WO2014095618A1

WO2014095618A1 - Enzyme treatment composition

Info

Publication number: WO2014095618A1
Application number: PCT/EP2013/076516
Authority: WO
Inventors: Ravine Anthony Gungabissoon
Original assignee: Unilever Plc; Unilever N.V.; Conopco, Inc., D/B/A Unilever
Priority date: 2012-12-20
Filing date: 2013-12-13
Publication date: 2014-06-26
Also published as: CN104884598A; EP2935550A1; BR112015013243A2; CN104884598B

Abstract

An enzymatic treatment composition comprising the combination of: • (iii) one or more enzymes; and • (iv) one or more arginine compounds.

Description

ENZYME TREATMENT COMPOSITION

This invention relates to enzymatic stain removal of stains from fabrics. In particular but not exclusively the invention relates to fabric stain removal composition for stain removal by direct application or pre-treatment of a stain on a stained fabric. Enzymes are used in detergent formulations to aid cleaning and stain removal.

In many climates and in developing countries, laundering is performed at cold or ambient temperatures. These temperatures are a challenge for laundry stain removal products which rely on enzymes which work optimally between 50 - 70 degrees. In the case of modern washing machines, stain removal mainly relies largely on the heating of water above ambient temperatures in the washing machine and this accounts for a large proportion of the laundry related

greenhouse gas footprint - which needs reducing for environmental reasons. The objective of the invention is to improve low / ambient temperature enzyme stain removal of stains on stained fabrics.

In a first aspect, the present invention provides a fabric stain-removal composition comprising the combination of:

(i) one or more enzymes; and

(ii) one or more arginine compounds.

Preferably said stains comprise biological material, preferably proteinaceous and/or starch material deposited on fabric.

Preferably the composition is ambient-active. Preferably the composition comprises one or more surfactants.

In a second aspect, the invention provides a process for removing a stain from a stained fabric substrate, comprising the step of treating the stain with the composition of the first aspect of the invention.

Preferably the method takes place in ambient conditions.

Preferably the method of the second aspect comprises the step of applying said composition directly to the stained fabric, with or without the addition of water. The step of applying the composition may itself be a main washing process or it may be as a pre-step (as a pre-treatment) to a further, subsequent ('main') washing process steps. Such further, subsequent washing steps main comprise any manual washing process or any washing process in a fabric washing machine.

Accordingly, in a third aspect the invention provides a fabric stain removal treatment device comprising a storage chamber for storing the composition of the first aspect of the invention and a treatment member for applying said composition directly to a substrate, preferably using the method of the second aspect.

In a further aspect the invention provides use of an arginine compound in the enzymatic removal of fabric stains. With the invention, the arginine compound enhances the enzymatic removal of stains present on fabric and is effective at lower temperatures. This offers improved laundry (i.e. fabric) cleaning of stained fabrics in regions where ambient water washing is prevalent. Improved washing performance at lower

temperatures may help inhibit the adoption of hot water washing in these countries, a rising trend as standards of living increase and more people are able to afford washing machines. The invention provides enzymatic performance of proteinaceous and/or starch based soil and/or stains in an ambient temperature cleaning processes (with low temperature wash liquor) without serious

consideration to the temperature sensitivity of the enzyme during storage. The enzyme can therefore be selected more freely, on the basis of other

considerations.

The arginine compound improves the removal of based stains at ambient temperature and thus may significantly reduce the energy cost for each wash.

As used herein, the term "substrate" includes fabric, clothing etc.

As used herein, the term "arginine compound" is intended to include any suitable arginine compound including stereoisomeric and racemic forms, derivatives, and substituted derivatives, salts thereof, and any mixtures thereof.

Preferably the arginine compound is present in any wash liquor in a concentration in the range 0.01 mg/ml - 10mg/ml and more preferably in the range 0.01 - 0.32 mg/ml, more preferably 0.08-0.16 mg/ml.

Preferably the arginine compound is present in any composition of the invention in a concentration in the range 40 mg - 5000 mg per dose, preferable 320mg - 4000 mg per dose. The composition may be provided as a single dose format or as multiple dose, free flowing format (powder, liquid, gel, paste etc) which is measured out by the consumer using a dosing device. The dose may range from 10 ml to 100ml.

For pre-treatment devices for localised stain treatment / pre-treatment,

concentration within the composition may be higher and the concentration per dose higher than in main wash formulations, so may in the range 300- 5000 mg per dose, preferably 500 mg - 2000 mg per dose. Pre-treatment device dose levels may vary from 0.1 - 10ml.

The term "ambient-active" is intended to mean less that 25 degrees Celcius and preferably 22 degrees Celcius or less, more preferably 15 degrees or less but always greater than 1 degree Celcius and "active" means effective in achieving stain removal.

As used herein the term "treatment" in the context of enzymatic fabric treatment composition preferably means cleaning and more preferably stain removal.

Preferably "stain removal" is measured in terms of Remission units or a Remission index. "Stain removal" is preferably shown when there is a remission equal to or greater than 2 Remission units and more preferably greater or equal to 5 units. This is represents effective stain removal for a visible (by the human eye) effect.

As used herein the term "enzyme" includes enzyme variants (produced, for example, by recombinant techniques) are included. Examples of such enzyme variants are disclosed, e.g., in EP 251 ,446 (Genencor), WO 91/00345 (Novo Nordisk), EP 525,610 (Solvay) and WO 94/02618 (Gist-Brocades NV).

All percentages mentioned herein are by weight calculated on the total

composition, unless specified otherwise. The abbreviation 'wt%' is to be understood as % by weight of the total composition.

Preferably the pre-treatment composition is ambient-active. Accordingly, the temperature of the wash liquor step of aqueous washing process is therefore less than 40°C and preferably less than 30°C and more preferably less than 25°C and more preferably less than 22°C further more preferably 15°C or less at all times during the washing but excluding drying. Encouraging low temperature wash liquor is advantageous environmentally and financially.

Accordingly, the enzymatic treatment composition is preferably packaged with instructions to treat a substrate at low temperatures using the composition for example in the method described herein, the low temperatures being preferably less than 40 °C, more preferably less than 30°C even more preferably less than 25°C preferably at 22°C or less most preferably at 15 degrees °C. The invention is especially advantageous for the particular situation where one requires enzymatic cleaning of stains in a ambient temperature cleaning

processes (i.e.with ambient temperature wash liquor) but where compositions are unavoidably stored at higher temperatures. Psychrophilic enzymes are effective at low temperatures but are sensitive to raised temperatures due to their flexibility. Mesophilic (and thermophilic) enzymes are stable at raised temperatures, but have reduced performance in low temperature washing conditions. The invention affords low temperature enzymatic cleaning of a substrate using mesophilic enzymes. Accordingly, the enzyme system preferably comprises a mesophilic or

thermophilic enzyme system. The enzyme system may even be a mesophilic and/or thermophilic enzyme system with the exclusion of pyschrophilic enzymes.

Enzymes may be from animal, vegetable, bacterial origin (derived from bacteria) or fungal origin (derived from fungus) however enzymes from bacterial origin are preferred. Chemically modified or protein engineered mutants are included.

Genes encoding such enzymes can be transferred from one host to a preferred expression production host which may or may not be the same as the original host. The one or more enzymes preferably comprises a protease.

Preferred proteases are serine proteases or metallo proteases, preferably an alkaline microbial protease or a trypsin-like protease.

Commercially available protease enzymes include Alcalase™, Savinase™, Primase™, Duralase™, Dyrazym™, Esperase™, Everlase™, Polarzyme™, and Kannase™, (Novozymes A S), Maxatase™, Maxacal™, Maxapem™,

Properase™, Purafect™, Purafect OxP™, FN2™, and FN3™ (Genencor

International Inc.).

In the case of protease enzymes, the arginine compound is underivatised arginine and/or homo-arginine and more preferably underivatised arginine. The one or more enzymes preferably comprises an amylase.

Suitable amylases (alpha and/or beta) include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, alpha-amylases obtained from Bacillus, e.g. a special strain of B. Iicheniformis, described in more detail in GB 1 ,296,839, or the Bacillus sp. strains disclosed in WO 95/026397 or WO 00/060060.

Commercially available amylases are Duramyl™, Termamyl™, Termamyl Ultra™, Natalase™, Stainzyme™, Fungamyl™ and BAN™ (Novozymes A/S), Rapidase™ and Purastar™ (from Genencor International IncCommercially available amylases include Stainzyme™ (Novozymes).

The one or more enzymes may comprises a protease in combination with an amylase and the arginine compound is underivitised arginine. The enzymes are preferably present at 0.001 - 5%wt more preferably 0.01 - 3%.

The composition preferably comprises further enzymes. The composition preferably comprises a lipase; the preferred lipases including so called ' first wash' lipases which comprise a polypeptide having an amino acid sequence which has at least 90 percent sequence identity with the wild-type lipase derived from Humicola lanuginosa strain DSM 4109 and compared to said wild-type lipase, comprises a substitution of an electrically neutral or negatively charged amino acid within 15 A of E1 or Q249 with a positively charged amino acid; and may further comprise:

(I) a peptide addition at the C-terminal;

(II) a peptide addition at the N-terminal;

(III) the following limitations:

I. comprises a negatively charged amino acid in position E210 of said wild-type lipase;

II comprises a negatively charged amino acid in the region corresponding to positions 90-101 of said wild-type lipase; and

III comprises a neutral or negatively charged amino acid at a position corresponding to N94 of said wild-type lipase; and/or

IV has a negative charge or neutral charge in the region corresponding to positions 90-101 of said wild-type lipase; and

v. mixtures thereof.

These are available under the Lipex™ brand from Novozymes. A similar enzyme from Novozymes but believed to fall outside of the above definition has been disclosed by Novozymes under the name Lipoclean™ and this is also preferred. Other possible lipases include lipases from Humicola (synonym Thermomyces), e.g. from other H. lanuginosa (T. lanuginosus) strains or from H. insolens, a Pseudomonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes, P.

cepacia, P. stutzeri, P. fluorescens, Pseudomonas sp. strain SD 705 (WO

95/06720 and WO 96/27002), P. wisconsinensis, a Bacillus lipase, e.g. from B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1 131 , 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422).

Commercially available lipase enzymes include Lipolase™ and Lipolase Ultra™, and the Bacterial enzyme, Lipomax ® ex Genecor. This is a bacterially derived Lipase, of variant M21 L of the lipase of Pseudomonas alcaligenes as described in WO 94/25578 to Gist-Brocades (M. M.M.J. Cox, H.B.M. Lenting, L.J.S.M.

Mulleners and J.M. van der Laan). The composition preferably comprises a phospholipase classified as EC 3.1 .1 .4 and/or EC 3.1 .1 .32. As used herein, the term phospholipase is an enzyme which has activity towards phospholipids. Phospholipids, such as lecithin or

phosphatidylcholine, consist of glycerol esterified with two fatty acids in an outer (sn-1 ) and the middle (sn-2) positions and esterified with phosphoric acid in the third position; the phosphoric acid, in turn, may be esterified to an amino-alcohol. Phospholipases are enzymes which participate in the hydrolysis of phospholipids. Several types of phospholipase activity can be distinguished, including

phospholipases A1 and A2 which hydrolyze one fatty acyl group (in the sn-1 and sn-2 position, respectively) to form lysophospholipid; and lysophospholipase (or phospholipase B) which can hydrolyze the remaining fatty acyl group in

lysophospholipid. Phospholipase C and phospholipase D (phosphodiesterases) release diacyl glycerol or phosphatidic acid respectively.

The composition preferably comprises a cutinase. classified in EC 3.1 .1 .74. The cutinase used according to the invention may be of any origin. Preferably cutinases are of microbial origin, in particular of bacterial, of fungal or of yeast origin.

The composition preferably comprises a cellulase include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulases produced from Humicola insolens, Thielavia terrestris, Myceliophthora thermophila, and Fusarium oxysporum disclosed in US 4,435,307, US 5,648,263, US 5,691 ,178, US 5,776,757, WO 89/09259, WO 96/029397, and WO 98/012307. Commercially available cellulases include Celluzyme™, Carezyme™, Endolase™, Renozyme™ (Novozymes A/S), Clazinase™ and Puradax HA™ (Genencor International Inc.), and KAC-500(B)™ (Kao Corporation). The composition preferably comprises peroxidases/oxidases, especially of bacterial origin. Chemically modified or protein engineered mutants are included. An example of an oxidative bacterium is, but not limited to, are Aeromonas sp wherefrom oxidases can be sourced. The composition preferably comprises a pectate lyase (also called

polygalacturonate lyases) include pectate lyases that have been cloned from different bacterial genera such as Erwinia, Pseudomonas, Klebsiella and

Xanthomonas, as well as from Bacillus subtilis (Nasser et al. (1993) FEBS Letts. 335:319-326) and Bacillus sp. YA-14 (Kim et al. (1994) Biosci. Biotech. Biochem. 58:947-949). Purification of pectate lyases with maximum activity in the pH range of 8-10 produced by Bacillus pumilus (Dave and Vaughn (1971 ) J. Bacteriol. 108:166-174), B. polymyxa (Nagel and Vaughn (1961 ) Arch. Biochem. Biophys. 93:344-352), B. stearothermophilus (Karbassi and Vaughn (1980) Can. J.

Microbiol. 26:377-384), Bacillus sp. (Hasegawa and Nagel (1966) J. Food Sci. 31 :838-845) and Bacillus sp. RK9 (Kelly and Fogarty (1978) Can. J. Microbiol. 24:1 164-1 172) have also been described. Any of the above, as well as divalent cation-independent and/or thermostable pectate lyases, may be used in practicing the invention. In preferred embodiments, the pectate lyase comprises the pectate lyase disclosed in Heffron et al., (1995) Mol. Plant-Microbe Interact. 8: 331 -334 and Henrissat et al., (1995) Plant Physiol. 107: 963-976. Specifically

contemplated pectatel lyases are disclosed in WO 99/27083 and WO 99/27084. Other specifically contemplated pectate lyases (derived from Bacillus

licheniformis) are disclosed in US patent no. 6,284,524 (which document is hereby incorporated by reference). Specifically contemplated pectate lyase variants are disclosed in WO 02/006442, especially the variants disclosed in the Examples in WO 02/006442 (which document is hereby incorporated by reference). Examples of commercially available alkaline pectate lyases include BIOPREP™ and

SCOURZYME™ L from Novozymes A/S, Denmark. The composition preferably comprises a mannanase: Examples of mannanases (EC 3.2.1 .78) include mannanases of bacterial and fungal origin. In a specific embodiment the mannanase is derived from a strain of the filamentous fungus genus Aspergillus, preferably Aspergillus niger or Aspergillus aculeatus (WO 94/25576). WO 93/24622 discloses a mannanase isolated from Trichoderma reseei. Mannanases have also been isolated from several bacteria, including Bacillus organisms. For example, Talbot et al., Appl. Environ. Microbiol., Vol.56, No. 1 1 , pp. 3505-3510 (1990) describes a beta-mannanase derived from Bacillus stearothermophilus. Mendoza et al., World J. Microbiol. Biotech., Vol. 10, No. 5, pp. 551 -555 (1994) describes a beta-mannanase derived from Bacillus subtilis. JP-A-03047076 discloses a beta-mannanase derived from Bacillus sp. JP-A-

63056289 describes the production of an alkaline, thermostable beta-mannanase. JP-A-63036775 relates to the Bacillus microorganism FERM P-8856 which produces beta-mannanase and beta-mannosidase. JP-A-08051975 discloses alkaline beta-mannanases from alkalophilic Bacillus sp. AM-001 . A purified mannanase from Bacillus amyloliquefaciens is disclosed in WO 97/1 1 164. WO 91/18974 describes a hemicellulase such as a glucanase, xylanase or

mannanase active. Contemplated are the alkaline family 5 and 26 mannanases derived from Bacillus agaradhaerens, Bacillus licheniformis, Bacillus halodurans, Bacillus clausii, Bacillus sp., and Humicola insolens disclosed in WO 99/64619. Especially contemplated are the Bacillus sp. mannanases concerned in the Examples in WO 99/64619.

Examples of commercially available mannanases include Mannaway™ available from Novozymes A/S Denmark.

The enzyme and any perfume/fragrance or pro-fragrance present may show some interaction and should be chosen such that this interaction is not negative. Some negative interactions may be avoided by encapsulation of one or other of enzyme and pro-fragrance and/or other segregation within the product.

Surfactant

The composition preferably comprises a surfactant. The surfactant may be a synthetic surfactant or a biosurfactant which is mircrobially synthesized e.g. from bacteria, fungi or other microbe. The

biosurfactant preferably comprises a microbial ly-derived biosurfactant. Preferably it comprises a glycolipid biosurfactant which may be a rhamnolipid or sophorolipid or trehalolipid or a mannosylerythritol lipid (MEL). Alternatively, the biosurfactant may advantageously comprise a cellobiose, peptide based biosurfactants, lipoproteins and lipopeptides e.g. surfactin, fatty acids e.g. corynomucolic acids (preferably with hydrocarbon chain C12-C14) , phospholipids e.g.

Phosphatidylethanolamine produced by Rhodococcus erythropolis grown on n- alkane resulted in the lowering of interfacial tension between water and

hexadecane to less than 1 mN m-1 and CMC of 30 mg L-1 (Kretschner et al., 1982) and Spiculisporic acid; polymeric biosurfactants including emulsan, liposan, mannoprotein and polysaccharide-protein complexes. Preferably the

biosurfactant comprises a rhamnolipid. The composition according to the invention comprises a surfactant, preferably a detersive surfactant. By a detersive surfactant we mean that the surfactant, or at least one surfactant of any surfactant mixture, provides a detersive, i.e. cleaning effect to textile fabrics treated as part of a laundering process. Other surfactants, which may or may not be detersive surfactants, can be used as part of the composition.

The detersive surfactant is present by weight in the laundry detergent

compositions at a level of from 3 to 85% by weight, preferably from 3 to 60% by weight, more preferably from 3 to 40% by weight, most preferably from 3 to 35% by weight. Additional surfactants can also be incorporated in the laundry compositions of the invention; these may be detersive or non-detersive

surfactants.

Preferably the detersive surfactant comprises anionic surfactant, nonionic surfactant or a mixture of the two. More preferably the detersive surfactant mixture comprises anionic and nonionic surfactants. Cationic surfactant may optionally be present as part of the detersive surfactant.

If present, anionic surfactant is present at a level of from 0.1 to 95% by weight, preferably from 1 to 50% by weight, more preferably from 1 .5 to 25% by weight based on total weight of surfactants present. Nonionic surfactant, if present, is incorporated at a level of from 0.1 to 95% by weight, preferably from 1 to 50% by weight, more preferably from 1 .5 to 25% by weight based on total weight of surfactants present. If a detersive surfactant mixture is used that incorporates both anionic and nonionic surfactants, then preferably the ratio of anionic surfactant to nonionic surfactant is from 10:1 to 1 :10.

Nonionic surfactant

For the purposes of this disclosure, 'nonionic surfactant' shall be defined as amphiphilic molecules with a molecular weight of less than about 10,000, unless otherwise noted, which are substantially free of any functional groups that exhibit a net charge at the normal wash pH of 6-1 1 .

Any type of nonionic surfactant may be used. Highly preferred are fatty acid alkoxylates, especially ethoxylates, having an alkyl chain of from C₈-C₃₅, preferably C8-C30, more preferably Cio-C2₄, especially C10-C18 carbon atoms, and having preferably 3 to 25, more preferred 5 to 15 ethylene oxide groups, for example, Neodols from Shell (The Hague, The Netherlands); ethylene

oxide/propylene oxide block polymers which may have molecular weight from 1 ,000 to 30,000, for example, Pluronic (trademark) from BASF (Ludwigshafen, Germany); and alkylphenol ethoxylates, for example Triton X-100, available from Dow Chemical (Midland, Mich., USA).

Other nonionic surfactants considered within the scope of this invention include condensates of alkanolamines with fatty acids, such as cocamide DEA, polyol- fatty acid esters, such as the Span series available from Uniqema (Gouda, The Netherlands), ethoxylated polyol-fatty acid esters, such as the Tween series available from Uniqema (Gouda, The Netherlands), alkylpolyglucosides, such as the APG line available from Cognis (Dusseldorf, Germany) and n- alkylpyrrolidones, such as the Surfadone series of products marketed by ISP (Wayne, N.J ., USA). Anionic surfactant

'Anionic surfactants' are defined herein as amphiphilic molecules comprising one or more functional groups that exhibit a net anionic charge when in aqueous solution at the normal wash pH of between 6 and 1 1 .

Preferred anionic surfactants are the alkali metal salts of organic sulphur reaction products having in their molecular structure an alkyl radical containing from about 6 to 24 carbon atoms and a radical selected from the group consisting of sulphonic and sulphuric acid ester radicals.

Although any anionic surfactant hereinafter described can be used, such as alkyl ether sulphates, soaps, fatty acid ester sulphonates, alkyl benzene sulphonates, sulphosuccinate esters, primary alkyl sulphates, olefin sulphonates, paraffin sulphonates and organic phosphate; preferred anionic surfactants are the alkali and alkaline earth metal salts of fatty acid carboxylates, fatty alcohol sulphates, preferably primary alkyl sulfates, more preferably they are ethoxylated, for example alkyl ether sulfates; and alkylbenzene sulfonates or mixtures thereof.

Cationic, amphoteric surfactants and/or zwitterionic surfactants

Also cationic, amphoteric surfactants and/or zwitterionic surfactants may be present in the compositions according to the invention.

Preferred cationic surfactants are quaternary ammonium salts of the general formula R₁R₂RsR₄N⁺ X^", for example where Ri is a Ci₂-Ci alkyl group, R₂ and R₃ are methyl groups, R is a 2-hydroxyethyl group, and X^" is a chloride ion. This material is available commercially as Praepagen (Trade Mark) HY from Clariant GmbH, in the form of a 40% by weight aqueous solution.

In a preferred embodiment the composition according to the invention comprises an amphoteric or zwitterionic surfactant. Amphoteric surfactants are molecules that contain both acidic and basic groups and will exist as zwitterions at the normal wash pH of between 6 and 11. Preferably an amphoteric or zwitterionic surfactant is present at a level of from 0.1 to 20% by weight, more preferably from 0.25 to 15% by weight, even more preferably from 0.5 to 10% by weight.

Suitable zwitterionic surfactants are exemplified as those which can be broadly described as derivatives of aliphatic quaternary ammonium, sulfonium and phosphonium compounds with one long chain group having about 8 to about 18 carbon atoms and at least one water solubilizing radical selected from the group consisting of sulfate, sulfonate, carboxylate, phosphate or phosphonate. A general formula for these compounds is:

Ri(R₂)xY⁺R₃Z- wherein Ri contains an alkyl, alkenyl or hydroxyalkyi group with 8 to 18 carbon atoms, from 0 to 10 ethylene-oxy groups or from 0 to 2 glyceryl units; Y is a nitrogen, sulfur or phosphorous atom; R₂ is an alkyl or hydroxyalkyi group with 1 to 3 carbon atoms; x is 1 when Y is a sulfur atom and 2 when Y is a nitrogen or phosphorous atom; R₃ is an alkyl or hydroxyalkyi group with 1 to 5 carbon atoms and Z is a radical selected from the group consisting of sulfate, sulfonate, carboxylate, phosphate or phosphonate.

Preferred amphoteric surfactants are amine oxides, for example coco dimethyl amine oxide. Preferred zwitterionic surfactants are betaines, and especially amidobetaines. Preferred betaines are C₈ to Ci₈ alkyl amidoalkyl betaines, for example coco amido betaine. These may be included as co-surfactants, preferably present in an amount of from 0 to 10 wt %, more preferably 1 to 5 wt %, based on the weight of the total composition. Preferred amphoteric or zwitterionic surfactants for incorporation in the

composition according to the present invention are betaine surfactants. Examples of these are mentioned in the following list.

The sulfatobetaines, such as 3-(dodecyldimethylammonium)-1 -propane sulfate; and 2-(cocodimethylammonium)-1 -ethane sulfate.

The sulfobetaines, such as: 3-(dodecyldimethyl-ammonium)-2-hydroxy-1 -propane sulfonate; 3-(tetradecyl-dimethylammonium)-1 -propane sulfonate; 3-(Ci2-Ci₄ alkyl- amidopropyldimethylammonium)-2-hydroxy-1 -propane sulfonate; and 3-

(cocodimethylammonium)-l -propane sulfonate.

The carboxybetaines, such as (dodecyldimethylammonium) acetate (also known as lauryl betaine); (tetradecyldimethylammonium) acetate (also known as myristyl betaine); (cocodimethylammonium) acetate (also known as coconut betaine);

(oleyldimethylammonium) acetate (also known as oleyl betaine);

(dodecyloxymethyldimethylammonium) acetate; and (cocoamido- propyldimethylammonium) acetate (also known as cocoamido-propyl betaine or

CAPB).

The sulfoniumbetaines, such as: (dodecyldimethylsulfonium) acetate; and 3- (cocodimethyl-sulfonium)-l -propane sulfonate.

The phosphoniumbetaines, such as 4-(trimethylphosphonium)-1 -hexadecane sulfonate; 3-(dodecyldimethylphosphonium)-1 -propanesulfonate; and

2-(dodecyldimethylphosphonium)-1 -ethane sulfate.

The compositions according to the present invention preferably comprise carboxybetaines or sulphobetaines as amphoteric or zwitterionic surfactants, or mixtures thereof. Especially preferred is lauryl betaine.

The treatment composition may comprise other ingredients commonly found in detergent liquids. Especially polyester substantive soil release polymers, hydrotropes, opacifiers, colorants, perfumes, other enzymes, other surfactants, microcapsules of ingredients such as perfume or care additives, softeners, polymers for anti redeposition of soil, bleach, bleach activators and bleach catalysts, antioxidants, pH control agents and buffers, thickeners, external structurants for rheology modification, visual cues, either with or without functional ingredients embedded therein and other ingredients known to those skilled in the art.

The invention will now be further described with reference to the following non- limiting examples in which

Figure 1 is a bar chart displaying the average RFU readings from Example 1 for replicates 1 -4 from Table 1 vs arginine concentration. Error bars display standard deviation between the four replicates for each concentration;

Figure 2 is a bar chart displaying the average RFU readings from Example 2 for replicates 1 -4 from Table 2 vs arginine concentration. Error bars display standard deviation between the four replicates for each concentration.

Examples

All values throughout are wt% of the total base composition A which is enzyme free, so that the only enzymes in each Example are those specifically referred to as added. Detergent Composition A

Ingredient % by weight of total Composition A

Non-ionic surfactant Neodol 25-7 6.2

Anionic surfactant LAS acid 11.8

Anionic surfactant SLES 3EO 6.5

Laurie Fatty Acid P5908 5.2

Glycerol 5.0

Monopropylene Glycol 9.0

Citric acid 3.9

Minors 2.0

Water balance to 100

Wherein:

Neodol 25-7 ex. Shell = C12-C15 alcohol 7-ethoxylate

LAS acid = Cio-Ci₄ alkyl benzene sulphonic acid;

SLES = C12-C13 alcohol 3-ethoxylate sulphate, Na salt: = sodium lauryl ether sulphate (with on average 3 ethylene oxide groups);

Example 1 : The effect of arginine on Savinase activity Example 1 Reagents:

- Reaction buffer: 50 mM Tris.HCI (2-Amino-2-hydroxymethyl-1 ,3-propanediol hydrochloride Trizma® hydrochloride ex. Sigma Aldrich)(pH 8.0)

- Savinase 16L (ex Novozymes).

- BODIPY TR-X dye conjugated casein substrate solution prepared as follows: 1 mg of BODIPY TR-X dye labelled casein substrate (Invitrogen, Cat No. E6639) was thoroughly dissolved in 200 μΙ of 0.1 M sodium bicarbonate buffer (pH 8.5). This 200 μΙ was then added to 19.8 ml of 50 mM T s.HCI (pH 8.0) to make a working solution of 10 Mg/ml. Fresh substrate was prepared shortly before each assay and stored in the dark at room temperature until required. - DL-Arginine (Sigma Cat No. 1 1020, EC Number: 230-571 -3)

Example 1 Experimental Protocol

The following reaction mixtures were prepared in a 300 μΙ well / 96 well microtitre (microwell) plate:

Test Mixture:

Savinase 16L (2.6 Mg/ml): 100 μΙ

BODIPY TR-X dye labelled casein substrate (10 Mg/ml) 80 μΙ

DL-arginine (0.15 to 1 M) 20 μΙ

Control Mixture (No arginine): Savinase 16L (2.6 Mg/ml): 100 μΙ

BODIPY TR-X dye labelled casein substrate (10 Mg/ml) 80 μΙ

50mM Tris.HCI (pH 8.0) 20 μΙ

In both cases the substrate was added last and the reactions incubated at 22 degrees for 1 hour. Protease-catalyzed hydrolysis releases highly fluorescent BODIPY TR-X dye-labelled peptides. The accompanying increase in

fluorescence is proportional to protease activity. Fluorescence intensity was measured in a fluorescence microplate reader set for excitation at 485 nm and emission detection at 530 nm. Example 1 Results

Table 1 : Relative fluorescent units (RFU) measured for 1 .3 μς/ιτιΙ Savinase and 4 μς/ιτιΙ BODIPY TR-X dye labelled casein substrate with a range arginine

concentrations. Four replicates were carried out for each concentration in parallel.

The results are shown in Figure 1

Arginine increases relative Savinase activity up to a maximum of 51.4 % at 0.25 M. Example 2 The effect of arginine on Stainzyme activity Example 2 Reagents:

- Reaction buffer: 50 mM MOPS (pH 6.9)

- Stainzyme 12L (Novozymes).

- BODIPY FL dye conjugated DQ starch substrate solution: 1 mg of BODIPY FL dye conjugated DQ starch substrate (Invitrogen, Cat No. E33651 ) was thoroughly dissolved in 100 ul of 50 mM sodium acetate buffer (pH 4.0). This 100 ul was added to 900 ul of 50 mM MOPS (pH 6.9) to make a working solution of 10 ug/ml. Fresh substrate was prepared shortly before each assay and stored in the dark at room temperature until required.

- Arginine (Sigma Cat No. 1 1020, EC Number: 230-571 -3)

Example 2 Protocol:

The following reaction mixtures were prepared in a 300 μΙ 96 well plate: Test Mixture:

Stainzyme 12L (0.34 ug/ml) 80 μΙ

BODIPY FL dye conjugated DQ starch substrate (10 μς/ιτιΙ) 100 μΙ

DL-arginine (0.15 to 1 M) 20 μΙ Control Mixture (No arginine):

Stainzyme 12L (0.34 ug/ml) 80 μΙ

BODIPY FL dye conjugated DQ starch substrate (10 μg/ml) 100 μΙ

50 mM MOPS (pH 6.9) 20 μΙ In both the Control and the Test Mixture the substrate was added last and the reactions incubated at 22 degrees for 30 mins. Degradation of the substrate by amylase releases highly fluorescent fragments . The accompanying increase in fluorescence is proportional to amylase activity. Fluorescence intensity was measured in a fluorescence microplate reader set for excitation at 485 nm and emission detection at 520 nm. Example 2 Results

Table 2: Relative fluorescent units (RFU) measured for Stainzyme and BODIPY FL dye conjugated DQ starch substrate with a range DL-arginine concentrations. Four replicates were carried out for each concentration in parallel.

The results of Table 2 are shown in Figure 2. Arginine increases relative Stainzyme activity up to a maximum of 14.3 % at 0.06 M

Example 3 End-point Stain Removal Assays

Example 3 Reagents:

- The following soiled cloth samples were hole punched into discs and transferred to 300 μΙ 96 well plates:

Protease sensitive stain: EMPA1 17: blood/milk/ink (Testfabrics Inc.)

Amylase sensitive stain: CS27: potato starch, coloured (Testfabrics Inc.) - Laundry Enzymes (Novozymes):

(1 ) Savinase 16L

(2) Stainzyme 12L

- Composition A

- DL-Arginine (Sigma Cat No. 1 1020, EC Number: 230-571 -3) Example 3 Protocol :

- The stained cloth was pre-rinsed to remove any residual free stain:

- 200 μΙ of distilled water was added to each well

- Plates shaken at 900 rpm for 10 mins

- Wash liquor removed

Reaction mixtures: Test Mixture:

Enzyme (Stainzyme / Savinase) 5 mg/L 20 μΙ

Example composition A 5 mg/L 100 μΙ

Arginine dilution^* 20 μΙ

Distilled water 60 μΙ

^*Arginine diluted to the following mg/ml concentrations in distilled water: 0.08, 0.16, 0.32, 0.64, 1 .28, 2.56 and 5.12 Control Mixture:

Enzyme (Stainzyme / Savinase) 5 mg/L 20 μΙ

Example c A: 5 mg/L 100 μΙ

Distilled water 80 μΙ

- In both cases the enzyme was added last. Reactions were incubated at 22 degrees for 1 hour with shaking at 900 rpm.

- The cloth was rinsed by adding 200 μΙ of distilled water to each well followed by shaking at 900 rpm for 5 minutes. The wash liquor was then removed. This procedure was repeated four consecutive times.

- The cloth was dried for 3 hours at 40 degrees

- After drying, the stain plates were digitally scanned and their deltaE measured. This value is used to express cleaning effect and is defined as the colour difference between a white cloth and that of the stained cloth after being washed. Mathematically, the definition of deltaE is: deltaE = [ (ΔΙ_) ² + (Aa) ² + (Ab) ² ] ^{1 2} wherein ΔΙ_ is a measure of the difference in darkness between the washed and white cloth; Aa and Ab are measures for the difference in redness and yellowness respectively between both cloths. From this equation, it is clear that the lower the value of deltaE, the whiter the cloth will be. With regard to this colour

measurement technique, reference is made to Commission International de I'Eclairage (CIE); Recommendation on Uniform Colour Spaces, colour difference equations, psychometric colour terms, supplement no. 2 to CIE Publication, no. 15, Colormetry, Bureau Central de la CIE, Paris 1978. Example 3 Results

In the tables below the cleaning effect is expressed in the form of a stain removal index (SRI):

SRI = 100 - deltaE.

The higher the SRI the cleaner the cloth, SRI = 100 (white). Table 3.1 : End-point stain removal assays using EMPA1 17 stained cloth treated with Savinase and a range of arginine concentrations in composition A. Four replicates were performed in parallel on the same 96 well plate. The plates were scanned and the deltaE and SRI values calculated.

The results of Table 3.1 are shown in Figure 3.1 . Table 3.2: End-point stain removal assays using CS27 stained cloth treated with Stainzyme and a range of DL-arginine concentrations in composition A. Four replicates were performed in parallel on the same 96 well plate. The plates were scanned and the deltaE and SRI values calculated. Rep 1 Rep 2 Rep 3 Rep 4 Average

Arginine

(mg/ml) SRI SRI SRI SRI SRI STDEV

0 73.0 71 .5 71 .8 73.0 72.3 0.8

0.08 76.3 74.2 76.1 76.4 75.7 1 .0

0.16 79.3 78.2 80.1 79.1 79.2 0.8

0.32 83.0 81 .6 82.5 82.7 82.4 0.6

0.64 84.2 82.3 82.9 83.6 83.2 0.8

1 .28 85.6 84.0 84.3 84.7 84.7 0.7

2.56 86.1 84.7 85.3 86.2 85.6 0.7

5.12 86.5 85.4 86.0 86.0 86.0 0.4

The results of Table 3.2 are shown in Figure 3.2.

These results demonstrate that the arginine compound, DL-arginine can enhance the stain removal activity of Savinase and Stainzyme in a dose dependent manner.

Claims

1 . A fabric stain removal composition comprising the combination of:

(i) one or more enzymes; and

(ii) one or more arginine compound compounds.

2. A fabric stain removal composition according to claim 1 characterised in that it is ambient-active.

3. A fabric stain removal composition according to claim 1 characterised in that it comprises one or more surfactants.

4. A fabric stain removal composition according to claim 1 characterised in that the one or more enzymes comprises a protease.

5. A fabric stain removal composition according to claim 1 characterised in that the one or more enzymes comprises an amylase.

6. A process for removing a stain from a stained fabric , comprising the step of treating the stain with the composition of any of claims 1 -5.

7. A process according to claim 6 characterised in that method takes place in ambient conditions.

8. A process according to claim 7 or 8 characterised in that the composition is applied directly to the stain, said step optionally being a pre-treatment step.

9. Use of an arginine compound in combination with one or enzymes, said one or more enzymes being preferably a protease and/or an amylase, in the removal of a stain from a stained fabric.