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WO1999023887A1 - Activite antimicrobienne des lacasses - Google Patents

Activite antimicrobienne des lacasses Download PDF

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Publication number
WO1999023887A1
WO1999023887A1 PCT/DK1998/000477 DK9800477W WO9923887A1 WO 1999023887 A1 WO1999023887 A1 WO 1999023887A1 DK 9800477 W DK9800477 W DK 9800477W WO 9923887 A1 WO9923887 A1 WO 9923887A1
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WO
WIPO (PCT)
Prior art keywords
alkyl
laccase
group
antimicrobial
enhancers
Prior art date
Application number
PCT/DK1998/000477
Other languages
English (en)
Inventor
Charlotte Johansen
Anders Hjelholt Pedersen
Claus Crone Fuglsang
Original Assignee
Novo Nordisk A/S
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novo Nordisk A/S filed Critical Novo Nordisk A/S
Priority to AU97379/98A priority Critical patent/AU9737998A/en
Priority to DE69835370T priority patent/DE69835370T2/de
Priority to CA002308181A priority patent/CA2308181A1/fr
Priority to EP98951297A priority patent/EP1030561B1/fr
Priority to JP2000520002A priority patent/JP2001522784A/ja
Priority to BR9814855-9A priority patent/BR9814855A/pt
Publication of WO1999023887A1 publication Critical patent/WO1999023887A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38654—Preparations containing enzymes, e.g. protease or amylase containing oxidase or reductase
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
    • A61K8/22—Peroxides; Oxygen; Ozone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A61K8/602—Glycosides, e.g. rutin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/66—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/005—Antimicrobial preparations
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
    • C09D5/00—Coating compositions, e.g. paints, varnishes or lacquers, characterised by their physical nature or the effects produced; Filling pastes
    • C09D5/02—Emulsion paints including aerosols
    • C09D5/024—Emulsion paints including aerosols characterised by the additives
    • C09D5/025—Preservatives, e.g. antimicrobial agents
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
    • C09D5/00—Coating compositions, e.g. paints, varnishes or lacquers, characterised by their physical nature or the effects produced; Filling pastes
    • C09D5/14—Paints containing biocides, e.g. fungicides, insecticides or pesticides
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/0005—Other compounding ingredients characterised by their effect
    • C11D3/0078—Compositions for cleaning contact lenses, spectacles or lenses
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/20—Organic compounds containing oxygen
    • C11D3/2003—Alcohols; Phenols
    • C11D3/2006—Monohydric alcohols
    • C11D3/2034—Monohydric alcohols aromatic
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/48—Medical, disinfecting agents, disinfecting, antibacterial, germicidal or antimicrobial compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/52—Stabilizers
    • A61K2800/524—Preservatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q5/00—Preparations for care of the hair

Definitions

  • the present invention relates to a method of antimicrobial treatment using a combination of a laccase enzyme and an enhancer capable of killing or inhibiting microorganisms, more specifically microorganisms present in laundry, on hard surface, on skin, teeth or mucous membranes; and for preserving food products, cosmetics, paints, coatings, etc., the composition comprising a Laccase enzyme, an enhancing agent acting as an electron donor.
  • WO 94/04127 discloses stabilized dentifrice compositions which are capable of producing antimicrobially effective concentrations of hypothiocyanite ions.
  • the compositions contain an oxidase capable of producing hydrogen peroxide and a peroxidase enzyme capable of oxidizing thiocyanate ions, which are normally present in saliva, to antimicrobial hypothiocyanite ions.
  • Suitable peroxidases include lactoperoxidase, myeloperoxidase, salivary peroxidase and chloro-peroxidase .
  • EP-A-0 500 387 enzymatic antimicrobial compositions comprising a haloperoxidase, e.g. myeloperoxidase, eosinophil oxidase, lactoperoxidase and chloroperoxidase, which selectively binds to and inhibits the growth of target microorganisms in the presence of peroxide and halide.
  • a haloperoxidase e.g. myeloperoxidase, eosinophil oxidase, lactoperoxidase and chloroperoxidase
  • WO 95/27046 discloses an antimicrobial composition comprising a Vanadium chloroperoxidase, halide ions, and hydrogen peroxide or a hydrogen peroxide-generating agent.
  • Laccase is an enzyme catalyzing the oxidation of substrates according to the general formula:
  • the enzyme has been used in the paper and pulp industry (WO 94/29510, for bleaching purposes in laundry washing (WO 91/05839, EP 91610032, DE4008894, JP-A64-60693) , but the use of the enzyme in for antimicrobial purposes was not suggested.
  • WO 96/10079 discloses a method for oxidizing a compound with a phenol oxidizing enzyme in the presence of an enhancer of the general formula:
  • WO 97/28257 and WO 97/00038 discloses a methods for inactivating a microorganism or virus with polyphenol oxidases derived from the bacterium of the genus Bacillus in the presence of oxygen and an enhancer.
  • the object of this invention is to provide a method for antimicrobial treatment of microbial cells or microorganisms, i.e. for disinfection or preservation, which is easy to use and an effective alternative to the known disinfecting and preserving methods.
  • Treatment of microorganisms and/or viruses comprising treating said microorganisms and/or viruses with an effective amount of fungal Laccase enzymes and an effective amount of one or more enhancers in the presence of oxygen (0 2 ) , the enhancer being of the formula:
  • an enzymatic antimicrobial method comprising treatment of microorganisms and/or viruses with effective amounts of Laccase and said enhancers in the presence of oxygen (0 2 ) or a source of oxygen (0 2 ) .
  • the invention is useful wherever antimicrobial treatment is needed, for example for the preservation of food, beverages, cosmetics, contact lens products, food ingredients paints or enzyme compositions; for antimicrobial treatment of e.g. on human or animal skin, hair, oral cavity, mucous membranes, wounds, bruises or in the eye; for antimicrobial treatment of laundry; and for antimicrobial treatment in connection with hard surface cleaning or disinfection.
  • the present invention provides a method for antimicrobial treatment of microorganisms and/or viruses present on laundry and/or in a liguid used for soaking, washing or rinsing the laundry, e.g. in a washing machine; a method for antimicrobial treatment of microorganisms and/or viruses on or in human or animal skin, hair, oral cavities, mucous membranes, teeth, eyes, wounds, bruises; a method for antimicrobial treatment of microorganisms and/or viruses on or in a cosmetic product; a method for antimicrobial treatment of microorganisms and/or viruses on or in contact lenses and a method of antimicrobial treatment of microorganisms and/or viruses present on or in a hard surface.
  • Figure 1 shows the antimicrobial activity of different laccase systems against P. aeruginosa and S. epidermidis .
  • Enzyme concentration used was 3 mg/L and enhancer concentration used was: 0.1 mM.
  • Figure 2 shows a response surface plot for the bactericidal activity (given as the logarithm to the number of killed cfu per ml) of rMtL and MeS against Pseudomonas aeruginosa at pH 6, 40°C.
  • Figure 3 shows the bactericidal activity (given as the logarithm to the number of killed cfu per ml) of laccases and methylsyringate against Staphylococcus epidermidis depending on pH.
  • Figure 5 shows a response surface plot for the bactericidal activity on Pseudomonas aeruginosa (given as the logarithm to the number of killed cfu per ml) of rPpL (1 mg/L) and methylsyringate and acetosyringon at pH 6, 40°C.
  • antimicrobial is to be understood as bactericidal, bacteriostatic, fungicidal or fungistatic.
  • bactericidal is to be understood as capable of killing bacterial cells.
  • bacteriostatic is to be understood as capable of inhibiting bacterial growth, i.e. inhibiting growing bacterial cells.
  • fungicidal is to be understood as capable of killing fungal cells.
  • fungistatic is to be understood as capable of inhibiting fungal growth, i.e. inhibi- ting growing fungal cells.
  • microorganism denotes a virus, a bacterium or cells thereof, a fungus or cells thereof.
  • hard surface as used herein relates to any surface which is essentially non-permeable for microorganisms.
  • hard surfaces are surfaces made from metal, e.g. stainless steel alloys, plastics/synthetic polymers, rubber, board, glass, wood, paper, textile, concrete, rock, marble, gypsum and ceramic materials which optionally may be coated, e.g. with paint, enamel, polymers and the like.
  • the key reaction in the antimicrobial effect of the combined Laccase/enhancer system of the present invention is the oxidation of essential protein and enzyme sulphydryl groups or other cellular sites.
  • Laccase enzymes are able to catalyze 0 2 -dependent oxidation of an electron-donor, e.g. an enhancer.
  • the oxidized enhancer make an electrophilic attack on microbial components, resulting in chemical modification of essential enzymes, transport systems, and other functional components.
  • Sulphydryl groups are especially susceptible to electrophilic attack, and are usually present in higher amounts than other easily oxidized groups.
  • Aromatic amino acid residues are also susceptible to attack.
  • Most aspects of antimicrobial action can be correlated with chemical modification of these nucleophilic components.
  • Antimicrobial activity is favored by influences that increase the stability of the oxidized agent, provided that these influences do not interfere with their electrophilic character, or their ability to penetrate microbial membranes.
  • 0 2 itself is an oxidizing agent, the 0 2 molecule is stabilized and reacts only slowly with biological materials.
  • the laccase catalyzed oxidation of the enhancer preserves the oxidizing power 0 2 into forms that react
  • the reaction catalyzed by laccase can be written as:
  • AH p and A are reduced and oxidized forms of suitable electron donors; or, in case of enhancers/agents of the formula:
  • the oxygen can be supplied from air either by diffusion or
  • the antimicrobial effect may be obtained against various microorganisms like bacteria, fungi and/or virus.
  • the antimicrobial effect may be greater towards certain strains over other strains, e.g. bacteria over fungi or vice versa or gram positive bacteria over gram negative or vice versa .
  • the enzyme Preferred are the below mentioned enzymes, especially recombinant and/or substantially purified enzymes.
  • laccases include enzymes comprised by the enzyme classification E.C. 1.10.3.2.
  • the laccase employed is derived from a strain of Polyporus sp. , in particular a strain of Polyporus pinisitus or Polyporu ⁇ versicolor, or a strain of Myceliophthera sp. , e .g. M. thermophila or a strain of Rhizoctonia sp. , in particular a strain of Rhizoctonia praticola or Rhizoctonia solani , or a strain of a Rhus sp. , in particular Rhus vernicif ra .
  • the oxidoreductase is a laccase, such as a Polyporus sp.
  • laccase especially the Polyporus pinisitus laccase (also called Trametes villosa laccase) described in WO 96/00290 (from Novo Nordisk Biotech., inc.) or a Myceliophthera sp. laccase especially the Myceliophthera thermophila laccase described in WO 95/33836 (from Novo Nordisk
  • the laccase may be a Scytalidium sp. laccase, such as the S . thermophilium laccase described in WO 95/33837 (from Novo Nordisk Biotech inc.) or a Pyricularia sp. laccase, such as the Pyricularia oryzae laccase which can be purchased from SIGMA under the trade name SIGMA no. L5510, or a Coprinus sp. laccase, such as a C. cinereus laccase , especially a C. cinereus IFO 30116 laccase, or a Rhizoctonia sp. laccase, such as a Rh . solani laccase, especially the neutral Rh. solani laccase described WO
  • the laccase may also be derived from a fungi such as
  • Collybia Fomes , Lentinus , Pleurotus , Aspergillus , Neurospora , Podospora , Phlebia , e . g. P. radiata (WO 92/01046), Coriolus sp. , e.g. C. hirsitus (JP 2-238885), or Botryti ⁇ .
  • enhancers acting as electron donors for laccases for various purposes are known (e.g. WO 94/12620, WO 94/12621, WO 95/01626 and WO 96/00179) .
  • One or more enhancers being of the formula:
  • Combinations of said enhancers may suitably be applied sequentially or simultaneously and may have additional synergistic effects, as different microorganisms may show different sensitivity towards specific enhancers.
  • a specific embodiment of the invention relates to an antimicrobial composition comprising a laccase enzyme (EC 1.10.3.2) and at least two different enhancers of the formula:
  • A, B and C may be substituents as mentioned vide supra , and the laccase and the enhancers are present in the composition in antimicrobially effective amounts.
  • the composition may be a solid granular or powder formulation or it may be a liquid.
  • a solid granular composition may prepared as disclosed in US 4,106,991, US 4,661,452, WO 97/31088 or WO 95/33039 and may optionally be coated by methods known to the art.
  • a liquid composition may be prepared as a stabile liquid composition by addition of conventional stabilizers, a slurry composition or a composition with the enzyme in a protected form.
  • Protected enzymes may be prepared according to the method disclosed in EP 238,216 A or WO 97/41215.
  • Said enhancers are commercially available or can be made by methods known to the art.
  • the Laccase may be present in the medium to be antimicrobially treated i.e. washing liquor, cosmetics, food or beverages; or in the medium used for antimicrobial treatment of other items i.e. washing liquor, disinfecting liquid, preserving liquid, foot bath etc. in 0.00001-100 mg/L, preferably 0.001-10 mg/L, e.g. 0.1-5 mg/L.
  • Laccase may be present in the medium to be antimicrobially treated i.e. washing liquor, cosmetics, food or beverages; or in the medium used for antimicrobial treatment of other items i.e. washing liquor, disinfecting liquid, preserving liquid, foot bath etc. in 0.00001-500 mM, preferably 0.0001-5 mM, e.g. 0.001-0.050 mM.
  • the medium in which the catalytic reaction between the laccase and the enhancer is intended to occur is preferably aqueous and may be a liquid, an aerosol, a gel, a paste or a slurry, depending on the intended use of the invention.
  • the laccase and the enhancer may also be comprised, either separately or together, in solid formulations intended for preparation of said mediums.
  • the antimicrobial efficiency of the invention depends, inter alia , on the properties of the medium, which thus may be matched to the intended use of the invention.
  • the properties to be matched may, inter alia , be solubility or mobility of the laccase, the enhancer and/or 0 2 , the rate of the catalytic reaction, which then again depends on e.g. pH, temperature and buffer of the medium.
  • the medium may also affect the half-life of radical, which the enhancer forms upon the catalytic reaction with the laccase and 0 2 .
  • radical the enhancer forms upon the catalytic reaction with the laccase and 0 2 .
  • the half-life of the radical depends on, inter alia , the pH, the temperature and the buffer of the medium.
  • the medium may also comprise auxiliary agents such as wetting agents, thickening agents, buffer, stabilizers, perfume, colorants, fillers, chelators and the like (e.g. from a detergent composition) .
  • auxiliary agents such as wetting agents, thickening agents, buffer, stabilizers, perfume, colorants, fillers, chelators and the like (e.g. from a detergent composition) .
  • Useful wetting agents are surfactants, i.e. non-ionic, anionic, amphoteric or zwitterionic surfactants.
  • Laccases are, among other reasons, well suited in the context of the invention since they catalyze oxidation by molecular oxygen.
  • reactions which take place in vessels open to the atmosphere (or in other reaction vessels into which air - or for that matter another oxygen-containing gas - is introduced) and which involve an oxidase as enzyme will be able to utilize gaseous oxygen as oxidant; it may, however, be desirable to forcibly aerate the liquid medium during the reaction to ensure an adequate supply of oxygen.
  • the pH in the medium employed should normally be in the range of 5-11, often preferably in the range 6-10, e.g. 6.5-8.5.
  • temperatures in the range of 10-65°C, more preferably 30-50°C, should be employed. Treatment times
  • Treatment times depends, inter alia, on the treatment type, the type of item to be treated, the properties of the medium, e.g. temperature and pH and the type and amounts of enzyme and enhancer employed.
  • treatment times may be in the range of depending of the expected lifetime of the item to be preserved.
  • treatment times in the range of 1-120 minutes may be employed. In many cases a treatment time in the range of 5-20 minutes will be suitable.
  • the present invention provides a method for antimicrobial treatment of microorganisms or viruses present on laundry and/or in a liquid used for soaking, washing or rinsing the laundry, e.g. in a washing machine.
  • the laccase and the enhancer may be incorporated in a detergent composition.
  • the detergent compositions according to the present invention comprise a surfactant system, wherein the surfactant can be selected from non-ionic and/or anionic and/or cationic and/or ampholytic and/or zwitterionic and/or semi-polar surfactants.
  • the surfactant is typically present at a level from 0.1% to 60% by weight.
  • the surfactant is preferably formulated to be compatible with enzyme components present in the composition.
  • the surfactant is most preferably formulated in such a way that it promotes, or at least does not degrade, the stability of any enzyme in these compositions.
  • Preferred systems to be used according to the present invention comprise as a surfactant one or more of the non-ionic and/or anionic surfactants described herein.
  • Polyethylene, polypropylene, and polybutylene oxide condensates of alkyl phenols are suitable for use as the non- ionic surfactant of the surfactant systems of the present invention, with the polyethylene oxide condensates being preferred.
  • These compounds include the condensation products of alkyl phenols having an alkyl group containing from about 6 to about 14 carbon atoms, preferably from about 8 to about 14 carbon atoms, in either a straight chain or branched-chain configuration with the alkylene oxide.
  • the ethylene oxide is present in an amount equal to from about 2 to about 25 moles, more preferably from about 3 to about 15 moles, of ethylene oxide per mole of alkyl phenol.
  • non-ionic surfactants of this type include IgepalTM CO-630, marketed by the GAF Corporation; and TritonTM X-45, X- 114, X-100 and X-102, all marketed by the Rohm & Haas Company. These surfactants are commonly referred to as alkylphenol alkoxylates (e.g., alkyl phenol ethoxylates) .
  • alkylphenol alkoxylates e.g., alkyl phenol ethoxylates
  • the condensation products of primary and secondary aliphatic alcohols with about 1 to about 25 moles of ethylene oxide are suitable for use as the non-ionic surfactant of the non-ionic surfactant systems of the present invention.
  • the alkyl chain of the aliphatic alcohol can either be straight or branched, primary or secondary, and generally contains from about 8 to about 22 carbon atoms.
  • About 2 to about 7 moles of ethylene oxide and most preferably from 2 to 5 moles of ethylene oxide per mole of alcohol are present in said condensation products.
  • non-ionic surfactants of this type include TergitolTM 15-S-9 (The condensation product of C 1;L -Ci 5 linear alcohol with 9 moles ethylene oxide) , TergitolTM 24-L-6 NMW (the condensation product of C 12 -Ci 4 primary alcohol with 6 moles ethylene oxide with a narrow molecular weight distribution) , both marketed by Union Carbide Corporation; NeodolTM 45-9 (the condensation product of C 14 -C ⁇ 5 linear alcohol with 9 moles of ethylene oxide) , Neodol 23-3 (the condensation product of C 12 -Ci 3 linear alcohol with 3.0 moles of ethylene oxi 4de) , NeodolTM 45-7 (the condensati4on product of C 4 -C 15 linear alcohol with 7 moles of ethylene oxide) , NeodolTM 45-5 (the condensation product of C 1 -C 15 linear alcohol with 5 moles of ethylene oxide) marketed by Shell Chemical Company, KyroTM EOB (the condensation product of C 13
  • alkylpolysaccharides disclosed in US 4,565,647, having a hydrophobic group containing from about 6 to about 30 carbon atoms, preferably from about 10 to about 16 carbon atoms and a polysaccharide, e.g. a polyglycoside, hydrophilic group containing from about 1.3 to about 10, preferably from about 1.3 to about 3, most preferably from about 1.3 to about 2.7 saccharide units.
  • Any reducing saccharide containing 5 or 6 carbon atoms can be used, e.g., glucose, galactose and galactosyl moieties can be substituted for the glycosyl moieties (optionally the hydrophobic group is attached at the 2-, 3-, 4-, etc. positions thus giving a glucose or galactose as opposed to a glycoside or galactoside) .
  • the intersaccharide bonds can be, e.g. , between the one position of the additional saccharide units and the 2-, 3-, 4-, and/or 6- positions on the preceding saccharide units.
  • the preferred alkylpolyglycosides have the formula
  • R 2 is selected from the group consisting of alkyl, alkylphenyl, hydroxyalkyl, hydroxyalkylphenyl, and mixtures thereof in which the alkyl groups contain from about 10 to about 18, preferably from about 12 to about 14, carbon atoms; n is 2 or 3, preferably 2; t is from 0 to about 10, preferably 0; and x is from about 1.3 to about 10, preferably from about 1.3 to about 3, most preferably from about 1.3 to about 2.7.
  • the glycosyl is preferably derived from glucose. To prepare these compounds, the alcohol or alkylpolyethoxy alcohol is formed first and then reacted with glucose, or a source of glucose, to form the glycoside (attachment at the 1-position) .
  • the additional glycosyl units can then be attached between their 1- position and the preceding glycosyl units 2-, 3-, 4-, and/or 6- position, preferably predominantly the 2-position.
  • the condensation products of ethylene oxide with a hydrophobic base formed by the condensation of propylene oxide with propylene glycol are also suitable for use as the additional non-ionic surfactant systems of the present invention.
  • the hydrophobic portion of these compounds will preferably have a molecular weight from about 1500 to about 1800 and will exhibit water insolubility.
  • polyoxyethylene moieties to this hydrophobic portion tends to increase the water solubility of the molecule as a whole, and the liquid character of the product is retained up to the point where the polyoxyethylene content is about 50% of the total weight of the condensation product, which corresponds to condensation with up to about 40 moles of ethylene oxide.
  • examples of compounds of this type include certain of the commercially available PluronicTM surfactants, marketed by BASF.
  • PluronicTM surfactants also suitable for use as the non-ionic surfactant of the non-ionic surfactant system of the present invention, are the condensation products of ethylene oxide with the product resulting from the reaction of propylene oxide and ethylenediamine.
  • the hydrophobic moiety of these products consists of the reaction product of ethylenediamine and excess propylene oxide, and generally has a molecular weight of from about 2500 to about 3000.
  • This hydrophobic moiety is condensed with ethylene oxide to the extent that the condensation product contains from about 40% to about 80% by weight of polyoxyethylene and has a molecular weight of from about 5,000 to about 11,000.
  • Examples of this type of non-ionic surfactant include certain of the commercially available TetronicTM compounds , marketed by BASF .
  • non-ionic surfactant of the surfactant systems of the present invention are polyethylene oxide condensates of alkyl phenols, condensation products of primary and secondary aliphatic alcohols with from about 1 to about 25 moles of ethyleneoxide, alkylpolysaccharides, and mixtures hereof. Most preferred are C 8 -C 1 alkyl phenol ethoxylates having from 3 to 15 ethoxy groups and C 8 -C 18 alcohol ethoxylates (preferably C 10 avg.) having from 2 to 10 ethoxy groups, and mixtures thereof.
  • Highly preferred non-ionic surfactants are polyhydroxy fatty acid amide surfactants of the formula
  • R 1 is H, or R 1 is C ⁇ - 4 hydrocarbyl, 2-hydroxyethyl, 2- hydroxypropyl or a mixture thereof, R is C 5 _ 3! hydrocarbyl, and Z is a polyhydroxyhydrocarbyl having a linear hydrocarbyl chain with at least 3 hydroxyls directly connected to the chain, or an alkoxylated derivative thereof.
  • R 1 is methyl
  • R 2 is straight Cn- 15 alkyl or C 16 _ ⁇ 8 alkyl or alkenyl chain such as coconut alkyl or mixtures thereof
  • Z is derived from a reducing sugar such as glucose, fructose, maltose or lactose, in a reductive amination reaction.
  • Highly preferred anionic surfactants include alkyl alkoxylated sulphate surfactants.
  • Examples hereof are water soluble salts or acids of the formula RO(A) m S03M wherein R is an unsubstituted C 10 -C- 24 alkyl or hydroxyalkyl group having a C 10 - C 24 alkyl component, preferably a C 12 -C 20 alkyl or hydroxyalkyl, more preferably C 1 -C 18 alkyl or hydroxyalkyl, A is an ethoxy or propoxy unit, m is greater than zero, typically between about 0.5 and about 6 , more preferably between about 0.5 and about 3 , and M is H or a cation which can be, for example, a metal cation (e.g., sodium, potassium, lithium, calcium, magnesium, etc.), ammonium or substituted-ammonium cation.
  • R is an unsubstituted C 10 -C- 24 alkyl or hydroxyalkyl
  • Alkyl ethoxylated sulphates as well as alkyl propoxylated sulphates are contemplated herein.
  • Specific examples of substituted ammonium cations include methyl-, dimethyl, trimethyl-ammonium cations and quaternary ammonium cations such as tetramethyl-ammonium and dimethyl piperdinium cations and those derived from alkylamines such as ethylamine, diethylamine, triethylamine, mixtures thereof, and the like.
  • Exemplary surfactants are C 12 -C ⁇ 8 alkyl polyethoxylate (1.0) sulphate (C 12 -C 18 E(1.0)M) , C 12 -C 18 alkyl polyethoxylate (2.25) sulphate (C 1 -C 18 (2.25)M, and C 12 -C 18 alkyl polyethoxylate (3.0) sulphate (C 12 -C 18 E(3.0)M) , and C 12 -C 18 alkyl polyethoxylate (4.0) sulphate (C 12 -C 18 E(4.0)M) , wherein M is conveniently selected from sodium and potassium.
  • Suitable anionic surfactants to be used are alkyl ester sulphonate surfactants including linear esters of C 8 -C 2 Q carboxylic acids (i.e., fatty acids) which are sulphonated with gaseous S0 3 according to "The Journal of the American Oil Chemists Society", 52 (1975), pp. 323-329.
  • Suitable starting materials would include natural fatty substances as derived from tallow, palm oil, etc.
  • alkyl ester sulphonate surfactant especially for laundry applications, comprise alkyl ester sulphonate surfactants of the structural formula:
  • R is a C 8 -C 20 hydrocarbyl, preferably an alkyl, or combination thereof
  • R 4 is a Ci-Cg hydrocarbyl, preferably an alkyl, or combination thereof
  • M is a cation which forms a water soluble salt with the alkyl ester sulphonate.
  • Suitable salt-forming cations include metals such as sodium, potassium, and lithium, and substituted or unsubstituted ammonium cations, such as monoethanolamine, diethonolamine, and triethanolamine.
  • R 3 is C 10 -C 16 alkyl
  • R 4 is methyl, ethyl or isopropyl.
  • the methyl ester sulphonates wherein R 3 is CXO-CD ⁇ alkyl.
  • alkyl sulphate surfactants which are water soluble salts or acids of the formula ROSO 3 M wherein R preferably is a C 1Q -C 2 4 hydrocarbyl, preferably an alkyl or hydroxyalkyl having a C 10 -C 2 o alkyl component, more preferably a C 12 -C 18 alkyl or hydroxyalkyl, and M is H or a cation, e.g. , an alkali metal cation (e.g. sodium, potassium, lithium) , or ammonium or substituted ammonium (e.g.
  • R preferably is a C 1Q -C 2 4 hydrocarbyl, preferably an alkyl or hydroxyalkyl having a C 10 -C 2 o alkyl component, more preferably a C 12 -C 18 alkyl or hydroxyalkyl
  • M is H or a cation, e.g. , an alkali metal cation (e.g. sodium, potassium
  • alkylamines such as ethylamine, diethylamine, triethylamine, and mixtures thereof, and the like.
  • alkyl chains of C 12 -C 16 are preferred for lower wash temperatures (e.g. below about 50°C) and ' C 16 -C 18 alkyl chains are preferred for higher wash temperatures (e.g. above about 50°C) .
  • anionic surfactants useful for detersive purposes can also be included in the laundry detergent compositions of the present invention. Theses can include salts (including, for example, sodium, potassium, ammonium, and substituted ammonium salts such as mono- di- and triethanolamine salts) of soap, C 8 - C 22 primary or secondary alkanesulphonates, Cs ⁇ c 24 olefinsulphonates, sulphonated polycarboxylic acids prepared by sulphonation of the pyrolyzed product of alkaline earth metal citrates, e.g., as described in British patent specification No.
  • salts including, for example, sodium, potassium, ammonium, and substituted ammonium salts such as mono- di- and triethanolamine salts
  • C 8 - C 22 primary or secondary alkanesulphonates Cs ⁇ c 24 olefinsulphonates
  • sulphonated polycarboxylic acids prepared by sulphonation of the pyrolyzed product of alkaline earth
  • alkylpolyglycolethersulfates (containing up to 10 moles of ethylene oxide) ; alkyl glycerol sulphonates, fatty acyl glycerol sulphonates, fatty oleyl glycerol sulphates, alkyl phenol ethylene oxide ether sulphates, paraffin sulphonates, alkyl phosphates, isethionates such as the acyl isethionates, N- acyl taurates, alkyl succinamates and sulphosuccinates, monoesters of sulphosuccinates (especially saturated and unsaturated C 12 -C 18 monoesters) and diesters of sulphosuccinates (especially saturated and unsaturated C ⁇ -C ⁇ 2 diesters) , acyl sarcosinates, sulphates of alkylpolysaccharides such as the sulphates of
  • Alkylbenzene sulphonates are highly preferred. Especially preferred are linear (straight-chain) alkyl benzene sulphonates (LAS) wherein the alkyl group preferably contains from 10 to 18 carbon atoms .
  • LAS linear (straight-chain) alkyl benzene sulphonates
  • the laundry detergent compositions of the present invention typically comprise from about 1% to about 40%, preferably from about 3% to about 20% by weight of such anionic surfactants.
  • the laundry detergent compositions of the present invention may also contain cationic, ampholytic, zwitterionic, and semi-polar surfactants, as well as the non-ionic and/or anionic surfactants other than those already described herein.
  • Cationic detersive surfactants suitable for use in the laundry detergent compositions of the present invention are those having one long-chain hydrocarbyl group.
  • cationic surfactants include the ammonium surfactants such as alkyltrimethylammonium halogenides, and those surfactants having the formula:
  • R 2 is an alkyl or alkyl benzyl group having from about 8 to about 18 carbon atoms in the alkyl chain, each R is selected form the group consisting of -CH 2 CH 2 -, -CH 2 CH(CH 3 ) -, CH 2 CH(CH 2 OH)-, -CH 2 CH 2 CH 2 -, and mixtures thereof; each R 4 is selected from the group consisting of C 1 -C 4 alkyl, C 1 -C hydroxyalkyl, benzyl ring structures formed by joining the two R 4 groups, -CH 2 CHOHCHOHCOR 6 CHOHCH 2 OH, wherein R 6 is any hexose or hexose polymer having a molecular weight less than about 1000, and hydrogen when y is not 0; R 5 is the same as R 4 or is an alkyl chain, wherein the total number of carbon atoms or R 2 plus R 5 is not more than about 18; each y is from 0 to about 10, and the sum
  • R]_ is Cs-Cxe alkyl
  • each of R 2 , R 3 and R 4 is independently C 1 -C 4 alkyl, C 1 -C 4 hydroxy alkyl, benzyl, and - ( c 2 H 4 ⁇ )x H where x has a value from 2 to 5
  • X is an anion.
  • R , R 3 or R 4 should be benzyl.
  • the preferred alkyl chain length for Ri is C 12 -C 15/ particularly where the alkyl group is a mixture of chain lengths derived from coconut or palm kernel fat or is derived synthetically by olefin build up or 0X0 alcohols synthesis.
  • Preferred groups for R 2 R 3 and R 4 are methyl and hydroxyethyl groups and the anion X may be selected from halide, methosulphate, acetate and phosphate ions.
  • Suitable quaternary ammonium compounds of formulae (i) for use herein are: coconut trimethyl ammonium chloride or bromide; coconut methyl dihydroxyethyl ammonium chloride or bromide; decyl triethyl ammonium chloride; decyl dimethyl hydroxyethyl ammonium chloride or bromide; c l2 - l5 dimethyl hydroxyethyl ammonium chloride or bromide; coconut dimethyl hydroxyethyl ammonium chloride or bromide; myristyl trimethyl ammonium methyl sulphate; lauryl dimethyl benzyl ammonium chloride or bromide; lauryl dimethyl (ethenoxy) 4 ammonium chloride or bromide; choline esters (compounds of formula (i) wherein R x is
  • cationic surfactants useful herein are also described in US 4,228,044 and in EP 000 224.
  • the laundry detergent compositions of the present invention typically comprise from 0.2% to about 25%, preferably from about 1% to about 8% by weight of such cationic surfactants.
  • Ampholytic surfactants are also suitable for use in the laundry detergent compositions of the present invention. These surfactants can be broadly described as aliphatic derivatives of secondary or tertiary amines, or aliphatic derivatives of heterocyclic secondary and tertiary amines in which the
  • 10 aliphatic radical can be straight- or branched-chain.
  • One of the aliphatic substituents contains at least about 8 carbon atoms, typically from about 8 to about 18 carbon atoms, and at least one contains an anionic water-solubilizing group, e.g. carboxy, sulphonate, sulphate. See US 3,929,678 (column 19, lines 18-35)
  • the laundry detergent compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1% to about 10% by weight of such ampholytic surfactants.
  • Zwitterionic surfactants are also suitable for use in laundry detergent compositions. These surfactants can be broadly described as derivatives of secondary and tertiary amines, derivatives of heterocyclic secondary and tertiary amines, or derivatives of quaternary ammonium, quaternary phosphonium or
  • laundry detergent compositions of the present invention typically comprise from 0.2% to about
  • Semi-polar non-ionic surfactants are a special category of non-ionic surfactants which include water-soluble amine oxides containing one alkyl moiety of from about 10 to about 18 carbon
  • Semi-polar non-ionic detergent surfactants include the amine oxide surfactants having the formula:
  • R 3 is an alkyl, hydroxyalkyl, or alkyl phenyl group or mixtures thereof containing from about 8 to about 22 carbon atoms
  • R 4 is an alkylene or hydroxyalkylene group containing from about 2 to about 3 carbon atoms or mixtures thereof
  • x is from 0 to about 3
  • each R 5 is an alkyl or hydroxyalkyl group containing from about 1 to about 3 carbon atoms or a polyethylene oxide group containing from about 1 to about 3 ethylene oxide groups.
  • the R 5 groups can be attached to each other, e.g. , through an oxygen or nitrogen atom, to form a ring structure .
  • amine oxide surfactants in particular include C 10 - C 18 alkyl dimethyl amine oxides and C 8 -C 12 alkoxy ethyl dihydroxy ethyl amine oxides .
  • the laundry detergent compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1% to about 10% by weight of such semi-polar non-ionic surfactants.
  • compositions according to the present invention may further comprise a builder system.
  • a builder system Any conventional builder system is suitable for use herein including aluminosilicate materials, silicates, polycarboxylates and fatty acids, materials such as ethylenediamine tetraacetate, metal ion sequestrants such as aminopolyphosphonates, particularly ethylenediamine tetramethylene phosphonic acid and diethylene triamine pentamethylenephosphonic acid.
  • phosphate builders can also be used herein.
  • Suitable builders can be an inorganic ion exchange material, commonly an inorganic hydrated aluminosilicate material, more particularly a hydrated synthetic zeolite such as hydrated zeolite A, X, B, HS or MAP.
  • inorganic ion exchange material commonly an inorganic hydrated aluminosilicate material, more particularly a hydrated synthetic zeolite such as hydrated zeolite A, X, B, HS or MAP.
  • Another suitable inorganic builder material is layered
  • silicate e.g. SKS-6 (Hoechst) .
  • SKS-6 is a crystalline layered silicate consisting of sodium silicate (Na 2 Si 2 0 5 ) .
  • Suitable polycarboxylates containing one carboxy group include lactic acid, glycolic acid and ether derivatives thereof as disclosed in Belgian Patent Nos. 831,368, 821,369 and
  • Polycarboxylates containing two carboxy groups include the water-soluble salts of succinic acid, malonic acid, (ethylenedioxy) diacetic acid, maleic acid, diglycollic acid, tartaric acid, tartronic acid and fumaric acid, as well as the ether carboxylates described in German Offenle-enschrift
  • Polycarboxylates containing three carboxy groups include, in particular, water-soluble citrates, aconitrates and citraconates as well as succinate derivatives such as the
  • Polycarboxylates containing four carboxy groups include oxydisuccinates disclosed in British Patent No. 1,261,829, 1,1, 2, 2, -ethane tetracarboxylates, 1, 1, 3 , 3-propane tetracarboxylates containing sulpho substituents include the sulphosuccinate derivatives disclosed in British Patent Nos.
  • Alicyclic and heterocyclic polycarboxylates include cyclopentane-cis , cis-cis-tetracarboxylates , cyclopentadienide pentacarboxylates, 2 , 3 , 4 , 5-tetrahydro-f ran - cis, cis, cis- tetracarboxylates , 2 , 5-tetrahydro-furan-cis, discarboxylates, 2 , 2 , 5 , 5 , -tetrahydrofuran - tetracarboxylates, 1, 2 , 3 , 4 , 5, 6-hexane - hexacarboxylates and carboxymethyl derivatives of polyhydric alcohols such as sorbitol, mannitol and xylitol.
  • Aromatic polycarboxylates include mellitic acid, pyromellitic acid and the phthalic acid derivatives disclosed in British Patent
  • the preferred polycarboxylates are hydroxy- carboxylates containing up to three carboxy groups per molecule, more particularly citrates.
  • Preferred builder systems for use in the present compositions include a mixture of a water-insoluble aluminosilicate builder such as zeolite A or of a layered silicate (SKS-6) , and a water-soluble carboxylate chelating agent such as citric acid.
  • a water-insoluble aluminosilicate builder such as zeolite A or of a layered silicate (SKS-6)
  • a water-soluble carboxylate chelating agent such as citric acid.
  • a suitable chelator for inclusion in the detergent compositions in accordance with the invention is ethylenediamine-N,N'-disuccinic acid (EDDS) or the alkali metal, alkaline earth metal, ammonium, or substituted ammonium salts thereof, or mixtures thereof.
  • EDDS compounds are the free acid form and the sodium or magnesium salt thereof. Examples of such preferred sodium salts of EDDS include Na EDDS and Na 4 EDDS. Examples of such preferred magnesium salts of EDDS include MgEDDS and Mg EDDS.
  • the magnesium salts are the most preferred for inclusion in compositions in accordance with the invention.
  • Preferred builder systems include a mixture of a water- insoluble aluminosilicate builder such as zeolite A, and a water soluble carboxylate chelating agent such as citric acid.
  • builder materials that can form part of the builder system for use in granular compositions include inorganic materials such as alkali metal carbonates, bicarbonates, silicates, and organic materials such as the organic phosphonates , amino polyalkylene phosphonates and amino polycarboxylates .
  • inorganic materials such as alkali metal carbonates, bicarbonates, silicates
  • organic materials such as the organic phosphonates , amino polyalkylene phosphonates and amino polycarboxylates .
  • suitable water-soluble organic salts are the homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated form each other by not more than two carbon atoms. Polymers of this type are disclosed in GB-A-1, 596, 756.
  • salts are polyacrylates of MW 2000-5000 and their copolymers with maleic anhydride, such copolymers having a molecular weight of from 20,000 to 70,000, especially about 40,000.
  • Detergency builder salts are normally included in amounts of from 5% to 80% by weight of the composition. Preferred levels of builder for liquid detergents are from 5% to 30%.
  • Enzymes Preferred detergent compositions in addition to the enzyme preparation of the invention, comprise other enzyme (s) which provides cleaning performance and/or fabric care benefits.
  • enzymes include proteases, lipases, cutinases, amylases, cellulases, peroxidases, other oxidases (e.g. laccases) .
  • protease suitable for use in alkaline solutions can be used.
  • Suitable proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically or genetically modified mutants are included.
  • the protease may be a serine protease, preferably an alkaline microbial protease or a trypsin-like protease.
  • alkaline proteases are subtilisins, especially those derived from Bacillus. e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279) .
  • trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270.
  • Preferred commercially available protease enzymes include those sold under the trade names Alcalase, Savinase, Primase, Durazym, and Esperase by Novo Nordisk A/S (Denmark) , those sold under the trade name Maxatase, Maxacal, Maxapem, Properase, Purafect and Purafect OXP by Genencor International, and those sold under the trade name Opticlean and Optimase by Solvay Enzymes.
  • Protease enzymes may be incorporated into the compositions in accordance with the invention at a level of from 0.00001% to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.01% to 0.2% of enzyme protein by weight of the composition.
  • Lipases Any lipase suitable for use in alkaline solutions can be used. Suitable lipases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included. Examples of useful lipases include a Humicola lanuginosa lipase, e.g., as described in EP 258 068 and EP 305 216, a Rhizomucor iehei lipase, e.g., as described in EP 238 023, a Candida lipase, such as a C. antarctica lipase, e.g., the C. antarctica lipase A or B described in EP 214 761, a Pseudomonas lipase such as a P.
  • a Humicola lanuginosa lipase e.g., as described in EP 258 068 and EP 305 216
  • Rhizomucor iehei lipase e.g., as described in EP 2
  • alcali ⁇ enes and P. pseudoalcaligenes lipase e.g., as described in EP 218 272, a P. cepacia lipase, e.g., as described in EP 331 376, a P. stutzeri lipase, e.g., as disclosed in GB 1,372,034, a P. fluorescens lipase, a Bacillus lipase, e.g., a B. subtilis lipase (Dartois et al., (1993), Bioche ica et Biophysica acta 1131, 253-260) , a B. stearo- thermophilus lipase (JP 64/744992) and a B. pumilus lipase (WO 91/16422) .
  • a Bacillus lipase e.g., a B. subtilis lipase (Dartois et al., (1993), Bio
  • cloned lipases may be useful, including the Penicillium camembertii lipase described by Yamaguchi et al., (1991), Gene 103, 61-67), the Geotricum candidu lipase (Schimada, Y. et al. , (1989), J. Biochem. , 106, 383-388) , and various Rhizopus lipases such as a R. delemar lipase (Hass, M.J et al. , (1991), Gene 109, 117-113), a R. niveus lipase (Kugi iya et al., (1992), Biosci. Biotech. Biochem.
  • Rhizopus lipases such as a R. delemar lipase (Hass, M.J et al. , (1991), Gene 109, 117-113), a R. niveus lipase (Kugi iya e
  • lipolytic enzymes such as cutinases may also be useful, e.g., a cutinase derived from Pseudomonas mendocina as described in WO 88/09367, or a cutinase derived from Fusarium solani pisi (e.g. described in WO 90/09446) .
  • a cutinase derived from Pseudomonas mendocina as described in WO 88/09367 or a cutinase derived from Fusarium solani pisi (e.g. described in WO 90/09446) .
  • lipases such as Ml LipaseTM, Luma fastTM and LipomaxTM (Genencor) , LipolaseTM and Lipolase UltraTM (Novo Nordisk A/S) , and Lipase P "Amano" (A ano Pharmaceutical Co.
  • the lipases are normally incorporated in the detergent composition at a level of from 0.00001% to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.01% to 0.2% of enzyme protein by weight of the composition.
  • Amylases Any amylase ( ⁇ and/or ⁇ ) suitable for use in alkaline solutions can be used. Suitable amylases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included. Amylases include, for example, ⁇ -amylases obtained from a special strain of B. licheniformis. described in more detail in GB 1,296,839. Commercially available amylases are DuramylTM, TermamylTM, FungamylTM and BANTM (available from Novo
  • amylases are normally incorporated in the detergent composition at a level of from 0.00001% to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.01% to 0.2% of enzyme protein by weight of the composition.
  • Cellulases Any cellulase suitable for use in alkaline solutions can be used. Suitable cellulases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included. Suitable cellulases are disclosed in US 4,435,307, which discloses fungal cellulases produced from Humicola insolens . Especially suitable cellulases are the cellulases having colour care benefits. Examples of such cellulases are cellulases described in European patent publication No. 0 495 257.
  • cellulases include CelluzymeTM produced by a strain of Humicola insolens, (Novo Nordisk A/S) , and KAC-500(B)TM (Kao Corporation).
  • Cellulases are normally incorporated in the detergent composition at a level of from 0.00001% to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.01% to 0.2% of enzyme protein by weight of the composition.
  • Peroxidases /Oxidases Peroxidase enzymes are used in combination with hydrogen peroxide or a source thereof (e.g. a percarbonate, perborate or persulphate) . Oxidase enzymes are used in combination with oxygen. Both types of enzymes are used for "solution bleaching", i.e. to prevent transfer of a textile dye from a dyed fabric to another fabric when said fabrics are washed together in a wash liquor, preferably together with an enhancing agent as described in e.g. WO 94/12621 and WO 95/01426. Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically or genetically modified mutants are included.
  • Peroxidase and/or oxidase enzymes are normally incorporated in the detergent composition at a level of from 0.00001% to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.01% to 0.2% of enzyme protein by weight of the composition.
  • Mixtures of the above mentioned enzymes are encompassed herein, in particular a mixture of a protease, an amylase, a lipase and/or a cellulase.
  • the enzyme of the invention is normally incorporated in the detergent composition at a level from 0.00001% to 2% of enzyme protein by weight of the composition, preferably at a level from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level from 0.01% to 0.2% of enzyme protein by weight of the composition.
  • Bleaching agents Additional optional detergent ingredients that can be included in the detergent compositions of the present invention include bleaching agents such as PB1, PB4 and percarbonate with a particle size of 400-800 microns. These bleaching agent components can include one or more oxygen bleaching agents and, depending upon the bleaching agent chosen, one or more bleach activators. When present oxygen bleaching compounds will typically be present at levels of from about 1% to about 25%. In general, bleaching compounds are optional added components in non-liquid formulations, e.g. granular detergents.
  • the bleaching agent component for use herein can be any of the bleaching agents useful for detergent compositions including oxygen bleaches as well as others known in the art.
  • the bleaching agent suitable for the present invention can be an activated or non-activated bleaching agent.
  • oxygen bleaching agent that can be used encompasses percarboxylic acid bleaching agents and salts thereof. Suitable examples of this class of agents include magnesium monoperoxyphthalate hexahydrate, the magnesium salt of eta-chloro perbenzoic acid, 4-nonylamino-4-oxoperoxybutyric acid and diperoxydodecanedioic acid. Such bleaching agents are disclosed in US 4,483,781, US 740,446, EP 0 133 354 and US
  • Highly preferred bleaching agents also include 6- nonylamino-6-oxoperoxycaproic acid as described in US 4,634,551.
  • bleaching agents that can be used encompasses the halogen bleaching agents.
  • hypohalite bleaching agents include trichloro isocyanuric acid and the sodium and potassium dichloroisocyanurates and N- chloro and N-bromo alkane sulphonamides .
  • Such materials are normally added at 0.5-10% by weight of the finished product, preferably 1-5% by weight.
  • the hydrogen peroxide releasing agents can be used in combination with bleach activators such as tetra-acetylethylene- diamine (TAED) , nonanoyloxybenzenesulfonate (NOBS, described in US 4,412,934), 3 , 5-trimethyl-hexsanoloxybenzenesulfonate (ISONOBS, described in EP 120 591) or pentaacetylglucose (PAG) , which are perhydrolyzed to form a peracid as the active bleaching species, leading to improved bleaching effect.
  • bleach activators such as tetra-acetylethylene- diamine (TAED) , nonanoyloxybenzenesulfonate (NOBS, described in US 4,412,934), 3 , 5-trimethyl-hexsanoloxybenzenesulfonate (ISONOBS, described in EP 120 591) or pentaacetylglucose (PAG) ,
  • bleach activators C8(6- octanamido-caproyl) oxybenzene-sulphonate, C9 (6-nonanamido caproyl) oxybenzenesulphonate and CIO (6-decanamido caproyl) oxybenzenesulphonate or mixtures thereof.
  • acylated citrate esters such as disclosed in European Patent Application No. 91870207.7.
  • bleaching agents including peroxyacids and bleaching systems comprising bleach activators and peroxygen bleaching compounds for use in cleaning compositions according to the invention are described in application USSN 08/136,626.
  • the hydrogen peroxide may also be present by adding an enzymatic system (i.e. an enzyme and a substrate therefore) which is capable of generation of hydrogen peroxide at the beginning or during the washing and/or rinsing process.
  • an enzymatic system i.e. an enzyme and a substrate therefore
  • Such enzymatic systems are disclosed in European Patent Publication EP 0 537 381.
  • Bleaching agents other than oxygen bleaching agents are also known in the art and can be utilised herein.
  • One type of non-oxygen bleaching agent of particular interest includes photo-activated bleaching agents such as the sulphonated zinc and/or aluminium phthalocyanines. These materials can be deposited upon the substrate during the washing process. Upon irradiation with light, in the presence of oxygen, such as by hanging clothes out to dry in the daylight, the sulphonated zinc phthalocyanine is activated and, consequently, the substrate is bleached.
  • Preferred zinc phthalocyanine and a photo-activated bleaching process are described in US 4,033,718.
  • bleachaching agents may also comprise a manganese catalyst.
  • the 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.
  • Suds suppressors Another optional ingredient is a suds suppressor, exemplified by silicones, and silica-silicone mixtures.
  • Silicones can generally be represented by alkylated polysiloxane materials, while silica is normally used in finely divided forms exemplified by silica aerogels and xerogels and hydrophobic silicas of various types. Theses materials can be incorporated as particulates, in which the suds suppressor is advantageously releasably incorporated in a water-soluble or water-dispersible, substantially non surface-active detergent impermeable carrier. Alternatively the suds suppressor can be dissolved or dispersed in a liquid carrier and applied by spraying on to one or more of the other components.
  • a preferred silicone suds controlling agent is disclosed in US 3,933,672.
  • Other particularly useful suds suppressors are the self-emulsifying silicone suds suppressors, described in German Patent Application DTOS 2,646,126.
  • An example of such a compound is DC-544, commercially available form Dow Corning, which is a siloxaneglycol copolymer.
  • Especially preferred suds controlling agent are the suds suppressor system comprising a mixture of silicone oils and 2-alkyl-alkanols. Suitable 2-alkyl- alkanols are 2-butyl-octanol which are commercially available under the trade name Isofol 12 R.
  • Such suds suppressor system are described in European Patent Publication EP 0 593 841.
  • Especially preferred silicone suds controlling agents are described in European Patent Application No. 92201649.8.
  • Said compositions can comprise a silicone/ silica mixture in combination with fumed nonporous silica such as Aerosil .
  • the suds suppressors described above are normally employed at levels of from 0.001% to 2% by weight of the composition, preferably from 0.01% to 1% by weight.
  • Other components may be employed such as soil-suspending agents, soil-releasing agents, optical brighteners, abrasives, bactericides, tarnish inhibitors, coloring agents, and/or encapsulated or non-encapsulated perfumes.
  • encapsulating materials are water soluble capsules which consist of a matrix of polysaccharide and polyhydroxy compounds such as described in GB 1,464,616.
  • Suitable water soluble encapsulating materials comprise dextrins derived from ungelatinized starch acid esters of substituted dicarboxylic acids such as described in US 3,455,838. These acid-ester dextrins are, preferably, prepared from such starches as waxy maize, waxy sorghum, sago, tapioca and potato. Suitable examples of said encapsulation materials include N-Lok manufactured by National Starch. The N-Lok encapsulating material consists of a modified maize starch and glucose. The starch is modified by adding mono-functional substituted groups such as octenyl succinic acid anhydride.
  • Anti-redeposition and soil suspension agents suitable herein include cellulose derivatives such as methylcellulose, carboxymethylcellulose and hydroxyethylcellulose, and homo- or co-polymeric polycarboxylic acids or their salts.
  • Polymers of this type include the polyacrylates and maleic anhydride-acrylic acid copolymers previously mentioned as builders, as well as copolymers of maleic anhydride with ethylene, methylvinyl ether or methacrylic acid, the maleic anhydride constituting at least 20 mole percent of the copolymer. These materials are normally used at levels of from 0.5% to 10% by weight, more preferably form 0.75% to 8%, most preferably from 1% to 6% by weight of the composition.
  • Preferred optical brighteners are anionic in character, examples of which are disodium 4 , 4 ' -bis- (2-diethanolamino-4- anilino -s- triazin-6-ylamino) stilbene-2 :2 ' disulphonate, disodium 4, - 4 ' -bis- (2-morpholino-4-anilino-s-triazin-6- ylamino-stilbene-2:2 • - disulphonate, disodium 4,4' - bis- (2,4- dianilino-s-triazin-6-ylamino) stilbene-2 :2 ' - disulphonate, monosodium 4 , ,4'' - bis- (2 , 4-dianilino-s-tri-azin-6 ylamino) stilbene-2-sulphonate, disodium 4,4' -bis- (2-anilino-4- (N-methyl-N-2-hydroxyethylamino) -s-tri
  • polyethyleneglycols particularly those of molecular weight 1000-10000, more particularly 2000 to 8000 and most preferably about 4000. These are used at levels of from 0.20% to 5% more preferably from 0.25% to 2.5% by weight. These polymers and the previously mentioned homo- or co-polymeric polycarboxylate salts are valuable for improving whiteness maintenance, fabric ash deposition, and cleaning performance on clay, proteinaceous and oxidizable soils in the presence of transition metal impurities.
  • Soil release agents useful in compositions of the present invention are conventionally copolymers or terpolymers of terephthalic acid with ethylene glycol and/or propylene glycol units in various arrangements. Examples of such polymers are disclosed in US 4,116,885 and 4,711,730 and EP 0 272 033.
  • a particular preferred polymer in accordance with EP 0 272 033 has the formula:
  • PEG is -(OC 2 H 4 )0-
  • PO is (OC 3 H 6 0)
  • T is (pOOC 6 H 4 CO) .
  • modified polyesters as random copolymers of dimethyl terephthalate, dimethyl sulphoisophthalate, ethylene glycol and 1, 2-propanediol, the end groups consisting primarily of sulphobenzoate and secondarily of mono esters of ethylene glycol and/or 1, 2-propanediol.
  • the target is to obtain a polymer capped at both end by sulphobenzoate groups, "primarily", in the present context most of said copolymers herein will be endcapped by sulphobenzoate groups.
  • some copolymers will be less than fully capped, and therefore their end groups may consist of monoester of ethylene glycol and/or 1, 2-propanediol, thereof consist “secondarily” of such species.
  • the selected polyesters herein contain about 46% by weight of dimethyl terephthalic acid, about 16% by weight of 1,2- propanediol, about 10% by weight ethylene glycol, about 13% by weight of dimethyl sulfobenzoic acid and about 15% by weight of sulphoisophthalic acid, and have a molecular weight of about 3.000.
  • the polyesters and their method of preparation are described in detail in EP 311 342.
  • Fabric softening agents can also be incorporated into laundry detergent compositions in accordance with the present invention. These agents may be inorganic or organic in type. Inorganic softening agents are exemplified by the smectite clays disclosed in GB-A-1 400898 and in US 5,019,292. Organic fabric softening agents include the water insoluble tertiary amines as disclosed in GB-A1 514 276 and EP 0 011 340 and their combination with mono C 12 -C 1 quaternary ammo- nium salts are disclosed in EP-B-0 026 528 and di-long-chain amides as disclosed in EP 0 242 919.
  • smectite clay levels of smectite clay are normally in the range from 5% to 15%, more preferably from 8% to 12% by weight, with the material being added as a dry mixed component to the remainder of the formulation.
  • Organic fabric softening agents such as the water-insoluble tertiary amines or dilong chain amide materials are incorporated at levels of from 0.5% to 5% by weight, normally from 1% to 3% by weight whilst the high molecular weight polyethylene oxide materials and the water soluble cationic materials are added at levels of from 0.1% to 2%, normally from 0.15% to 1.5% by weight.
  • the detergent compositions according to the present invention may also comprise from 0.001% to 10%, preferably from 0.01% to 2%, more preferably form 0.05% to 1% by weight of polymeric dye- transfer inhibiting agents.
  • Said polymeric dye-transfer inhibiting agents are normally incorporated into detergent compositions in order to inhibit the transfer of dyes from colored fabrics onto fabrics washed therewith. These polymers have the ability of complexing or adsorbing the fugitive dyes washed out of dyed fabrics before the dyes have the opportunity to become attached to other articles in the wash.
  • Especially suitable polymeric dye-transfer inhibiting agents are polyamine N-oxide polymers, copolymers of N-vinyl- pyrrolidone and N-vinylimidazole, polyvinylpyrrolidone polymers, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof.
  • the detergent composition according to the invention can be in liquid, paste, gels, bars or granular forms.
  • 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.
  • waxy coating materials are poly (ethylene oxide) products (polyethyleneglycol, PEG) with mean molecular weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids.
  • film- forming coating materials suitable for application by fluid bed techniques are given in GB 1483591.
  • Granular compositions according to the present invention can also be in "compact form", i.e. they may have a relatively higher density than conventional granular detergents, i.e. form 550 to 950 g/1; in such case, the granular detergent compositions according to the present invention will contain a lower amount of "Inorganic filler salt", compared to conventional granular detergents; typical filler salts are alkaline earth metal salts of sulphates and chlorides, typically sodium sulphate; "Compact" detergent typically comprise not more 5 than 10% filler salt.
  • the liquid compositions according to the present invention can also be in "concentrated form", in such case, the liquid detergent compositions according to the present invention will contain a lower amount of water, compared to conventional liquid detergents. Typically, the water content of the
  • 10 concentrated liquid detergent is less than 30%, more preferably less than 20%, most preferably less than 10% by weight of the detergent compositions.
  • compositions of the invention may for example, be formulated as hand and machine laundry detergent compositions
  • compositions suitable for use in the pre-treatment of stained fabrics including laundry additive compositions and compositions suitable for use in the pre-treatment of stained fabrics, rinse added fabric softener compositions, and compositions for use in general household hard surface cleaning operations and dishwashing operations.
  • TAS Sodium tallow alkyl sulphate
  • XYEZS C lx - C l ⁇ sodium alkyl sulphate condensed with an average of Z moles of ethylene oxide per mole
  • Non-ionic C ⁇ 3 - C 15 mixed ethoxylated/propoxylated fatty alcohol with an average degree of ethoxylation of 3.8 and an average degree of propoxylation of 4.5 sold under the trade name Plurafax LF404 by BASF GmBH
  • CFAA c l2 ⁇ c l4 alkyl N-methyl glucamide
  • TFAA c l6 ⁇ c l8 alkyl N-methyl glucamide
  • NaSKS-6 Crystalline layered silicate of formula ⁇ -Na 2 Si 2 0 5
  • Carbonate Anhydrous sodium carbonate Phosphate: Sodium tripolyphosphate MA/AA: Copolymer of 1:4 maleic/acrylic acid, average molecular weight about 80,000
  • Poly- acrylate Polyacrylate homopolymer with an average molecular weight of 8,000 sold under the trade name PA30 by BASF GmBH
  • Zeolite A Hydrated Sodium Aluminosilicate of formula Na 12 (A10 2 Si0 2 ) 12 .27H0 having a primary particle size in the range from 1 to 10 micrometers
  • Perborate Anhydrous sodium perborate monohydrate bleach, empirical formula NaB0 .H 2 0
  • TAED Tetraacetyl ethylene diamine
  • CMC Sodium carboxymethyl cellulose
  • DETPMP Diethylene triamine penta (methylene phosphonic acid) , marketed by Monsanto under the Trade name Dequest 2060
  • EDDS Ethylenediamine-N, N' -disuccinic acid, [S,S] isomer in the form of the sodium salt Suds 25% paraffin wax Mpt 50°C, 17% hydrophobic silica, 58%
  • HMWPEO High molecular weight polyethylene oxide
  • TAE 25 Tallow alcohol ethoxylate (25)
  • a granular fabric cleaning composition in accordance with the invention may be prepared as follows: 0
  • Detergent Example II 45 A compact granular fabric cleaning composition (density
  • Enzyme of the invention 0.1
  • Granular suds suppressor 3.5 water/minors Up to 100%
  • Granular fabric cleaning compositions in accordance with the invention which are especially useful in the laundering of coloured fabrics were prepared as follows:
  • Granular fabric cleaning compositions in accordance with the invention which provide "Softening through the wash” capability may be prepared as follows:
  • Enzyme of the invention 0.10 0.05
  • Heavy duty liquid fabric cleaning compositions in accordance with the invention may be prepared as follows:
  • Enzyme of the invention 0.10 0.05
  • the invention provides a method for antimicrobial treatment of microorganisms or viruses present on or in human or animal skin, hair, oral cavities, mucous membranes, teeth, eyes,
  • the invention may be useful for disinfection, e.g. treatment of acne or other skin infections, infections in the eye or the mouth, microbial growth on feet, in armpits; teeth (oral care) , wounds, bruises and the like.
  • the treatment may be
  • the invention may be useful for preservation of food, beverages, cosmetics such as lotions, creams, gels, ointments, soaps, shampoos, conditioners, antiperspirants, deodorants, mouth wash, contact lens products, foot bath products; enzyme formulations, or food ingredients.
  • the invention may be applied to the unpreserved food, beverages, cosmetics, food ingredients in an amounts effective for obtaining the desired antimicrobial effect.
  • the invention may be useful for cleaning and/or antimicrobial treatment of contact lenses.
  • the present invention provides a method, which is useful for antimicrobial treatment of any hard surface as defined earlier.
  • the treatment may be applied for general disinfection purposes, e.g. disinfection of hospital wards, operation rooms, rooms for food processing or other facilities, which require disinfection.
  • the hard surface can also be a process equipment member of a cooling tower, a water treatment plant, a dairy, a processing plant for food or food additives, a chemical or pharmaceutical process plant.
  • the hard surface may also be a medical device or a water sanitation equipment.
  • the invention provides an antimicrobial method, which is useful in a conventional cleaning-in-place (C- I-P) system.
  • the laccase and the enhancer employed in the context of the invention may, through a medium, be brought in contact with the surface in question and should be present in amounts, which is has an antimicrobial effect.
  • paint is construed as a substance comprising a solid coloring matter dissolved or dispersed in a liquid vehicle such as water, organic solvent and/or oils, which when spread over a surface, dries to leave a thin colored, decorative and/or protective coating.
  • a liquid vehicle such as water, organic solvent and/or oils
  • isothiazoliones such as 5-chlor-2-methyl-4-thia-zoli-3-on, has been added to the paint as biocides at dosages in the range of about 0.05-0.5% to inhibit/prevent microbial growth in the paint.
  • the invention provides a method for conservation of a paint comprising contacting said paint with a laccase and one or more enhancers of the invention. Further the invention provides a paint composition comprising a laccase and one or more enhancers of the invention.
  • the paint is preferably a water based paint, i.e. the solids of the paint is dispersed in an aqueous solution.
  • the paint may contain 0-20 % organic solvent, preferable 0-10%, e.g. 0-5%.
  • the enzyme may be added the paint in an amount of 0.0001- 100 mg active enzyme protein per litre paint, preferable 0.001-
  • the enhancer may be added in an amount of 10-500 ⁇ M, preferably 25-250 ⁇ M, e.g. 100 ⁇ M of the paint composition.
  • Antibacterial activity of laccases with different enhancers The antimicrobial activity (i.e. bactericidal activity) of recombinant Myceliophthera laccase (rMtL, WO 95/33836) and recombinant Polyporus laccase (rPpL, WO 96/00290) , was tested at pH 6 by use of methylsyringate (MeS) , cinnamic acid, chlorogenic acid, syringaldehyd and ppt as enhancer. Bactericidal activity was evaluated in 0.05 M MES-buffer (2- [N-morpholino]ethanesulfonic acid) (pH 6), which were flushed with air for 5 min before filter sterilisation.
  • MES-buffer 2- [N-morpholino]ethanesulfonic acid
  • the bactericidal activity was determined against Pseudomonas aeruginosa (ATCC10145) and Staphylococcus epidermidis (DSM20042) .
  • Cells were grown in Tryptone Soya Broth (Oxoid CM129) at 30°C for 24 h and diluted in the MES-buffer to a final cell concentration of approximately 10 4 cfu/ml.
  • the cell suspensions were mixed with laccase (3 mg/L) and enhancer (0.1 mM) for 20 min at 40°C.
  • the bactericidal activity was determined by incubation in Malthus. Detection times measured by the Malthus instrument were converted to cfu/ml by a calibration curve.
  • a detection time (dt) will be recorded.
  • the dt's were converted to colony counts by use of a calibration curve relating cfu/ml to dt (Johansen et al. 1995. Journal of Applied Bacteriology 78:297-303, Johansen et al. 1997, Applied and Environmental Microbiology 63:3724-3728).
  • rMtL/MeS or rPpL/MeS The bactericidal activity of rMtL/MeS or rPpL/MeS was particularly pronounced against S . epidermidis (Fig. 1) which was 100% killed (pH 6) by the rPpL/MeS and only a few cells survived the treatment with rMtL/MeS. Whereas the bactericidal activity against P. aeruginosa was observed as a cell reduction of 1-2 log units (Fig. 1) .
  • Antimicrobial activity i.e. bactericidal activity
  • Myceliophthera laccase (rMtL) and methylsyringate (MeS) was determined using a 3 2 factorial design at pH 6 at 40 °C in MES- buffer as described in example 1, however, a higher cell concentration was used (10 7 - 10 8 cfu/ml) .
  • Tested rMtL levels was 0 - 2,5 - 5 mg/L and MeS levels was 0 - 5 - 10 mM.
  • Bactericidal activity against P. aeruginosa ATCC10145 was obtained at MeS concentrations between 5 - 10 mM.
  • bactericidal activity obtained at high MeS concentrations at pH 6 was very close to a 100% bactericidal activity (Fig. 2). At pH 4, a 100% bactericidal activity was obtained.
  • the Gram-positive strain, S . epidermidis was killed 100% by rMtL/MeS treatment at pH 5-6 and MeS concentrations above 0.5 mM. MeS was not bactericidal against the two test-organisms, in the used concentrations, unless combined with laccase.
  • Antimicrobial activity i.e. bactericidal activity
  • rMtL and rPpL with MeS as enhancer was determined at pH 4 - 10 by use of the following buffer systems; homopipes (pH 4 and 5) , MES (pH 6), HEPES (pH 7 and 8), HEPES/CAPS (pH 9) and CAPS (pH 10).
  • Bactericidal activity was evaluated against Staphylococcus epidermidis at 40 °C as described in Example 1. Both rPpL and rMtL have bactericidal activity at low pH (Fig.
  • Bactericidal activity can be obtained at different conditions (pH, temperature etc.) by selecting a laccase and an enhancer, which are active at the particular desired conditions.
  • Antimicrobial activity i.e. bactericidal activity
  • Acetosyringon, methylsyringate, ethylsyringate, butylsyringate and laurylsyringate were evaluated as electron-donors in the concentration of 0.2 mM.
  • Acetosyringon, methylsyringate, ethylsyringate and butylsyringate resulted in a 100% bactericidal activity against S . epidermidis when combined with Polyporus laccase. Whereas the bactericidal activity obtained with Myceliophthera laccase and acetosyringon was decreased compared to both the activity of Polyporus laccase and the activity obtained with the other syringate compounds. The different syringate-compounds were also evaluated against Pseudomonas aeruginosa which are more resistant than S . epidermidis . Acetosyringon was found to be the most bactericidal enhancer against P. aeruginosa .
  • the sensitivity of various microorganisms depends on the used enhancer, thus a broad spectrum of antimicrobial activity may be obtained by combining the different enhancers and applying them simultaneously.
  • the total antimicrobial activity against a mixed culture of different microorganisms is expected to be significantly increased if a mix of enhancers is used.
  • the enhancers are tested in a sub- lethal concentration (less than 100 % bactericidal activity) , and tested in different combinations against microorganisms with different physiology. Synergistic effects may be determined by a multi-factorial experiment with a laccase and enhancers like eg acetosyringon, methylsyringate, ethylsyringate, butylsyringate and laurylsyringate.
  • Antimicrobial activity of laccases with two enhancers was determined against Pseudomonas aeruginosa (ATCC 10145) and Staphylococcus epidermidis (DSM 20042) at pH 6 as described in example 1. Methylsyringate and acetosyringon was used as enhancers and the laccase was rPpL (1 mg/L) and the antimicrobial activity was determined by use of a 3 2 factorial experimental design.
  • Acetosyringon resulted in a cell reduction of P . aeruginosa of approximately 1.5 log units
  • methylsyringate resulted in a cell reduction of approximately 3 log units
  • the combination resulted in a total kill of the test organism corresponding to a cell reduction of approximately 6-7 log units (Fig. 5) .
  • Conservation of a paint may be achieved by preparing a paint sample comprising the enzymatic antimicrobial system by adding an amount of laccase and enhancer, e.g. 1 mg Myceliophthera laccase and 10 ⁇ moles of methylsyringate (sterile filtered) , to 100 mL sterile water based paint in a sterile jar. To this paint sample and to a 100 mL sterile reference sample of paint without the enzyme system is now added 200 ⁇ L 10 cfu/ml mixed culture sample of Pseudomonas sp. and Bacillus sp . The cultures are mixed into the paints and the jars are closed and placed at 30 or 40 °C.
  • laccase and enhancer e.g. 1 mg Myceliophthera laccase and 10 ⁇ moles of methylsyringate (sterile filtered)

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Abstract

L'invention concerne un procédé destiné au traitement antimicrobien des micro-organismes et/ou des virus qui consiste à les traiter avec une quantité efficace d'une Lacasse fongique et d'un ou de plusieurs renforçateurs, en présence de l'oxygène O2, les renforçateurs correspondant à la formule (I).
PCT/DK1998/000477 1997-11-10 1998-11-04 Activite antimicrobienne des lacasses WO1999023887A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
AU97379/98A AU9737998A (en) 1997-11-10 1998-11-04 Antimicrobial activity of laccases
DE69835370T DE69835370T2 (de) 1997-11-10 1998-11-04 Antimicrobielle aktivität von laccasen
CA002308181A CA2308181A1 (fr) 1997-11-10 1998-11-04 Activite antimicrobienne des lacasses
EP98951297A EP1030561B1 (fr) 1997-11-10 1998-11-04 Activite antimicrobienne des lacases
JP2000520002A JP2001522784A (ja) 1997-11-10 1998-11-04 ラッカーゼの抗菌活性
BR9814855-9A BR9814855A (pt) 1997-11-10 1998-11-04 Processo para tratamento antimicrobiano de microrganismos e/ou vìrus, e, composição antimicrobiana.

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DK127397 1997-11-10
DK9801144 1998-09-10
DKPA199801144 1998-09-10
DK1273/97 1998-09-10

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AU (1) AU9737998A (fr)
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CA (1) CA2308181A1 (fr)
WO (1) WO1999023887A1 (fr)

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WO2001049255A3 (fr) * 1999-12-30 2002-02-21 Univ Louisville Res Found Procedes et compositions permettant d'inhiber l'adherence des les micro-organismes
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US6518231B2 (en) 2000-12-18 2003-02-11 Unilever Home & Personal Care Usa, Division Of Conopco, Inc. Enhancement of air bleaching catalysts
US6818149B2 (en) 2000-12-15 2004-11-16 Unilever Home & Personal Care Usa, Division Of Conopco, Inc. Ligand and complex for catalytically bleaching a substrate
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US7063970B1 (en) 1999-05-06 2006-06-20 Norozymes A/S Enzymatic preservation of water based paints
US9040275B2 (en) 2007-12-14 2015-05-26 Novozymes A/S Laccase for detoxification of feed products
US9308264B2 (en) 2000-11-08 2016-04-12 Fxs Ventures, Llc Ophthalmic contact lens solutions containing forms of vitamin B
US9492582B2 (en) 2000-11-08 2016-11-15 Fxs Ventures, Llc Ophthalmic and contact lens solutions containing simple saccharides as preservative enhancers
US9492581B2 (en) 2000-11-08 2016-11-15 Fxs Ventures, Llc Ophthalmic and contact lens solutions containing simple saccharides as preservative enhancers

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EP2252163A2 (fr) * 2008-03-05 2010-11-24 Novozymes A/S Détoxication d' aflatoxin dans les produits d'alimentation
CN103122170B (zh) * 2013-03-10 2015-04-08 山东蓝图家具制造有限公司 一种用于异形件的油漆及其浸涂工艺
CN105395393A (zh) * 2015-11-25 2016-03-16 广州丹奇日用化工厂有限公司 具有去痘印功效的护肤品组合物及祛痘膏
SG11201806124QA (en) * 2016-01-22 2018-08-30 Novapharm Res Australia Pty Ltd Sanitising composition
JP2019094261A (ja) * 2016-03-29 2019-06-20 国立大学法人 東京大学 アフラトキシン産生阻害剤及びアフラトキシン汚染防除方法

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US6355461B2 (en) * 1996-04-29 2002-03-12 Novozymes A/S Non-aqueous, liquid, enzyme-containing compositions
EP1336415A3 (fr) * 1998-08-06 2004-07-28 NDT, Inc. Lentilles de contact et solutions ophtalmiques
EP1336415A2 (fr) * 1998-08-06 2003-08-20 NDT, Inc. Lentilles de contact et solutions ophtalmiques
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US9492581B2 (en) 2000-11-08 2016-11-15 Fxs Ventures, Llc Ophthalmic and contact lens solutions containing simple saccharides as preservative enhancers
US9585394B2 (en) 2000-11-08 2017-03-07 Fxs Ventures, Llc Ophthalmic contact lens solutions containing forms of vitamin B
US9308264B2 (en) 2000-11-08 2016-04-12 Fxs Ventures, Llc Ophthalmic contact lens solutions containing forms of vitamin B
US9492582B2 (en) 2000-11-08 2016-11-15 Fxs Ventures, Llc Ophthalmic and contact lens solutions containing simple saccharides as preservative enhancers
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AU9737998A (en) 1999-05-31
CA2308181A1 (fr) 1999-05-20
BR9814855A (pt) 2000-10-03
CN1278703A (zh) 2001-01-03
CN1177536C (zh) 2004-12-01
JP2001522784A (ja) 2001-11-20

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