TECHNICAL FIELD

The present invention relates to the field of laundry detergent compositions. More in particular, it relates to an enzymatic laundry detergent composition in solid form for the promotion of hygiene and the prevention of malodour. Furthermore, it relates to a process for washing fabrics by means of the enzymatic laundry detergent

composition and to a process for manufacturing said enzymatic laundry detergent composition.

BACKGROUND OF THE INVENTION

Modern laundry detergent compositions have become very effective at cleaning soiled fabrics. Among the remaining challenges are the promotion of hygiene and the prevention of malodour, which may occur under certain circumstances, especially when reduced wash times, lower temperatures and more neutral pH values are used. It is believed that such conditions may be less effective to remove microbes or to prevent their metabolism in the wash process or on the fabric. The microbes can then be retained on the fabric or can be distributed across garments in the washing process and they can subsequently contribute to the generation of malodour on the garment during wear or storage.

Suitable solutions to prevent microbial activity and or achieve microbial kill have traditionally involved the application of high pH and elevated temperatures, as well as strong chemicals such as bleaches. An example is the application of sodium percarbonate with a bleach activator such as TAED. However, this bleach system is very inefficient at low temperature and a more neutral to low pH. In order to reduce the environmental impact of laundry washing processes, lower washing temperatures at more neutral pH values have been proposed. For the same reason, the use of strong chemicals has clearly diminished over the last decade. Moreover, there exist many formulations that are bleach free, for instance colour care formulations.

We find it important to develop laundry detergent systems that have a reduced environmental impact. Moreover, there is a constant need for new or alternative laundry detergent composition and fabric washing process providing improved hygiene and having a reduced tendency for malodour.

It is therefore an object of the present invention to provide such a laundry detergent composition and fabric washing process, providing improved hygiene and having a reduced tendency for malodour.

It is a further object of the invention to provide a process for the preparation of such laundry detergent compositions. Still a further object of the invention is to provide a process for the manufacture of such laundry detergent compositions

It has now been surprisingly found that this and further objects of the invention may be achieved by the enzymatic laundry detergent composition in solid form according to the invention comprising one or more surfactants, a source of hydrogen peroxide and a perhydrolase, wherein the composition is essentially free from substrates for the perhydrolase.

It was surprisingly found that when such laundry detergent composition was used in a washing process, an effective inhibition of bacterial metabolism could be observed. In particular, the bacteria that are found on the axilla region are effectively inactivated and /or killed so that one of the main causes of laundry malodour is removed. DEFINITION OF THE INVENTION

According to a first aspect of the invention, there is provided an enzymatic laundry detergent composition in solid form comprising (a) one or more surfactants, (b) a source of hydrogen peroxide and (c) a perhydrolase, wherein the composition is essentially free from substrates for the perhydrolase.

According to a second aspect of the invention, there is provided a process for laundering textile fabrics, characterised in that it comprises the step of immersing the fabrics in a wash liquor comprising water in which the enzymatic laundry detergent composition according to the invention is dissolved or dispersed.

According to a third aspect of the invention, there is provided a process for manufacturing an enzymatic laundry detergent composition according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, all percentages quoted are percentages by weight (wt.%) unless otherwise stated.

In a first aspect, the present invention relates to a laundry detergent composition in solid form. Preferably, it is in the form of a powder. It may also take the form of compressed powder or a tablet. The surfactant

The composition comprises, as a first ingredient, at least one surface active ingredient or surfactant. Depending on the type of laundry detergent, the surfactants are present in an amount of 0.1 to 60 % by weight of the composition. Fabric washing powders typically comprise from 20% to 45% by weight of one or more surfactants. Surfactants are well-known to those skilled in the art. Many suitable detergent- active compounds are available and are fully described in the literature, for example, in "Surface Active Agents" Vol. 1 , by Schwartz & Perry, Interscience 1949, Vol. 2 by Schwartz, Perry & Berch, Interscience 1958, in the current edition of "McCutcheon's Emulsifiers and Detergents" published by Manufacturing

Confectioners Company or in "Tenside Taschenbuch", H. Stache, 2nd Edn., Carl Hauser Verlag, 1981 . Preferably the surfactants used are saturated.

Examples of surfactants include anionic surfactants such as alkylbenzene sulphonates, branched or linear alkyl benzene sulphonates, primary and secondary alcohol sulphates, particularly C8-C16 primary alcohol sulphates; alkyl ether sulphates, olefin sulphonates, including alpha olefin sulphonates, alkane sulphonates, alkyl xylene sulphonates, dialkyl sulphosuccinates, and alkyl carboxylates. These may be present as sodium, potassium, calcium or

magnesium salts or mixtures of these. Sodium salts are generally preferred.

The surfactant is preferably a sulphonate or sulphate anionic surfactant or a combination thereof. More preferably, the anionic surfactant is linear alkylbenzene sulphonate or primary alkyl sulphate. Blends of these two types of anionic surfactants are especially preferred. The linear alkyl benzene sulphonate may be present as sodium, potassium, or alkaline earth metal salts, or mixtures of these salts. Sodium salts are generally preferred.

The surfactant may also comprise a nonionic surfactant, preferably an ethoxylated alcohol nonionic surfactant with an average degree of ethoxylation ranging from about 3 to 9, preferably from about 3 to 7. The alcohol may be derived from natural or synthetic feedstock. Preferred alcohol feedstocks are coconut and palm kernel, predominantly C12-C14, and oxo C12-C15 alcohols. The nonionic surfactant is suitably present in an amount of from 1 to 20 wt.%, preferably from 1 to 10, more preferably from 2 to 6 wt.%, most preferably from 3 to 5 wt.%, based on the weight of the total composition. Additional surfactants may comprise other nonionics such as alkylpolyglucosides, polyhydroxyamides (glucamide), methyl ester ethoxylates and glycerol

monoethers. Also cationic, amphoteric surfactants and/or zwitterionic surfactants may be present. Preferred cationic surfactants are quaternary ammonium salts of the general formula RiR ₂ R3R ₄ N+ X., for example where Ri is a C-12-Cu alkyl group, R ₂ and R ₃ are methyl groups, R ₄ 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 wt.% aqueous solution. 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 C18 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.

The laundry detergent may also contain a biological based surfactant, as sourced and generated via microbial fermentation. Such biosurfactants that could be used are described in Pattanathu et al. Biotechnology 7 (2) 360-370 (2008) and

Muthusamy et al. Current Science Vol. 94 N°6 736-747 (2008).

Preferred are compositions comprising blends of anionic and non-ionic surfactants in a weight ratio of 20 : 1 to 1 : 20, preferably from 20 : 1 to 1 : 1 . . The source of hydrogen peroxide

The laundry detergent composition of the invention comprises, as a second ingredient, a source of hydrogen peroxide. This is preferably based on peroxy bleach compounds, for example, inorganic persalts or organic peroxyacids, capable of yielding hydrogen peroxide in aqueous solution. Suitable peroxy bleach compounds include organic peroxides such as urea peroxide, and inorganic persalts such as the alkali metal perborates, percarbonates, perphosphates, persilicates and persulphates. Preferred inorganic persalts are sodium perborate monohydrate and tetrahydrate, and sodium percarbonate. Especially preferred is sodium percarbonate having a protective coating against destabilisation by moisture. Sodium percarbonate having a protective coating comprising sodium metaborate and sodium silicate is disclosed in GB-A-2 123 044 (Kao). The source of hydrogen peroxide is suitably present in an amount of from 5 to 35 wt.%, preferably from 10 to 25 wt.%.

Alternatively, one may employ an enzymatic hydrogen peroxide-generating system. The enzymatic hydrogen peroxide-generating system may in principle be chosen from the various enzymatic hydrogen peroxide-generating systems which have been disclosed in the art. For example, one may use an amine oxidase and an amine, an amino acid oxidase and an amino acid, cholesterol oxidase and cholesterol, uric acid oxidase and uric acid or a xanthine oxidase with xanthine. In the latter system, superoxide is generated which decomposes to give hydrogen peroxide. Preferably, however, the combination of a C1-C4 alkanol oxidase and a Ci-C ₄ alkanol is used, and especially preferred is the combination of methanol oxidase and ethanol. The methanol oxidase is preferably isolated from a catalase- negative Hansenula polvmorpha strain, (see for example EP-A-244 920

(Unilever)).

The perhydrolase

The laundry detergent composition of the invention comprises, as a third component a perhydrolase. As used herein, the term "perhydrolase" refers to any enzyme that is capable of exhibiting perhydrolase activity. Perhydrolysis is the generation of peroxycarboxylic acids from peroxygen compounds and the corresponding esters or amides.

Enzymatic perhydrolase activity is typically accompanied by hydrolase activity. Preferably the perhydrolases of the present invention have a high ratio of perhydrolase to hydrolase activity, and thereby produce a high ratio of peracid relative to acid products. The high perhydrolysis to hydrolysis ratio makes these perhydrolases very suitable for use in the compositions of the present invention. Preferably, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio of at least about 1 : 1 , more preferably 5: 1 , 10: 1 , or greater. The perhydrolase activity of an enzyme sample can be measured using the assay described below in example 1 .

Suitable perhydrolases are described in US-A-2005/124012 (Henkel) disclosing an enzymatic bleaching system comprising a combination of certain oxidases and perhydrolases. Further suitable perhydrolases are classified as members of the carbohydrate esterase family 7 (CE-7) based on the conserved structural features. Such perhydrolases are described in US-A-2008/0176299 and US-A-2010048448 (DuPont) which disclose a process for producing peroxycarboxylic acids from carboxylic acid esters, wherein carboxylic acid esters are reacted with an inorganic peroxide, such as hydrogen peroxide, in the presence of a specific enzyme catalyst having perhydrolysis activity.

Still further suitable perhydrolases are described in WO-A-2008/140988 (Danisco) which discloses stable compositions comprising a perhydrolase enzyme, a hydrogen peroxide source, and an ester substrate that efficiently generate aqueous peracid solutions. More suitable perhydrolases are described in WO-A- 2010/030769 (Danisco) that relates to compositions and methods for enzymatic bleaching of textiles.

The perhydrolase may be in the form of a partially or completely purified enzyme, but one can also employ the whole microbial cell, permeabilised microbial cell(s), one or more components of a microbial cell extract, of a micro-organism

producing and expressing said perhydrolase. They can also be protein variants, fragments, epitopes or peptides derived thereof.

Especially preferred are the carbohydrate esterase family 7 (CE7) as exemplified in US-A-2008/176299 and US-A-2010/048448. Enzymes with perhydrolase activity can also be obtained through protein engineering and diversity screening e.g. US-A-2005/281773, WO-A-2006/133079, US-A-2006/286651 , or via peptides or fragments thereof e.g. US-A-2007/244021 , or targeted towards a desired substrate specificity e.g. US-A-2007/167344.

Most preferred perhydrolases are the perhydrolases from in US-A-2008/176299 that comprises CE-7 signature motif that aligns with a reference sequence SEQ ID NO: 2 using CLUSTALW, said signature motif comprising: i) an RGQ motif at amino acid positions 1 18-120 of SEQ ID NO:2; ii) a GXSQG motif at amino acid positions 179-183 of SEQ ID NO:2; and iii) an HE motif at amino acid positions 298-299 of SEQ ID NO:2; and wherein said enzyme comprises at least 30 percent amino acid identity to SEQ ID NO: 2.

Specific examples of preferred perhydrolases are acetyl xylan esterase from

Thermotoga maritima MSB 8 as described in WO-A-2010/019544 (DuPont) and having the amino acid sequence SEQ ID NO: 16 of said patent application, and including variants derived from said enzyme, which are also disclosed in WO-A- 2010/019544. These enzymes require an acyl donor (fatty acid - FA) and a peroxide source as substrates, leading to the generation of peracetic acid (PAA).

It was surprisingly found that the enzymatic laundry detergent composition in solid form according to the present invention exhibits an effective inhibition of bacterial metabolism, in particular of the bacteria that are found on the axilla region, which are believed to be one of the main causes of laundry malodour. This inhibition is surprising because the enzymatic laundry detergent composition in solid form according to the present invention is essentially free from substrate for the perhydrolase. Without wishing to be bound by any theory, it is believed that the miniscule amounts of triglycerides present in the soil on the fabrics may be already sufficient to generate sufficient peracid to effectively inhibit the growth of microorganisms, both in the wash liquor and on the garments. It is an important element that composition is essentially free from substrates for the perhydrolase. In particular, the composition should be essentially free from ester substrates, more in particular from carboxylic acid esters. By essentially free we mean that the total amount of ester substrates is less than about 2 % by weight of the total composition, preferably less than about 0.5 % by weight of the total composition. It will be understood that the composition may also be completely free from ester substrates.

In use, the pH of the enzymatic laundry detergent compositions according to the invention is preferably in the range of 6 to 1 1 , preferably from 7.5 to 9.5. This means that an aqueous solution of the composition of 4 g/l should have a pH value in that range.

The enzymatic laundry detergent composition of the invention may optionally comprise a number of optional ingredients which provide cleaning performance, fabric care and/or sanitation benefits. Among these optional ingredients are:

Detergency builder

The compositions of the invention may contain a detergency builder. Preferably the builder is present in an amount of from 1 to less than 80 wt.% based on the weight of the total composition. More preferably the amount of builder is from 1 to 60 wt.%. Builders are well-known to those skilled in the art. Many suitable builder compounds are available. Examples are zeolites, sodium tripolyphosphate, layered silicate, sodium carbonate, sodium bicarbonate, burkeite and sodium silicate The builder may be selected from strong builders such as phosphate builders, aluminosilicate builders and mixtures thereof. However, strong builders are preferably present in an amount not exceeding 5 % by weight and most preferably strong builders are absent. One or more weak builders such as calcite/carbonate, beryllium/carbonate, citrate or polymer builders may be additionally or alternatively present. The phosphate builder (if present) may for example be selected from alkali metal, preferably sodium, pyrophosphate, orthophosphate and tripolyphosphate, and mixtures thereof. The aluminosilicate (if present) may be, for example, selected from one or more crystalline and amorphous aluminosilicates, for example, zeolites as disclosed in GB-A-1 473 201 (Henkel), amorphous aluminosilicates as disclosed in GB-A-1 473 202 (Henkel) and mixed crystalline/amorphous aluminosilicates as disclosed in GB-A-1 470 250 (Procter & Gamble); and layered silicates as disclosed in EP- A-164 514 (Hoechst). The alkali metal aluminosilicate may be either crystalline or amorphous or mixtures thereof, having the general formula: 0.8 1 .5 Na20. AI203. 0.8 6 Si02.

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 Si02 units (in the formula above). Both the amorphous and the crystalline materials can be prepared readily by reaction between sodium silicate and sodium aluminate, as amply described in the literature. Suitable crystalline sodium aluminosilicate ion exchange detergency builders are described, for example, in GB-A-1 429 143 (Procter & Gamble). The preferred sodium aluminosilicates of this type are the well known commercially available zeolites A and X, and mixtures thereof.

The zeolite may be the commercially available zeolite 4A now widely used in laundry detergent powders. However, according to a preferred embodiment of the invention, the zeolite builder incorporated in the compositions of the invention is maximum aluminium zeolite P (zeolite MAP) as described in EP-A-384 070 (Unilever). Zeolite MAP is defined as an alkali metal aluminosilicate of the zeolite P type having a silicon to aluminium ratio not exceeding 1 .33, preferably within the range of from 0.90 to 1 .33, and more preferably within the range of from 0.90 to 1 .20. Suitably zeolite MAP may be used, having a silicon to aluminium ratio not exceeding 1 .07, more preferably about 1 .00. The calcium binding capacity of zeolite MAP is generally at least 150 mg CaO per g of anhydrous material.

A bleach stabiliser (heavy metal sequestrant) may also be present. Suitable bleach stabilisers include ethylenediamine tetraacetate (EDTA) and the

polyphosphonates such as Dequest (Trade Mark), EDTMP.

The laundry detergent composition of the invention may also comprise one or more further enzymes, which provide cleaning performance, fabric care and/or sanitation benefits.

These further enzymes are suitably selected from the group consisting of glycosyl hydrolases (mannanase, glucanase, hemicellulase, cellulase, amylase, glycosidase, lysozyme, exopolysaccharidase, chitinase, ligninase), lactonase, transferase, amidase, protease, lipase, phospholipase, esterase, cutinase, polyesterase, oxidoreductases (laccase, peroxidase, pyranose oxidase, haloperoxidase) and lysostaphin.

Preferred further enzymes are protease, lipase, esterase (cutinase and

polyesterase) and glycosyl hydrolases such as mannanase and amylase. The presence of a protease is especially preferred. Moreover, it may be considered as surprising that the perhydrolase is capable of performing its function in the presence of a protease, which would be expected to rapidly deactivate the perhydrolase. The protease is preferably a serine protease, in particular a subtilase.

Suitable members of these enzyme classes are described in Enzyme

nomenclature 1992: recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the nomenclature and classification of enzymes, 1992, ISBN 0-12-227165-3, Academic Press. The most recent information on the nomenclature of enzymes is available on the Internet through the ExPaSy Molecular Biology Server (http://us.expasy.org/). Examples of commercially available bacterial lipases, cutinases or esterases are LumafastTM and LipomaxTM or variants thereof. The composition may also contain commercial enzymes such as Mannaway™, Natalase™, Renozyme™, Ovozyme™, CelluClean™, Polarzyme™, Stainzyme™, Coronase™,

Pectaaway™, Pectawash™, Termamyl Ultra™ (all from Novozymes) and FN-3, FN-4, Purafect Prime™, Properase™ (all from Genencor)

The compositions of the invention may also contain maltogenic a-amylases as described in WO-A-02/02726 page 5, line 14 to page 1 1 line 29. Commercially available maltogenic a-amylases are Novamyl™ and Maltogenase™ from

Novozymes.

In the compositions of the invention, all enzymes are usually employed in amounts of about 0.001 to about 1 .0 wt.%, preferably from about 0.002 to about 0.3% by weight, more preferably from about 0.02 to about 0.1 % by weight, based on the amount of pure enzyme. The enzyme is preferably present in the form of enzyme granules (containing only a small amount of pure enzyme) in amounts of from about 0.1 to about 10.0 wt.%, preferably from about 0.2 to about 3% by weight, more preferably from about 0.2 to about 1 % by weight.

The detergent compositions of the invention may further comprise one or more of the following optional ingredients selected from soap, sequestrants, cellulose ethers and esters, cellulosic polymers, other antiredeposition agents, sodium chloride, calcium chloride, sodium bicarbonate, other inorganic salts, dyes, fluorescers, photobleaches, polyvinyl pyrrolidone, other dye transfer inhibiting polymers, foam controllers, lather control agents or lather boosters as appropriate, acrylic and acrylic/maleic polymers, citric acid, soil release polymers, silicone, fabric conditioning compounds, coloured speckles such as blue speckles, and perfume. This list is not intended to be exhaustive. Suitable lather boosters for use in the present invention include cocamidopropyl betaine (CAPB), cocomonoethanolamide (CMEA) and amine oxides. Preferred amine oxides are of the general form:

CH ₃

I

CH ₃ (CH ₂ ) _n -N— >0

I

CH ₃ where n is from 7 to 17. A suitable amine oxide is Admox (Trademark) 12, supplied by Albemarle.

In addition to the optional ingredients discussed above, the compositions of the invention may optionally contain other active ingredients to enhance performance and properties.

For example, the compositions of the invention may contain from 0 to 85 wt.% of an inorganic non-builder salt, such as sodium sulphate, sodium sesquicarbonate, sodium chloride, calcium chloride, magnesium chloride and calcite, preferably from 0 to 60 wt.%, preferably from 0 to 40 wt.%, based on the weight of the total composition.

The compositions of the invention may contain a polycarboxylate polymer. These include homopolymers and copolymers of acrylic acid, maleic acid and

acrylic/maleic acids. The publication 'Polymeric Dispersing Agents, Sokalan', a printed publication of BASF Aktiengesellschaft, D-6700 Ludwigshaven, Germany describes organic polymers which are useful. Preferably, the polycarboxylate polymer is selected from the group consisting of sodium polyacrylate, sodium acrylate maleate and mixtures thereof. Examples of suitable polymers include Sokalan CP5, ex BASF polyacrylate, namely maleic acid-acrylic acid copolymer, with a sodium salt. A second aspect of the present invention is a process for laundering textile fabrics, characterised in that it comprises the step of immersing the fabrics in a wash liquor comprising water in which the enzymatic laundry detergent

composition according to the invention is dissolved or dispersed. The process may be carried out by machine or hand. Preferably, the enzymatic laundry detergent composition is used in a concentration of approximately 0.1 to 100 g/l, more preferably from approximately 0.5 to 50 g/l. In an especially preferred embodiment, approximately 1 to 10 g/l is used. The process is preferably carried out at a temperature of about 5 to 70 ^° C, more preferably from 10 to 50 ^° C, most preferably from about 15 to 40 ^° C,

Form of the composition

The enzymatic laundry detergent composition of the invention may be in any convenient solid form, e.g., a bar, a tablet, a powder, prills or flakes. It is preferably in the form of a powder having a mean particle size between 200 and 800 micrometer. Alternatively, the compositions may be in tablet form. The compositions can be formulated for use as hand wash or machine wash detergents.

Preparation of the compositions

A third aspect of the present invention is a process for manufacturing the enzymatic laundry detergent composition according to the invention. The solid or granular compositions of the invention may be prepared by any suitable process. Powders of low to moderate bulk density may be prepared by spray-drying a slurry, and optionally post-dosing (dry-mixing) further ingredients. "Concentrated" or "compact" powders may be prepared by mixing and granulating processes, for example, using a high-speed mixer/granulator, or other non-tower processes. Tablets may be prepared by compacting powders, especially "concentrated" powders. The choice of processing route may be in part dictated by the stability or heat-sensitivity of the surfactants involved, and the form in which they are available. In all cases, all ingredients may be added separately.

The invention will now be further illustrated by the following, non-limiting Examples.

EXAMPLE 1

METHOD AND MATERIALS

Procedure for measuring PAA and Hydrogen Peroxide in 96 Well MTP's Reagents required:

ammonium molybdate (16 g/L),

potassium iodide solution, (50 g/L)

catalase (0.50 g/L in borax buffer)

borax buffer (19.06 g/L sodium tetraborate)

sulfuric acid (0.1 M)

peractic acid solution (for calibration; goes in sample plate: columns 1 and 12) (normally 39% in acetic acid)

The solutions must be prepared on the day of the assay, except for the borax buffer and ammonium molybdate solution, which can be prepared in advance. After preparing the solutions for the assay, they are transferred into motherplate troughs, along with a water motherplate for dilution (filled columns 2-1 1 ). The troughs are then placed on the Tecan deck in the positions required by the programme. Absorbance is measured at 360nm.

For the sample plate

Peracetic acid solution is added into columns 1 and 12, for calibration of the assay. The concentrations used are 0, 0.25, 0.50, 1 .0 mM and this is

repeated 4 times in total in the end columns. The readings from these wells will be used to construct a calibration curve later.

The middle 80 wells (columns 2-1 1 ) are used for the sample solutions. In the validation exercise, these wells were filled with mixtures of hydrogen peroxide (0, 2.5, 5.0, 7.5 10 mM) and TAED (0, 0.125, 0.25, 0.5 mM) (forming peracetic acid in situ). The experiment was repeated 3 times, with 2 independent operators.

Measuring Perhydrolase activity of an enzyme sample:

Enzyme sample is diluted to a concentration of 0.1 to 10 ppm and mixed with 1 .5mM hydrogen peroxide and Triacetin from 0.1 to 10 mM. Peracid species generated are then detected as per the standard plate.

Reaction mixture in MTP (containing enzyme, hydrogen peroxide and triacetin) to be determined is divided into two samples-one for PAA measurement and one for total active oxygen measurement. For PAA, catalase in borax buffer is added and the mixture is allowed to stand for 2 minutes (this destroys any hydrogen peroxide present in the sample). To this sample, sulfuric acid (0.1 M) and potassium iodide solution (50g/L) are added, before measurement of visible absorbance at 360nm. For the total active oxygen determination, ammonium molybdate (16g/L), sulfuric acid (0.1 M) and potassium iodide solution (50g/L) are added, before

measurement of visible absorbance at 360nm. Columns 1 and 12 in the MTP are used for the calibration solutions of PAA (0, 0.1 , 0.2, 0.3mM); columns 2-1 1 are used for the samples to be determined.

Volumes Used:

PAA

Kl (125μΙ_); H2S04 (100μΙ_); Catalase/borax (100μΙ_); Sample (75μΙ_)

Total [O]

1. Calibration (columns 1&12): Ammonium molybdate (25μΙ_); Kl

(125μΙ_); H2S04 (100μΙ_); Sample (25μΙ_)

2. Assay (columns 2-10): Water (800μΙ_); Ammonium molybdate

(25μΙ_); Kl (125μΙ_); H2S04 (100μΙ_); Sample (25μΙ_)

Water is added to columns 2-10 to allow for the difference in concentration of active [O] between columns 1 & 12 and 2-10. This way, the calibration solutions in columns 1 & 12 can be used for both PAA and Total [O] See Figure 1.

Test samples

Swatches of polyester Coolmax™ (approximately 10x15 cm) were inserted into cotton T-shirts and then worn by female panellists for a period of 16-24 hours. Once harvested, they were placed on store at -20°C until use. The perhydrolase used was the acetyl xylan esterase from Thermotoqa maritima MSB 8 described in WO-A-2010/019544 (Du Pont) and having the amino acid sequence SEQ ID

c

NO: 16 of said patent application and denoted below as "Perhydolase". These enzymes require an acyl donor (fatty acid - FA) and a peroxide source as substrates, leading to the generation of peracetic acid (PAA). For the purpose of these examples, the substrate and source of fatty acid / ester utilised is triacetin and the peroxide source is hydrogen peroxide solution.

Test Formulations

Stocks:

Sterile diluent was used in preparing the stock solutions. This consisted of 2.3g/L of laundry liquid detergent diluted in sterile distilled water (SDW) at 12°FH (2: 1 Ca:Mg ions).

Stock solutions prepared as listed in the table below:

Textile samples to test:

The centre of each swatch was cut into four pieces and treated as a separate textile sample. Each sample to be used was weighed and the level of "wash" solution required was calculated (liquor : cloth ratio is 6 : 1 ). Four samples from each textile piece was obtained using a fabric punch (approximately 0.3 cm ² ) and placed into 96-well microplates containing an oxygen sensitive fluorophore (BD™ Oxygen Biosensor System). These samples are referred to as the pre-wash samples. Final "wash" volumes in test:

The centre of each swatch was cut into four pieces and treated as a separate textile sample. Each sample to be used was weighed and the level of "wash" solution required was calculated (liquor: cloth ratio is 6: 1 ).

Mixes:

Dilutions of each stock (1 : 10) were prepared in the relevant volume of laundry base at room temperature (20°C). These consisted of the following components and levels (table 2 below):

Test combinations

Laundry Liquid Base (LLB) alone

Perhydrolase (2ppm), Triacetin (2mM),

H ₂ O ₂ (10mM), LLB

H ₂ O ₂ (10mM), LLB

Percarbonate (0.06%), LLB

Perhydrolase(2ppm), H ₂ 0 ₂ (10mM), LLB

Treatment of textile samples

The textile samples were submerged immediately. No agitation was employed except one turn of the sample tube to ensure mixing. The samples were left at room temperature (20°C) for 30 min. Textile samples were then placed onto sterile kimwipe (10 sec) inserted into a sterile metal tray, to remove excess liquid, and then placed into SDW (same volumes as wash liquor). Tubes were inverted once and left for 1 min at 20°C. This rinse was repeated, and textile samples left on sterile kimwipe and placed into the incubator (37°C) for 2 hours to dry.

Four samples from each textile piece was obtained using a fabric punch

(approximately 0.3cm ² ) and placed into 96-well microplates containing an oxygen sensitive fluorophore (BD™ Oxygen Biosensor System). These samples are referred to as the post-wash samples. Bacterial media (200μΙ of liquid media comprising 12.5% Coryne broth and 87.5% tryptone soya broth) was added to each well.

Detecting respiration from the remaining viable cells

A sterile, plastic sheet was placed on top of each plate. Plates were then incubated at 37°C for 20 hours. Bacterial respiration within each well was determined using a Genios plate reader and the Tecan workstation was employed to transfer the plates between the plate reader and the incubator. The data generated was represented as time required to detect bacterial respiration (mid way through the exponential phase of the "growth" curve for the control i.e. no treatment).

RESULTS

Data is represented below as the maximum difference between the pre and post wash samples (see equation below) that can be obtained. 100% represents no metabolism detected after treatment i.e. total bacterial kill.

Equation 1

Percentage of max = difference / maximum x 100

Where:

Difference = "Pre" minus "Post"

Maximum that could be obtained = 20h minus "Pre"

NB: 100% equates to no re-growth obtained

LLB: Laundry liquid base (dilute in use) The data show that the dilute laundry detergent base reduces the metabolism of bacteria present in natural soil by 28.9%. The addition of the peroxide donor from percarbonate increases this efficacy, whilst providing peroxide from a hydrogen peroxide solution decreases the efficacy observed from the dilute base alone. The inclusion of the perhydrolase enzyme along with peroxide via solution however significantly increases the performance of the mixture, resulting in the total reduction of bacterial metabolism (100% reduction). The data also show that there is a potential for other components of the natural soil (i.e. sebum) to provide a suitable alternative to triacetin when using the perhydrolase enzyme.