WO1997031088A1

WO1997031088A1 - Coated peroxidase-containing preparation, and compositions comprising such a preparation

Info

Publication number: WO1997031088A1
Application number: PCT/DK1997/000079
Authority: WO
Inventors: Ture Damhus; Kirsten Bøegh LEVRING
Original assignee: Novo Nordisk A/S
Priority date: 1996-02-20
Filing date: 1997-02-20
Publication date: 1997-08-28
Also published as: AU1719597A

Abstract

A peroxidase-containing preparation which is well suited for incorporation, e.g., in a detergent composition for use in laundry washing consists of peroxidase-containing granules which are coated with a substantially water-insoluble coating. Whilst the coating may comprise an enhancer, the granules per se contain no enhancer.

Description

COATED PEROXIDASE-CONTAINING PREPARATION, AND COMPOSITIONS COMPRISING SUCH A PREPARATION

FIELD OF THE INVENTION

The present invention relates, inter alia , to a coated peroxidase-containing granulate preparation, to a composition comprising such a preparation and an enhancer { vide infra) , and to a detergent composition comprising a granulate preparation or composition of the invention. The invention further relates to a method of fabric washing (i.e. washing of textile or fabric articles, e.g. laundry articles such as articles of clothing and the like) wherein the fabric articles are washed in an aqueous washing medium to which has been added a preparation or a composition of the invention.

BACKGROUND OF THE INVENTION

The concept of dye-transfer inhibition (DTI), i.e. inhibition of the transfer of dye (e.g. dye originating from a dyed fabric undergoing washing) or other colorant to a fabric during washing, is described in, e.g., WO 91/05839, which discloses that the inclusion of an enzyme with peroxidase activity in the wash liquor, in the presence of hydrogen peroxide, or a peroxide precursor or source (e.g. a perborate or percarbonate) , can inhibit dye transfer, and that the dye-transfer inhibitory effect can be enhanced by the addition of "another oxidisable substrate" (see page 10, lines 7-19, of WO 91/05839) , the first oxidisable substrate being the dye in question. For the sake of brevity, an oxidisable substance or species of this type which gives rise to enhanced dye-transfer inhibitory effect when used in conjunction with a peroxidase will be referred to hereinafter in the present specification with claims as an "enhancer" . Such substances are also known as "mediators" or "accelerators". In the course of studying the behaviour of a number of peroxidase-containing compositions in the context of the present invention, an investigation was made of the effect of encapsulating or coating peroxidase in granule form with various coating materials which have been employed in the art, e.g. a water-dissolvable material such as a polyethylene glycol (PEG) . However, it was observed that when such coated granules were incorporated in finished detergent compositions comprising a peroxide source, the storage stability of the peroxidase was often too poor (i.e. the rate of loss of peroxidase activity during storage was too high) .

Moreover, it has been observed that the inclusion of a peroxidase together with a peroxide source and an enhancer in cleaning compositions, such as detergent compositions for laundry washing or for dishwashing, can be problematical when the composition in question further contains other enzymes, e.g. proteases, lipases, cutinases, amylases and/or cellulases, added e.g. for the purpose of aiding in the removal of various kinds of soil or stain from the article (s) to be washed or cleaned. Thus, the rather aggressive oxidizing effect of peroxidase/peroxide/enhancer systems can result in the inactivation, during the wash cycle, of other enzymes present before those enzymes have been able to perform their intended function to a satisfactory extent.

SUMMARY OF THE INVENTION

It has now surprisingly been found that peroxidases in the form of peroxidase-containing granules (in the following sometimes denoted "peroxidase granulate" or "peroxidase-containing granulate") which have been appropriately coated, more particularly with a coating which is substantially insoluble (or of low solubility) in water, exhibit very satisfactory storage stability. Moreover, it also appears that the use of such a coated peroxidase granulate as a peroxidase source in washing/cleaning compositions of the types mentioned above, e.g. detergent compositions for laundry washing, results in significant reduction or elimination of the above-described, unwanted, oxidative side-effects of peroxidase systems with respect to inactivation of other enzymes present before they have exerted their intended effect.

An aspect of the present invention thus relates to a peroxidase- containing preparation in the form of peroxidase-containing granules (i.e. a "granulate") coated with a substantially water- insoluble or poorly water-soluble coating. The peroxidase- containing granules in question do not contain an enhancer [i.e. an enhancer as described above in the context of dye-transfer inhibition (vide supra)] .

Moreover, peroxidase-containing preparations of the invention are, inter alia, useful in connection with the inhibition of dye- transfer (i.e. transfer of dye or other colorant to fabric) and/or bleaching of stains during fabric washing (e.g. laundry washing) . It is believed that the dissolution/disintegration characteristics conferred upon preparations of the invention by the substantially water-insoluble coating extend the usefulness of preparations of the invention in the context of dye-transfer inhibition to cases in which the dye-releasing ("dye-bleeding") fabric or textile which is to be washed is one which exhibits relatively slow release of dye under the washing conditions in question (such a fabric or textile sometimes being referred to as a "slow bleeder") .

For DTI or bleaching (e.g. stain-bleaching or textile bleaching) purposes, an enhancer [e.g. one of the enhancers mentioned below, very suitably a substantially water-insoluble or poorly water- soluble enhancer, such as an enhancer of the syringic acid ester type or the phenothiazine type (e.g. methyl syringate or 10-phenothiazinepropionic acid)] may, for example, very suitably be incorporated in the coating material which coats the peroxidase-containing granules in a preparation of the invention.

In some cases it may, however, be advantageous that the preparation per se, as a whole, does not contain an enhancer (i.e. that the coating also contains no enhancer) . Thus, a peroxidase-containing preparation of the invention may, alternatively, be employed as a component of a composition which is suitable for use, for example, as a dye-transfer inhibitory composition or a stain-bleaching composition and which further contains an enhancer as a separate component [e.g. enhancer in the form of (optionally coated) granules, or in some other form] .

Accordingly, another aspect of the present invention relates to a composition which comprises a peroxidase-containing preparation according to the invention and an enhancer. Such compositions may suitably further comprise a source of peroxide (e.g. a source of hydrogen peroxide) , such as e.g. a percarbonate and/or a perborate.

A peroxidase-containing preparation or composition of the invention may suitably be employed, for example, as an additive which is incorporated in a detergent composition. A further aspect of the present invention thus relates to the use of such a preparation or composition according to the invention in the manufacture of a detergent formulation for washing, especially laundry washing. The invention also encompasses detergent compositions comprising a peroxidase-containing preparation or composition of the invention.

The invention further relates to a method of fabric or laundry washing, wherein fabric articles or articles of laundry are washed in an aqueous washing medium to which has been added a peroxidase-containing preparation, a peroxidase- and enhancer- containing composition, or a detergent composition according to the invention. A peroxide source will normally also have been introduced into the washing medium in question, typically either as a constituent of a peroxidase- and enhancer-containing composition of the invention, or as a constituent of a detergent composition of the invention.

DETAILED DESCRIPTION OF THE INVENTION Peroxidases

Peroxidase enzymes (EC 1.11.1) employed in the context of the invention may very suitably be, e.g., any peroxidase comprised by the enzyme classification EC 1.11.1.7; peroxidase fragments, ex¬ hibiting peroxidase activity, as well as synthetic or semisynthetic peroxidase derivatives [e.g. with porphyrin ring systems, or microperoxidases (see, e.g., US 4,077,768, EP 0 537 381, WO 91/05858 and WO 92/16634)] are also relevant in the context of the invention. Suitable peroxidases are obtainable from microbial, plant and animal sources.

Preferred peroxidases in the context of the invention are peroxidases obtainable from plants (e.g. horseradish peroxidase or soy bean peroxidase) or from microorganisms such as fungi or bacteria. Some preferred fungi include strains belonging to the subdivision Deuteromycotina, class Hyphomycetes, e.g. Fusarium,

Humicola , Tricoderma , Myrothecium, Verticillu , Arthrotnyces , Cal - dario yces, Ulocladium, Embellisia, Cladosporium or Dreschlera, in particular Fusarium oxysporum (DSM 2672) , Humicola insolens,

Trichoderma resii , Myrothecium verrucana (IFO 6113) , Verticillum alboatrum, Verticillum dahlie, Arthromyces ra oεus (FERM P-7754),

Caldariomyces fu ago, 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. 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 5 verticillium ssp. verticillium

Other preferred bacteria include Bacillus pumilus (ATCC 12905) , Bacillus stearothermophilus , Rhodobacter sphaeroides, Rhodomonas palustri , Streptococcus lactis, Pseudomonas purrocinia (ATCC 10 15958) or Pseudomonas fluorescens (NRRL B-ll) .

Further preferred bacteria include strains belonging to Myxococcus, e.g. M. virescenε .

15 Other relevant peroxidases are "haloperoxidases" (see, e.g., US 4,937,192), such as chloride peroxidases (EC 1.11.1.10), bromide peroxidases, and iodide peroxidases (EC 1.11.1.8) .

Other potential sources of useful particular peroxidases are 20 listed in B.C. Saunders et al. , Peroxidase, London, 1964, pp. 41- 43.

The peroxidase may furthermore be one which is producible by a method comprising cultivating a host cell transformed with a 25 recombinant DNA vector which carries a DNA sequence encoding said peroxidase as well as DNA sequences encoding functions permitting the expression of the DNA sequence encoding the peroxidase, in a culture medium under conditions permitting the expression of the peroxidase and recovering the peroxidase from the culture.

30

A suitable recombinantly produced peroxidase is a peroxidase derived from a Coprinus sp., in particular C. macrorhizus or C. cinereus according to WO 92/16634.

35 As already indicated to some extent above, the term peroxidase as employed in the context of the invention embraces substances possessing peroxidase activity, such as peroxidase-active fragments derived from cytochro es, hemoglobin or peroxidase enzymes, and synthetic or semisynthetic derivatives thereof, e.g. iron porphins, iron porphyrins and iron phthalocyanine and 5 derivatives thereof.

The peroxidase employed in a preparation of the invention will very suitably often be a Coprinus peroxidase, a Myxococcus peroxidase, or a horseradish peroxidase.

10

Preferred embodiments of a peroxidase-containing preparation according to the invention contain peroxidase in an amount of from 0.01 to 100 mg enzyme protein/g of preparation, preferably 0.1-20 mg enzyme protein/g of preparation.

15

Enhancers

The enhancer in a preparation or composition of the invention can be any suitable peroxidase enhancer. Examples of enhancers include the following: halide ions (e.g. chloride and bromide) ;

20 certain metal ions (e.g. Mn²⁺) ; phenolic species [e.g. acetosyringone, syringaldehyde, syringic acid, alkyl syringates (such as methyl, ethyl, propyl, butyl, hexyl or octyl syringate) , ethyl 3- (4-hydroxy-3, 5-dimethoxyphenyl)acrylate, p-hydroxycinnamic acid, 2,4-dichlorophenol, vanillin,

25 7-hydroxycoumarin, 6-hydroxy-2-naphthoic acid, and p-hydroxy- benzenesulfonate] ; 2,2 ' -azino-bis (3-ethylbenzothiazoline-6- sulfonate) (ABTS; see, e.g., WO 94/12620) ; and 10-methyl-, 10- ethyl- and 10-propylphenothiazine (see, e.g., WO 94/12621) . Other suitable enhancers are disclosed in, e.g., WO 94/12619, WO

30 94/12620 and WO 94/12621.

Enhancers of the syringate, phenoxazine or phenothiazine type are generally very suitable in the context of the invention, and some preferred enhancers are acetosyringone, methyl syringate, 35 10-phenothiazinepropionic acid, 10-ethylpheno-thiazine-4- carboxylic acid, 10-phenoxazinepropionic acid and 10- methylphenoxazine (described in WO 94/12621) . As demonstrated in working examples herein ( vide infra) , the use, in fabric washing, of compositions according to the invention which comprise such enhancers appears to result in, for example, very satisfactory dye-transfer inhibition.

Enhancer is suitably present in a composition of the invention in an amount of from 10^"8 to 10^"1 mol/g of preparation, e.g. from 10^"7 to 2 'lO^'2 mol/g, such as from 10^"7 to 10^"2 mol/g of preparation. Amounts in the range of from 10^"5 to 10^"3 mol/g of preparation will often be appropriate. When enhancer is incorporated in the coating of a peroxidase-containing preparation of the invention, it may suitably be incorporated in an amount in the range of 1- 25% by weight (calculated on the basis of the weight of the uncoated, dry granulate) , more preferably 2-15% by weight, such as an amount in the range of 5-10% by weight (calculated in the manner in question) .

Coating

Suitable coatings are coatings which, under washing conditions, ensure appropriate release of peroxidase whilst ensuring satisfactory storage stability of the preparation or composition of the invention. As already mentioned, preferred coatings are coatings which are substantially insoluble (or poorly soluble) in water. Coatings which are appropriate, e.g., in the context of dye-transfer inhibition or stain bleaching in washing media may, for example, comprise substances (coating agents) selected from the following: tallow; hydrogenated tallow (e.g. hydrogenated beef tallow) ; partially hydrolyzed tallow; hydrogenated oils of plant origin (e.g. hydrogenated palm oil) ; fatty acids and fatty alcohols of natural and synthetic origin; long-chain fatty acid mono-, di- and triesters of glycerol (e.g. glycerol monostearate) ; ethoxylated fatty alcohols; latexes; hydrocarbons of melting point in the range of 50-80°C; and waxes.

Melt-coating agents are a preferred class of coating agents which can be used without dilution with water. Reference may be made to Controlled Release Systems : Fabrication Technology, Vol . I, CRC Press, 1988, for further information on appropriate coatings.

The coating may suitably further comprise substances such as clays (e.g. kaolin) , titanium dioxide, pigments, salts (such as calcium carbonate) and the like. The person skilled in the art will be aware of further coating constituents of relevance in the present context. As already mentioned, an enhancer may be incorporated in the coating in certain embodiments of a peroxidase-containing preparation of the invention.

The coating will normally be applied to the peroxidase-containing granulate in an amount in the range of 3-50% by weight (calculated on the basis of the weight of the uncoated, dry granulate) , such as in the range of 5-50% w/w, e.g. 8-40% w/w. An amount in the range of 10-40% by weight will often be appropriate. The amount of coating to be applied to the granulate will depend to a considerable extent on the nature and composition of the desired coating.

Granules

The peroxidase-containing granules (i.e. disregarding any coating) of a preparation of the invention may suitably further contain various granulation aids, binders, fillers, lubricants, polymers and the like. Examples hereof include cellulose (e.g. cellulose in fibre or microcrystalline form) , dextrins (e.g. yellow dextrin) , polyvinylpyrrolidone, polyvinylalcohol, cellulose derivatives (such as CMC or hydroxypropylcellulose) , gelatin, salts (e.g. sodium sulfate, sodium chloride, calcium sulfate or calcium carbonate) , titanium dioxide, talc and clays

(e.g. kaolin or bentonite) .

Other materials of relevance for incorporation in granules of the type in question are described, for example, in EP 0 304 331 Bl, and will be well known to persons skilled in the art. Methods and apparatus for producing enzyme-containing granulates are likewise well known to the skilled person (see, e.g., EP 0 304 331 Bl) . Compact granulates - produced, e.g., using apparatus comprising knives as described in Example 1 in US Patent No. 4,106,991 - constitute very suitable granulates in the context of the invention.

Detergent composition

As already mentioned, the present invention also relates to a detergent composition. The detergent composition will normally contain between 0.01 and 25% by weight (w/w) of a peroxide- and enhancer-containing preparation or composition of the invention. Numerous embodiments of such a detergent composition will contain between 0.01 and 10% w/w of a peroxide- and enhancer-containing preparation or composition of the invention, such as between 0.1 and 2% w/w thereof. A detergent composition according to the invention may be in any convenient form, e.g. in the form of granules, granules/powder, or paste containing a preparation or composition of the invention.

A detergent composition of the invention will further comprise a source of peroxide (e.g. a source of hydrogen peroxide) . The peroxide source in question may (as already mentioned above) be present, for example, as a component of a peroxidase- and enhancer-containing composition of the invention which is used in the formulation of the detergent composition, or it may be incorporated in the detergent composition as a separate ingredient thereof.

Suitable peroxide sources are well known, and include, e.g., sodium perborates and sodium percarbonate. Such peroxide sources are incorporated into certain types of detergent formulations as bleaching systems directed at oxidizable stains in laundry.

A detergent composition according to the invention comprises one or more surfactants, each of which may be anionic, nonionic, cationic, or zwitterionic. It will usually contain 0-50% of anionic surfactant such as linear alkylbenzenesulfonate (LAS) , alpha-olefinsulfonate (AOS) , alkyl sulfate (fatty alcohol sulfate) (AS) , alcohol ethoxysulfate (AEOS or AES) , secondary alkanesulfonates (SAS) , alpha-sulfo fatty acid methyl esters, alkyl- or alkenylsuccinic acid or soap. It may also contain 0-40% of nonionic surfactant such as alcohol ethoxylate (AEO or AE) , carboxylated alcohol ethoxylates, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, or polyhydroxy alkyl fatty acid amide (e.g. as described in WO 92/06154) .

A detergent composition according to the invention may additionally comprise one or more other enzymes, such as an amylase, a lipase, a cutinase, a protease, a cellulase or an oxidase (e.g. a laccase) .

Enzymes (e.g. proteases) incorporated in the detergent composition according to the invention may be stabilized by means of conventional stabilizing agents, e.g. a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, a boric acid derivative such as, e.g., an aromatic borate ester, or an organoboronic or -borinic acid, and the detergent according to the invention may be formulated as described in, e.g., WO 92/19709 and WO 92/19708.

Where appropriate, enzymes may be incorporated in the detergent composition in the form of, e.g., a non-dusting granulate or a protected enzyme. Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and 4,661,452 (both to Novo Industri A/S) and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molecular weights of 1000 to 20000. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in patent GB 1483591. Protected enzymes may be prepared according to the method disclosed in EP 238,216. A detergent composition according to the invention may contain 1- 65% of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, citrate, nitrilotriacetic acid (NTA) , ethylenediaminetetraacetic acid (EDTA) , diethylenetriaminepentaacetic acid (DTMPA) , alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst) . A detergent composition according to the invention may also be unbuilt, i.e. essentially free of detergent builder.

A detergent composition according to the invention may comprise one or more polymers. Examples are carboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP) , polyethyleneglycol (PEG) , poly(vinyl alcohol) (PVA) , polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.

A detergent composition according to the invention may further comprise a peracid-forming bleach activator such as tetraacetylethylenediamine (TAED) or nonanoyloxybenzenesulfonate (NOBS) . Alternatively, the detergent composition may comprise a peroxyacid, e.g. a peroxyacid of the amide, imide or sulfone type.

A detergent composition according to the invention may also contain other conventional detergent ingredients such as e.g. fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti- soil redeposition agents, dyes, bactericides, optical brighteners, or perfume.

The pH (measured in aqueous solution at use concentration) will usually be neutral or alkaline, e.g. in the range of 7-11.

Particular forms of detergent composition according to the invention include: 1) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising

Linear alkylbenzenesulfonate (cal¬ 7 - 12% culated as acid)

Alcohol ethoxysulfate (e.g. C₁₂._lβ alcohol, 1-2 EO) or alkyl sulfate 1 - 4% (e.g. C_lβ._lβ)

Alcohol ethoxylate (e.g. C_14-1S alco¬ hol, 5 - 9% 7 EO)

Sodium carbonate (as Na₂C0₃) 14 - 20%

Soluble silicate (as Na₂0,2Si0₂) 2 - 6%

Zeolite (as NaAlSiO 15 - 22%

Sodium sulfate (as Na₂S0₄) 0 - 6%

Sodium citrate/citric acid (as C₆H₅Na₃0₇/C₆H₈0₇) 0 - 15%

Sodium perborate (as NaB0₃.H₂0) 11 - 18%

TAED 2 - 6%

Carboxymethylcellulose 0 - 2%

Polymers (e.g. maleic/acrylic acid copolymer, PVP, PEG) 0 - 3%

Enzymes (calculated as pure enzyme 0.00001 - 2% protein)

Enhancer and other ingredients (e.g. suds suppressors, perfume, optical up to 5% brightener, photobleach)

2) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising

Linear alkylbenzenesulfonate (cal¬ culated as acid) 6 - 11%

Alcohol ethoxysulfate (e.g. C_12-18 alcohol, 1-2 EO or alkyl sulfate 0 - 3% (e.g. C₁₆._lβ)

Alcohol ethoxylate (e.g. of C_l2-ιs alcohol, 3EO-7EO or C₁₄.₁₅ alcohol, 3 - 9% 7 EO)

Sodium carbonate (as Na₂C0₃) 15 - 21%

Soluble silicate (as Na₂0,2Si0₂) 1 - 4%

Zeolite (as NaAlSiO 24 - 34%

Sodium sulfate (as Na₂S0₄) 4 - 10%

Sodium citrate/citric acid (as C₆H₅Na₃0₇/C₆H₈0₇) 0 - 15%

Carboxymethylcellulose 0 - 2%

Polymers (e.g. maleic/acrylic acid copolymer, PVP, PEG) 1 - 6%

Enzymes (calculated as pure enzyme 0.00001 - 2% protein)

Enhancer and other ingredients (e.g. up to 5% suds suppressors, perfume)

3) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising

Linear alkylbenzenesulfonate (cal¬ 5 - 9% culated as acid)

Alcohol ethoxylate (e.g. C_{12 15} alco¬ hol, 7 - 14% 7 EO)

Soap as fatty acid (e.g. C₁₆_₂₂ fatty 1 - 3% acid)

Sodium carbonate (as Na₂C0₃) 10 - 17%

Soluble silicate (as Na₂0,2Si0₂) 3 - 9%

Zeolite (as NaAlSiO 23 - 33%

Sodium sulfate (as Na₂S04) 0 - 4%

Sodium perborate (as NaB0₃.H₂0) 8 - 16%

TAED 2 - 8% Phosphonate (e.g. EDTMPA) 0 - 1%

Carboxymethylcellulose 0 - 2%

Polymers (e.g. maleic/acrylic acid copolymer, PVP, PEG) 0 - 3%

Enzymes (calculated as pure enzyme 0.00001 - 2% protein)

Enhancer ingredients (e.g. suds suppressors, perfume, optical up to 5% brightener)

4) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising

Linear alkylbenzenesulfonate (cal¬ 8 12% culated as acid)

Alcohol ethoxylate (e.g. C_{12 1S} alco¬ hol, 10 25% 7 EO)

Sodium carbonate (as Na₂C0₃) 14 22%

Soluble silicate (as Na₂0,2Si0₂) 1 5%

Zeolite (as NaAlSiO 25 35%

Sodium sulfate (as Na₂S0₄) 0 10%

Carboxymethylcellulose 0 2%

Polymers (e.g. maleic/acrylic acid copolymer, PVP, PEG) 1 3%

Enzymes (calculated as pure enzyme 0.00001 - 2% protein)

Enhancer and other ingredients (e.g. up to 5% suds suppressors, perfume)

5) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising

Fatty alcohol sulfate 5 10%

Ethoxylated fatty acid monoethanol¬ 3 9% amide

Soap as fatty acid 0 3%

Sodium carbonate (as Na₂C0₃) 5 10%

Soluble silicate (as Na₂0,2Si0₂) 1 4%

Zeolite (as NaAlSiO 20 40%

Sodium sulfate (as Na₂S0₄) 2 8%

Sodium perborate (as NaB0₃.H₂0) 12 18%

TAED 2 7%

Polymers (e.g. maleic/acrylic acid 1 5% copolymer, PEG)

Enzymes (calculated as pure enzyme 0.00001 - 2% protein)

Enhancer and other ingredients (e.g. optical brightener, suds suppressors, up to 5% perfume)

6) A detergent composition formulated as a granulate comprising

Linear alkylbenzenesulfonate (calculated as acid) 8 - 14%

Ethoxylated fatty acid monoethanol¬ 5 - 11% amide

Soap as fatty acid 0 - 3%

Sodium carbonate (as Na₂C0₃) 4 - 10%

Soluble silicate (as Na₂0,2Si0₂) 1 - 4%

Zeolite (as NaAlSi0₄) 30 - 50%

Sodium sulfate (as Na₂S0₄) 3 - 11%

Sodium citrate (as C₆H₅Na₃0₇) 5 - 12%

Polymers (e.g. PVP, maleic/acrylic 1 - 5% acid copolymer, PEG)

Enzymes (calculated as pure enzyme 0.00001 - 2% protein)

Enhancer and other ingredients (e.g. up to 5% suds suppressors, perfume) 7) A detergent composition formulated as a granulate comprising

Linear alkylbenzenesulfonate (calculated as acid) 6 - 12%

Nonionic surfactant 1 - 4%

Soap as fatty acid 2 - 6%

Sodium carbonate (as Na₂C0₃) 14 - 22%

Zeolite (as NaAlSiO 18 - 32%

Sodium sulfate (as Na₂S0₄) 5 - 20%

Sodium citrate (as C₆H_sNa₃0₇) 3 - 8%

Sodium perborate (as NaB0₃.H₂0) 4 - 9%

Bleach activator (e.g. NOBS or TAED) 1 - 5%

Carboxymethylcellulose 0 - 2%

Polymers (e.g. polycarboxylate or 1 - 5% PEG)

Enzymes (calculated as pure enzyme 0.00001 - 2% protein)

Enhancer and other ingredients (e.g. up to 5% optical brightener, perfume)

8) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising

Anionic surfactant (linear alkylbenzenesulfonate, alkyl sulfa¬ te, alpha-olefinsulfonate, alpha- 25 - 40% sulfo fatty acid methyl esters, alkanesulfonates, soap)

Nonionic surfactant (e.g. alcohol 1 - 10% ethoxylate)

Sodium carbonate (as Na₂C0₃) 8 - 25%

Soluble silicates (as Na₂0, 2Si0₂) 5 - 15%

Sodium sulfate (as Na₂S0₄) 0 - 5%

Zeolite (as NaAlSi0₄) 15 - 28%

Sodium perborate (as NaB0₃.4H₂0) 0 - 20%

Bleach activator (TAED or NOBS) 0 - 5%

Enzymes (calculated as pure enzyme 0.00001 - 2% protein)

Enhancer and other ingredients (e.g. up to 3% perfume, optical brighteners)

9) Detergent formulations as described in 1) - 8) wherein all or part of the linear alkyIbenzenesulfonate is replaced by (C₁₂-C_lβ) alkyl sulfate.

10) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising

( i2"^c _ιβ) alkyl sulfate 9 - 15%

Alcohol ethoxylate 3 - 6%

Polyhydroxy alkyl fatty acid amide 1 - 5%

Zeolite (as NaAlSi0₄) 10 - 20%

Layered disilicate (e.g. SK56 from 10 - 20% Hoechst)

Sodium carbonate (as Na₂C0₃) 3 - 12%

Soluble silicate (as Na₂0,2Si0₂) 0 - 6%

Sodium citrate 4 - 8%

Sodium percarbonate 13 - 22%

TAED 3 - 8% Polymers (e.g. polycarboxylates and 0 - 5% PVP=

Enzymes (calculated as pure enzyme 0.00001 - 2% protein)

Enhancer and other ingredients (e.g. optical brightener, photo bleach, up to 5% perfume, suds suppressors)

11) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising

(C₁₂-C_1B) alkyl sulfate 4 - 8%

Alcohol ethoxylate 11 - 15%

Soap 1 - 4%

Zeolite MAP or zeolite A 35 - 45%

Sodium carbonate (as Na₂C0₃) 2 - 8%

Soluble silicate (as Na₂0,2Si0₂) 0 - 4%

Sodium percarbonate 13 - 22%

TAED 1 - 8%

Carboxymethyl cellulose 0 - 3%

Polymers (e.g. polycarboxylates and 0 - 3% PVP)

Enzymes (calculated as pure enzyme 0.00001 - 2% protein)

Enhancer and other ingredients (e.g. up to 3% optical brightener, phosphonate, perfume)

12) Detergent formulations as described in 1) - 11) which contain a stabilized or encapsulated peracid, either as an additional component or as a substitute for already specified bleach systems.

10

13) Detergent compositions as described in 1) , 3) , 5) , 7) and 8) wherein perborate is replaced by percarbonate.

14) Detergent compositions as described in 1), 3), 5) , 7) , 8),

15 10) and 11) which additionally contain a manganese catalyst or other metal-based catalyst. A manganese catalyst may, e.g., be one of the compounds described in "Efficient manganese catalysts for low-temperature bleaching", Nature 369, 1994, pp. 637-639. Other suitable catalysts containing manganese, copper, cobalt or zinc are described in WO 95/30681.

It is at present contemplated that a preparation or composition of the invention will typically be incorporated in the detergent composition of the invention in an amount corresponding to 0.001- 5 mg (calculated as pure enzyme protein) of peroxidase per liter of wash liquor, preferably an amount corresponding to 0.01-2 mg/1, such as 0.1-1 mg/1.

Storage stability testing

In order to reduce the period of time required to complete storage stability testing, i.e. testing of the retention of peroxidase activity as a function of time, of detergent compositions comprising a peroxide source and peroxidase in the form of a peroxidase-containing preparation according to the invention, the tests may suitably be performed as accelerated tests. In such tests, peroxidase-containing preparations are mixed with the other ingredients of the detergent composition, and the mixtures (i.e. the final detergent compositions) are stored under "stressed" conditions (i.e. conditions of elevated temperature and/or elevated relative humidity) for various periods of time (days or weeks) . Residual peroxidase activity in the stored detergent compositions is determined using either a standard analytical enzyme activity assay (e.g. a peroxidase assay based on a chromogenic substrate) or a performance test (e.g. a dye-transfer test performed under specified conditions which simulate washing conditions) .

Compatibility testing

A suitable way to examine the influence of peroxidase/peroxide/enhancer system (as provided by the present invention) on the washing performance of other (i.e. non- peroxidase) detergent enzymes is to carry out a full wash cycle and monitor the enzymatic activity of these other enzymes (determined by an appropriate analytical assay) in samples withdrawn from the washing liquor at selected times during the wash cycle.

Alternatively, changes in overall performance of the other 5 enzymes may be assessed by evaluating their effect on technical test fabrics in a washing cycle in which the peroxidase/peroxide/enhancer system is present in the washing liquor. Such test fabrics are well known in the field of detergency testing; thus, e.g., fabrics soiled with starch or 10 proteinaceous substances may be used for evaluating the performance of amylases and proteases, respectively.

The invention is further illustrated in the following:

15 EXAMPLE 1. Preparation of a peroxidase-containing preparation (coated peroxidase-containing granulate) of the invention

A) Granulation: 2.0 kg of cellulose fibres (Arbocel™ BC 200) , 0.9 kg of kaolin, 1.2 kg of yellow dextrin (TACKIDEX™ G 155) and 20 10.1 kg of sodium sulfate (all dry components) were mixed in a Lόdige mixer.

The above mixed dry components were sprayed, with continuous mixing, with 2.1 kg of liquid peroxidase concentrate (71 mg

25 enzyme protein/g; Coprinus peroxidase produced as described in

Example 1 in EP 505 311) to which had been added (and dissolved) 0.5 kg of sucrose and 0.8 kg of water.

During and after spraying, a compact granulate was formed by 30 means of the knives described in Example 1 in US Patent No. 4,106,991.

When the granulation was finished, the granulate was dried on a fluidized bed. The dry granulate was sieved, and the product 35 fraction of size between 300 and 1000 μm was separated for coating. B) Coating: Granulate prepared as described above was coated with different coatings based on (1) glycerol monostearate, (2) hydrogenated palm oil and (3) hydrogenated beef tallow, 5 respectively, as described in the following. All percentages are weight percentages relative to the dry, uncoated granulate.

(la) : Granulate prepared as in "A)", above, was heated to 80°C in a Lόdige mixer, and 5% of glycerol monostearate (also heated to 10 80°C) was added under continuous mixing. When the glycerol monostearate was distributed, the granulate was powdered with 6.7% of organoclay (Claytone™ AF) and 6.7% of titanium dioxide under continuous mixing.

is After cooling, the coated granulate was sieved and the fraction in the size range 300-1100 μm was collected for use.

(lb) : Granulate prepared as in "A)", above, was heated to 80°C in a Lδdige mixer, and 3% of glycerol monostearate (also heated to 20 80°C) was added under continuous mixing. When the glycerol monostearate was distributed, the granulate was powdered with 3.5% of titanium dioxide and 3.5% of kaolin, and then with a further 3% of glycerol monostearate (heated to 80°C) under continuous mixing.

25

After cooling, the coated granulate was sieved and the fraction in the size range 300-1200 μm was collected for use.

(2) : Granulate prepared as in "A)", above, was heated to 80°C in 30 a Lδdige mixer, and 5% of hydrogenated palm oil (also heated to 80°C) was added under continuous mixing. When the hydrogenated palm oil was distributed, the granulate was powdered with 5% of titanium dioxide and 5% of kaolin, and then with a further 5% of hydrogenated palm oil (heated to 80°C) under continuous mixing. 35 Finally the granulate was powdered with 4.5% of titanium dioxide and 4.5% of kaolin. After cooling, the coated granulate was sieved and the fraction in the size range 300-1200 μm was collected for use.

(3) : Granulate prepared as in "A)", above, was heated to 80°C in a Lδdige mixer, and 3% of hydrogenated beef tallow (also heated to 80°C) was added under continuous mixing. When the hydrogenated beef tallow was distributed, the granulate was powdered with 3.5% of titanium dioxide and 3.5% of kaolin, and then with a further 3% of hydrogenated beef tallow (heated to 80°C) under continuous mixing.

EXAMPLE 2. Compatibility of peroxidase-containing preparations (coated peroxidase-containing granulates) of the invention with another detergent enzyme, and dye-transfer inhibitory perform¬ ance of the preparations

In order to determine the compatibility of coated peroxidase- containing granulates of the present invention with another de¬ tergent enzyme, a washing trial was performed in which the per¬ formance of the detergent protease Alcalase™ in the presence of three different preparations according to the invention and - for comparison purposes - a "traditional" peroxidase-containing granulate, respectively, was examined.

The test material used for assessing protease performance was cotton (Style #400 from Test Fabrics, Inc.; bleached, but with no optical brightener added) soiled with grass juice. After be¬ ing soaked with the grass juice, the material had been air-dried and kept in a freezer for ca. 18 months. Before being employed in the present wash trial, the grass-soiled fabric was pre- rinsed with demineralized water in a washing machine at 30°C. The wash liquor was prepared using a model powder detergent of the following composition:

Ingredient %w/w

Zeolite P 28

Sodium carbonate, anhydrous 19

Sodium hydrogencarbonate 19 1

LAS 7 . 1 Sodium sulfate, anhydrous 6 , . 1

Alkali metal orthosilicate 4 .. 0

Copolymer of acrylic and maleic acids 3 . . 5

Alcohol ethoxylates (C₁₂-C_1S, 3EO-7EO) 3 . , 0

Soap 3 . 0 Carboxymethylcellulose 1 . . 5

Antifoam agent (Dehydran™ 760) 1 . . 0

Phosphonate 0 , . 5

Water ad 100

The powder detergent was dosed to 5 g/1 in tap water adjusted with demineralized water to a hardness of l2°dH, and the pH of this detergent solution was adjusted to 10.0 with sodium hydrox¬ ide.

The wash trial was carried out in a Terg-o-tometer at 40°C and a stirring rate of 70 rotations per minute. Each pot was charged with 1 litre of wash liquor, and the ingredients specified below were added, always in rapid succession (within 1 minute) , in the order in which they are listed.

One part of the wash trial was carried out for the purpose of examining the compatibility issue (comparing - as mentioned above - three peroxidase-containing granulates of the invention with a "traditional", coated peroxidase-containing granulate). The other part of the trial was carried out in order to estab¬ lish that that the three granulates of the invention in question exhibit satisfactory dye-transfer inhibition (DTI) properties. The peroxidase-containing granulates all contained Coprinus per¬ oxidase as in the granulates described in Example 1 ( vide su¬ pra) .

Granulate A: a traditional "T-granulate" coated with poly(ethylene glycol) (PEG) with a mean molar mass of 4000. The preparation of PEG-coated granulates of this type is well known in the art (see, for example, US 4,106,991) .

Granulate B: a granulate prepared in the manner described in Ex¬ ample 1, with a coating of the same type as in "(3)" of part "B) " of Example 1.

Granulate C: a granulate prepared in the manner described in Ex¬ ample 1, with a coating of same type as in " (2)" of part "B) of Example 1.

Granulate D: a granulate prepared in the manner described in Ex- ample 1, with a coating of the same type as in " (lb) " of part "B) " of Example 1.

A solution of the purified peroxidase was also employed as a control.

All four granulates as well as the peroxidase solution were dosed to a level of 0.2 mg of active enzyme protein per liter of wash liquor (based on a fresh analytical determination of the activity of the peroxidase preparations, and the specific activ- ity of the enzyme) . In addition, hydrogen peroxide was dosed to each wash liquor to a concentration of 200 μM, to¬ gether with enhancer [10-phenothiazinepropionic acid (PPT) , pre- dissolved in ethanol] to a concentration of 10 μM.

Protease compatibility

The protease preparation employed was the commercially available product Alcalase™ 2.5L, type DX (Novo Nordisk A/S, Bagsvaerd, Denmark) , dosed in all cases to a level of 0.5 mg of active en¬ zyme protein per litre of wash liquor. A preparatory experiment had shown that the protease performance as measured below is roughly proportional to dosage in the range 0 to 0.5 mg active enzyme protein per litre.

The following components were added to the Terg-o-tometer pots:

Pot #1: wash liquor (w.l.) alone Pot #2 w.l. with PPT, peroxidase solution, and H₂0₂ Pot #3 w.l. with PPT, granulate A, and H₂0₂ Pot #4 w.l. with PPT, granulate B, and H₂0₂ Pot #5 w.l. with PPT, granulate C, and H₂0₂ Pot #6 w.l. with PPT, granulate D, and H₂0₂

Pot #7: w.l. with Alcalase™

Pot #8: w.l. with PPT, peroxidase solution, H₂0₂, and

Alcalase™

Pot #9: w.l. with PPT, granulate A, H₂0₂, and Alcalase™ Pot #10: w.l. with PPT, granulate B, H₂0₂, and Alcalase TM Pot #11: w.l. with PPT, granulate C, H₂0₂, and Alcalase™ Pot #12: w.l. with PPT, granulate D, H₂0₂, and Alcalase™

and in each case, as the last addition, 5 swatches (size 7 cm X 7 cm) of the grass-soiled fabric described above.

After washing for 30 minutes in the Terg-o-tometer, the grass- soiled swatches were rinsed for 10 minutes in cold tap water and air-dried in the dark. The remission R at 460 nm was then meas- ured using a Macbeth Color Eye 7000 remission spectrophotometer. The increase in percentage remission (ΔR) relative to that for the fabric in Pot #1 was taken as a measure of protease perform¬ ance. The protease compatibility part was run in duplicate (runs I and II, respectively) , and the results obtained (average over the values for 4 swatches from each pot) are given below:

Pot # ΔR (average over 4 swatches)

I I I

1 0 0

2 - 1 - 1 3 - 3 0

4 0 0

5 2 1

6 1 0

7 16 15 8 2 4

9 4 3

10 7 12

11 15 16

12 5 7

In general, only changes (ΔR) in remission units of 2-3 or more are visible. Whilst the experimental uncertainty with dissolved components in these experiments is typically around 1-2 remis¬ sion units, the repeatability of treatments involving granulates may be poorer owing to the small amount of granulate weighed out for each pot.

The results shown above for pots #2 to #6 thus demonstrate that for all practical purposes the peroxidase-containing prepara- tions employed do not themselves influence the test fabric. In the treatments involving the protease, it is clearly seen that the effect (a change of about 15 remission units) of the prote¬ ase alone (pot #7) is effectively inhibited by the dissolved, free peroxidase (pot #8) and the traditionally coated granulate (pot #9) , whereas the results for the granulates prepared ac¬ cording to the present invention (pots #10-12) - particularly for granulate C (pot #11) - demonstrate significantly improved protease compatibility of these preparations according to the invention.

Peroxidase performance (dve-transfer inhibition) In order to examine the DTI effect of peroxidase-containing preparations of the invention compared to that of a tradition¬ ally coated peroxidase-containing granulate or of predissolved peroxidase, the peroxidase preparations described above were subjected to a wash in which dye transfer was monitored by in- eluding dye-bleeding fabric as well as tracer fabric on which transferred dye could be detected.

Experimental conditions were the same as for the protease com¬ patibility part of the wash trial, unless otherwise indicated. In each Terg-o-tometer pot, 4 swatches (size 7 cm X 7cm) of "bleeder" fabric dyed with Direct Blue 1 (from Textile Innova¬ tors) were washed together with 2 tracer swatches (cotton, Style #400 from Test Fabrics Inc.) . For reference purposes, 6 swatches of tracer fabric were washed in detergent solution alone in the absence of bleeder fabric.

Pot #1: wash liquor (w.l.) alone with bleeder and tracer fabric

Pot #2: w.l. with PPT, peroxidase solution, and H₂0₂ + bleeder + tracer

Pot #3: w.l. with PPT, granulate A, and H₂0₂ + bleeder + tracer

Pot #4: w.l. with PPT, granulate B, and H₂0₂ + bleeder + tracer Pot #5: w.l. with PPT, granulate C, and H₂0₂ + bleeder + tracer

Pot #6: w.l. with PPT, granulate D, and H₂0₂ + bleeder + tracer

Pot #7: w.l. with tracer fabric alone (reference)

After washing for 15 minutes, the swatches were rinsed and dried as above. The remission spectrum of the tracers was measured and the Hunter color difference ΔE (average over the tracers in the pot) with respect to the (average of) reference tracers from pot #7 was calculated. The results are given below:

Pot # ΔE

1 25

2 16

3 8

4 7 5 18

6 8

In the latter table, the lower the value of ΔE, the better the result. ΔE differences of 2-3 units may typically be perceived visually, and standard deviations are typically in the range of 1-4 ΔE units. It is thus apparent that the performance of the four granulates almost equals (pot #5) or exceeds (pots #3, #4 and #6) the performance of the predissolved peroxidase (pot #2) , and thus that preparations of the invention exhibit satisfactory DTI properties.

Claims

1. A peroxidase-containing preparation in the form of peroxidase- containing granules coated with a substantially water-insoluble coating, said granules containing no enhancer.

2. A preparation according to claim 1, wherein said coating comprises an enhancer.

3. A preparation according to claim 2, wherein said enhancer is selected from the group consisting of: methyl syringate; 10-phenothiazinepropionic acid; 10-ethylphenothiazine-4- carboxylic acid; 10-methylphenoxazine; and 10-phenoxazinepropionic acid.

4. A preparation according to any one of claims 1-3, wherein peroxidase is the only enzymatic component.

5. A preparation according to any one of claims 1-4, wherein said coating comprises a coating agent selected from the group consisting of: tallow; hydrogenated tallow; hydrogenated palm oil; partially hydrolyzed tallow; glycerol monostearate; fatty acids and fatty alcohols of natural and synthetic origin; ethoxylated fatty alcohols; hydrocarbons of melting point in the range of 50-80°C; and waxes.

6. A preparation according to any one of claims 1-5, comprising a peroxidase obtainable from a Coprinus or Myxococcus species, or a horseradish peroxidase.

7. A preparation according to any one of the preceding claims, wherein peroxidase is present in an amount in the range of 0.01 to 100 mg enzyme protein/g of preparation, preferably 0.1 to 20 mg enzyme protein/g of preparation.

8. A composition comprising a preparation according to any one of claims 1-7 and an enhancer.

9. A composition according to claim 8, further comprising a peroxide source.

10. A composition according to claim 8 or 9, wherein said enhancer is selected from the group consisting of: methyl syringate; 10-phenothiazinepropionic acid; 10-ethylphenothiazine- 4-carboxyiic acid; 10-methylphenoxazine; and 10-phenoxazinepropionic acid.

10

11. A composition according to any one of claims 8-10, wherein said enhancer is present in an amount in the range of 10^"7 to 2 -10^"2 mol/g of composition.

15 12. A detergent composition comprising a preparation according to any one of claims 1-7 or a composition according to any one of claims 8-11.

13. A detergent composition according to claim 12, containing 20 between 0.01 and 10% w/w, preferably between 0.1 and 2% w/w, of said preparation or composition.

14. A method of laundry washing, wherein articles of laundry are washed in an aqueous washing medium to which has been added a

25 preparation according to any one of claims 1-7, a composition according to any of claims 8-11, or a detergent composition according to claim 12 or 13.