Field of Invention

The present Invention provides and enzymatic and dispersant formulation for use in domestic laundry.

Background of Invention

Laundry detergent formulations containing a high fraction of anionic surfactant relative to non-ionic surfactant are ubiquitous. Metalloprotease enzymes are used in laundry detergent formulations to remove protein containing stains from fabrics.

WO2013/087286 (Unilever) discloses liquids formulations containing alkyl ether carboxylic acids, betaines, anionic surfactant, non-ionic surfactant for providing softening benefits.

US 5 269 960 discloses stable liquid, aqueous enzyme detergent comprising a mixture of nonionic and anionic surfactants, enzymes and a calcium ion source as an enzyme stabilizer, and an alkyl ether carboxylate as a phase stabilizer. There is a need to increase stain removal in laundry formulations containing a high fraction of anionic surfactant relative to non-ionic surfactant.

Summary of the Invention

Surprisingly the combination of a metalloprotease with specific alkyl ether carboxylic acid, provides enhanced stain removal. In one aspect the present invention provides a laundry detergent composition comprising:

(i) from 5 to 50 wt% of a surfactant, other than an alkyi ether carboxylic acid dispersant, selected from: anionic and non-ionic surfactants, preferably from 6 to 30 wt%, more preferably 8 to 20 wt%; wherein the weight fraction of non-ionic surfactant anionic surfactant is from 0 to 0.3, preferably 0 to 0.15, most preferably 0.05 to 0.12; from 0.5 to 20 wt%, preferably 1 .5 to 10 wt%, most preferably 2.5 to 5 wt% of an alkyi ether carboxylic acid dispersant of the following structure:

R-(OCH ₂ CH ₂ )n-OCH ₂ -COOH,

wherein:

R is selected from saturated and mono-unsaturated C10 to C26 linear or branched alkyi chains, preferably C12 to C24 linear or branched alkyi chains, most preferably a C16 to C20 linear alkyi chain; n is selected from 5 to 20, preferably 7 to 13, more preferably 8 to 12, most preferably 9.5 to 10.5; and,

(iii) from 0.0005 to 0.2wt% of a metallo protease enzyme, preferably from 0.002 to 0.02 wt%.

The wt% of anionic surfactants are calculated as the sodium salt. The wt% of the alkyi ether carboxylic acid dispersant is calculated as the COOH form. In a further aspect the present invention provides a domestic method of treating a textile, the method comprising the steps of:

(i) treating a textile with from 1 g/L of an aqueous solution of the laundry detergent composition as defined in any one of the preceding claims; and,

(ii) allowing said aqueous laundry detergent solution to remain in contact with the textile for 10 minutes to 2 days then rinsing and drying the textile.

Detailed Description of the Invention Detergent format

Preferably the laundry detergent formulation is a non-phosphate built laundry detergent formulation, i.e., contains less than 1 wt% of phosphate. Preferably powder laundry detergent formulations are predominantly carbonate built. Powders, should preferably give an in use pH of 9.5-1 1. Most preferably the laundry detergent is an aqueous liquid laundry detergent, preferably with a pH of from 7 to 9.

Protease

Metallo Protease enzymes (E.C 3.4.24) hydrolyse bonds within peptides and proteins, in the laundry context this leads to enhanced removal of protein or peptide containing stains.

Metalloproteases (metallopeptidases or metalloproteinases) is a class of hydrolases which cleave peptide bonds by the action of a water molecule which is activated by complexing to at least one bivalent metal ions belonging to the group of zinc, manganese, cobalt, nickel or copper ions, preferably zinc.

According to the classification of proteases based on protein structure and homology implemented in the MEROPS database (http://merops.sanger.ac.uk/cgi- bin/family_index?type=P), metalloproteases are found in 14 different families. Preferably the protease is selected from the M4, M7 or M35 family, more preferably an M4 metalloprotease, most preferably a neutral metalloprotease.

The metallo-proteases that may be used in this invention includes any of those which may be used in a homecare application. These metallo-proteases are, for example, derived from bacterium selected from the group consisting bacillus amyloliquefaciens, bacillus subtilis, bacillus stearothermophilus, and bacillus thermoproteolyticus, and fungi selected from the group consisting Aspergillus oryzae and Aspergillus niger. The term "M4 Metalloprotease Family" or "M4 Metalloprotease" or "M4" as used herein means a polypeptide falling into the M4 metalloprotease family according to Rawlings et al., Biochem. J., 290, 205-218 (1993) and as further described in MEROPS - (Rawlings et al. , MEROPS: the peptidase database, Nucl Acids Res, 34 Database issue, D270-272, 2006). The M4 metalloproteases are neutral metalloproteases containing mainly endopeptidases. All peptidases in the family bind a single, catalytic zinc ion. M4 metalloprotease family members include the common HEXXH motif, where the histidine residues serve as zinc ligands and glutamate is an active site residue. M4 metalloproteases have a pH optimum mainly at neutral pH. The M4 metalloprotease family includes, e.g., Neutrase™

(Novozymes) (classified as MEROPS subclass M04.014), Thermolysin, Bacillolysin, vibriolysin, pseudolysin, Msp peptidase, coccolysin, aureolysin, vimelysin, lambda toxin neutral peptidase B, PA peptidase (Aeromonas- type), griselysin, stearolysin, Mprlll

(Alteromonas sp. strain 0-7), pap6 peptidase, neutral peptidase (Thermoactinomyces-type), ZmpA peptidase (Burkholderia sp.), zpx peptidase, PrtS peptidase (Photorhabdus luminescens), protealysin, ZmpB peptidase (Burkholderia sp.). The M4 metalloprotease family of polypeptides have been further characterized and presently includes, according to MEROPS, at least twenty-two subclasses for which a distinct MEROPS ID (i.e., an identifier of the formula M04.xxx) has been assigned, as well as non-peptidase homologues and unassigned peptidases. The term "Thermolysin-Like Metalloprotease" as used herei n means (a) an M4

metalloprotease of the MEROPS subclass M04.001 ; (b) an M4 metalloprotease of the MEROPS subclass M04.018; (c) an M4 metalloprotease of the MEROPS subclass M04.021 ; (d) an M4 metal loprotease having an active cleft motif: TG[TS] [QS] D N GGVH [Tl] ; (e) an M4 metalloprotease having an active cleft motif: DPDHSKRYTG[TS][QS]DNGGVH[TI]NSGI; and (f) an M4 metalloprotease having an active cleft motif: NT[TS][QS]DNGGVH[TI]NSGI. In these motifs, the accepted lUPAC single letter amino acid abbreviation is employed. Also in the above motifs, the use of brackets indicates alternative amino acid choices at a particular position. The Thermolysin-Like Metalloproteases are described, e.g., in Barret et al. (1998) Handbook of proteolytic enzymes. Academic Press, pp. 350-369. The amino acid sequences of several TLPs have been determined, and the three dimensional structures of several TLPs have been solved. Focus has been on increasing the thermostability of the TLP and a number of publications describe the thermostability of Bacillus TLPs, e.g., Veltman et al., (1998) Biochemistry 37(15):5312-9. The TLPs consist of an alpha-helical C- terminal domain and an N-terminal domain mainly consisting of beta-strands. The domains are connected by a central alpha-helix. This helix is located at the bottom of the active site cleft and contains several of the catalytically important residues such as four substrate binding pockets S2, Si , Si' and S2' have been identified (Hangauer et al. (1984)

Biochemistry 23:5730-5741 )

The term "M7 Metalloprotease Family" or "M7 Metalloprotease" or "M7" or "snapalysin family" as used herein means a polypeptide falling into the M7 metalloprotease family according to Rawlings et al., Biochem. J., 290, 205-218 (1993) and as further described in MEROPS - (Rawlings et al., MEROPS: the peptidase database, Nucl Acids Res, 34

Database issue, D270- 272, 2006). The protease family M7 contains a

metalloendopeptidase, snapalysin. Snapalysin is active at neutral pH. The only known activity is cleavage of proteins of skimmed milk to form clear plaques around the growing bacterial colonies. The Zinc is bound by two histidines and an aspartate in an

HEXXHXXGXXD sequence motif; the glutamate is a catalytic residue. The M7 proteases have clear signal peptides recognized by the SignalP prediction program. They also all have a propeptide that is cleaved off.

The term "M35 M etalloprotease Fami ly" or "M 35 Metal loprotease" or "M 35" or

"deuterolysin family" as used herein means a polypeptide falling into the M35

metalloprotease family according to Proteolysis in Cell Function, pp13-21 , IOS Press, Amsterdam (1997), Rawlings et al., Biochem. J., 290, 205-218 (1993) and as further described in MEROPS - (Rawlings et al., MEROPS: the peptidase database, Nucl Acids Res, 34 Database issue, D270- 272, 2006). Family M35 members contain two zinc binding histidines and a catalytic glutamate in an HEXXH motif. There is a third zinc ligand, an Asp, found in a GTXDXXYG motif C-terminal to the His zinc ligands (see the Alignment). For this reason the peptidases in this family are sometimes termed "aspzincins", although peptidases in which the third ligand of zinc is Asp also occur in families M6, M7 and M64. AlkyI Ether Carboxylic acid

In the context of the current invention alkyl ether carboxylic acid dispersants are not included as anionic surfactants. Weights of alkyl ether carboxylic acid are calculated as the protonated form, R-(OCH2CH2)n-OCH2COOH. They may be used as salt version for example sodium salt, or amine salt.

The alkyl chain may be linear or branched, preferably it is linear.

The alkyl chain may be aliphatic or contain one cis-double bond.

The alkyl chain is most preferably CH ₃ (CH ₂ )7CH=CH(CH ₂ )8-

The alkyl ether carboxylic acid is most preferably of the structure: CH ₃ (CH2)7CH=CH(CH2)8(OCH2CH2)ioOCH ₂ COOH.

Alkyl ether carboxylic acid are available from Kao (Akypo ®), Huntsman (Empicol®) and Clariant (Emulsogen ®)

Surfactants

The laundry composition comprises anionic charged surfactant (which includes a mixture of the same). Suitable anionic detergent compounds which may be used are usually water-soluble alkali metal salts of organic sulphates and sulphonates having alkyl radicals containing from about 8 to about 22 carbon atoms, the term alkyl being used to include the alkyl portion of higher alkyl radicals. Examples of suitable synthetic anionic detergent compounds are sodium and potassium alkyl sulphates, especially those obtained by sulphating higher Cs to Cis alcohols, produced for example from tallow or coconut oil, sodium and potassium alkyl Cg to C20 benzene sulphonates, particularly sodium linear secondary alkyl C10 to C15 benzene sulphonates; and sodium alkyl glyceryl ether sulphates, especially those ethers of the higher alcohols derived from tallow or coconut oil and synthetic alcohols derived from petroleum.

The anionic surfactant is preferably selected from: linear alkyl benzene sulphonate; alkyl sulphates; alkyl ether sulphates; soaps; alkyl (preferably methyl) ester sulphonates, and mixtures thereof.

The most preferred anionic surfactants are selected from: linear alkyl benzene sulphonate; alkyl sulphates; alkyl ether sulphates and mixtures thereof. Preferably the alkyl ether sulphate is a C12-C14 n-alkyl ether sulphate with an average of 1 to 3EO (ethoxylate) units. Sodium lauryl ether sulphate is particularly preferred (SLES). Preferably the linear alkyl benzene sulphonate is a sodium Cn to C15 alkyl benzene sulphonates. Preferably the alkyl sulphates is a linear or branched sodium C12 to C18 alkyl sulphates. Sodium dodecyl sulphate is particularly preferred, (SDS, also known as primary alkyl sulphate).

The level of anionic surfactant in the laundry composition is preferably from (i) 5 to 50 wt% negatively charged surfactant, preferably the level of negatively charged surfactant is from 6 to 30 wt%, more preferably 8 to 20 wt%. Preferably two or more anionic surfactant are present, preferably linear alkyl benzene sulphonate together with an alkyl ether sulphate.

Non-ionic surfactant may be present in the surfactant mix. Suitable nonionic detergent compounds which may be used include, in particular, the reaction products of compounds having an aliphatic hydrophobic group and a reactive hydrogen atom, for example, aliphatic alcohols, acids or amides, especially ethylene oxide either alone or with propylene oxide. Preferred nonionic detergent compounds are the condensation products of aliphatic Cs to C18 primary or secondary linear or branched alcohols with ethylene oxide.

Preferably the alkyl ethoxylated non-ionic surfactant is a Cs to C18 primary alcohol with an average ethoxylation of 7EO to 9EO units. Builders or Complexinq Agents

Builder materials may be selected from 1 ) calcium sequestrant materials, 2) precipitating materials, 3) calcium ion-exchange materials and 4) mixtures thereof.

Examples of calcium sequestrant builder materials include alkali metal polyphosphates, such as sodium tripolyphosphate and organic sequestrants, such as ethylene diamine tetra-acetic acid. Examples of precipitating builder materials include sodium orthophosphate and sodium carbonate.

Examples of calcium ion-exchange builder materials include the various types of water- insoluble crystalline or amorphous aluminosilicates, of which zeolites are the best known representatives, e.g. zeolite A, zeolite B (also known as zeolite P), zeolite C, zeolite X, zeolite Y and also the zeolite P-type as described in EP-A-0,384,070.

The composition may also contain 0-65 % of a builder or complexing agent such as ethylenediaminetetraacetic acid, diethylenetriamine-pentaacetic acid, alkyl- or

alkenylsuccinic acid, nitrilotriacetic acid or the other builders mentioned below. Many builders are also bleach-stabilising agents by virtue of their ability to complex metal ions.

Zeolite and carbonate (carbonate (including bicarbonate and sesquicarbonate) are preferred builders for powder detergents.

The composition may contain as builder a crystalline aluminosilicate, preferably an alkali metal aluminosilicate, more preferably a sodium aluminosilicate. This is typically present at a level of less than 15%w. Aluminosilicates are materials having the general formula: 0.8-1 .5 M ₂ 0. AI2O3. 0.8-6 Si02 where M is a monovalent cation, preferably sodium. These materials contain some bound water and are required to have a calcium ion exchange capacity of at least 50 mg CaO/g. The preferred sodium aluminosilicates contain 1.5-3.5 S1O2 units in the formula above. They can be prepared readily by reaction between sodium silicate and sodium aluminate, as amply described in the literature. The ratio of surfactants to alumuminosilicate (where present) is preferably greater than 5:2, more preferably greater than 3:1 .

Alternatively, or additionally to the aluminosilicate builders, phosphate builders may be used. In this art the term 'phosphate' embraces diphosphate, triphosphate, and phosphonate species. Other forms of builder include silicates, such as soluble silicates, metasilicates, layered silicates (e.g. SKS-6 from Hoechst).

Preferably the laundry detergent formulation is a non-phosphate built laundry detergent formulation, i.e., contains less than 1 wt% of phosphate. Preferably the powder laundry detergent formulation is carbonate built.

Fluorescent Agent

The composition preferably comprises a fluorescent agent (optical brightener). Fluorescent agents are well known and many such fluorescent agents are available commercially.

Usually, these fluorescent agents are supplied and used in the form of their alkali metal salts, for example, the sodium salts. The total amount of the fluorescent agent or agents used in the composition is generally from 0.005 to 2 wt %, more preferably 0.01 to 0.1 wt %. Preferred classes of fluorescer are: Di-styryl biphenyl compounds, e.g. Tinopal (Trade Mark) CBS-X, Di-amine stilbene di-sulphonic acid compounds, e.g. Tinopal DMS pure Xtra and Blankophor (Trade Mark) HRH, and Pyrazoline compounds, e.g. Blankophor SN. Preferred fluorescers are: sodium 2 (4-styryl-3-sulfophenyl)-2H-napthol[1 ,2-d]triazole, disodium 4,4'- bis{[(4-anilino-6-(N methyl-N-2 hydroxyethyl) amino 1 ,3,5-triazin-2-yl)]amino}stilbene-2-2' disulfonate, disodium 4,4'-bis{[(4-anilino-6-morpholino-1 ,3,5-triazin-2-yl)]amino} stilbene-2-2' disulfonate, and disodium 4,4'-bis(2-sulfostyryl)biphenyl.

It is preferred that the aqueous solution used in the method has a fluorescer present. When a fluorescer is present in the aqueous solution used in the method it is preferably in the range from 0.0001 g/l to 0.1 g/l, preferably 0.001 to 0.02 g/l. Perfume

Preferably the composition comprises a perfume. The perfume is preferably in the range from 0.001 to 3 wt %, most preferably 0.1 to 1 wt %. Many suitable examples of perfumes are provided in the CTFA (Cosmetic, Toiletry and Fragrance Association) 1992 International Buyers Guide, published by CFTA Publications and

OPD 1993 Chemicals Buyers Directory 80th Annual Edition, published by Schnell Publishing Co.

It is commonplace for a plurality of perfume components to be present in a formulation. In the compositions of the present invention it is envisaged that there will be four or more, preferably five or more, more preferably six or more or even seven or more different perfume components.

In perfume mixtures preferably 15 to 25 wt% are top notes. Top notes are defined by Poucher (Journal of the Society of Cosmetic Chemists 6(2):80 [1955]). Preferred top-notes are selected from citrus oils, linalool, linalyl acetate, lavender, dihydromyrcenol, rose oxide and cis-3-hexanol.

Perfume and top note may be used to cue the whiteness benefit of the invention.

It is preferred that the laundry treatment composition does not contain a peroxygen bleach, e.g., sodium percarbonate, sodium perborate, and peracid.

Polymers

The composition may comprise one or more further polymers. Examples are

carboxymethylcellulose, poly (ethylene glycol), polyvinyl alcohol), polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers. Polymers present to prevent dye deposition, for example poly(vinylpyrrolidone), poly(vinylpyridine-N-oxide), and poly(vinylimidazole), are preferably absent from the formulation.

Further Enzymes

One or more further enzymes are preferred present in a laundry composition of the invention and when practicing a method of the invention.

Preferably the level of each enzyme in the laundry composition of the invention is from 0.0001 wt% to 0.1 wt% protein.

The further enzyme is preferably selected from: amylases lipases; and, cellulases.

Any enzyme present in the composition may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in e.g. WO 92/19709 and WO 92/19708.

Shading Dyes Shading dyes are preferably present in the formulation at a level from 0.002 to 0.2 wt%.

Dyes are described in Color Chemistry Synthesis, Properties and Applications of Organic Dyes and Pigments, (H Zollinger, Wiley VCH, Zurich, 2003) and, Industrial Dyes Chemistry, Properties Applications. (K Hunger (ed), Wiley-VCH Weinheim 2003).

Shading Dyes for use in laundry detergents preferably have an extinction coefficient at the maximum absorption in the visible range (400 to 700nm) of greater than 5000 L mol ^"1 cm ^-1 , preferably greater than 10000 L mol ^"1 cm ^-1 . The dyes are blue or violet in colour. Preferred shading dye chromophores are azo, azine, anthraquinone, and triphenylmethane.

Azo, anthraquinone, phthalocyanine and triphenylmethane dyes preferably carry a net anionic charged or are uncharged. Azine preferably carry a net anionic or cationic charge. Blue or violet shading dyes deposit to fabric during the wash or rinse step of the washing process providing a visible hue to the fabric. In this regard the dye gives a blue or violet colour to a white cloth with a hue angle of 240 to 345, more preferably 250 to 320, most preferably 250 to 280. The white cloth used in this test is bleached non-mercerised woven cotton sheeting.

Shading dyes are discussed in WO2005/003274, WO2006/032327(Unilever),

WO 2006/032397 (Unilever), WO2006/045275 (Unilever), WO06/027086 (Unilever), WO 2008/017570 (Unilever), WO 2008/141880 (Unilever), WO2009/132870 (Unilever), WO 2009/141 173 (Unilever), WO 2010/099997 (Unilever), WO 2010/102861 (Unilever), WO 2010/148624 (Unilever), WO2008/087497 (P&G), WO201 1/01 1799 (P&G),

WO2012/054820 (P&G), WO2013/142495 (P&G), and WO2013/151970 (P&G).

Mono-azo dyes preferably contain a heterocyclic ring and are most preferably thiophene dyes. The mono-azo dyes are preferably alkoxylated and are preferably uncharged or anionically charged at pH=7. Alkoxylated thiophene dyes are discussed in WO/2013/142495 and WO/2008/087497. Preferred examples of thiophene dyes are shown below:

Bis-azo dyes are preferably sulphonated bis-azo dyes. Preferred examples of sulphonated bis-azo compounds are direct violet 7, direct violet 9, direct violet 1 1 , direct violet 26, direct violet 31 , direct violet 35, direct violet 40, direct violet 41 , direct violet 51 , Direct Violet 66, direct violet 99 and alkoxylated versions thereof. Alkoxylated bis-azo dyes are discussed in WO2012/054058 and WO2010/151906.

An example of an alkoxylated bis-azo dye

Azine dye are preferably selected from sulphonated phenazine dyes and cationic phenazine dyes. Preferred examples are acid blue 98, acid violet 50, dye with CAS-No 72749-80-5, acid blue 59, and the phenazine dye selected from:

wherein: X ₃ is selected from: -H; -F; -CH ₃ ; -C ₂ H ₅ ; -OCH ₃ ; and, -OC ₂ H ₅ ;

X ₄ is selected from: -H; -CH ₃ ; -C ₂ H ₅ ; -OCH ₃ ; and, -OC ₂ H ₅ ; Y ₂ is selected from: -OH; -OCH ₂ CH ₂ OH; -CH(OH)CH ₂ OH; -OC(0)CH ₃ ; and, C(0)OCH ₃ .

The shading dye is present is present in the composition in range from 0.0001 to

0.5 wt %, preferably 0.001 to 0.1 wt%. Depending upon the nature of the shading dye there are preferred ranges depending upon the efficacy of the shading dye which is dependent on class and particular efficacy within any particular class. As stated above the shading dye is a blue or violet shading dye.

A mixture of shading dyes may be used. The shading dye is most preferably a reactive blue anthraquinone dye covalently linked to an alkoxylated polyethyleneimine. The alkoxylation is preferably selected from ethoxylation and propoxylation, most preferably propoxylation. Preferably 80 to 95 mol% of the N-H groups in the polyethylene imine are replaced with iso-propyl alcohol groups by propoxylation.

Preferably the polyethylene imine before reaction with the dye and the propoxylation has a molecular weight of 600 to 1800.

An example structure of a preferred reactive anthraquinone covalently attached to a propoxylated polyethylene imine is:

(Structure I).

Preferred reactive anthraquinone dyes are: Reactive blue 1 ; Reactive blue 2; Reactive blue 4; Reactive blue 5; Reactive blue 6; Reactive blue 12; Reactive blue 16; reactive blue 19;

Reactive blue 24 ; Reactive blue 27; Reactive blue 29; Reactive blue 36; Reactive blue 44;

Reactive blue 46 ; Reactive blue 47; reactive blue 49; Reactive blue 50; Reactive blue 53;

Reactive blue 55; Reactive blue 61 ; Reactive blue 66; Reactive blue 68; Reactive blue 69;

Reactive blue 74; Reactive blue 86; Reactive blue 93; Reactive blue 94; Reactive bluel 01 ; Reactive bluel 03; Reactive bluel 14; Reactive bluel 17; Reactive bluel 25; Reactive blue141 ; Reactive blue142; Reactive blue 145; Reactive blue 149; Reactive blue 155;

Reactive blue 164; Reactive blue 166; Reactive blue 177; Reactive blue 181 ; Reactive blue

185; Reactive blue 188; Reactive blue 189; Reactive blue 206; Reactive blue 208; Reactive blue 246; Reactive blue 247; Reactive blue 258; Reactive blue 261 ; Reactive blue 262; Reactive blue 263; and Reactive blue 172.

The dyes are listed according to Colour Index (Society of Dyers and Colourists/American Association of Textile Chemists and Colorists) classification. Experimental Example 1 An aqueous liquid laundry detergent was prepared of the following formulation:

The formulation was used to wash eight 5x5cm EMPA 1 17 stain monitor (blood/milk/ink stain on polycotton) in a tergotometer set at 200rpm. A 20 minute wash was conducted in 800ml of 26° French Hard water at 35°C, with 2.3g/L of the formulation. To simulate oily soil (12.5g) of an SBL2004 soil strip (ex Warwick Equest) was added to the wash liquor.

Once the wash had been completed the cotton monitors were rinsed once in 400ml clean water, removed dried and the colour measured on a reflectometer and expressed as the CIE L ^* a ^* b ^* values.

Equivalent Formulations but with the addition of 8.7wt% alkyl ether carboxylic acid, wherein the alkyl group was cis-9-octadecene, were tested. The average number of ethoxy groups was varied from 2 to 10.

Experiments were repeated with and without the addition of an M4 family protease

(classified as MEROPS subclass M04.014) to the wash liquor. (Neutrase® ex Novozymes). The enzyme was added to give 0.0043 wt% pure active protein to the formulation.

95% confidence limits are also given calculated from the standard deviation on the measurements from the 8 monitors. Without protease With protease Expected

L ^* 95% L ^* 95% L ^*

Control 49.49 0.75 50.51 0.66 -

10EO 56.77 0.52 61.31 0.52 57.78

Inclusion of the metalloprotease and alkyl ether carboxylic acid to the formulation increases the stain removal as seen by higher L ^* values. The combination of metalloprotease with the alkyl ether carboxylic acid gives a greater increase in L ^* than expected from the effect of the protease and alkyl ether carboxylic acid alone where a value of 57.78 is expected and 61.31 obtained.