cigarlet or cigar).It is alternatively possible to use cutting implant to provide tobacco gas colloidal sol to generate substrate in based on the smoking product distilled or electrically heated cigarette smoking system.Alternatively, it is possible to cutting implant is used as from volume (roll-your-own) product, or pine soot product, for instance, it is used in tobacco pipe.

Tobacco-containing material can mix in multiple consumable product (such as tobacco product).Also include the method for preparing such tobacco product.Tobacco product includes, but is not limited to goods and the smokeless tobacco product that smoking product maybe can aspirate, including on-fuel product, the product of heating with produce aerocolloidal product.The non-limitative example of the goods that smoking product maybe can aspirate includes medicated cigarette, cigarlet, cigar and pipe tobacco.The non-limitative example of smokeless tobacco product includes chewing tobacco, Folium Nicotianae preparatum and is used in the substrate produced in aerocolloidal product.Smokeless tobacco product can include any form of Nicotiana tabacum L., including as dry granule, fragment, microgranule, powder or slurry, it is deposited on other composition, is blended in other composition, surrounded by other composition or otherwise combine with other composition, other composition described is arbitrary form, such as thin slice, film, tab, foam or pearl.The liquid contents of smokeless tobacco product can be comprised in a device or be encapsulated in the forms such as such as pearl, to hinder the interaction with water soluble package paper.Described wrapping paper can be shaped as bag partially or even wholly to encapsulate the compositions comprising Nicotiana tabacum L. or to work to keep together multiple tabs of Nicotiana tabacum L., pearl or thin slice as binding agent.The exemplary material for constructing wrapping paper includes film composition, and it includes HPMC, CMC, pectin, alginate, pulullan and film-forming polymer that other commercially available arrives, edible.Other packaging material can include the preformed capsule from gelatin, HPMC, starch/carrageenin or other manufacture of materials being obtained commercially.Such packaging material can include Nicotiana tabacum L. as composition.The wrapping paper of per os disintegrate can not can be made up of woven or non-woven fabric, be made up of coated or not coated paper, or by that bore a hole or otherwise there is the plastic foil of porous form.Wrapping paper can comprise correctives or coloring agent.Utilize known to the skilled any method in commercial packing field, the method such as including such as blister package (wherein parcel can use Vertical form/filling/sealing and packing machine to be formed), it is possible to will be assembled together without smoke product and wrapping paper.

When compared with the consumable product being derived from comparison Tobacco plant material, these can smoking article and the NNK without the binding matrix in smoke product and aerosol thereof measure can be low by least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% and 100%, for instance low by about 200% or 300%.The amount of free NNK is likely to substantially not change.

These smoking article, amount without the NNK of the binding matrix in the gentle colloidal sol of smoke product etc. can be about 3500ng/g or less or about 3000ng/g or less or about 2500ng/g or less or about 2000ng/g or less or about 1500ng/g or less or about 1000ng/g or less or about 500ng/g or less.

These can smoking article and the amount without the free NNK in smoke product etc. can be about 330ng/g or less, about 300ng/g or less, about 250ng/g or less, about 200ng/g or less, about 150ng/g or less, about 100ng/g or less or about 50ng/g or less.

These can smoking article, amount without the NNN in the gentle colloidal sol of smoke product etc. can be about 1700ng/g or less, about 1500ng/g or less, about 1300ng/g or less, about 1100ng/g or less, about 1000ng/g or less or about 500ng/g or less.

These can smoking article and the amount without the nicotine in smoke product etc. can be about 2600ng/g or less, about 2300ng/g or less or about 2100ng/g or less or about 2000ng/g or less or about 1900ng/g or less, or about 1800ng/g or less.

These can smoking article and the amount without the lignin in smoke product etc. can be about the 6% of gross dry weight of about 6.5% or plant tissue less, drying of gross dry weight of drying plant tissue, about the 5% of gross dry weight of drying plant tissue, the gross dry weight of about 4% or drying plant tissue of the gross dry weight of drying plant tissue about 3% or less.

In one embodiment, these can smoking article and the amount without the NNK of the binding matrix in smoke product etc. be about 3500ng/g or less, the amount of free NNK is about 300ng/g or less, the amount of NNN is about 1700ng/g or less, about the 6.4% or less of the gross dry weight content that the amount of lignin is drying plant tissue, and the amount of nicotine be about 2600 μ g or less.

In one embodiment, can smoking article or include about 3500ng/g or the NNK of less binding matrix without smoke product etc..Suitably, the amount of free NNK is about 300ng/g or less.Suitably, the amount of NNN is about 1700ng/g or less.Suitably, about the 6.4% or less of the gross dry weight content that amount is drying plant tissue of lignin.Suitably, the amount of nicotine is about 2600 μ g or less.Suitably, mean diameter is about 0.5mm or bigger.

In another embodiment, can smoking article or include about 1900ng/g or the NNK of less binding matrix without smoke product etc..Suitably, the amount of free NNK is about 250ng/g or less.Suitably, the amount of NNN is about 1270ng/g or less.Suitably, about the 4.4% or less of the gross dry weight content that amount is drying plant tissue of the lignin in this drying plant tissue.Suitably, the amount of nicotine is about 2300 μ g or less.Suitably, mean diameter is at about 0.85mm and about between 1mm.

In another embodiment, can smoking article or include about 1600ng/g or the NNK of less binding matrix without smoke product etc..Suitably, the amount of free NNK is about 200ng/g or less.Suitably, the amount of NNN is about 1100ng/g or less.Suitably, about the 3% or less of the gross dry weight content that amount is drying plant tissue of the lignin in this drying plant tissue.Suitably, the amount of nicotine is about 2100 μ g or less.Suitably, average grain is more than about 1mm.

Tobacco-containing material can be derived from tobacco plant, and described tobacco plant includes the plant of Nicotiana, Nicotiana not of the same race, including Folium Nicotianae rusticae (N.rustica) and Nicotiana tabacum (N.tabacum).Tobacco-containing material can be derived from the kind of Nicotiana kind, is commonly referred to as flue kind or light color (bright) kind, burley kind, dark kind and spice Nicotiana tabacum L./Turkish tobacco kind.In certain embodiments, described tobacco-containing material is derived from burley, Virginia tobacco, the Nicotiana tabacum L. of flue baking, the Nicotiana tabacum L. dried, the Nicotiana tabacum L. of baking, spice Nicotiana tabacum L. or dark tobacco plant.nullIn certain embodiments，Described tobacco-containing material is derived from one or more in such as following kind: Nicotiana tabacum AA37-1、Nicotiana tabacum B13P、Nicotiana tabacum Xanthi (Mitchell-Mor)、Nicotiana tabacum KTD#3 hybrid 107、Nicotiana tabacum Bel-W3、Nicotiana tabacum 79-615、Nicotiana tabacum SamsunHolmesNN、Derive from the F4 of hybridization Nicotiana tabacum BU21x Nicotiana tabacum HojaParado、97th is、Nicotiana tabacum KTRDC#2 hybrid 49、Nicotiana tabacum KTRDC#4 hybrid 110、Nicotiana tabacum burley 21、Nicotiana tabacum PM016、Nicotiana tabacum KTRDC#5KY160SI、Nicotiana tabacum KTRDC#7FCA、Nicotiana tabacum KTRDC#6TN86SI、Nicotiana tabacum PM021、Nicotiana tabacum K149、Nicotiana tabacum K326、Nicotiana tabacum K346、Nicotiana tabacum K358、Nicotiana tabacum K394、Nicotiana tabacum K399、Nicotiana tabacum K730、Nicotiana tabacum KY10、Nicotiana tabacum KY14、Nicotiana tabacum KY160、Nicotiana tabacum KY17、Nicotiana tabacum KY8959、Nicotiana tabacum KY9、Nicotiana tabacum KY907、Nicotiana tabacum MD609、Nicotiana tabacum McNair373、Nicotiana tabacum NC2000、Nicotiana tabacum PG01、Nicotiana tabacum PG04、Nicotiana tabacum P01、Nicotiana tabacum P02、Nicotiana tabacum P03、Nicotiana tabacum RG11、Nicotiana tabacum RG17、Nicotiana tabacum RG8、Nicotiana tabacum SpeightG-28、Nicotiana tabacum TN86、Nicotiana tabacum TN90、Nicotiana tabacum VA509、Nicotiana tabacum AS44、Nicotiana tabacum BanketA1、Nicotiana tabacum BasmaDramaB84/31、Nicotiana tabacum BasmaIZichnaZP4/B、Nicotiana tabacum BasmaXanthiBX2A、Nicotiana tabacum Batek、Nicotiana tabacum BesukiJember、Nicotiana tabacum C104、Nicotiana tabacum Coker319、Nicotiana tabacum Coker347、Nicotiana tabacum CriolloMisionero、Nicotiana tabacum PM092、Nicotiana tabacum Delcrest、Nicotiana tabacum Djebel81、Nicotiana tabacum DVH405、Nicotiana tabacum GalpaoComum、Nicotiana tabacum HB04P、Nicotiana tabacum HicksBroadleaf、Nicotiana tabacum KabakulakElassona、Nicotiana tabacum PM102、Nicotiana tabacum KutsageE1、Nicotiana tabacum KY14xL8、Nicotiana tabacum KY171、Nicotiana tabacum LABU21、Nicotiana tabacum McNair944、Nicotiana tabacum NC2326、Nicotiana tabacum NC71、Nicotiana tabacum NC297、Nicotiana tabacum NC3、Nicotiana tabacum PVH03、Nicotiana tabacum PVH09、Nicotiana tabacum PVH19、Nicotiana tabacum PVH2110、Nicotiana tabacum RedRussian、Nicotiana tabacum Samsun、Nicotiana tabacum Saplak、Nicotiana tabacum immaba、Nicotiana tabacum Talgar28、Nicotiana tabacum PM132、Nicotiana tabacum Wislica、Nicotiana tabacum Yayaldag、Nicotiana tabacum NC4、Nicotiana tabacum TRMadole、Nicotiana tabacum PrilepHC-72、Nicotiana tabacum PrilepP23、Nicotiana tabacum PrilepPB156/1、Nicotiana tabacum PrilepP12-2/1、Nicotiana tabacum YakaJK-48、Nicotiana tabacum YakaJB125/3、Nicotiana tabacum TI-1068、Nicotiana tabacum KDH-960、Nicotiana tabacum TI-1070、Nicotiana tabacum TW136、Nicotiana tabacum PM204、Nicotiana tabacum PM205、Nicotiana tabacum Basma、Nicotiana tabacum TKF4028、Nicotiana tabacum L8、Nicotiana tabacum TKF2002、Nicotiana tabacum TN90、Nicotiana tabacum GR141、Nicotiana tabacum Basmaxanthi、Nicotiana tabacum GR149、Nicotiana tabacum GR153 and Nicotiana tabacum PetitHavana.Disclosed compositions and method can be applicable to any species of Nicotiana, including Folium Nicotianae rusticae and Nicotiana tabacum (such as LAB21, LNKY171, TI1406, Bath agate, Galpao, Perique, Beinhart1000-1 and Petico).nullOther species include acaulescence Nicotiana tabacum L. (N.acaulis)、Point leaf Nicotiana tabacum L. (N.acuminata)、The point many Henbanes of leaf (N.acuminatavar.multiflora)、Africa Nicotiana tabacum L. (N.africana)、Floral leaf Nicotiana tabacum L. (N.alata)、Embrace stem Nicotiana tabacum L. (N.amplexicaulis)、Allan Zi Shi Nicotiana tabacum L. (N.arentsii)、Gradually narrow leaf Nicotiana tabacum L. (N.attenuata)、Bei Namoteshi Nicotiana tabacum L. (N.benavidesii)、Ben Saimushi Nicotiana tabacum L. (N.benthamiana)、Nicotiana bigelovi (N.bigelovii)、Rich Nicotiana tabacum L. (N.bonariensis) inside、Hole uncured tobacco (N.cavicola)、Ke Lifulanshi Nicotiana tabacum L. (N.clevelandii)、Lobus cardiacus Nicotiana tabacum L. (N.cordifolia)、Umbrella bed Nicotiana tabacum L. (N.corymbosa)、Di Bainashi Nicotiana tabacum L. (N.debneyi)、Wood wool Nicotiana tabacum L. (N.excelsior)、Fu Erjiteshi Nicotiana tabacum L. (N.forgetiana)、Medicated cigarette grass (N.fragrans)、Powder blue smoke grass (N.glauca)、Viscous Nicotiana tabacum L. (N.glutinosa)、Gu Tesi Bi Shi Nicotiana tabacum L. (N.goodspeedii)、Brother Xi Shi Nicotiana tabacum L. (N.gossei)、Hybridization Nicotiana tabacum L. (N.hybrid)、Because of ancient youngster bar Nicotiana tabacum L. (N.ingulba)、Slips card Michaelis Nicotiana tabacum L. (N.kawakamii)、Nai Teshi Nicotiana tabacum L. (N.knightiana)、Lang Shi Nicotiana tabacum L. (N.langsdorffii)、Acuminate Nicotiana tabacum L. (N.linearis)、Long Henbane (N.longiflora)、Beach Nicotiana tabacum L. (N.maritima)、Especially big pipe Nicotiana tabacum L. (N.megalosiphon)、Mo Xishi Nicotiana tabacum L. (N.miersii)、Nightjasmine Nicotiana tabacum L. (N.noctiflora)、Naked stem Nicotiana tabacum L. (N.nudicaulis)、Ou Busite Nicotiana tabacum L. (N.obtusifolia)、West Nicotiana tabacum L. (N.occidentalis)、West subspecies perfume (or spice) mustard Nicotiana tabacum L. (N.occidentalissubsp.hesperis)、Ear Nicotiana tabacum L. (N.otophora)、Circular cone Nicotiana tabacum L. (N.paniculata)、Few Henbane (N.pauciflora)、Petunia shape Nicotiana tabacum L. (N.petunioides)、Blue Folium Jasmini sambac Nicotiana tabacum L. (N.plumbaginifolia)、Kua Deruiwushi Nicotiana tabacum L. (N.quadrivalvis)、Lei Mengdeshi Nicotiana tabacum L. (N.raimondii)、Ripple edge Nicotiana tabacum L. (N.repanda)、Lotus throne Nicotiana tabacum L. (N.rosulata)、Lotus throne subspecies are because of ancient youngster bar Nicotiana tabacum L. (N.rosulatasubsp.ingulba)、Roundleaf Nicotiana tabacum L. (N.rotundifolia)、Sai Teshi Nicotiana tabacum L. (N.setchellii)、Intend like Nicotiana tabacum L. (N.simulans)、Folium Solani Melongenae Nicotiana tabacum L. (N.solanifolia)、Si Peigecishi Nicotiana tabacum L. (N.spegazzinii)、Si Tuoketongshi Nicotiana tabacum L. (N.stocktonii)、Fragrant and sweet Nicotiana tabacum L. (N.suaveolens)、U.S. Henbane (N.sylvestris)、Intend spike Nicotiana tabacum L. (N.thyrsiflora)、Velvet tobacco (N.tomentosa)、Fine hair shape Nicotiana tabacum L. (N.tomentosiformis)、Triangle leaf Nicotiana tabacum L. (N.trigonophylla)、Bright (N.umbratica)、Ripple leaf Nicotiana tabacum L. (N.undulata)、Quiver a mao Nicotiana tabacum L. (N.velutina)、Sequence leaf Nicotiana tabacum L. (N.wigandioides) and Henbane (N.xsanderae).

It is also contemplated herein use tobacco cultivation mutation and excellent tobacco cultivation mutation.Useful especially Nicotiana tabacum kind includes burley tobaccos type, deep type, fire-cured tobacco type and Oriental type tobacco.nullThe non-limitative example of kind or variety is: the non-limitative example of kind or variety is BD64、CC101、CC200、CC27、CC301、CC400、CC500、CC600、CC700、CC800、CC900、Coker176、Coker319、Coker371Gold、Coker48、CD263、DF911、DT538LCGalpao Nicotiana tabacum L.、GL26H、GL350、GL600、GL737、GL939、GL973、HB04P、HB04PLC、HB3307PLC、Hybridization 403LC、Hybridization 404LC、Hybridization 501LC、K149、K326、K346、K358、K394、K399、K730、KDH959、KT200、KT204LC、KY10、KY14、KY160、KY17、KY171、KY907、KY907LC、KTY14xL8LC、LittleCrittenden、McNair373、McNair944、msKY14xL8、Narrow leaf Madole、Narrow leaf MadoleLC、NBH98、N-126、N-777LC、N-7371LC、NC100、NC102、NC2000、NC291、NC297、NC299、NC3、NC4、NC5、NC6、NC7、NC606、NC71、NC72、NC810、NCBH129、NC2002、NealSmithMadole、OXFORD207、PD7302LC、PD7309LC、PD7312LC ' ' Periq ' e' Nicotiana tabacum L.、PVH03、PVH09、PVH19、PVH50、PVH51、R610、R630、R7-11、R7-12、RG17、RG81、RGH51、RGH4、RGH51、RS1410、Speight168、Speight172、Speight179、Speight210、Speight220、Speight225、Speight227、Speight234、SpeightG-28、SpeightG-70、SpeightH-6、SpeightH20、SpeightNF3、TI1406、TI1269、TN86、TN86LC、TN90、TN97、TN97LC、TND94、TND950、TR(TomRosson)Madole、VA309、VA359、AA37-1、B13P、Xanthi(Mitchell-Mor)、Bel-W3、79-615、SamsunHolmesNN、No. KTRDC2 hybridization 49、White rib 21、KY8959、KY9、MD609、PG01、PG04、PO1、PO2、PO3、RG11、RG8、VA509、AS44、BanketA1、Bath agate DramaB84/31、Bath agate IZichnaZP4/B、Bath agate XanthiBX2A、Batek、BesukiJember、C104、Coker347、CriolloMisionero、Delcrest、Djebel81、DVH405、nullComum、HB04P、Hicks broad-leaved、KabakulakElassona、KutsageE1、LABU21、NC2326、NC297、PVH2110、Red Russia、Samsun、Saplak、Simmaba、Talgar28、Wislica、Yayaldag、PrilepHC-72、PrilepP23、PrilepPB156/1、PrilepP12-2/1、YakaJK-48、YakaJB125/3、TI-1068、KDH-960、TI-1070、TW136、Bath agate、TKF4028、L8、TKF2002、GR141、Bath agate xanthi、GR149、GR153、PetitHavana.Even if not specializing herein, it is also considered that above-mentioned low conversion subvariety.

There is provided following example illustratively, not as restriction.Except as otherwise noted, the present invention adopts routine techniques and the method for molecular biology and Plant Physiology.

Embodiment

Embodiment 1

For the method analyzing free and that substrate the combines NNK in Nicotiana tabacum L.

By shaking at around room temperature about 1 hour, with about 30mLTris-HCl buffer (50mM；PH7.4) aliquot (such as, about 750mg) of tobacco sample is extracted.Add internal controls (100ng/mLNNK-d4).Use the sample (0.4mL) of 0.2 μM of filter filtrated extract, and use ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to analyze NNK content.The sample concentration calculated from these extract concentrations is corresponding to " free NNK " concentration sample.Process after extracting mixture (such as, by heating to about 130 DEG C of maintenances about 4 hours) and the aliquot of filtrated extract, measure NNK concentration again by UPLC-MS/MS." NNK that substrate combines " concentration is the difference between " total NNK " and " free NNK " concentration.

A kind of alternative method that " total NNK " extracts include with dense HCl acidifying extract mixture (such as, 3mL37%HCl add 30mL in) and at 80 DEG C incubation 48 hours.By by NaOH solution (6N, 40 μ L) and magnesium magma (10% before filtration and UPLC are analyzed；40 μ L) it is added in the extract of 320 μ L and acidic extraction thing.

Embodiment 2

UPLC analyzes

The post used is WatersAcquityBEHC18,1.7 μm, 2.1 × 50mm.The eluent used is: (A) ammonium hydrogen carbonate (10mM；It is adjusted to pH9.8 with ammonia)+2% (v/v) acetonitrile；(B) acetonitrile.The gradient used is 0min-5%B；0.5min-5%B；3.3min-18.3%B.The flow used is 0.5mL/min.The column temperature used is 50 DEG C.

Embodiment 3

MS/MS methodology

Use following MRM to change, WatersTQ spectrometer carries out this and analyzes: NNK:208.2 → 122.2；Time of staying 100ms；NNK-d4:212.2 → 126.2；Time of staying 100ms；Capillary voltage: 0.6kV；Cone voltage: 25V；Impact energy: 11eV；Source temperature: 120 DEG C；Desolvation temperature: 400 DEG C；Desolvation air-flow: 800L/h.

Embodiment 4

The distribution of the NNK of the binding matrix in the lignifying of white rib stem and non-woodization tissue

The middle beniol of about 2 grams of drying burleys is received and distributed and is separated into internal lignified tissue (the 36% of gross dry weight) and outside non-woodization tissue (the 64% of gross dry weight).In each in these samples, analyzed the concentration of free NNK and total NNK by UPLC-MS, as described above.The NNK of binding matrix is calculated as the difference between free NNK and total NNK concentration.Photometry is used to quantify content of lignin ((2002) J.Chem.Ecol., the 28,2483-2501 see Brinkmann et al.) based on the derivatization using TGA.

The result of Fig. 1 shows the distribution of the lignin in free NNK, the NNK of binding matrix and the lignifying (L) of drying white rib stem and non-wood (NL) tissue.Fig. 2 is the cross-sectional view of display lignifying (L) and the moisture drying white rib stem stalk of non-wood (NL) tissue.Use phloroglucinol that lignified tissue is dyed redness.Result in Fig. 3 is shown in and uses sodium nitrite solution to carry out the lignifying of arteries and veins in green after nitrosylation and the NNK content of the binding matrix of non-woodization tissue.

The NNK of these results display binding matrix is distributed mainly in the lignified tissue of burley tobaccos grass blade and middle arteries and veins.

Embodiment 5

Enrichment by the part of the NNK content with low combination of the confession rib stem of lyophilizing, grinding and apart

The sample of white rib stem (52g) uses water (350mL) humidification and lyophilizing.Resulting materials (12g) in part through use steel ball (2 balls, diameter is 2cm；300rpm；Vibration 15min) is ground, and uses vibrating sieving machine to be separated into the fragment having more than 1mm to the different-grain diameter less than 0.25mm.

Free and the NNK of binding matrix, NNN, lignin and nicotine content in the screening part of the white rib stem of lyophilizing are ground in Fig. 4 and Biao 1 display.The analysis instruction content of lignin (the % dry weight as every part) of the NNK dissociated with binding matrix in Fig. 5 and Biao 1 and the NNK content of binding matrix are having minimizing in more than the part of the particle diameter of 0.5, including having 0.5mm to 0.85mm, 0.85mm to 1mm and the part of the particle diameter more than 1mm.Fig. 5 shows that content of lignin associates very well with the NNK of binding matrix, it is thus determined that the common location of the NNK of lignin and binding matrix.

Embodiment 6

The location of the NNK precursor of the binding matrix in the white rib stem of normal pulse in green TN90

TN90 measures in the non-sclerenchyma of the heavy wall pipe of arteries and veins the Relative distribution of the NNK of binding matrix.The NNK of prediction binding matrix combines the higher concentration of this precursor to lignin prediction lignifying sclerenchyma.In testing second, research free and binding matrix NNK Relative distribution in the non-sclerenchyma of heavy wall pipe of (drying) white rib stem.

Materials and methods

Arteries and veins in green

The middle arteries and veins of 15 ripe TN90 leaves (only major part) is separation in sclerenchyma (S) (middle arteries and veins " " center ") and non-sclerenchyma (NS) manually.Both of which lyophilizing and grinding well.Reach three times (room temperature 1 hour) by using 40mL methanol/water (1:3) to extract 1g and insoluble material (being appointed as SW and NSW) weighed determine the water-fast part of bi-material after freeze drying.Following methods is used to measure vacation oxidation nicotine (PON) and the nicotine analysis of S and NS (n=5).By at room temperature using containing the PON-methyl-d as internal standard (200ng/mL)₃Methanol/water (4:1) vibrate 45 minutes and extract fine powdered vegetable material (～20mg).After filtration (0.2 μm) sample stands LC-MS analysis, use following condition: chromatographic column Ultra Performance Liquid Chromatography instrument BEHC18 chromatographic column (1.7 μm, 50 × 2.1mm；Water)；Chromatogram column temperature: 50 DEG C；Eluant uses the NH with acetonitrile₃(98:2, v/v；Eluant A) aqueous carbonic acid ammonia ammonium is adjusted to pH9.8；Acetonitrile (eluant B)；Gradient: 0min 0%B, 0.5mL/min；0.5min 0%B, 0.5mL/min；6min 30%B, 0.5mL/min；MS detects: PONm/z179.2 à m/z106.1；PON-methyl-d₃m/z182.2àm/z106.1；UV detects: 260nm.Eluting PON and nicotine after 2.6 minutes and 4.1 minutes respectively.For the quantization of nicotine, use peak value and the external calibration at 260nm place.In order to estimate the content of the NNK precursor of the binding matrix in S, NS, SW and NSW, by using the vibration of 10mL water, being centrifuged and wash/be centrifuged the aliquot (～20mg at room temperature cultivating 4 hours these materials of nitrosation for four times in NaNO2 solution (1.5mL (in water 10mg/mL))；N=5).Then, using method described herein, the centrifugal deposition thing of each nitrosation sample is at 4mLTris-HCl buffer (50mMpH7.5；Under 100ng/mL, there is NNK-d4 and NNN-d4) in occupy, through hot-pressing processing (carrying out at 130 DEG C 4 hours) and analyze NNK content.

Drying white rib stem

White rib stem sample (2g=is about 5cm tetra-) is manually separated into outside (non-wood) tissue (CNS) and internal (lignifying) partly (CS).Two samples in mixer mill fine gtinding (Lay speed (Retsch) " Tissuelyzer " reach 2.5 minutes, 50s-1).This measure causes CNS and CS powder respectively 1183mg and 651mg.1.5mL TRIS buffer (+IS) is at room temperature used to extract the free NNK determining in two samples after about 50mg aliquot (n=5) reaches 1 hour.The autoclave of about 50mg aliquot (n=5) in 5mL TRIS buffer (+IS) determines total NNK after extracting (carrying out at 130 DEG C 4 hours).

Result

The result of this experiment is shown in table 2 and Fig. 6.After nitrosation and washing, comparing the outer layer (NS) of middle arteries and veins, the concentration of the NNK of the binding matrix of high 7 times is found in sclerenchyma (S).PON and nicotine relatively high twice in S in NS.The result of these artificial nitrosation experiment is by the NNK level confirmation of the combination in the lignifying (CS) of commercial drying white rib stem sample and non-wood (CNS) part.Due to free NNK high twice in CS, the NNK of binding matrix is high 7 times in CS.Relatively CNS is high and less than NNK in CS for nicotine and NNN level.

Conclusion

There is the NNK precursor of high concentration binding matrix in green in the lignifying sclerenchyma of arteries and veins, this shows that the NNK of binding matrix covalently or noncovalently links to lignin.

Any publication that is that quote in this article or that describe provides disclosed in before the submission date of the application for information about.Statement herein should not be construed as to be admitted: inventor loses prior to such disclosed qualification.The all publications mentioned in superincumbent description are incorporated herein by.Without departing from the scope and spirit of the present invention, the various modifications and variations of the present invention will be readily apparent to one having ordinary skill.Although the invention has been described in connection with specific preferred embodiments, it is to be understood that, it is desirable to the invention of protection should not be unduly limited to such being embodied as.It practice, cytobiology, molecular biology and Plant Physiology or different improvement the about the apparent described pattern for realizing the present invention of technical staff in field are intended in the scope of following claims.

Table 1

Table 2

Claims

1. a method for the amount of the NNK of the binding matrix in the drying Tobacco plant material of minimizing, described method includes lignified tissue and non-wood separate tissue, it is preferred that wherein adopt chemical mode and/or mechanical system to reduce the amount of lignin.

2. method according to claim 1, described method comprises the steps:

A () provides drying Tobacco plant material；

B () is by the lignified tissue in described drying Tobacco plant material and non-wood separate tissue；And

C () obtains the drying Tobacco plant material that the amount of the amount minimizing of wherein described lignin compared with the described drying Tobacco plant material provided in step (a) and the NNK of described binding matrix reduces.

3. method according to claim 2, after step (a), wherein there is another step of the amount measuring at least NNK of binding matrix, and optionally, after step (b), wherein there is another step of the described amount of the NNK measuring at least binding matrix.

4. method according to claim 3, another step (d) that the level of level and the NNK of the binding matrix measured after step (b) that wherein said method includes the NNK by least binding matrix measured after step (a) compares, the described amount of the NNK of the binding matrix in the described tobacco-containing material wherein obtained in step (b) is compared the described amount reducing the NNK indicating the binding matrix in described tobacco-containing material of the described tobacco-containing material provided in step (a) and is reduced.

5. reducing a method for the formation of the NNK of binding matrix in the drying process of Tobacco plant material, described method includes the amount of minimizing lignin therein before drying, it is preferable that described method comprises the steps:

A () provides not drying Tobacco plant material；

(b) before drying by the lignified tissue in described not drying Tobacco plant material and non-wood separate tissue；

The described Tobacco plant material provided in (c) drying step (b)；And

D () obtains the drying Tobacco plant material that the described amount of the wherein NNK of binding matrix compared with the comparison that the amount of wherein lignin has reduced reduces.

6. method according to claim 5, after step (a), wherein there is another step of the described amount of the NNK measuring at least binding matrix, and optionally, after step (b), wherein there is another step of the described amount of the NNK measuring at least binding matrix, and optionally, after step (c), wherein preferably there is another step of the described amount of the NNK measuring at least binding matrix

Another step that wherein the described level of described level and the NNK of at least binding matrix measured after step (b) and/or step (c) that described method includes the NNK by least binding matrix measured after step (a) compares after step (c) or step (d), the described amount of the NNK of the binding matrix in the described tobacco-containing material wherein obtained in step (b) or step (c) is compared the described amount reducing the NNK indicating the binding matrix in described tobacco-containing material of the described tobacco-containing material provided in step (a) and is reduced.

7. the method according to any one in aforementioned claim, wherein preferably described Tobacco plant material is treated to expand non-wood plant tissue, wherein by by expansion and the plant tissue of on-expansible separate based on its different densities and/or its varying strength and/or its different-grain diameter reduce lignin amount and/or

Wherein by removing at least vascular bundle or xylem or lignified sclerenchyma from described vegetable material or its two or more combination reduces the described amount of lignin；And/or

The described vegetable material wherein provided in step (a) includes plant master pulse or axis or plant haulm or its two or more combination or is made up of plant master pulse or axis or plant haulm or its two or more combination or is substantially made up of plant master pulse or axis or plant haulm or its two or more combination.

8. that obtained by method according to any one of claim 1 to 7 or available Tobacco plant material.

9. the use of Tobacco plant material, wherein the comparison Tobacco plant material of the Nicotiana tabacum L. that the described amount of lignin therein compares the level of the minimizing for manufacturing the NNK with binding matrix reduces, and wherein the described level of the NNK of binding matrix compares described comparison minimizing.

10. the method for producing reconstituted tobacco, described method comprises the steps:

A () carries out the method according to any one in claim 1 to 7；

B the described tobacco-containing material deriving from step (a) is manufactured reconstituted tobacco by ()；With

C described reconstituted tobacco is optionally mixed in tobacco product by ().

11. that obtained by method according to claim 10 or available reconstituted tobacco.

12. for the method preparing the Nicotiana tabacum L. cutting implant as Nicotiana tabacum L., described method comprises the steps:

A () carries out the method according to any one in claim 1 to 8；With

B () rolling and cutting are used as the described tobacco-containing material of Nicotiana tabacum L. cutting implant.

13. the drying Tobacco plant material of the lignin of the minimizing level of the comparison Tobacco plant material reduced containing the described amount comparing wherein lignin, and the described amount of the wherein NNK of binding matrix is preferably about 3500ng/g or less.

The amount of wherein free NNK is less than approximately 330ng/g, and optionally, wherein NNN content is less than approximately 1700ng/g, and optionally, wherein nicotine content is less than approximately 2610 μ g/g；And/or

Wherein said drying Tobacco plant material includes plant cortex (such as outside plant cortex), is made up of plant cortex (such as outside plant cortex), is substantially made up of plant cortex (such as outside plant cortex)；And/or

Wherein vascular bundle or xylem or lignified sclerenchyma or its combination are created substantially absent in described drying plant tissue.

14. tobacco product or a reconstituted tobacco product, form including the drying vegetable material described in Tobacco plant material according to claim 8 or claim 13, drying vegetable material described in Tobacco plant material according to claim 8 or claim 13 or substantially drying vegetable material described in Tobacco plant material according to claim 8 or claim 13 forms.

15. blend the Nicotiana tabacum L. being wherein blended with the dissimilar Nicotiana tabacum L. of an at least two method to form tobacco composition, described method comprises the steps:

A () provides the first drying Tobacco plant material and reduces the amount of wherein lignin；

B () measures described described total NNK of the first drying Tobacco plant material and/or the NNK content of binding matrix and selects the NNK content of wherein said total NNK and/or binding matrix to compare the drying Tobacco plant material that the described first drying Tobacco plant material provided in step (a) reduces；

C () provides the described described total NNK of the first drying Tobacco plant material obtained in relatively step (b) and/or the NNK of binding matrix the second drying Tobacco plant material of the NNK content with higher total NNK and/or binding matrix, and optionally measure the NNK content of the total NNK in described second drying Tobacco plant material and/or binding matrix；

D first and second drying Tobacco plant material described from step (b) He (c) is blended together by (), and optionally measure the NNK content of the described total NNK in described blending Tobacco plant material and/or binding matrix；And

E () obtains described total NNK of wherein said blending Tobacco plant material and/or the NNK content of the binding matrix blending Tobacco plant material lower than the described second drying Tobacco plant material provided in step (c), optionally, wherein step (a) and (b) carry out after step (c).