Felby, Claus (Kiselvej 7E 2th Herlev, DK-2730, DK)
Berker, Otto (Elmevej 6 Bjerringbro, DK-8850, DK)
|1.||A process for preparing a cork article, the method comprising: i) treating cork fragments with a phenol oxidising enzyme, and ii) pressing the treated cork fragments into a cork article.|
|2.||A process according to claim 1, wherein the cork article is in the form of a board, a panel or a laminate.|
|3.||A process according to claims 1 or 2, wherein step ii) is performed at a temperature above 70°C, preferably above 80°C, more preferably above 90°C, even more prefera bly above 100°C, most preferably above 110°C, in particular above 150°C, such as above 175°C.|
|4.||A process according to claims 1 or 2, wherein step ii) is performed at a temperature in the interval of from 70°C to 300°C, preferably in an interval of from 80°C to 250°C, more preferably in an interval of from 90°C to 240°C, even more preferably in an inter val of from 100°C to 230°C, most preferably in an interval of from 110°C to 220°C.|
|5.||A process according to any of the preceding claims, wherein the treated cork frag ments are dried before pressing.|
|6.||A process according to any of the preceding claims, wherein the density of the cork article is at the most 400 kg/m3, preferably at the most 350 kg/m3, such as at the most 300 kg/m3, in particular at the most 250 kg/m3, such as at the most 200 kg/m3.|
|7.||A process according to any of the preceding claims, wherein the process is carried out in the absence of binders, in particular in the absence of uric formaldehyde resins, melamine formaldehyde resins, phenol formaldehyde resins and diisocyanates.|
|8.||A process according to any of the preceding claims, wherein the cork fragments are treated with an oxidising agent prior to treatment with a phenol oxidising enzyme.|
|9.||A process according to claim 8, wherein the oxidizing agent is a peroxide, preferably hydrogen peroxide.|
|10.||A process according to any of the preceding claims, wherein the cork fragments are in the form of granules and/or crumbs.|
|11.||A process according to any of the preceding claims, wherein the phenol oxidising enzyme is a phenolic oxidase or a peroxidase.|
|12.||A process according to claim 11, wherein the phenolic oxidase is selected from the group consisting of catechol oxidase, laccase and mixtures thereof.|
|13.||A process according to claim 12, wherein the phenolic oxidase is a laccase.|
|14.||A cork article obtainable by the process defined in any of claims 113.|
|15.||A cork article according to claim 14, wherein the cork article is obtained by the process defined in any of claims 113.|
|16.||A process for conglutinating cork fragments, which comprises the step of treating the cork fragments with a phenol oxidising enzyme.|
|17.||A process according to claim 16, wherein the process is carried out as defined in any of claims 113.|
Background of the Invention Cork is bark from the cork oak, e. g. Quercus suber L, which grows predominately in countries near the Mediterranean Sea.
The production of cork stoppers commences with the stripping of the reproduction bark from the tree to provide cork slabs. These slabs are stored for up to two years.
Usually, the slabs are then graded and often bundled, boiled in water, and stacked once more.
The further production steps vary according to the actual cork stopper type, the intended use thereof, and any special demands the end-user might have.
Natural cork stoppers are inter alia used as closures for wine bottes. Such stoppers are cut or punched generally in the cork slab longitudinal plane.
Natural cork slices or disks are used inter alia in the production of laminate cork stoppers, which are typically used as closures for champagne bottes. Moreover, cork slices are used for sealing purposes at the inner bottom of various screw caps. Slices are usually cut perpendicular to the longitudinal direction of the cork slab.
About 50% of the slab material can be used for natural cork stoppers or slices. The remaining material, viz. cork pieces or crumbs of varying size, is generally used for preparing granulate cork, or may be used directly in the heating system of the factory as an energy source.
Granulate cork can be used for various purposes, among other things in the production of agglomerated stoppers. To this end the crumbs are ground, cleaned and classifie into various particle ranges. Binders, and if desired other additives such as plasticizers and cutting aids, are mixed with the granulated cork of the desired particle size, and the composition is moulded or extruded into the desired shape. Optionally, the thus formed product is then polished.
Granulate cork may, however, also be used for making boards, insulating materials etc. Whenever the granulate cork is to be used as cork stoppers, cork boards or other formed articles, the granulate must be glued (or conglutinated) with a binder in form of a synthetic resin and formed into the desired form under pressure and heat.
In the field of gluing together cork particles (or fragments) to form boards, etc. the cork (typically cork granules and/or crumbs) are normally conglutinated with binders, such as uric formaldehyde resins, melamine formaldehyde resins, phenol formaldehyde resins and/or diisocyanates as binding components.
The use of synthetic resins of the above-mentioned type is, however, generally undesirable from an environmental and/or safety point of view, since many such adhesives are directly toxic or can, at a later stage, give rise to release of toxic and environmentally harmful substances.
The present inventors have now surprisingly found that a phenol oxidising enzyme may be used to conglutinate cork fragments, in particular to conglutinate cork granules and/or cork crumbs, thereby avoiding the use of the above-mentioned hazardous binders.
WO 99/58309 discloses a method for reducing the off taste in wine by treating cork stoppers, or alternatively the cork slices from which the stoppers are produced, with a phenol oxidising enzyme.
US 5,505,772 discloses a process for conglutinating wood fragments via activation of the lignin in the middle lamella of the wood fibres by incubating the wood fragments with a phenol oxidising enzyme.
WO 95/07604 discloses a method of producing fibreboard having improved mechanical properties by adding a phenol oxidising enzyme system, and WO 95/09946 discloses a process for producing linerboard or corrugated medium having increased strength by treatment with a phenol oxidising enzyme system.
Summary of the Invention It is an object of the present invention to provide a process for preparing cork articles, such as a cork boards, by conglutinating cork fragments, such as cork granulats and/or crumbs. This object is achieved by treating the cork fragments with a phenol oxidising enzyme, followed by pressing the treated cork fragments into a cork article.
Thus, in a first aspect, the present invention relates to a process for preparing a cork article, the method comprising: i) treating cork fragments with a phenol oxidising enzyme, and ii) pressing the treated cork fragments into a cork article.
In a second aspect, the invention relates to a cork article obtainable by, in particular obtained by, the process according to the invention.
In a third aspect the present invention relates to a process for conglutinating cork fragments, which comprises the step of treating the cork fragments with a phenol oxidising enzyme.
Detailed Description of the Invention Cork and cork articles The composition of a cork material depends on its growth conditions, however a typical natural cork contains around 16% (w/w) lignin, 4% (w/w) tannins, and various
amounts of organic compounds, such as resorcinol, hydroquinone, salicylic acid, and sterols.
When used herein, the term"cork fragment"includes fragments of bark from trees, in particular from cork oaks, whatever the physical form thereof. In a particular preferred embodiment, the cork fragments to be treated is in the form of crumbs, granules or mixtures thereof.
Crumbs may be defined as small cork particles/fragments of various shape and being at the most 50 mm in the longest dimension, preferably at the most 40 mm, such as at the most 30 mm, in particular at the most 20 mm, e. g. at the most 10 mm, such as at the most 5 mm in the longest dimension.
Granules typically have a substantially spherical or angular shape. The dimensions (diameter) of such granules will in general be at the most 50 mm in the longest dimension, preferably at the most 40 mm, such as at the most 30 mm, in particular at the most 20 mm, e. g. at the most 10 mm, such as at the most 5 mm in the longest dimension.
In the present context, a"cork article"is a shaped article, which contains cork. In other words: A cork article is the result of performing one or more process steps for which cork fragments, e. g. granulate cork, are used as a raw material. A typical cork article is an article of commerce. Preferred cork articles or products are formed articles. In particular, board shaped materials, which can be obtained by the process according to the invention, can be pre-formed in any desired mould, and then processed with appropriate pressing devices in a known manner, e. g. into boards such as insulating boards, panels, laminates, formed elements for the packing industry etc. In the present context, cork closures or cork closure components, e. g. cork stoppers, cork slices or cork disks are also considered as being"cork articles".
The cork articles obtained by the process according to the invention typically have a density of at the most 400 kg/m3, preferably at the most 350 kg/m3, such as at the most 300 kg/m3, in particular at the most 250 kg/m3, such as at the most 200 kg/m3.
Phenol oxidising enzyme An oxidation is an electron transfer reaction between two reactants: A donor looses an electron, an acceptor gains the electron; one of the reactants is oxidised (the electron donor), the other reactant is reduced (the acceptor). Enzymes catalysing such reactions are called oxidoreductases.
Phenol oxidising enzymes constitute a subgroup of the oxidoreductases and when used herein, the term"phenol oxidising enzyme"is intended to cover any oxidoreductase acting on phenols and related substances (such as substituted phenols) as donors and with oxygen or hydrogen peroxide as acceptors. This definition includes enzymes derived from animals, plants and microorganisms, as well as mutants and variants thereof, which retain their phenol oxidising enzymatic activity.
Generally, when used herein, the term"phenol oxidising enzyme"includes whatever compounds necessary for the actual enzyme to work, i. e. for instance an appropriate acceptor. Such acceptor may or may not be naturally present in the reaction system.
However, whenever it is desirable to underline the presence of the acceptor, the concept of a"phenol oxidising enzyme system"can be used, viz. to mean a phenol oxidising enzyme plus its acceptor.
Other components, such as activators etc., are included in the concept of a"phenol oxidising enzyme system"to the extent such components are desirable for the enzyme to work optimally under the actual conditions. This optimisation of the enzyme- catalyse reaction is a matter of routine for the skilled man, once a specific enzyme has been selected.
Examples of suitable activators are described in WO 95/01426, which is hereby incorporated by reference. These activators may be described by the below general formula:
The definition of the R1 to R10 and A groups can be found in WO 95/010426 (see pp. 9 to 11).
Specifically contemplated compounds encompassed by the above formula include the following: 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonate (ABTS); 6-hydroxy-2- naphtoic acid; 7-methoxy-2-naphtol; 7-amino-2-naphthalene sulfonic acid; 5-amino-2- naphthalene sulfonic acid; 10- methylphenothiazine; 10-phenothiazine-propionic acid (PPT); N-hydroxysuccinimide-10- phenothiazine-propionate; benzidine; 3,3'-dimethoxybenzidine; 3,3', 4'-hydroxy-4-biphenylcarboxylic acid; 4-amino-4'- methoxystilbene; 4,4'-diaminostilbene-2, 2'-disulfonic acid; 4,4'-diaminodiphenylamine; triphenylamine; 10-ethyl-4- phenothiazinecarboxylic acid; 10-ethylphenothiazine; 10-propylphenothiazine; 10- isopropylphenothiazine; methyl-10-phenothiazinepropionate; 10-phenylphenothiazine; 10-allylphenothiazine; 10-phenoxazinepropionic acid (POP); 10- (3- (4-methyl-1- piperazinyl) propyl) phenothiazine; 10- (2-pyrrolidinoethyl) phenothiazine; 10-methyl- phenoxazine; iminostilbene; 2- (p-aminophenyl)-6-methylbenzothiazole-7-sulfonic acid; N-benzylidene-4-biphenylamine; 5-amino-2-naphthalenesulfonic acid; 7-methoxy-2- naphtol; 4,4'-dihydroxybenzophenone; N- (4- (dimethylamino) benzylidene)-p-anisidine; 3- methyl-2-benzothiazolinone (4- (dimethylamino) benzylidene) hydrazone; 2-acethyl-10- methylphenothiazine; 10- (2-hydroxyethyl) phenothiazine; 10- (2-hydroxyethyl) phenoxa- zine; 10- (3-hydroxypropyl) phenothiazine; 4,4'-dimethoxy-N-methyl-diphenylamine, vanil- lin azine.
Other activators contemplated include 4-hydroxybenzoic acid, L-tyrosine, syringate ac- ids, ferulic acid, sinapic acid, Chlorogenic acid, caffeic acid and esters thereof.
Still further examples include the organic compounds described in WO 96/10079, which is hereby incorporated by reference. These activators may be described by the below general formula:
wherein A is a group, such as-D,-CH=CH-D,-CH=CH-CH=CH-D,-CH=N-D,-N=N-D, or -N=CH-D, in which D is selected from the group consisting of-CO-E,-SO2-E,-N-XY, and -N+-XYZ, in which E may be-H,-OH,-R, or-OR, and X and Y and Z may be identical or different and selected from-H and-R; R being a C,-C, 6 alkyl, preferably a Cl-C, alkyl, which alkyl may be saturated or unsaturated, branched or unbranched and optionally substituted with a carboxy, sulfo or amino group; and B and C may be the same or dif- ferent and selected from CmH2m+,, wherein 1 sm <_5.
Specific compounds covered by the above formula are acetosyringone, syringaldehyde, methylsyringate, syringic acid, ethylsyringate, propylsyringate, butylsyringate, hexylsy- ringate, octylsyringate and ethyl 3- (4-hydroxy-3, 5-dimethoxyphenyl).
Other suitable activators are vanillic acid, NHA, HOBT, PPO and violoric acid.
In the present context, the term"phenol"or"phenols"means any compound which comprises at least one phenolic ring structure, i. e. an aromatic ring structure, in particular a benzene ring structure or a condensed benzene ring structure, with at least one OH-substituent at a carbon ring atom. The phenolic ring structure may, in addition to the OH-substituent, contain other substitituents, including more OH-groups.
Examples of classes of compounds which fall within this definition of"phenol"include (mono) phenols, as well as polyphenols, such as di-, tri-, tetra-, penta-and hexaphenols. Also comprised in this definition are tannins (see e. g. Grant & Hackh's
Chemical Dictionary, 5t edition, McGraw-Hill Book Company, 1987, p. 574, hereby incorporated by reference).
Preferred phenol oxidising enzymes In a preferred embodiment of the present invention, the phenol oxidising enzyme is a phenolic oxidase or a peroxidase.
Examples of preferred phenolic oxidases are enzymes of classes EC 1.13.-.- ; EC 1.14.-.-and EC 1.10.3.-, in particular any of the classes EC 126.96.36.199-188.8.131.52.
Examples of preferred referred peroxidases are enzymes of the class EC 184.108.40.206.
(Enzyme Nomenclature, 1992, Published for the International Union of Biochemistry and Molecular Biology (IUBMB) by Academic Press, Inc.; 1992).
The group EC 220.127.116.11 comprises peroxidases, catalysing oxidation reactions in which a donor is oxidised, and wherein hydrogen peroxide is acting as the acceptor.
The group EC 1.10.3.- comprises enzymes acting on di-phenols and related substances as donors with oxygen as acceptor. Preferred enzymes of this class are: Catechol oxidases (EC 18.104.22.168); laccases (also namede urishiol oxidases, EC 22.214.171.124); and o-aminophenol oxidases (EC 126.96.36.199). Monophenols, however, are also very good substrates.
The group EC 188.8.131.52 comprises monophenol monooxygenase (also sometimes named tyrosinase, phenolase, monophenol oxidase, or cresolase).
The phenol oxidising enzymes are preferably purified, viz. only minor amounts of other proteins being present. The expression"other proteins"relate in particular to other enzymes. Preferably, the enzymes are at least 75% (w/w) pure, more preferably at least 80%, at least 85%, at least 90% or even at least 95% pure. In a still more preferred embodiment the phenol oxidising enzyme is at least 98% pure, such as at least 99% pure.
Preferred phenol oxidising enzymes are listed below. Any enzymatically active variants or mutants thereof are also preferred phenol oxidising enzymes. The activities thereof can be measured by any method known in the art.
Suitable peroxidases may be any peroxidase enzyme comprised by the enzyme classification (EC 184.108.40.206), or any fragment derived therefrom, exhibiting peroxidase activity. Preferably, the peroxidase is derived from plants (e. g. horseradish or soybean peroxidase) or microorganisms such as fungi or bacteria. Some preferred fungi include strains belonging to the subdivision Deuteromycotina, class Hypho-mycetes, e. g., Fusarium, Humicola, Trichoderma, Myrothecium, Verticillum, Arthromyces, Caldariomyces, Ulocladium, Embellisia, Cladosporium or Dreschlera, in particular Fusarium oxysporum (DSM 2672), Humicola insolens, Trichoderma resii, Myrothecium verrucana (IFO 6113), Verticillium alboatrum, Verticillum dahlie, Arthromyces ramosus (FERM P-7754), Caldariomyces fumago, Ulocladium chartarum, Embellisia alli or Dreschiera 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. NA-12) or Trametes (previously called Polyporus), e. g. T. versicolor (e. g. PR4 28-A).
Further preferred 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 (ATTC 23965), Strep-tomyces thermoviolaceus (IFO 12382) or Streptoverticillum verticillium ssp. verticillium.
Still other preferred bacteria include Bacillus pumilus (ATCC 12905), Bacillus stearothermophilus, Rhodobacter sphaeroides, Rhodomonas palustri, Streptococcus lactis, Pseudomonas purrocinia (ATCC 15958) or Pseudomonas fluorescens (NRRL B-11).
Further preferred bacteria include strains belonging to Myxococcus, e. g., M. virescens.
Particularly, 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, or a variant thereof, e. g., a variant as described in WO 94/12621.
Laccase enzymes of microbial and plant origin are well known. A suitable microbial laccase enzyme may be derived from bacteria or fungi (including filamentous fungi and yeasts) and suitable examples include a laccase derivable from a strain of Neurospora, e. g., N. crassa, Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus, Trametes, e. g., T. villosa and T. versicolor, Rhizoctonia, e. g., R. solani, Coprinus, e. g. C. plicatilis and C. cinereus, Psatyrella, Myceliophthora, e. g. M. thermophila, Scytalidium, Polyporus, e. g., P. pinsitus, Phlebia, e. g., P. radita (WO 92/01046), or Coriolus, e. g., C. hirsutus (JP 2-238885), in particular laccases obtainable from Trametes, Myceliophthora, Scytalidium or Polyporus.
A suitable catechol oxidase may be 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 (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).
Treatment with phenol oxidising enzyme and pressing The treatment of cork fragments with a phenol oxidising enzyme can be performed in various ways. Basically, a composition comprising such enzyme (the enzyme preparation, be it liquid or dry) is applied to or brought into contact with the cork fragments. The enzyme preparation is preferably liquid. Preferred methods of treating cork fragments with liquid enzyme preparations include dipping, spraying, immersing or injecting.
As wili be acknowledged by the skilied person, cork is non-fibrous. It is composed of tiny closely packed cells that are tetrakaidecahedral (or 14-sided). Six of the faces are quadrilateral and eight are hexagonal. This shape provides optimum packing of the cells without extra void space, which accounts for the excellent gasketing and flotation characteristics of cork. The cells are typically in the range of 0.025-0.050 mm in the longest dimension. The cell walls are highly resistant to water, most organic liquids, and all but strong acid and alkaline solutions.
It is well known (vide supra) that phenol oxidising enzymes may be used to improve the strength of paper, boards, etc. prepared from a fibrous pulp. In these cases the enzyme has access to the lignin present in the middle lamella between the individual fibres and, consequently, a relatively large amount of lignin is susceptible to enzyme- catalyse cross-linking. Cork fragments, such as cork granules and/or crumbs, however, are not fibrous materials. Thus, only limited amounts of lignin, namely the iignin present on the surface of the individual cork fragments, are susceptible to cross- linking. The present inventors have now surprisingly found that, despite the relatively low amount of accessible lignin in cork fragments as compared to the relatively larger amount of accessible lignin present in a fibrous pulp, it is possible to obtain cross- linking of lignin which, in turn, leads to conglutination (or adhesion) of the cork fragments.
Another interesting aspect of the present invention therefore relates to a process for conglutinating (or gluing together) cork fragments, which comprises the step of treating the cork fragments with a phenol oxidising enzyme. Preferably the cork fragments to be conglutinated (or glued together) is in the form of granules and/or crumbs.
As will be understood from the above discussion, the lignin present on the surface of the cork fragments is cross-linked, thereby given rise to adhesion between individual cork fragments, such as cork granules and/or crumbs. Consequently, a further interesting aspect of the present invention relates to a process for cross-linking lignin present on the surface of cork fragments, in particular on the surface of cork granules and/or cork crumbs. As explained above, the macroscopic effect of the cross-linking is an improved adherence between individual cork fragments.
The interaction between the enzyme and the cork fragments may be enhanced, and the enzymatic effect on the cork fragments thus improved, by any means, which improve the contact between enzyme and cork and/or the access of the enzyme to cork surface areas.
The following is a non-exclusive list of means improving such contact and/or access ("contact-improving means"): Means which minimise the water repelling effect of the cork surface, e. g. surface tension-lowering compounds and compositions; organic solvents or treatment with an oxidising agent; mechanical means such as ultrasonic treatment, aeration, stirring, vacuum or pressure. Any combination of these means may be used.
In a preferred embodiment, suitable solvents, preferably an alcohol such as ethanol, are added to the enzyme-containing treatment liquid.
Thus, in a preferred embodiment of the present invention, the enzyme treatment takes place in a liquid comprising water and ethanol. A preferred amount of ethanol is in the range of 1-30%, preferably 2-25%, more preferably 3-20%, even more preferably 5- 15% (all percentages in vol/vol). Consequently, a preferred amount of water is in the range of 70-99%, preferably 75-98%, more preferably 80-97%, even more preferably 85-95%.
In another interesting embodiment of the invention, the enzymatic treatment takes place in an ultrasonic bath.
In a particular interesting embodiment of the invention, the cork fragments are treated with an oxidising agent prior to treatment with a phenol oxidising enzyme. Any com- monly used oxidising agent, which will be known to the person skilled in the art, may be used. Preferably, the oxidising agent is a peroxide, in particular hydrogen peroxide.
The use of peroxide is particular advantageous as small residual amounts of peroxide may easily be removed by a peroxide degrading enzyme, e. g. a catalase, such as Catazyme (available from Novo Nordisk A/S). A suitable concentration of hydrogen
peroxide in the enzyme-containing treatment liquid will be in the range of from 0.1 % to 10% (w/w), such as in the range of from 1% to 5% (w/w).
The step of treating cork fragments with a phenol oxidising enzyme can be performed at any step in the preparation of cork articles. Preferably, the treatment should take place following the granulation and before compression (pressing) of the particles into formed articles.
Appropriate conditions, under which the treatment of cork fragments with a phenol oxidising enzyme should occur, is dependent on the characteristics of the enzyme of choice.
Examples of typical conditions are listed below. In general, any of the below- mentioned conditions may constitute the basis for further optimisation of the process.
A generally preferred pH of the liquid, wherein the enzymatic treatment of the cork fragments takes place, is pH 3-10, preferably 3.5-9, more preferably 4-8, still more preferably 4-7.
A generally preferred temperature in the enzymatic treatment step of the invention is 10-90°C, preferably 20-80°C, more preferably 30-70°C, still more preferably 40-65°C.
The preferred temperature will, of course, be dependent on the temperature optimum and the temperature stability of the enzyme in question.
A generally preferred enzymatic treatment time is 5 minutes to 5 hours, preferably 5 minutes to 4 hours, more preferably 15 minutes to 3 hours, still more preferably 15 minutes to 1 hour.
The concentration of oxygen as acceptor (relevant to the use of phenolic oxidases only, viz. e. g. laccase) is not critical. At 25°C and in a normal atmosphere, water has an equilibrium concentration of oxygen of around 200! 1M, which is usually fully sufficient for the enzyme reactions to occur in a satisfactory way. If desired, the concentration of oxygen in the liquid can be increased, e. g. to saturation.
The concentration of hydrogenperoxide as acceptor (relevant to the use of peroxidase only) is generally not critical. However, the selected peroxidase enzyme could be sensible to the concentration hydrogenperoxide (thereby loosing activity). Preferably, the concentration range of hydrogenperoxide is 0.010-10 mM, more preferably 0.020- 8 mM, still more preferably 0.05-5 mM, even more preferably 0.100-2.5 mM.
Generally, a preferred dosage of the phenol oxidising enzyme to dry cork is 0.01- 1000 ppm (w/w) enzyme protein, preferably 0.1-500 ppm (w/w), more preferably 0.2- 200 ppm (w/w). The amount of enzyme protein can be measured using any method known in the art. These dosage values are preferably based on purified enzyme protein, purified being defined as indicated above.
Before being pressed into the desired shape, the enzyme-treated cork fragments are preferably dried so that the treated cork fragments have an absoute moisture content of at the most 40%, i. e. a moisture content, which is at the most 40% by weight based on the moisture-containing cork fragments. Preferably, the enzyme-treated cork fragments are dried so that the absolute moisture content is at the most 30%, such as the most 20%, more preferably at the most 15%. The drying step may be performed by any method known in the art, e. g. heating, evaporation under reduced pressure, a combination thereof, as well as other method known to the person skilled in the art.
Compression (or pressing) of the enzyme-treated cork fragments into the desired cork article may be carried out in a discontinuous operation as well as in continuous operation processes, the so-called pass-through process or conti-machines. The specific conditions, including specific equipment to be used, will be well-known to the skilled person.
In order to obtain sufficient conglutination, the pressing step must be performed at ele- vated temperatures, in particular at temperature above 70°C. Preferably, the pressing step is performed at a temperature above 80°C, more preferably above 90°C, even more preferably above 100°C, most preferably above 110°C, in particular above 120°C, such as above 150°C, e. g. above 175°C.
On the other hand, the temperature should not be too high due to the risk of carboni- zation. Thus, suitable temperature intervals under which the pressing may take place are in the interval of from 70°C to 300°C, preferably in the interval of from 80°C to 250°C, more preferably in the interval of from 90°C to 240°C, even more preferably in the interval of from 100°C to 230°C, most preferably in the interval of from 110°C to 220°C, in particular in the interval of from 120°C to 220°C.
As will be understood by the skilled person, one particular advantage of the process described herein is that cork articles may be prepared from cork fragments (or, in other words, cork may be conglutinated) by the process according to the invention in the ab- sence of binders, in particular in the absence of uric formaldehyde resins, melamine formaldehyde resins, phenol formaldehyde resins and diisocyanates.
The present invention is further illustrated by the following non-limiting examples. As it appears from the results provided herein, it is possible to obtain cork articles, which have excellent properties, and which have been prepared by the process described herein.
EXAMPLES Example 1: Treatment of Cork Granules with a Phenol Oxidising Enzyme 30 kg cork granules (about 1-10 mm) was treated with 280 I water and 17 130% H202 in a rotating drum. The liquid was drained after 30 minutes and replace by another 300 1 of water and 300 ml Catazyme@ (Novo Nordisk A/S, Denmark) in order to re- move residual amounts of H202. The granules were treated for 30 minutes after which the liquid was drained. Another 300 1 of water was added, pH adjusted to 5.5. with acetic acid, and 3.7 g of Myceliophthora thermophila laccase was added. The granules were treated for 50 minutes at room temperature after which the liquid was drained and the granules dried by forced air to a water content of about 10-15%. 10 mm boards were made by pressing a layer of granules to stops at 200°C for 9 minutes. Af- ter a resting period of 10 minutes, the board was cut into boards of 50 x 100 cm. The resulting boards had a density of 180 kg/m3.
A similar control board was made without the addition of enzyme.
The enzyme-treated boards were cutable and able to carry their own weight. The con- trol board, however, disintegrated into the granules, which clearly shows that congluti- nation was obtained by the enzyme treatment.
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