Classifications

• • A—HUMAN NECESSITIES
  • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
  • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
  • A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
  • A24B15/18—Treatment of tobacco products or tobacco substitutes
  • A24B15/20—Biochemical treatment
• • A—HUMAN NECESSITIES
  • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
  • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
  • A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
  • A24B15/18—Treatment of tobacco products or tobacco substitutes
  • A24B15/24—Treatment of tobacco products or tobacco substitutes by extraction; Tobacco extracts

Definitions

• This invention relates to the preparation and treatment of tobacco. More specifically, the invention relates to such processes in which a tobacco material is treated with a phenol oxidising enzyme to provide an improved tobacco material.
• a process for removing phenolic compounds from tobacco which makes use of solid adsorbents such as alumina is disclosed in US 3,561,451.
• US 5,601,097 discloses the use of another insoluble adsorbent, viz. polyvinylpolypyrrolidone (PVPP) , in such process.
• PVPP polyvinylpolypyrrolidone
• these methods are disadvantageous in being relatively non-selective.
• GB 2069814 relates to a method of changing the structure of tobacco, changing its chemical composition and improving its sensorial feature by submitting tobacco to the action of enzymes selected from oxidoreductases (e.g. monophenol monooxygenase, EC 1.14.18.1), lyases, hydrolases and microorganisms constituting a source of such enzymes.
• oxidoreductases e.g. monophenol monooxygenase, EC 1.14.18.1
• lyases e.g. monophenol monooxygenase, EC 1.14.18.1
• hydrolases e.g. hydrolases and microorganisms constituting a source of such enzymes.
• the present invention provides a process for preparing a tobacco product, which process comprises treating a tobacco material with a phenol modifying enzyme, preferably a phenol oxidizing enzyme, and most preferably a polyphenol oxidizing enzyme.
• the invention provides a method for reducing the amount of phenolic compounds in a tobacco material, in which method the tobacco material is treated with a phenol oxidising enzyme .
• an improved tobacco material having a reduced content of phenolic compounds is achieved by treating a tobacco extract with a phenol oxidising enzyme.
• the invention relates to a process for preparing a tobacco product, which comprises the steps of extracting a tobacco material with a solvent to provide an extract and a tobacco residue; and treating the extract with a phenol oxidising enzyme.
• the invention relates to a method for improving the customer compliance, such as, e.g. improving the smoking pleasure of the consumer, e.g. by modifying the chemical composition, flavour, aroma, taste and/or colour thereby increasing the versatility of the tobacco products on the market.
• the invention relates to the use of a phenol oxidising enzyme in the preparation or treatment of tobacco. In one embodiment, the invention relates to the use of a laccase in the treatment of tobacco.
• the invention relates to tobacco materials obtainable, in particular obtained, by any of the processes described herein.
• the invention encompasses the final, ready- for-use tobacco products, as well as any extracts of a tobacco material having been treated by any of the herein claimed processes. These tobacco materials have a reduced amount of phenolic compounds.
• Figure 1 Binary gradient profile for HPLC analysis of phenols, see Methods and procedures in Examples.
• Figure 2 Spectra of nicotine before and after treatment with laccase, see Example 1.
• Figure 4 Spectra of rutin before and after treatment with laccase, see Example 1.
• Figure 5 Spectra of scopoletin before and after treatment with laccase, see Example 1.
• Figure 6 HPLC chromatogram of mixture of phenol standards: ru- tin, scopoletin, and chlorogenic acid. The two chromatograms are identical except for the scale, see Example 2.
• FIG. 7 HPLC chromatogram of tobacco extract. The two chromatograms are identical except for the scale, see Example 2.
• Figure 8 HPLC chromatogram of tobacco extract spiked with rutin, scopoletin, and chlorogenic acid. The two chromatograms are identical except for the scale, see Example 2.
• Figure 9 Series of HPLC chromatograms of samples of tobacco extract as a f nction of time when treated with 1.6 ⁇ g/ml TvL at pH 5.5 and 55°C. The two sets of chromatograms are identical except for the scale, see Example 2.
• Figure 10 Flow chart showing process-flow and indicating which samples have been prepared, see Example 8.
• Figure 12 Chemical structure of Scopoletin.
• FIG. 13 Chemical structure of Chlorogenic acid.
• the present invention provides a process for preparing a tobacco product, which process comprises the step of (i) treating a tobacco material with a phenol oxidising enzyme.
• tobacco material denotes the tobacco starting material for the various treatments in the present invention, whatever type, source or origin and whatever other kinds of treatments prior to the treatment of the present invention it has been subjected to.
• tobacco material includes, without limitation, tobacco solids and any solid form of tobacco, such as, e.g., cured tobacco (such as flue-cured tobacco) ; uncured tobacco (so-called green tobacco) ; dried, aged, cut, ground, stripped or shredded tobacco; tobacco scrap; expanded tobacco, fermented tobacco; reconstituted tobacco; whatever the source and whatever the grade, as well as any combination of these tobacco materials. Also tobacco blends are included.
• the tobacco material may be from any parts of the tobacco plant, such as stem, veins, scrap and waste tobacco, cuttings and the like as well as whole leaf and part thereof.
• the tobacco material used as a starting material for present invention is the lamina portion of the tobacco leaf.
• the tobacco material is in part or totally composed of cured tobacco.
• this expression covers the tobacco raw material entering a tobacco preparation or treatment process.
• the term tobacco material includes a tobacco extract or a tobacco extraction mixture of any solid form of tobacco; preferably, an extract from cured tobacco is used.
• the tobacco material used in the process of the invention may be from any tobacco species from which it is desired to make a tobacco product. Of particular interest is tobacco from the subgenus Nicotiana tabacum .
• tobacco product denotes the product resulting from any of the processes of the invention. Included are, without limitation, the final, ready-for-use tobacco products, as well as any extracts of a tobacco material where the extracts have been treated by any of the herein claimed processes.
• tobacco product includes the final products wherein the process according to the invention has been used, in particular the term includes tobacco articles for smoking such as, e.g., cigarettes, cigars, pipe tobacco, but also other kinds of tobacco product such as a tobacco extract and a tobacco for chewing, such as, e.g., chewing tobacco and tobacco chewing gum.
• the invention encompasses methods for preparing tobacco products, comprising the step of (i) treating, i.e. contacting, a tobacco material with a phenol modifying enzyme, preferably a phenol oxidizing enzyme, and most preferably a polyphenol oxidizing enzyme.
• a phenol modifying enzyme preferably a phenol oxidizing enzyme, and most preferably a polyphenol oxidizing enzyme.
• said enzyme is not monophenol monooxygenase (EC 1.14.18.1).
• the method of the invention comprises the steps of (ii) extracting a tobacco material with a solvent to provide an extraction mixture; (iii) separating the extraction mixture into a tobacco extract and a tobacco residue; where step (i) , i.e. treatment with the phenol oxidising enzyme, is performed during or after step (ii) and before step (iii), or step (i) is performed on the extract after step (iii) .
• the invention provides a process for preparing a tobacco product comprising the steps of (ii) extracting a tobacco material with a solvent to provide an extraction mixture; (iii) separating the extraction mixture into a tobacco extract and a tobacco residue; (i) treating the tobacco extract with a phenol oxidising enzyme to produce one or more oxidised phenolic compounds.
• the present invention provides a method for reducing the amount of phenolic compounds in tobacco, whereby the soluble phenolic compounds of the tobacco material are extracted into the liquid part of an extraction mixture, thereby facilitating the action of the phenol oxidising enzyme.
• the solvent used for the extraction step is preferably an aqueous solvent.
• mixtures of water and organic solvents may also be used to extract phenolic components such as lignin or other hydrophobic compounds, which are not soluble, or only slightly soluble, in water.
• the water soluble phenolic compounds of the tobacco material will partition into the aqueous extract, together with i.a. nicotine, soluble proteins, sugars, amino acids, pectins, inorganic salts and the surfactants used, if any.
• an aqueous solvent which comprises a major amount of water, viz. more than 30%, such as 50%, preferably more than 60%, more preferably more than 75%, still more preferably more than 90% and most preferably more than 95% water, such as more than 99% Water (% means weight percentage) to extract the tobacco material.
• the extraction of the tobacco material is performed with an aqueous solvent composed of 100% water.
• the aqueous solvent may comprise additional components other than water, such as, e.g. alcohols such as ethanol or methanol; or other water miscible solvents like dimethylpropylene Urea, N-methylpyrrolidone, acetone, propan-2- ol, propan-1-ol, ethyleneglycol dimethyl ether, ethyleneglycol monomethyl ether, tetrahydrofuran, 1-butanol, 2-butanol, isobutanol, tert-butanol, 1,4-dioxane, morpholine, dimethyl- formamide, diethylene glycol, dimethyl ether, dimethyl sulfoxide, diethylene glycol monomethyl ether, ethyleneglycol, diethyleneglycol, sulpholane, glycerol or triethanolamine .
• alcohols such as ethanol or methanol
• other water miscible solvents like dimethylpropylene Urea, N-methylpyrrolidone, acetone
• the solvent may comprise additional components, including, without limitation, surfactants (whether anionic such as sodium dodecylsulfate and sodium dodecylbenzenesulfonate, cationic or non-ionic); enzymes such as proteolytic enzymes, such as, e.g. SavinaseTM from Novo Nordisk A/S, Denmark.
• surfactants whether anionic such as sodium dodecylsulfate and sodium dodecylbenzenesulfonate, cationic or non-ionic
• enzymes such as proteolytic enzymes, such as, e.g. SavinaseTM from Novo Nordisk A/S, Denmark.
• the tobacco material may be extracted with both an organic solvent and an aqueous solvent in two different steps, to extract components which are not soluble or only slightly soluble in water or in aqueous solvents.
• the extraction of the tobacco material with the aqueous solvent may come before or after the extraction of the tobacco material with the organic solvent.
• the extraction of the tobacco material is performed with an aqueous solvent.
• a further extraction of the tobacco material with an organic solvent is performed before or after the aqueous extraction of the tobacco material.
• the organic solvent may be pure, water miscible organic solvents, such as alcohols, e.g.
• ethanol or methanol or other water miscible solvents like dimethylpropylene Urea, N-methylpyrrolidone, acetone, propan-2- ol, propan-1-ol, ethyleneglycol dimethyl ether, ethyleneglycol monomethyl ether, tetrahydrofuran, 1-butanol, 2-butanol, isobu- tanol, tert-butanol, 1,4-dioxane, morpholine, dimethylformamide, diethylene glycol dimethyl ether, dimethyl sulfoxide, diethylene glycol monomethyl ether, ethyleneglycol, diethyleneglycol, sulpholane, glycerol, triethanolamine, or pure, organic solvents not miscible with water, such as alcohols, aldehydes, ketones, ethers, alkanes, e.g.
• tetrahydrofuran THF
• diethylether diethylether
• methyl isobutyl ketone pentane, hexane or dioxane
• dichlo- romethane ethyl acetate
• cyclohexane ligroin
• petroleum ether toluene
• xylenes anisol
• an aqueous extraction step is performed as a liquid-liquid extraction of the organic extract in order to provide an aqueous phase which in the present context should be understood as being encompassed in the term aqueous extract of the tobacco material.
• the content of organic solvent may be decreased by conventional methods for removal of organic solvents, such as evaporation or freezing (cooling) .
• a tobacco material is first extracted with an aqueous solvent, preferably water. Subsequently the tobacco residue may be treated with an organic solvent to extract phenolic compounds which are soluble in the organic solvent but not in the aqueous solvent.
• the resulting organic extract i.e. the liquid portion separated from the tobacco residue, the tobacco residue being the solid portion of the extraction mixture
• the extraction step be performed under conditions maximising the extraction of soluble phenolic compounds.
• partial extraction is useful for ultimately reducing the concentration of phenolic compounds in the final tobacco product.
• extraction conditions are listed below. Generally, any of these conditions can be optimised using only routine experimentation by establishing a matrix of conditions and testing different points in the matrix.
• Suitable extraction process conditions are e.g. a temperature of 10-80°C, 30-80°C, such as 20-70°C, 30-70°C, 45-70°C, e.g. 35-60°C, such as 40-55°C, about 45-50°C, typically about 45°C; a pH of 3- 10, such as 4-9, 4-8, 5-8, 5-7, e.g. 5-6; an extraction time of 5 minutes to 24 hours, 1-24 hours, such as 5 minutes to 10 hours, 1 minute to 5 hours, 5 minutes to 5 hours, 5 min to 1 hour, 5 min to ⁇ hour, typically about 15 minutes.
• the solvent preferably aqueous, is advantageously added in an amount of 5- 200 times the amount of tobacco material (dry weight) , such as 5-100 times, more preferably as 5-50 times, most preferably 10- 50 times, typically about 40 times.
• the extraction advantageously takes place under stirring or other kind of mixing of the tobacco material and the solvent.
• the surfactant, if any, in the solvent used for extraction may be, without limitation, sodium alkylsulfonates, sodium alkylsul- fates, sodium or potassium salts of oarboxylic acids, sodium al- kylarylsulfonates, sodium alkylsulfosuccinates and mixtures of any of the foregoing.
• surfactants having a chain length of between 8 and 12 carbon atoms are considered surfactants having a chain length of between 8 and 12 carbon atoms.
• the surfactant is one or more of sodium dodecylsulfate, sodium dodecylbenzenesulfonate and sodium dioctylsulfosuccinate (Aerosol OT.TM).
• the surfactant is preferably added to the solvent at a concentration range of 0.01%-5% w/v solution.
• the tobacco residue is preferably separated from the extract either before or after (preferably before) the treatment with a phenol oxidising enzyme.
• the separation can be performed using any methods, including, without limitation, centrifugation, filtration, sedimentation, decanting or sieving or any combinations thereof. Particularly preferred methods of separation are filtration and centrifugation.
• the surfactants if any, are removed from the extract, said extract is preferably an aqueous extract. This may be done by cooling, e.g. to 4°C causing the surfactants to precipitate and/or precipitation using e.g. inorganic calcium or magnesium salt followed by e.g. centrifugation.
• tobacco residue refers to the solid tobacco material resulting from extraction of a "tobacco material” with whatever kind of extraction liquor followed by separation (e.g. by filtration or centrifugation) from the liquid fraction (the "tobacco extract”) .
• the tobacco residue representing the water insoluble portion of tobacco.
• the tobacco residue may be subjected to further treatment/processing such as drying.
• tobacco extract refers to the liquid fraction (being clarified or not) resulting from extraction of "tobacco material” with whatever kind of extraction liquor followed by separation
• the tobacco extract comprises soluble components of tobacco and is substantially free from tobacco solids.
• the tobacco extract may be subjected to treatment with other additives and/or other processing.
• extraction mixture refers to a suspension resulting from an extraction of a tobacco material; the suspension comprising a solid fraction and a liquid fraction.
• the tobacco material preferably in the form of an aqueous extraction mixture or aqueous extract, is treated with a phenol oxidising enzyme.
• This step (i) is described in greater detail in the following.
• phenol oxidising enzyme includes any oxidoreductase acting on phenols and related substances as donors, preferably with molecular oxygen or hydrogen peroxide as acceptor.
• the phenol oxidising enzyme of the invention are any enzyme capable of oxidising at least one phenolic compound.
• An oxidation is an electron transfer reaction between two reactants: A donor looses an electron (i.e. one or more electrons), an acceptor gains an electron (i.e. one or more electrons) ; one of the reactants is oxidised (the electron donor) , the other reactant is reduced (the acceptor) . Enzymes catalysing such reactions are called oxidoreductases .
• the phenol oxidising enzyme may be of any origin.
• the term "phenol oxidising enzyme” encompasses phenol oxidising enzymes derived from prokaryotic or eukaryotic organisms, such as animals, plants or microorganisms (such as e.g. bacteria or fungi - including filamentous fungi and yeast) .
• the process of the invention utilises a phenol oxidising enzyme derived from tobacco.
• derived means in this context that the enzyme may have been isolated from an organism where it is present natively, i.e. the identity of the amino acid sequence of the enzyme are corresponding to a native enzyme.
• derived also means that the enzymes may have been produced recombinantly in a host organism, the recombinant produced enzyme having either an identity corresponding to a native enzyme or having it a modified amino acid sequence, e.g. having one or more amino acids which are deleted, inserted and/or substituted, i.e. a recombinantly produced enzyme which is a mutant and/or a fragment of a native amino acid sequence.
• a native enzyme are included natural variants.
• derived includes enzymes produced synthetically by e.g. peptide synthesis.
• derived also encompasses enzymes which have been modified e.g. by glycosylation, phosphorylation etc., whether in vivo or in vitro .
• the term "obtainable” means in this context that the enzyme has an amino acid sequence corresponding to a native enzyme.
• the term encompasses an enzyme that has been isolated from an organism where it is present natively or one in which it has been expressed recombinantly in the same type of organism or another.
• the terms "obtainable” and “derived” refers to the identity of the enzyme and not the identity of the host organism in which it is produced recombinantly.
• an enzyme obtainable from an organism denotes an enzyme which has been obtained from the organism where it is produced natively or an enzyme that has been produced recombinantly in a host organism, where the recombinant enzyme has an amino acid sequence corresponding to a native enzyme.
• a phenol oxidising enzyme encompassed phenol oxidising enzymes (such as, e.g. a laccase) obtainable from animals, plants or microorganisms such as bacteria or fungi (including filamentous fungi and yeast) as well as mutants, fragments or variants thereof with phenol oxidising enzymatic activity.
• phenol oxidising enzymes such as, e.g. a laccase
• fungi including filamentous fungi and yeast
• mutants, fragments or variants thereof with phenol oxidising enzymatic activity comprises use of a phenol oxidising enzyme obtainable from tobacco.
• the phenol oxidising enzyme may be obtained from a microorganism by use of any suitable technique.
• a phenol oxidising enzyme preparation may be obtained by fermentation of a suitable microorganism and subsequent isolation of a phenol oxidising enzyme containing preparation from the resulting fermented broth or microorganism by methods known in the art.
• the phenol oxidising enzyme preparation is ob- tained by use of recombinant DNA techniques.
• Such method normally comprises cultivation of a host cell transformed with a recombinant DNA vector comprising a DNA sequence encoding the phenol oxidising enzyme in question and the DNA sequence being operationally linked with an appropriate expression signal such that it is capable of expressing the phenol oxidising enzyme in a culture medium under conditions permitting the expression of the enzyme and recovering the enzyme from the culture.
• the DNA sequence may also be incorporated into the genome of the host cell.
• the DNA sequence may be of genomic, cDNA or synthetic origin or any combinations of these, and may be isolated or synthesized in accordance with methods known in the art.
• Phenolic compounds/Phenols Phenolic compounds/Phenols :
• phenolic compounds and "phenols” refers to any compound which comprises at least one phenolic ring structure, i.e. an aromatic ring structure, in particular a benzene ring structure, with at least one OH- substituent at a ring C-atom, whatever other substituents, and whatever the number of condensed benzene rings.
• This definition in particular comprises (mono) phenols, as well as polyphenols, such as di-, tri-, tetra-, penta- and hexaphenols.
• monophenol encompasses a compound comprising one hydroxy group attached to an aromatic ring system.
• di-, tri-, tetra-, -penta and hexaphenols encompasses a compound comprising a total of two, tree, four, five or six hydroxy groups, respectively, and one or more aromatic ring systems, where the hydroxy groups are attached to the same or different aromatic rings.
• polyphenol refers to a compound comprising 2 or more hydroxy groups attached to one or more aromatic ring systems, as used herein such compounds are also termed “polyhydroxy phenols".
• polyphenol as used herein also encompasses a compound comprising one aromatic ring having at least two hydroxy groups attached.
• polyphenol as used herein also includes polymeric material based on phenolic mono- mers, as used herein such compounds are also termed “polymeric phenols”. The polymeric material may originate from polymerisation reactions of phenolic compounds
• Non-limiting examples of phenols relevant to the present invention include flavanoids, such as rutin (also named rutoside) , quercetin (also named 2- (3, 4-Dihydroxyphenyl) -3, 5, 7-trihydroxy- 4H-l-benzopyran-4-one; and 3, 3' , 4' , 5, 7-pentahydroxyflavone) , isoquercitrin (also named 2- (3, 4-Dihydroxyphenyl) -3- ( ⁇ -D-glyco- furanosyloxy) -5, 7-dihydroxy-4H-benzopyran-4-one; and 3, 3', 4', 5, 7-pentahydroxyflavone-3-glycoside; and quercetin-3-glycoside) , kaempferol (also named 3, 5, 7-Trihydroxy-2- (4-hydroxyphenyl) -4H- l-benzopyran-4-one; and 3, 4' , 5, 7-tetrahydroxyflavone)
• chlorogenic acid also named 3-o-caffeoylquinic acid and 3- [ [3- (3, 4-Dihydroxyphenyl) -1- oxo-2-propenyl] oxy] 1,4, 5-trihydroxycyclohexanecarboxylic acid
• rutin and scopoletin is of special interest.
• the chemical struc- ture of rutin, scopoletin and chlorogenic acid are shown in figures 11-13.
• An object of the present invention is to reduce the content of low molecular weight phenolic compounds in tobacco, in particular phenolic compounds that are extractable from tobacco.
• phenolic compounds extractable in an aqueous solvent such as, e.g., the compounds scopoletin, rutin and chlorogenic acid.
• extractable phenolic compounds refers to "soluble phenolic compounds", i.e. phenolic compounds which are capable of being extracted from a tobacco material by means of a solvent.
• One aspect of the invention relates to water soluble phenolic compounds, i.e.
• phenolic compounds capable of being extracted from a tobacco material by use of an aqueous solvent, the aqueous solvent being as defined herein, such as, e.g., pure water.
• aqueous solvent being as defined herein, such as, e.g., pure water.
• One embodiment relates to a process of the invention for reducing the concentration of at least one phenolic compound in a tobacco material wherein said phenolic compound (s) is/are soluble, preferably water soluble, phenolic compound(s).
• soluble phenolic compounds includes low molecular weight phenolic compounds.
• low molecular weight refers to compounds having a molecular weight of less than about 10,000 Da, preferably less than 5,000 Da, such as less than 2,000 Da, more preferably less than 1,000 Da.
• the term refers to pheno- lie "monomers", such as, e.g., the low molecular compounds scopoletin, rutin, and chlorogenic acid, that is phenolic compounds which can be polymerised into oligomers or polymers of the phenolic monomer. By polymerisation of the phenolic monomer the molecular weight of the phenolic compounds is increased.
• a polymerisation reaction - caused by the treatment with a phenol oxidising enzyme - proceeds until a high molecular weight phenolic material has been made that will allow separation from the tobacco material, preferably in the form of an extract, by means of e.g. ultrafiltration.
• the polymeric material becomes insoluble and precipitates.
• One embodiment of the invention relates to low molecular weight phenolic compounds.
• the phenolic compounds of the invention are low molecular weight phenolic compounds soluble in a solvent, preferably an aqueous solvent.
• Suitable phenol oxidising enzymes i.e. enzymes which act on phenolic compounds, such as polyphenols, high molecular weight as well as low molecular weight compounds, include, without limitation, peroxidases (EC 1.11.1.7), laccases (EC 1.10.3.2), bilirubin oxidases (EC 1.3.3.5), monophenol mono- oxygenases (EC 1.14.18.1) and catechol oxidases (EC 1.10.3.1).
• the phenol oxidising enzyme is a phenolic oxidase or a peroxidase.
• phenolic oxidases catalyses oxidation reactions in which a donor (in the present context a phenolic compound) is oxidised, molecular oxygen acting as the acceptor.
• Peroxidases are characterised by catalysing reactions in which a donor (in the present context a phenolic compound) is oxidised, hydrogen peroxide acting as the acceptor.
• the phenolic oxidases or peroxidases of the invention are phenolic oxidases and peroxidases capable of oxidising at least one phenolic compound.
• the peroxidase of the invention may also be termed a "phenolic peroxidase".
• the phenol oxidising enzyme reacts with its substrate by single electron transfer. Laccases are included in this group of enzymes performing 1- electron oxidations. This is in contrast to phenol oxidising en- zymes performing 2-electron oxidations.
• 1-electron oxidation and “single electron transfer is meant that the compound to be oxidised, in this case the phenolic compound, is oxidised by transfer of one electron or one electron charge equivalent, although the compound from an overall view may be further oxidised.
• 1-electron oxidation a radical is generated.
• the enzymes in consideration will oxidise the phenolic compounds via generation of a radical.
• phenolic compounds this means, that initially an electron is abstracted from the phenolic compound generating a phenoxy radical; this is described e.g. by Yaropolov A.I et al. (1994), Applied biochemistry and Biotechnology, 49, page 257- 280; and Thurston C.F. (1994), Microbiology, 140, page 19-26.
• EPR spectroscopy is a very sensitive and highly specific method of detecting radicals, and it can be used to analyse complex ma- trixes without extensive sample pre-treatment such as purification and concentration. It is a matter of routine for a person skilled in the art to perform such analysis.
• a more simple method to detect radical formation is to incubate the phenol oxidising enzyme with a substrate known to form a stable radical which can be detected by simple UV/visible spectroscopy, e.g.
• ABTS (2, 2' -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) , PPT (phenothiazine-10-propionic acid) ) or HEPO (10- (2-hydroxyethyl) phenoxazine) .
• enzymes capable of performing 1-electron oxidation are peroxidases (EC 1.11.1.7), laccases (EC 1.10.3.2), bilirubin oxidases (EC 1.3.3.5), and catechol oxidases (EC 1.10.3.1)
• Preferred phenolic oxidases are enzymes of classes EC 1.13.-.-; EC 1.14.-.- (such as, e.g., EC 1.14.18.1.) and EC 1.10.3.-, in particular any of the classes EC 1.10.3.1-1.10.3.8, i.e. EC 1.10.3.1, EC 1.10.3.2, EC 1.10.3.3, EC 1.10.3.4, EC 1.10.3.5, EC 1.10.3.6, EC 1.10.3.7 or EC 1.10.3.8.
• the phenolic oxidase is an enzyme corresponding to EC 1.10.3.- which comprises enzymes acting on diphenols and related substances as donors with oxygen as acceptor. Monophenols, however, are also very good substrates.
• Preferred enzymes of these classes are:
• Catechol oxidases (EC 1.10.3.1); laccases (alternative name urishiol oxidases, EC 1.10.3.2); and o-aminophenol oxidases
• the grouping EC 1.14.18.1 comprises monophenol monooxygenase
• the phenol oxidising enzyme is for the treatment of a tobacco extract by the process according to the invention is EC 1.14.18.1.
• Preferred phenolic oxidases are listed below, included herein are the phenolic oxidases obtainable from the organism in question and any phenol oxidising enzymatically active variants, fragments or mutants thereof. The activities thereof can be measured by any method known in the art.
• Laccase (EC 1.10.3.2) enzymes of microbial and plant origin are well known.
• a suitable microbial laccase enzyme may be derived from plants, bacteria or fungi (including filamentous fungi and yeast) and suitable examples include a laccase derived from a strain of Aspergillus, Neurospora , e.g. N. crassa , Podospora , Botrytis, Collybia , Fomes, Lentinus, Pleurotus, Trametes, e.g., T. villosa , previously called Polyporus pinsi tus, and T. versicolor, Rhizoctonia , e.g., R. solani, Coprinus, e.g. C.
• the phenol oxidising enzyme is a laccase derived from tobacco.
• a suitable catechol oxidase or monophenol monooxygenases may be derived from animals, plants or microorganism such as bacteria or fungi (including filamentous fungi and yeast) . Of particular interest is a catechol oxidase or a monophenol monooxygenase derived from tobacco. Examples of catechol oxidases include a catechol oxidase derived from Solanum melongena (Phytochemistry, 1980, 19(8), 1597-1600) or from tea (Phytochemistry, 1973, 12(8), 1947-1955). Polyphenol oxidase may be derived from molds (Hakko Kogaku Zasshi, 1970, 48(3), 154-160). A mammalian monophenol monooxygenase (tyrosinase) has been described
• the phenol oxidising enzyme is not monophenol monooxygenase (EC 1.14.18.1).
• Suitable peroxidases may be of class EC 1.11.1.-, e.g. EC 1.11.1.7, EC 1.11.1.13 and EC 1.11.1.14.
• Preferred peroxidases are enzyme of class EC 1.11.1.7.
• the group EC 1.11.1.7 comprises peroxidases, catalysing oxidation reactions in which a donor is oxidised, hydrogen peroxide acting as the acceptor.
• Preferred peroxidases are listed below, included herein are the peroxidases obtainable from the organism in question and any phenol oxidising enzymatically active variants, fragments or mutants thereof. The activities thereof can be measured by any method known in the art.
• the peroxidase may originate from any organism.
• the peroxidase is derived from plants (e.g. horseradish, soybean or tobacco) or microorganisms such as fungi (including filamentous fungi and yeast) or bacteria.
• fungi including filamentous fungi and yeast
• Some preferred fungi include strains belonging to the subdivision Deuteromycotina , class Hypho-mycetes, e.g., Fusarium, Humicola , Trichoderma , Myrothecium, Verticillum, Arthromyces, Caldariomyces ,
• Verticillium alboa trum Verticill um dahlie, Arthromyces ramosus (FERM P-7754), Caldariomyces fumago, Ulocladium chartarum, Embellisia alii or Dreschlera halodes .
• Other preferred fungi include strains belonging to the sub-division Basidiomycotina, class Basidiomycetes, e.g. Coprinus , Phanerochaete, Coriolus or Trametes, in particular Coprinus cinereus f. microsporus (IFO 8371), Coprinus macrorhizus, Phanerochaete chrysosporium (e.g.
• fungi include strains belonging to the sub-division Zygomycotina , class Mycoraceae, e.g. Rhizopus or Mucor, in particular Mucor hiemalis .
• Some preferred bacteria include strains of the order Actino-mycetales, e.g., Streptomyces spheroides (ATCC 23965), Strep-tomyces thermoviolaceus (IFO 12382) or Streptoverticillum verticillium ssp. verticillium .
• Bacillus pumilus ATCC 12905
• Bacillus stearothermophilus Rhodobacter sphaeroides
• Rhodomonas pal ustri Streptococcus lactis
• Pseudomonas purrocinia ATCC 15958
• Pseudomonas fluorescens NRRL B-ll
• Further preferred bacteria include strains belonging to Myxococcus, e.g., M. virescens .
• a recombinantly produced peroxidase is preferred, e.g., a peroxidase derived from a Coprinus sp., in particular C. macrorhizus or C. cinereus according to WO 92/16634 and WO 94/12621.
• the phenol oxidising enzyme may be purified, viz. only minor amounts of other proteins being present.
• the expression "other proteins" relate in particular to other enzymes.
• the term "purified” as used herein refers to removal of other components, particularly other proteins and most particularly other enzymes, that are present in the cell of origin of the phenol oxidising enzyme.
• the enzymes are at least 75% (w/w) pure, more preferably at least 80, 85, 90 or even at least 95% pure.
• the phenol oxidising enzyme is an at least 98% pure enzyme protein preparation.
• phenol oxidising enzyme includes whatever auxiliary compounds that may be necessary for the enzyme's catalytic activity, such as, e.g. an appropriate acceptor or cofactor, which may or may not be naturally present in the reaction system.
• phenol oxidising enzyme also includes components such as stabilisers, activators, preservatives, metal ions, buffers, surfactants, flocculants, chelating agents and dispersants that allow the enzyme to work optimally under the actual conditions. This optimization of the enzyme catalyzed reaction is a matter of routine experimentation for those of ordinary skill in the art.
• phenol oxidising enzyme also includes enhancers or mediators which facilitate and/or accelerate the enzymatic reaction, such as, e.g., described by Faure et al.
• a generally preferred pH is pH 3-11, such as 4-9, 4-8, such as 4-7 or 5-6, such as, e.g. about 5.5.
• a generally preferred temperature is 10-90°C, such as 10-80°C, preferably 10-70°C, more preferably 15-60°C, 20-60°C, 20-50°C, such as 30-60°C.
• a generally preferred treatment time is 1 minute to 5 hours, such as 5 minutes to 5 hours, preferably 1 minute to 4 hours, preferably 1 minute to 3 hours, such as 15 minutes to 3 hours, 1 minute to 1 hour, still more preferably 5 to 30 minutes.
• the concentration of oxygen as acceptor (relevant to the use of phenolic oxidases only, e.g., laccase) is generally not critical for the reaction as such, except that at high dosages of enzyme the supply of oxygen and thus the concentration of oxygen in the liquor might be rate limiting.
• molecular oxygen from the atmosphere will usually be present in sufficient quantity so that oxygen can be supplied to the process by means of surface aeration or intensive submerse aeration with atmospheric air. Alternatively, pure oxygen can be used for aeration.
• the source may be hydrogen peroxide or a hydrogen peroxide precursor for in situ production of hydrogen peroxide.
• treatment with peroxidase is performed in the presence of a hydrogen peroxide source.
• treatment in the context of peroxidase” encompasses the presence of a hydrogen peroxide source whenever such a source is required.
• a hydrogen peroxide source means a source of hydrogen peroxide being it hydrogen peroxide itself or a hydrogen peroxide precursor for in situ production of hydrogen peroxide.
• the treatment with peroxidase is performed in the presence of a hydrogen peroxide source selected from the group consisting of (1) hydrogen peroxide, (2) a hydrogen peroxide precursor, e.g. percarbonate or perborate, (3) a hydrogen peroxide generating enzyme system, e.g. an oxidase and its substrate, e.g. glucose oxidase and glucose, (4) and a peroxycarboxylic acid or a salt thereof.
• the hydrogen peroxide source may be added at the beginning of or during the process, e.g. in a concentration corresponding to 0.001-25 mM H 2 0 2 .
• the hydrogen peroxide source may be added continuously to maintain a substantially constant concentration of hydrogen peroxide.
• the concentration of hydrogen peroxide as acceptor is generally not critical. However, the selected peroxidase enzyme could be sensitive to hydrogen peroxide (loose activity) .
• concentration range of hydrogen peroxide is 0.010-lOmM, such as 0.020-8mM, 0.05-5mM, or 0.100-2.5mM. The appropriate range may depend on the enzyme in question and can be determined by the person skilled in the art.
• a preferred dosage of the phenol oxidising enzyme is 0.001-1000 mg enzyme protein per litre of the extract (impregnation liquid), such as 0.001-500 mg, 0.001-200 mg, 0.001-100 mg, 0.001-50 mg, preferably 0.01-100 mg, such as 0.01- 80 mg, 0.01-50 mg, 0.01-30 mg, 0.01-20, more preferably 0.1-20 mg/litre.
• dosages of phenol modifying enzyme protein per dry weight of tobacco is 1-1000 ⁇ g/g, such as 10-500 ⁇ g/g, such as 150 ⁇ g/g. These dosage values are preferably based on purified enzyme protein, purified being defined as indicated above.
• the phenol oxidising enzyme may be in any form suited for the use in question, such as e.g. in the form of a dry powder or granulate, a non-dusting granulate, a liquid, a stabilized liquid, or a protected enzyme.
• Granulates may be produced, e.g. as disclosed in US 4,106,991 and US 4,661,452 (both to Novo Industry A/S) , and may optionally be coated by methods known in the art.
• Liquid enzyme preparations may, for instance, be stabilized by adding stabilizers such as a sugar, a sugar alcohol or another polyol, lactic acid or another organic acid according to established methods.
• Protected enzymes may be prepared according to the method disclosed in EP 238,216.
• the tobacco material preferably in the form of an aqueous extract, is treated with a phenol oxidising enzyme.
• treatment in the context of a “phenol oxidising enzyme” is meant the addition to a tobacco material, the material being preferably in the form of an aqueous extract, of an effective amount of a phenol oxidising enzyme under conditions which the enzyme exerts its oxidizing activity, i.e. oxidising a phenolic compound from the tobacco material thereby providing an "oxidised phenolic compound”.
• treatment in the context of a phenol oxidising enzyme and a tobacco material encompasses contacting a tobacco material with a phenol oxidising enzyme under conditions which result in a reduction in the concentration of at least one phenolic compound in said material.
• contact in the context of "contacting a tobacco material with a phenol oxidising enzyme” is meant the addition of a phenol oxidising enzyme to a tobacco material .
• a process for preparing a tobacco product comprises (a) extracting a tobacco material with a solvent to provide an extraction mixture; and (b) separating the extraction mixture into a tobacco extract and a tobacco residue, contacting the tobacco extract with a phenol oxidising enzyme, such as a phenolic oxidase or a peroxidase, under conditions which result in a reduction in the concentration of at least one phenolic compound in said extract.
• a phenol oxidising enzyme such as a phenolic oxidase or a peroxidase
• an amount of enzyme which is effective in order to provide a tobacco product having a reduced content of a specific phenolic compound such as an at least 10% reduction, at least 20%, at least 50%, at least 75%, preferably 95 %, or even more preferred at least a 98% reduction, such as a 100 % reduction of the specific compound.
• the %-reduction being calculated as indicated below.
• a specific phenolic compound encompasses that by the enzyme treatment of the tobacco material there is obtained a re- duction in the amount of at a phenolic compound in said material. There may be a variation i the %-reduction for different phenolic compounds.
• the reduction of each phenolic compound corresponds to a %- reduction as indicated above.
• the enzyme treatment of a tobacco material provides a reduction on the concentration of at least one phenolic compound of said tobacco material, such as reduction on the concentration of at least two phenolic compounds or at least tree phenolic compounds in said tobacco material.
• the tobacco product obtained by a proc- ess according to the invention has a reduced content of at least one of chlorogenic acid, rutin or scopoletin, such as an at least 5% reduction, such as at least 10%, at least 20%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, preferably at least a 95 %, or even more preferred at least 98% a reduction, such as a 100 % reduction in the content of chlorogenic acid, rutin or scopoletin, respectively, as compared to the tobacco material before the treatment.
• the reduction may be monitored e.g. by HPLC analyses of extracts of the tobacco material before and after treatment with a phenol oxi- dising enzyme. The %-reduction being calculated as indicated below.
• the process according to the invention comprising 1) treatment with a phenol oxidising enzyme to provide an oxidation of at least one phenolic compound in said tobacco material, 2) further comprising the step of separating the oxidised phenolic compound (s) from the tobacco material - leads to a tobacco product having a reduced total content of phenolic compound.
• the reduction being e.g. at least a 5% reduc- tion, at least 10%, at least 20%, at least 40%, at least 50%, at least 60%, at least 75%, at least a 80% reduction; such as a reduction in the range 2%-95%, 5%-80%, 5%-50%, 5%-40%, 5%-30%.
• the resulting signal e.g. peak area or peak height or similar
• relative quantification by comparing the size of the resulting signal produced by the individual phenolic compound, before, during, and after the enzyme treatment, and use the development in the size of the signal to calculate the relative (that is the percentage) reduction of the individual phenolic compounds.
• the size of the resulting signal will normally correlate with the concentration observed in the sample if an appropriate range of concentration is examined (normally, this means that the concentration range used is in the range in which the signal correlates linearly with the concentration) .
• the process of the invention comprising treatment with a phenol oxidising enzyme, provides a tobacco material which by HPLC analysis of the tobacco material before and after the enzyme treatment shows reduction in a signal (peaks), corresponding to a phenolic compound, monitored by HPLC analyses.
• a signal peaks
• This is preferably a %-reduction of at least 5%, such as at least 10%, at least 20%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, preferably at least 95 %, or even more preferred least 98%, such as 100 %. This may be measured as described herein in the section "Examples".
• there is a reduction in at least one signal i.e. at least one phenolic compound
• at least 2 signals i.e. at least two phenolic compounds.
• the invention relates to a process for reducing the concentration of at least one phenolic compound in a tobacco material, which process comprising treating tobacco material with a phenol modifying enzyme, said tobacco material preferably being in the form of an extract of a tobacco material.
• the modification of the enzyme facilitates the removal of the modified phenolic compound, thereby leading to a reduction in the total content of phenolic compounds in the tobacco material.
• the phenol modifying enzyme is a phenol oxidising enzyme.
• the process according to the invention preferably comprises the step of (iv) separating the oxidized phenolic compound from the tobacco extract.
• oxidised phenolic compound encompasses the phenolic compounds which has been oxidised by the treatment with an enzyme according to the invention.
• the oxidised phenolic compound is preferably in a polymerised form.
• the oxidised phenolic compound is preferably in one or more of the forms precipitate, haze, dissolved or dispersed. Thus, a precipitate/haze may be generated during the treatment of tobacco extract with a phenol oxidising enzyme.
• the precipitate comprising the oxidised polymerised phenolic compounds is preferably separated from the extract.
• the precipitate/haze may be removed by any suitable method in the art, including, without limitation, centrifugation, filtration, ultrafiltration, sedimentation, flocculation, reverse osmosis, decanting or sieving. A combination of different methods and/or a repetition of one or more of the methods may also be used.
• the adsorption is performed with an adsorption means selected from the group consisting of Fuller's earth minerals such as attapulgite or bentonite, hydroxyapatite (tri-calcium phosphate) , PVPP, an anion exchange resin, a cation exchange resin, a hydrophobic resin and activated carbon, or any combination thereof.
• the process of the invention includes an adsorption step using, first, bentonite, which is followed by an adsorption step using hydroxy apatite
• tri-calcium phopshate (tri-calcium phopshate) .
• ultrafiltration is used, in particular for the dissolved or dispersed polymerised oxidised phenolic compounds.
• the extract is also treated with an insoluble adsorbent, step (vii) , preferably a water insoluble adsorbent.
• suitable insoluble adsorbents are hydroxyapatite (tri-calciumphophate) and Fuller's earth minerals such as attapulgite or bentonite.
• This step serves i.a. to remove soluble polypeptides such as proteins including the enzyme, as well as the polymerized phenolic reaction products of step (i) .
• Preferred insoluble adsorbents are bentonite and hydroxylapatite .
• the adsorbent can be simply suspended in the extract and subsequently separated by e.g. centrifugation, or it can be contained in a column through which the extract flows.
• the extract is treated with polyvinylpolypyrrolidone (PVPP) , step (viii) .
• PVPP polyvinylpolypyrrolidone
• the treatment with PVPP is omitted.
• treatment in the context of an adsorbent is meant contacting the extract with an adsorbent under conditions facilitating the adsorbtions, followed by the removal of the adsorbent if desired, the adsorbent being preferably an insoluble adsorbent.
• One embodiment of the invention relates to the removal of the modified phenolic compounds, the modified phenolic compound provided by treatment with a phenol modifying enzyme as described herein.
• the enzyme treatment is performed on an extraction mixture.
• the oxidised phenolic compounds is preferably separated from the extraction mixture, i.e. from tobacco solids ("tobacco residue") as well as the liquid part ("tobacco extract”) .
• This separation may be done by first making a crude separation, e.g. by filtration, that allows the solvent and the oxidised phenolic compounds, which may have formed a precipitate and/or haze, to be separated from the tobacco residue.
• the choice of method for separation is not critical as long as it allow to distinguish between the tobacco residue suspended in the solvent and the solid precipitate/haze that may have been formed by the oxidised phenolic compounds.
• the thus separated tobacco residue may be rinsed with water to remove additional oxidised phenolic compounds.
• the filtrate containing the oxidised phenolic compounds which will probably be present as a fine precipitate, and/or haze, and/or as oxidised phenolic compounds in colloidal solution may be further processed to sepa- rate the oxidised phenolic compounds from the extract. This may be done by means of separation such as filtration, centrifugation, ultra filtration, sedimentation etc. as already described herein.
• step (v) Inactivating and/or removal of the enzyme, step (v)
• the phenol oxidising enzyme may optionally be inactivated (e.g., by heating to 100°C and holding this temperature for, e.g., 10 minutes) and/or removed (e.g., by adsorption or precipitation) by any method suitable for inactivation and/or denaturation and/or precipitation and/or removing the particular enzyme in question) .
• the enzyme may be removed together with the precipitated oxidised phenolic compounds, e.g. by adsorption as described for the removal of the oxidised phenolic compounds.
• ultrafiltration is used for removal of the enzyme.
• the process of the invention in one embodiment comprises the step of inactivating and/or removing the phenol oxidising enzyme from an enzyme treated tobacco material, said tobacco material being, e.g., in the form of a tobacco extract.
• the enzyme used is immobilised and is easily and quantitatively removed, thereby facilitating the product of the invention to be substantially free from the added phenol oxidising enzyme.
• Common techniques for immobilisation of enzymes include, without limitation, 0 adsorption, covalent bonding and cross-linking onto carrier materials such as ion-exchange resins, artificial polymers, e.g. nylon, polyethylene imine, polystyrene, methacrylate, naturally occurring biopolymers and derivatives thereof, e.g.
• chitin chitosan, glyceryl chitosan, cellulose and derivatives thereof, 5 e.g., DEAE-cellulose, (ground/crushed-) egg shells, inorganic materials, e.g. Si0 2 , glass beads, bentonite, and other insoluble supports, as well as encapsulation in gels or
• immobilised enzymes exhibit little or even no leakage of enzyme during use, 0 resulting in a liquid fraction with no or very little enzyme when separated from the immobilised enzyme particles.
• a very specific and sensitive method for quantification of the amount of leaked enzyme is the use of immobilized enzyme which has been radioactively labelled prior to immobilisation, e.g. by
• a preferred way of separating the oxidised phenolic compounds, which may have produced a precipitate/haze, from the immobilised enzyme solids, as well as the solvent is to first make a crude separation, e.g. a filtration, that allows the solvent and the oxidised phenolic compounds, which may have formed a precipitate and/or haze, to be separated from the immobilised enzyme.
• a crude separation e.g. a filtration
• the choice of method for separation is not critical as long as it allow to distinguish between the immobilised enzyme suspended in the solvent and the solid precipitate/haze that may have been formed by the oxidised phenolic compounds.
• the thus separated immobilised enzyme may be rinsed with water to remove additional oxidised phenolic compounds prior to its possible re-use.
• the filtrate containing the oxidised phenolic compounds which will probably be present as a fine precipitate, a haze, and/or as oxidised phenolic compounds in colloidal solution, may be further treated to separate the extract from the oxidised phenolic compounds. This may be done by means of separation such as filtration, centrifugation, ultra filtration, sedimentation etc., as already described herein.
• the treatment by immobilised enzymes of the tobacco extract is performed in a column comprising the immobilised enzymes by passing the tobacco extract thought said column, preferably using down-flow.
• the direction of the flow may be change to up-flow with intervals to fluidize and thus rinse and remove any precipitate/haze formed.
• the phenol oxidising enzyme-treatment of a tobacco extract in column containing immobilised enzymes is done using the principle of fluidized bed utilising the differences in particle size and density of the immobilised enzyme particles and the precipitate/haze formed to continuously treat the extract and separate the oxidised phenolic compounds forming a precipitate/haze from the enzyme.
• the enzyme-treated extract is concentrated to a solids content of between 10-70%, such as 20-50%, typically 50%
• the tobacco extract can be dried to a solid (e.g. having a solid content of 90-100%, such as 100% dry weight) by means of conventional drying processes, such as freeze-drying, spray-drying or evaporation.
• a concentration step is made by a normal procedure for concentrating liquids, followed by a drying step by normal procedures.
• any result of any of the processes described above for the treatment of the extract is finally re-combined with the tobacco residue (step (ix) ) , which may or may not have been further processed or treated e.g. by drying or by extraction with an organic solvent, to provide an improved resulting tobacco product.
• the treated extract may be combined with any tobacco solid, but advantageously it is a tobacco solid with a low content of phenolic compounds, such as a tobacco residue resulting from an extraction process as described herein.
• the enzyme treated tobacco extract may be recombined with a tobacco solid, such as the tobacco residue, typically by spraying back the extract onto the tobacco residue, but the choice of method is not critical as any method suitable for recombining extract and a tobacco solid can be used.
• the recombined tobacco is dried by conventional methods.
• the treated tobacco extract is combined with a green tobacco, i.e. uncured tobacco, which may be used for tobacco chewing gum or the treated extract may be used to enrich the flavour of green tobacco by spraying tobacco extract on green tobacco leaves, see, e.g., US 5,845,647.
• a green tobacco i.e. uncured tobacco
• the treated extract may be used to enrich the flavour of green tobacco by spraying tobacco extract on green tobacco leaves, see, e.g., US 5,845,647.
• the enzyme treated extract may be recombined with other materials, such as cigarette paper, cigarette filters, tobacco cover sheets, or any other material than the tobacco residue, which will later be combined with the tobacco residue to make up the final tobacco product.
• the tobacco extract is dried to a solid content of e.g. 90-100%, such as 100% (dry weight) and thus essentially is to be regarded as a solid
• the dried tobacco extract may be recombined with the tobacco residue or with other materials, such as cigarette paper, cigarette filters, tobacco cover sheets to make up the final enzyme treated tobacco material, by direct blending/mixing of the dried tobacco extract with the material in question, if necessary mixed with additional binders, e.g. starch, to make the dried extract stick to the material.
• the tobacco is also treated with a proteolytic enzyme, step (x) .
• the tobacco material is first extracted with an aqueous solvent without the presence of enzymes, whether they are phenol oxidising enzymes or proteolytic enzymes, and preferably without surfactants.
• the extraction mixture is separated and the aqueous extract treated with a phenol oxidising enzyme, while the residue is subjected to a further extraction with a solvent comprising a protease and optionally a surfactant.
• the enzymatic treated extract and residue may subsequently be combined.
• the process of the invention may comprise a step where the tobacco material or the tobacco residue has been treated with a protease before the combination with an extract treated with a phenol oxidising enzyme .
• the tobacco material to be treated with a phenol oxidising enzyme is a protease treated tobacco material, thus being a tobacco material with a reduced content of protein.
• the solvent for extraction may comprise a protease.
• step (x) of proteolytic treatment it is preferred that the process of the invention further comprises the step of removing the protease in order to provide a tobacco product substantially free from the protease in question.
• both a laccase and a protease are used in the preparation of a tobacco product.
• the proteolytic enzyme if used, is preferably chosen from the group comprising bacterial and fungal enzymes. Of most interest for the purpose of this invention are the enzymes used commercially in the food and detergent industries which are available at low cost. Thus, Savinase . TM. , Neutrase . TM. , Enzobake.TM. or Alcalase.TM. available from Novo Inc. have been found to be ef- fective for protein removal from tobacco.
• the proteolytic enzymes may be added to the solution in the concentration range 0.0001%-5% w/w, such as 0.1%-5% w/w of the tobacco material.
• step (ii) is performed before any of the following steps.
• step (ii) followed by step (iii) is performed before any of the following step.
• Step (i) may be performed immediately after step: (ii) or e.g. following step (iii), (vii) , (viii) or (vi) .
• steps (v) , (vi) , (vii) or (viii) can be included in the process, in whatever sequence. They can be included once or repeated several times. They preferably always follow step (ii) and (iii) , however.
• step (vii) follows step (i) .
• step (viii) is not included. If included, step (viii) preferably follows steps (i) and (vii) .
• step (v) is not included. If included, step (v) always follows step (i) , and preferably also step (vii) .
• step (vi) is the final step.
• D (ii) , (iii) , (i) , (iv) ; or E: (ii) , (iii), (i), (iv) , (vi); or
• the process according to the invention further comprises the step of making a tobacco article for smoking.
• the process of the invention includes the following steps in the sequence indicated: (ii) extracting a tobacco material with a solvent to provide an extraction mixture; (iii) separating the extraction mixture into a tobacco extract and a tobacco residue; (i) treating the extract with a phenol oxidising enzyme; (iv) separating the oxidised phenolic compound from the tobacco extract; (vi) concentrating the extract; combining the thus treated extract with the tobacco residue; and preparing a tobacco article for smoking from the combined residue and extract.
• the process also comprises the step (v) of removing the enzyme, e.g., before step (vi) .
• a process for preparing a tobacco product with a reduced amount of phenolic compounds which process comprises the step of treating a tobacco material with a phenol oxidising enzyme.
• a method for reducing the amount of phenolic compounds in a tobacco material comprising treating an extract of a tobacco material with a phenol oxidising enzyme.
• the invention further relates to the use of a phenol oxidising enzyme in the treatment of a tobacco extract.
• a specific embodiment of the invention relates to the use of a phenol oxidising enzyme in the preparation of a tobacco product, where said phenol oxidising enzyme is not monophenol monooxygenase (EC 1.14.18.1) .
• a laccase in the preparation of a tobacco product.
• An even more preferred embodiment of the invention is the use of both a phenol oxidising enzyme, preferably a laccase, and a protease in the preparation of a tobacco product.
• the present invention relates to a tobacco material obtainable, in particular obtained, by any of the processes described herein.
• the invention thus encompasses the final, ready-for-use tobacco products, as well as any extracts of a tobacco material having been treated by any of the processes of the invention.
• a modified tobacco product having a reduced concentration of at least one phenolic compound relative to a tobacco material from which it is derived, wherein said product is produced by any of the herein described proc- esses comprising the step of treating, i.e. contacting, a tobacco material with a phenol oxidizing enzyme.
• the invention relates to a method for producing a tobacco product having an improved customer compliance, such as, e.g., a tobacco product according to the invention which gives the consumer an improved smoking pleasure.
• the invention thus relates to the use of a process according to the invention to provide a tobacco product having, e.g., a modified chemical composition, flavour, aroma, taste and/or colour.
• Tobacco material Virginia flue-cured tobacco (obtainable from Imperial Tobacco Ltd) . Approx. 85% d.m. (dry matter) , stored at 4°C.
• Laccase Liquid preparation of Trametes villosa laccase (TvL) (previously called Polyporus pinsitus laccase (PpL) ) , obtainable from Novo Nordisk A/S.
• the laccase can be prepared as disclosed in WO96/00290 (the laccase enzyme called lacl from a strain with pDSYlO) .
• Liquid preparation of Myceliopthora thermophila laccase (MtL) (obtainable from Novo Nordisk A/S) . All of the enzymes were in a purified form. Standards: Scopoletin (Aldrich #24,658-1), Chlorogenic acid (Merck #820319), Rutin (Aldrich #R230-3) and Nicotine (Aldrich 24, 658-1).
• Liquid chromatographic system E.g. Shimadzu SCL-6B System Con- troller, and two LC-6A Liquid Chromatograph (pumps) or Waters model 600 E
• Injector Shimadzu SIL 6B Autoinjector or Waters 715 Ultra Wisp.
• UV detector Shimadzu SPD-6A Photodiode Array UV-VIS detector or
• Bondapack C198 stainless steel column 30 cm x 4 mm i.d., 10 ⁇ m particle size.
• Guard column none or Guard-Pak from Waters packed with the same material.
• Table 1 A binary (linear) gradient profile used for the HPLC analysis of phenolic compounds.
• the samples Prior to HPLC analysis, the samples were filtered through a 0.45 ⁇ m filter or smaller, e.g. a sterile Sartorius Minisart 0.45 ⁇ m filter (#16555) or a Millipore Millex-GS 0.22 ⁇ m (#SBGS 0 25 SB) to remove the haze/precipitate normally formed during enzyme treatment. Normally, no dilution of the extract/sample was made before HPLC analysis.
• a 0.45 ⁇ m filter or smaller e.g. a sterile Sartorius Minisart 0.45 ⁇ m filter (#16555) or a Millipore Millex-GS 0.22 ⁇ m (#SBGS 0 25 SB)
• the tobacco is dried and sieved before analysis.
• the tobacco is dried at 65°C for 15 hours and sieved through a 40-80 meshscreen.
• 100-200 mg tobacco is weighed accurately into a 50 ml flask with septum. 5 ml of a 1:1 mixture of water and methanol and extracted in an ultrasonic bath for 20 minutes with occasional shaking.
• Temperature of the water in the ultrasonic bath is checked and ice is added if necessary to keep the water at room temperature.
• each of the standards was dissolved in 96% ethanol, and then mixed in a ratio and at a concentration which was within the range in the tobacco extract.
• For semi-quantification of the phenolic compounds in the tobacco extract and as a measure of development in concentration of the various compounds contained in the extract, decrease in peak area relative to the peak area before addition of enzyme was used.
• degree of reduction in total peak area of all peaks (not only the major ones) in the HPLC chromatogram with retention time Rt>3 min was used (named: "All peaks”) .
• the reducing sugars and the nicotine content of tobacco samples and freeze-dried tobacco extracts were determined using continuous flow analysis methods similar to the CORESTA (France, Cooperation Centre for Scientific Research Relative to Tobacco) rec- ommended methods N° 35 and N° 37.
• the total sugars were obtained after a hydrolysis step but the method is similar to the reducing sugars method.
• Preparation of tobacco extract for enzyme treatment Unless otherwise indicated, the tobacco was extracted at a laboratory scale with water according to the following procedure: 1000 ml demineralised water is heated to 45°C. 30 g tobacco (approx. 25 g d.m.) is added and the mixture stirred by a magnetic bar and occasionally with a stick. After 15 minutes the tobacco residue is separated from the tobacco extract by vacuum filtration (Whatman glass micro fibre GF/F 11 cm) . The filtered extract is then vacuum filtered at least once more or until it is completely clear. Yield was around 900 ml tobacco extract. The aqueous tobacco extract appeared as a red-brown liquor with a distinct smell of tobacco. pH of the tobacco extract was in the range 5.4-5.5 and it had a good buffering capacity as pH was very stable during processing and later enzyme treatment. No buffer was thus included in any of the trials. D.m. content of the extract was approximately 2.1%.
• Tobacco extract (typically 50-100 ml) was poured into a glass beaker and a magnetic bar was added. No pH adjustments were made, and no buffer was added.
• the tobacco extract was heated to and controlled at 55 °C during the entire enzyme treatment by means of a heating plate and a thermostat. Intensive submerse aeration was applied by blowing atmospheric air into the extract by means of a metal sinter (metal suction filter normally used for HPLC) .
• the enzyme in question (TvL or PpL) was added to the desired concentration. Shortly after addition of laccase, the colour of the tobacco extract turned dark brown to black, and with time the liq- uor became turbid and a precipitate formed. The liquor was sampled with intervals for HPLC analysis.
• EXAMPLE 1 Effect of laccase on scopoletin, rutin, chlorogenic acid and nicotine
• Tobacco constituents Nicotine, Chlorogenic acid, Rutin and Scopoletin.
• Enzyme Purified laccase derived from Trametes villosa , TvL.
• Apparatus HP8452 UV/Vis diode array spectrophotometer, 1 cm quarts cuvette.
• Buffer Sodium acetate (Merck)
• 96% ethanol was prepared of nicotine, chlorogenic acid, rutin, and scopoletin.
• a stock solution of 0.31 mg enzyme protein/ml of laccase in de-mineralised water was prepared.
• the HP diode array spectrophotometer was operated as per the manufacturer's instructions.
• a 1 cm quartz cuvette was used.
• As a blank was used 950 ⁇ l buffer mixed with 50 ⁇ l 96% ethanol.
• In a 1 cm quart cu- vette was mixed 900 ⁇ l 10 mM acetate buffer and 50 ⁇ l of the stock solution of the compound in question.
• a spectrum of the "native" compound was recorded in the range 190-700 nm.
• TvL stock solution 50 ⁇ l was added and carefully mixed, and spectra in the range 190-700 nm were recorded every 20 seconds for 5 minutes. This resultet in the following conditions during measurement: 9 mM Na-acetate buffer pH 5.0; 4.8% Ethanol; 0.025 mg/ml of tobacco constituent; 0.016 mg/ml laccase.
• Enzyme treatment Surface aeration was used. TvL enzyme was added to the tobacco extract to a concentration of approximately 1.6 ⁇ g/ml.
• the HPLC analyses of the tobacco extract before, during, and after enzyme treatment showed that the phenolic compounds rutin, scopoletin, and chlorogenic acid are present in the tobacco extract in addition to various other compounds as indicated in table 2.
• HPLC chromatograms of the tobacco extract before, during, and after enzyme treatment as well as chromatograms of the mixture of HPLC standards are shown in figure 6, 7, ,8, and 9.
• Table 2 Retention time of the 8 major peaks in the HPLC chromatogram of the aqueous tobacco extract.
• Enzyme treatment pH of the extract was adjusted with H 2 S0 4 or NaOH to pH 4, pH 5, pH 6, or pH 7.
• the extract was kept at ambi- ent temperature (approximately 20°C) during the entire enzyme treatment. Surface aeration was used.
• TvL enzyme was added to a concentration of approximately 1.6 ⁇ g/ml. Except for the tobacco extract adjusted to pH 7 the liquor became turbid and a precipitate formed with time after the addition of enzyme. The results are shown in table 3. From table 3 it is evident, that significant reduction in peak area for all peaks was obtained. From an overall point of view optimum pH at the conditions used is in the range pH 5-6.
• Table 3.1-3 pH profile for reduction in peak area for tobacco extract treated with 1.6 ⁇ g/ml TvL at ambient temperature and surface aeration for various periods of time in the pH range pH 4 - pH 7.
• CA Chlorogenic acid
• R Rutin
• S Scopoletin
• CA Chlorogenic acid
• R Rutin
• S Scopoletin
• CA Chlorogenic acid
• R Rutin
• S Scopoletin
• EXAMPLE 4 Laccase TvL treatment of tobacco extract at two different enzyme concentrations and different processing times
• Enzyme treatment TvL enzyme was added to a concentration of approximately 1.6 ⁇ g/ml or to a concentration of approximately 7.8 ⁇ g/ml. The results are shown in table 4 and table 5. It is evident, that using a dosage of TvL of 1.6 ⁇ g/ml, a longer process- ing time is needed compared to using a dosage of 7.8 ⁇ g/ml to obtain the same degree of reduction of the phenolic compounds.
• Table 4 Time profile for reduction (% remaining) in peak area for tobacco extract treated with approximately 1.6 ⁇ g/ml TvL at 55 °C and pH 5.4 using intensive, submerse aeration.
• CA Chlorogenic acid
• R Rutin
• S Scopoletin
• Table 5 Time profile for reduction (% remaining) in peak area for tobacco extract treated with approximately
• CA Chlorogenic acid
• R Rutin
• S Scopoletin
• MtL enzyme was added to a concentration of approximately 0.63 ⁇ g/ml or approximately 6.3 ⁇ g/ml. The results are shown in table 6 and table 7. It is obvious, that MtL can also be used to remove the phenolic compounds. It is evident, that using a dosage of MtL of approximately 0.63 ⁇ g/ml, longer processing time is needed compared to using a dosage of 6.3 ⁇ g/ml to obtain the same degree of reduction of the phenolic compounds, and that almost complete removal can be obtained with a dosage of approximately 0.63 ⁇ g/ml. 0.63 ⁇ g/ml % Remaining (peak area) after various
• Table 6 Residual amount (peak area) following treatment with approx. 0.63 ⁇ g/ml MtL at pH 5.4 and 55°C and intensive submerse aeration for various processing times, CA: Chlorogenic acid, R: Rutin, S: Scopoletin
• Table 7 Residual amount (% remaining) (peak area) following treatment with approx. 6.3 ⁇ g/ml MtL at pH 5.4 and 55°C and intensive submerse aeration for various processing times.
• CA Chlorogenic acid
• R Rutin
• S Scopoletin
• MtL enzyme was added at various concentrations in the range 2.5 ⁇ g/ml - 6.3 ⁇ g/ml. A fixed processing time of 20 minutes was used. After addition of laccase (but varying with the dosage applied) with time the liquor became turbid and a precipitate formed. The results are shown in table 8. It is obvious, that with increasing dosage of MtL decreasing processing time is needed to remove the phenolic compounds, and that almost complete removal can be obtained in less than 20 minutes. ⁇ g MtL /ml
• Table 8 Residual amount (% remaining) (peak area) following treatment with various enzyme dosages for 20 min- utes at pH 5.4 and 55 °C and intensive submerse aeration using MtL.
• CA Chlorogenic acid
• R Rutin
• S Scopoletin
• Table 9 Residual amount (% remaining) (peak area) following treatment with 3.8 ⁇ g/ml for 20 minutes at pH 5.4 and 55°C at various levels of p0 2 .
• CA Chlorogenic acid
• R Rutin
• S Scopoletin
• EXAMPLE 8 Laccase and bentonite treatment of tobacco Centrifugation: Beckman J-6B centrifuge equipped with JS-4.2 rotor. 1 Litre containers were used. Operated at 4200 rpm (approximately 5000 x g) for 10 minutes at room temperature. Freeze drying: Heto SICC CD 40. The extracts were frozen at - 45°C, and temperature gradually increased to 20°C during drying at 3 mbar.
• the tobacco was extracted at a laboratory scale with water according to the following procedure: 15 Litres de-mineralised wa- ter is heated to 45-46°C. 450 g tobacco (approx. 380 g d.m.) is added and the mixture is stirred manually with a stick. After 15 minutes the tobacco residue is separated from the aqueous tobacco extract by vacuum filtration. The tobacco residue was freeze dried and labelled "2; Freeze Dried Extracted Tobacco Residue" .
• the tobacco extract was centrifuged for 10 min at approx. 5000 x g to remove suspended particles/haze. Yield was around 13.5 Litres.
• the aqueous tobacco extract appeared as a red-brown liquor with a distinct smell of tobacco.
• pH of the tobacco extract was 5.5 and apparently it had quite a good buffering capacity as pH was very stable during processing and later enzyme treatment. No pH adjustment was made prior to enzyme processing, and no buffer was added.
• D.m. content of tobacco extract was approximately 1.4 % (approximately 10 g tobacco extract dried at 120 °C until constant weight for at least 120 s) .
• a sample of the tobacco extract was analysed by means of HPLC. 1.5 L of the tobacco extract was freeze dried and labelled "3; Tobacco Extract " .
• the "precipitate from enzyme treated tobacco extract” (solid material containing a little extract) was suspended in de- mineralised water and collected from the containers and freeze dried and labelled 5 : Precipi ta te From Enzyme Treated Tobacco Extract .
• Bentonite treatment The remaining 3 L of the 6 L "enzyme treated extract” was treated with bentonite (Aldrich #28,523-4): 3 g bentonite was suspended in approx. 200 ml "enzyme treated extract”. The extract/bentonite slurry was recombined with the remaining enzyme treated extract and incubated at 50-55°C for 15 minutes during stirring. After 15 minutes, a sample was analysed by means of HPLC.
• the precipitate (solid material containing enzyme generated precipitate as well as bentonite and a little extract) was suspended in de-mineralised water and collected from the containers, freeze dried, and labelled "7; Precipi ta te from Enzyme- and Bentonite Trea ted Tobacco Extract " .
• Table 11 shows the results from the Auto Analyzer analysis of reducing sugars and total sugars as well as of nicotine of the various solid tobacco fractions.
• table 12 the results from the Auto Analyzer analysis of re- ducing sugars and total sugars as well as of nicotine of the various tobacco extracts and fractions thereof is shown.
• Table 10 Remaining content (% remaining) (measured as peak area relative to the content in the extract) of the major peaks in the HPLC chromatograms as well as of all peaks with R t > 3 minutes.
• CA Chlorogenic acid
• R Rutin
• S Scopoletin
• Table 11 Content of reducing sugars, total sugars, and nicotine in "tobacco"/"tobacco residue”. All figures are % (w/w) and have been determined relative to 100% dry matter.
• R.S Reducing sugars
• T.S Total sugars
• Table 12 Content of reducing sugars, total sugars, and nicotine in the "tobacco extract” as well as in the various fractions of enzyme treated tobacco extract. All figures are % (w/w) and have been determined relative to 100% dry matter.
• R.S Reducing sugars
• T.S Total sugars.
• Table 13 Content of various tobacco extract components as a function of time during MtL treatment during the 3.0 kg/100 litre scale treatment.
• CA Chlorogenic acid
• R Rutin
• S Scopoletin
• Table 14 Content of various tobacco components in tobacco before (“raw tobacco”) and after extraction ("tobacco residue”) during the 3.0 kg/100 litre scale treatment.
• DWB Tobacco Dry Weight Basis
• CA Chlorogenic acid
• R Rutin
• S Scopoletin
• Table 15 Content of various tobacco extract components as a function of time during MtL treatment during the 6.0 kg/200 litre scale treatment.
• CA Chlorogenic acid
• R Rutin
• S Scopoletin
• Table 16 Content of various tobacco components in tobacco before (“raw tobacco) and after extraction ("tobacco residue") during the 6.0 kg/200 litre scale treatment.
• DWB Tobacco Dry Weight Basis
• CA Chlorogenic acid
• R Rutin
• S Scopoletin
• Samples of tobacco before and after extraction and recombination with the tobacco extract was analysed for nicotine, reducing sugars, total sugars by means of an Auto Analyzer according to the method previously described, and analysed for chlorogenic acid, rutin, and scopoletin by means of HPLC according the method previously described.
• the tobacco extract before and after MtL and bentonite treatment was analysed for chlorogenic acid, rutin, and scopoletin by means of HPLC according the method previously de- scribed. The results obtained are shown in table 17 and 18.
• Table 17 Analysis of phenolic compound content of tobacco extract before and after enzyme- and bentonite treatment.
• CA Chlorogenic acid
• R Rutin
• S Scopoletin Detection limit: CA: 2.43 ⁇ g/ml; R: 1.55 ⁇ g/ml; S: 0.50 ⁇ g/ml
• BDL Below detection limit
• Table 18 Content of various tobacco components in tobacco before and after extraction and recombination with the enzyme- and bentonite treated extract.

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