(12) United States Patent Kierullf et al.

US006298859B1

(10) Patent N0.: (45) Date of Patent:

US 6,298,859 B1 Oct. 9, 2001

(54) USE OF A PHENOL OXIDIZING ENZYME IN THE TREATMENT OF TOBACCO

(75) Inventors: J esper Vallentin Kierul?', Roskilde; Ole Bill Jorgensen, Farum; Tomas Tage Hansen, Allerod, all of (DK); Anthony James Knox, Ontario; Yves De Grandpré, Quebec, both of (CA)

(73) Assignee: Novozymes A/S, Bagsvaerd (DK)

( * ) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days.

(21) Appl. No.: 09/349,301

(22) Filed: Jul. 7, 1999

Related US. Application Data (60) Provisional application No. 60/092,134, ?led on Jul. 9,

1998.

(30) Foreign Application Priority Data

Jul. s, 1998

(51) Int. Cl.7 ........................... .. A24B 15/24; A24B 3/12; A24B 1/02

(52) US. Cl. ........................ .. 131/297; 131/308; 131/300; 131/309; 131/290

(58) Field of Search ................................... .. 131/290, 297,

131/298, 308, 300, 309, 310

(DK) ........................................... .. 1998 00905

(56) References Cited

U.S. PATENT DOCUMENTS

3,256,888 * 6/1966 De La Burde ..................... .. 131/141

3,561,451 * 2/1971 Jacin et al. .

3,599,645 8/1971 Johnson . 3,927,682 * 12/1975 Matsuyama et al. .......... .. 131/141 R

4,347,324 8/1982 Wildman et al. . 4,887,618 12/1989 Bernasek et al. . 4,941,484 7/1990 Clapp et al. . 5,311,886 5/1994 Grandpre et al. . 5,343,879 9/1994 Teague . 5,360,022 * 11/1994 Newton et al. .................... .. 131/297

5,601,097 * 2/1997 Grandpre et al. .

FOREIGN PATENT DOCUMENTS

2 069 814 *

64 5478 WO 92/01046 WO 92/16634 WO 94/12621 WO 96/00290

9/1981 (GB). 1/1989 (JP). 1/1992 (WO).

10/1992 (WO). 6/1994 (WO). 1/1996 (WO). OTHER PUBLICATIONS

STN International, File CAPLUS, Accession No. 1972151073, Doc. No. 771110753.* Yano et al., (1971) 113:171—173 (Japanese).* Schlotzhauer et al., (1992) 22(3) :231—238.* Coggon et al., (1973) Phytochernistry 12:1947—1955. Motoda et al., (1970) Ferment. Technol. 48(3):154—160. Hearing (1987) 142:155—165. Sharma et al., (1980) Phytochernistry 19:1597—1600. Lerch (1987) Methods in Enzymology 142:165—169. Abstract JP 2238885 A, ACC. N0. 90—331804.

* cited by examiner

Primary Examiner—Stanley S. Silverman Assistant Examiner—Dionne A. Walls (74) Attorney, Agent, or Firm—Jason I. Garbell Esq.; Elias J. Lambiris Esq.

(57) ABSTRACT

A process for preparing tobacco, Which process comprises the steps of treating a tobacco material With a phenol oxidising enzyme, such as by extracting tobacco With a solvent to provide an extract and a residue; and treating the extract With a phenol oxidising enzyme such as a laccase. An improved tobacco product having a reduced amount of phenolic compounds. This is an alternative or a supplement to a process in Which the phenolic compounds are adsorbed onto the insoluble carrier polyvinylpolypyrrolidone (PVPP). In preferred embodiments, the process includes further steps of removing the oxidised phenolic compound, adding adsor bents such as bentonite; removing and/or inactivating the enzyme; and concentrating the extract. Preferred phenol oxidising enzymes are peroxidases and laccases. The thus treated extract is advantageously re-combined With the tobacco residue and further processed to provide a tobacco article for smoking.

33 Claims, 14 Drawing Sheets

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US 6,298,859 B1 1

USE OF A PHENOL OXIDIZING ENZYME IN THE TREATMENT OF TOBACCO

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 USC 119 of Danish application PA 1998 00905 ?led Jul. 8, 1998, and of US. Provisional application 60/092,134 ?led Jul. 9, 1998, the contents of Which are fully incorporated herein by reference.

TECHNICAL FIELD

This invention relates to the preparation and treatment of tobacco. More speci?cally, the invention relates to such processes in Which a tobacco material is treated With a phenol oxidising enZyme to provide an improved tobacco material.

BACKGROUND ART

Processes for improving tobacco quality and varying tobacco ?avour are constantly being sought. With these overall goals in mind, efforts are currently spent on ia the removal of proteins and phenolic compounds such as polyphenols from tobacco, see eg US. Pat. No. 5,601,097, Which relates to a method for reducing protein and polyphe nol content of a tobacco material.

It has been reported that of the polyphenols present in tobacco leaves chlorogenic acid is predominant and that loW levels of chlorogenic acid in tobacco leaves leads to loW levels of the undesired component catechol in tobacco smoke, SchlotZhauer W.S. (1992), Journal of Analytical and Applied Pyrolysis. Vol 22, page 231—238. A process for removing phenolic compounds from

tobacco Which makes use of solid adsorbents such as alumina, is disclosed in US. Pat. No. 3,561,451. US. Pat. No. 5,601,097 discloses the use of another insoluble adsorbent, viZ. polyvinylpolypyrrolidone (PVPP), in such process. HoWever, 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 improv ing 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.

SUMMARY OF THE INVENTION

In a ?rst aspect, 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.

In one aspect, the invention provides a method for reduc ing the amount of phenolic compounds in a tobacco material, in Which method the tobacco material is treated With a phenol oxidising enZyme.

According to the invention, an improved tobacco material having a reduced content of phenolic compounds is achieved by treating a tobacco extract With a phenol oxidising enzyme.

In a further aspect, 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.

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2 In further aspects, 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, ?avour, aroma, taste and/or colour thereby increasing the versatility of the tobacco products on the market.

In a further aspect, 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.

In another aspect, the invention relates to tobacco mate rials obtainable, in particular obtained, by any of the pro cesses described herein. The invention encompasses the ?nal, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Binary gradient pro?le for HPLC analysis of phenols, see Methods and procedures in Examples.

FIG. 2: Spectra of nicotine before and after treatment With laccase, see Example 1.

FIG. 3: Spectra of chlorogenic acid before and after treatment With laccase, see Example 1.

FIG. 4: Spectra of rutin before and after treatment With laccase, see Example 1.

FIG. 5: Spectra of scopoletin before and after treatment With laccase, see Example 1.

FIG. 6: HPLC chromatogram of mixture of phenol stan dards: rutin, 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.

FIG. 8: HPLC chromatogram of tobacco extract spiked With rutin, scopoletin, and chlorogenic acid. The tWo chro matograms are identical except for the scale, see Example 2.

FIG. 9: Series of HPLC chromatograms of samples of tobacco extract as a function of time When treated With 1.6 pig/ml TvL at pH 5.5 and 55° C. The tWo sets of chromato grams are identical except for the scale, see Example 2.

FIG. 10: FloW chart shoWing process-?oW and indicating Which samples have been prepared, see Example 8.

FIG. 11: Chemical structure of Rutin.

FIG. 12: Chemical structure of Scopoletin.

FIG. 13: Chemical structure of Chlorogenic acid.

DETAILED DISCLOSURE OF THE INVENTION

The present invention provides a process for preparing a

tobacco product, Which process comprises the step of treating a tobacco material With a phenol oxidising enZyme.

The expression “tobacco material” as used herein 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. Thus, the expression “tobacco material” includes, Without limitation, tobacco solids and any solid form of tobacco, such as, e.g., cured tobacco (such as ?ue-cured tobacco); uncured tobacco (so-called green tobacco); dried, aged, cut, ground, stripped or shredded tobacco; tobacco scrap; expanded tobacco,

US 6,298,859 B1 3

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. Prefer ably the tobacco material used as a starting material for present invention is the lamina portion of the tobacco leaf. In a preferred embodiment, the tobacco material is in part or totally composed of cured tobacco. In particular, this expres sion covers the tobacco raW material entering a tobacco preparation or treatment process. In speci?c embodiments of the invention, 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.

The term “tobacco product” denotes the product resulting from any of the processes of the invention. Included are, Without limitation, the ?nal, 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. Thus, the term “tobacco product” includes the ?nal 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 treating, i.e. contacting, a tobacco material With a phenol modifying enZyme, preferably a phenol oxidiZing enZyme, and most preferably a polyphenol oxidiZing enZyme. In one embodi ment said enZyme is not monophenol monooxygenase (EC 1.14.18.1).

In preferred embodiments, 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 oxid ising enZyme, is performed during or after step (ii) and before step (iii), or step is performed on the extract after step (iii).

In one embodiment, 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; 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. HoWever, 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. By aqueous extraction the Water soluble phenolic com

pounds of the tobacco material Will partition into the aque

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4 ous extract, together With i.a. nicotine, soluble proteins, sugars, amino acids, pectins, inorganic salts and the surfac tants used, if any.

Thus, in general it is preferred that in the process of the invention an aqueous solvent is used 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. In one embodiment of the invention, the extraction of the tobacco material is performed With an aqueous solvent composed of 100% Water.

Accordingly, the aqueous solvent may comprise addi tional components other than Water, such as, eg alcohols such as ethanol or methanol; or other Water miscible sol vents 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, dimethylformamide, diethylene glycol, dim ethyl ether, dimethyl sulfoxide, diethylene glycol monom ethyl ether, ethyleneglycol, diethyleneglycol, sulpholane, glycerol or triethanolamine.

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, eg 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 extrac tion of the tobacco material With the aqueous solvent may come before or after the extraction of the tobacco material With the organic solvent. In one embodiment of the invention, the extraction of the tobacco material is per formed With an aqueous solvent. Optionally, 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 dimethyl propylene Urea, N-methylpyrrolidone, acetone, propan-2 ol, propan-1-ol, ethyleneglycol dimethyl ether, ethylenegly col monomethyl ether, tetrahydrofuran, 1-butanol, 2-butanol, isobutanol, tert-butanol, 1,4-dioxane, morpholine, dimethylformamide, diethylene glycol dim ethyl ether, dimethyl sulfoxide, diethylene glycol monom ethyl 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, methyl isobutyl ketone, pentane, hexane or dioxane, dichloromethane, ethyl acetate, cyclohexane, ligroin, petro leum ether, toluene, xylenes, anisol. At the stage of treatment With the phenol oxidising

enZyme the environment of the extraction mixture or the extract alone, i.e. substantially Without the tobacco residue, should be so that the enZyme is capable of being active. Normally, this requires that the enZymatic treatment is performed in an aqueous solvent phase in contrast to an organic solvent phase. If the extraction of the tobacco material is performed using an organic solvent, it is pre ferred that an aqueous extraction step is performed as a

US 6,298,859 B1 5

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. If a mixture of Water soluble organic solvents and Water is used, the content of organic solvent may be decreased by conventional methods for removal of organic solvents, such as evaporation or freezing (cooling).

In a preferred embodiment a tobacco material is ?rst extracted With an aqueous solvent, preferably Water. Subse quently 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) may be disregarded or used, preferably folloWing a liquid-liquid extraction to pro vide and aqueous phase, for combination With a tobacco material, such as eg a phenol oxidising enzyme-treated tobacco extract.

According to the invention it is preferred, but not required, that the extraction step be performed under con ditions maximising the extraction of soluble phenolic com pounds. HoWever even partial extraction is useful for ulti mately reducing the concentration of phenolic compounds in the ?nal tobacco product. Some examples of appropriate extraction conditions are listed beloW. Generally, any of these conditions can be optimised using only routine experi mentation by establishing a matrix of conditions and testing different points in the matrix.

Suitable extraction process conditions are eg a tempera ture 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 1/2 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 advan tageously 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 alkylsulfates, sodium or potassium salts of oarboxylic acids, sodium alkylarylsulfonates, sodium alkylsulfosuccinates and mixtures of any of the foregoing. In particular are considered surfactants having a chain length of betWeen 8 and 12 carbon atoms. In speci?c embodiments of the invention, the surfactant is one or more of sodium dodecylsulfate, sodium dodecylbenzenesulfonate and sodium dioctylsulfosuccinate (Aerosol OTTM). The surfac tant is preferably added to the solvent at a concentration range of 0.01%—5% W/v solution.

According the invention, having provided an extraction mixture, the tobacco residue is preferably separated from the extract either before or after (preferably before) the treat ment With a phenol oxidising enzyme. The separation can be performed using any methods, including, Without limitation, centrifugation, ?ltration, sedimentation, decanting or siev ing or any combinations thereof. Particularly preferred methods of separation are ?ltration and centrifugation.

It is preferred that the surfactants, if any, are removed from the extract, said extract is preferably an aqueous

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6 extract. This may be done by cooling, eg to 4° C. causing the surfactants to precipitate and/or precipitation using eg inorganic calcium or magnesium salt folloWed by eg cen trifugation. The term “tobacco residue” as used herein refers to the

solid tobacco material resulting from extraction of a “tobacco material” With Whatever kind of extraction liquor folloWed by separation (eg by ?ltration 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.

The term “tobacco extract” refers to the liquid fraction (being clari?ed or not) resulting from extraction of “tobacco material” With Whatever kind of extraction liquor folloWed by separation (eg by ?ltration or centrifugation) from the solid extraction material (“tobacco residue”). Accordingly, 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. The term “extraction mixture” as used herein refers to a

suspension resulting from an extraction of a tobacco mate rial; the suspension comprising a solid fraction and a liquid fraction. Phenol Oxidising Enzyme, Step

According to the invention, it is essential that the tobacco material, preferably in the form of an aqueous extraction mixture or aqueous extract, is treated With a phenol oxidis ing enzyme. This step is described in greater detail in the folloWing.

In the present context, the term “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 oxi dising 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 (ie one or more

electrons), an acceptor gains an electron (ie 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 oxi dising enzymes derived from prokaryotic or eukaryotic organisms, such as animals, plants or microorganisms (such as eg bacteria or fungi—including ?lamentous fungi and yeast). In one embodiment, the process of the invention utilises a phenol oxidising enzyme derived from tobacco.

The term “derived” means in this context that the enzyme may have been isolated from an organism Where it is present natively, ie the identity of the amino acid sequence of the enzyme are corresponding to a native enzyme. The term “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 modi?ed amino acid sequence, e.g. having one or more amino acids Which are

deleted, inserted and/or substituted, ie a recombinantly produced enzyme Which is a mutant and/or a fragment of a native amino acid sequence. Within the meaning of a native enzyme are included natural variants. Furthermore, the term “derived” includes enzymes produced synthetically by eg peptide synthesis. The term “derived” also encompasses enzymes Which have been modi?ed eg by glycosylation, phosphorylation etc., Whether in vivo or in vitro.

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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. With respect to recom binantly produced enZyme 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. The expression “an enZyme obtainable from an

organism”, Whether an eukaryotic or a prokaryotic 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 cor responding to a native enZyme.

Thus, the term “derived” in the context of “a phenol oxidising enZyme” encompassed phenol oxidising enZymes (such as, eg a laccase) obtainable from animals, plants or microorganisms such as bacteria or fungi (including ?la mentous fungi and yeast) as Well as mutants, fragments or variants thereof With phenol oxidising enZymatic activity. One embodiment of the invention comprises use of a phenol oxidising enZyme obtainable from tobacco.

Accordingly, the phenol oxidising enZyme may be obtained from a microorganism by use of any suitable technique. For instance, a phenol oxidising enZyme prepa ration may be obtained by fermentation of a suitable micro organism and subsequent isolation of a phenol oxidising enZyme containing preparation from the resulting fermented broth or microorganism by methods knoWn in the art. Preferably the phenol oxidising enZyme preparation is obtained by use of recombinant DNA techniques. Such method normally comprises cultivation of a host cell trans formed 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:

In the present context, the concept of “phenolic com pounds” and “phenols” refers to any compound Which comprises at least one phenolic ring structure, ie an aro matic 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 de?nition, in particular comprises (mono)phenols, as Well as polyphenols, such as di-, tri-, tetra-, penta- and hexaphenols.

The term “monophenol” encompasses a compound com prising one hydroxy group attached to an aromatic ring system.

The term di-, tri-, tetra-, -penta and hexaphenols encom passes a compound comprising a total of tWo, tree, four, ?ve or six hydroxy groups, respectively, and one or more aro matic ring systems, Where the hydroxy groups are attached to the same or different aromatic rings.

The term “polyphenol” as used herein refers to a com pound comprising 2 or more hydroxy groups attached to one

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8 or more aromatic ring systems, as used herein such com pounds are also termed “polyhydroxy phenols”. The term “polyphenol” as used herein also encompasses a compound comprising one aromatic ring having at least tWo hydroxy groups attached. The term “polyphenol” as used herein also includes polymeric material based on phenolic monomers, as used herein such compounds are also termed “polymeric phenols”. The polymeric material may originate from poly merisation reactions of phenolic compounds

Non-limiting examples of phenols relevant to the present invention include ?avanoids, such as rutin (also named rutoside), quercetin (also named 2-(3,4-Dihydroxyphenyl) 3,5,7-trihydroxy-4H-1-benZopyran-4-one; and 3,3‘,4‘,5,7 pentahydroxy?avone), isoquercitrin (also named 2-(3,4 Dihydroxyphenyl)-3-([3-D-glycofuranosyloxy)-5,7 dihydroxy-4H-benZopyran-4-one; and 3,3‘,4‘,5,7 pentahydroxy?avone-3-glycoside; and quercetin-3 glycoside), kaempferol (also named 3,5,7-Trihydroxy-2-(4 hydroxyphenyl)-4H-1-benZopyran-4-one; and 3,4‘,5,7 tetrahydroxy?avone), robinin (also named 3-[[6-O-Deoxy ot-L-mannopyranosyl)-D-galactopyranosyl]oxy]-7-[(6 deoxy-ot-L-mannopyranosyl-)oxy]-5-hydroxy-2-(4 hydroxyphenyl)-4H-1-benZopyran-4-one); coumarins, such as scopoletin (also named 7-Hydroxy-6-methoxy-2H-1 benZopyran-2-one, and 7-hydroxy-6-methoxycoumarin) and scopolin (also named scopoletin-7-glycoside); and caffetannins, such as isomers of cafeoylquinic acid (3-. 4-. 5-o- caffeoylquinic acid). According to the process of the invention, 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 structure of rutin, scopoletin and chlorogenic acid are shoWn in FIGS. 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. Of special interest are phenolic compounds extract able in an aqueous solvent such as, e.g., the compounds scopoletin, rutin and chlorogenic acid. The term “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 de?ned 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, prefer ably Water soluble, phenolic compound(s). The term “soluble phenolic compounds” includes loW

molecular Weight phenolic compounds. In speci?c embodi ments of the invention, 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. In one embodiment of the invention, the term refers to phenolic “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. In a preferred embodiment of the invention, a polymerisation reaction—caused by the treat ment With a phenol oxidising enZyme—proceeds until a high

US 6,298,859 B1

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. ultra?ltration. In a. preferred embodiment, the polymeric material becomes insoluble and precipitates.

One embodiment of the invention relates to loW molecular Weight phenolic compounds. In one embodiment, the phe nolic compounds of the invention are loW molecular Weight phenolic compounds soluble in a solvent, preferably an aqueous solvent. Preferred Phenol oxidising EnZymes

Examples of suitable phenol oxidising enZymes, i.e. enZymes Which act on phenolic compounds, such as polyphenols, high molecular Weight as Well as loW molecu lar Weight compounds, include, Without limitation, peroxi dases (EC 1.11.1.7), laccases (EC 1.10.3.2), bilirubin oxi dases (EC 1.3.3.5), monophenol monooxygenases (EC 1.14.181) and catechol oxidases (EC 1.10.3.1).

Preferably, the phenol oxidising enZyme is a phenolic oxidase or a peroxidase. Common to phenolic oxidases is that this group 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. To clarify this, the peroxidase of the invention may also be termed a “phenolic peroxi dase”.

In speci?c embodiments of the invention, 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 enZymes performing 2-electron oxidations. By the terms “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. In 1-electron oxidation a radical is generated. Thus, the enZymes in consideration Will oxidise the phenolic com pounds via generation of a radical. With respect to 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.

The radicals, Whether a phenoxy radical or an other radical, may be detected by EPR (Electron Paramagnetic Resonance) spectroscopy (also called ESR=Electron Spin Resonance spectroscopy). EPR spectroscopy is a very sen sitive and highly speci?c method of detecting radicals, and it can be used to analyse complex matrixes Without exten sive sample pre-treatment such as puri?cation and concen tration. 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).

Examples of enZymes capable of performing 1-electron oxidation are peroxidases (EC 1.11.1.7), laccases (EC

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10 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 1.14.181.) 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 enZyme classes (EC) mentioned herein are as de?ned

in EnZyme Nomenclature, 1992, Published for the Interna tional Union of Biochemistry and Molecular Biology (IUBMB) by Academic Press, Inc.

In speci?c embodiments of the invention, the phenolic oxidase is an enZyme corresponding to EC 1.10.3.- Which comprises enZymes acting on diphenols and related sub stances 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); lac cases (alternative name urishiol oxidases, EC 1.10.3.2); and o-aminophenol oxidases (EC 1.10.3.4). The grouping EC 1.14.18.1 comprises monophenol

monooxygenase (alternative name tyrosinase, phenolase, monophenol oxidase, cresolase). In speci?c embodiments, 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 organ ism 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 ?lamentous 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 pinsitus, and T versicolor, RhiZoctonia, e.g., R. solani, Coprinus, e.g. C. plicatilis and C. cinereus, Psatyrella, Myceliophthora, e.g. M. thermophila, Scytalidium, e.g. S. thermophilum, Polyporus, e.g., P. pinsitus, Phlebia, e.g., P. radita (WO 92/01046), or Coriolus, e.g., C. hirsutus (JP 2-238885), Rhus, e.g. R. vernicifera, Pycnoporus, e.g. R cinnabarious, in particular laccases derived from Trametes, Myceliophthora, Scytalidium or Polyporus. In a preferred embodiment, the phenol oxidising enZyme is a laccase derived from tobacco. A suitable catechol oxidase or monophenol monooxyge

nases may be derived from animals, plants or microorganism such as bacteria or fungi (including ?lamentous 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). Amammalian monophenol monooxygenase (tyrosinase) has been described (Methods EnZymol., 1987, 142, 154—165). Other suitable monophenol monooxygenases can be derived from tea leaves (Prikl. Biokhim. Mikrobiol., 1997, 33(1), 53—56), from Chlorella (Ukr. Bot. Zh., 1986, 43(5), 56—59) or from Neurospora crassa (Methods EnZymol., 1987, 142, 165—169).

In a speci?c embodiment of the invention the phenol oxidising enZyme is not monophenol monooxygenase (EC 1.14.18.1).