BR112021009789A2 - detergent composition, method of treating a fabric substrate and use of an esterase enzyme - Google Patents

Abstract

DETERGENT COMPOSITION, METHOD OF TREATMENT OF A TISSUE SUBSTRATE AND USE OF AN ESTERASE ENZYME. The present invention provides a detergent composition comprising: (i) from 1 to 60% by weight of a surfactant; and, (ii) from 0.0005 to 5% by weight of an esterase enzyme of the EC 3.1.1.1 enzyme class, having at least 60% sequence identity to any one of SEQ ID NOs: 1 to 4; to a method using the enzyme and the use of the enzyme to improve the cleaning of sebum stains on fabric.

Description

Descriptive Report of Patent of Invention DETERGENT COMPOSITION, TREATMENT METHOD OF A

TISSUE SUBSTRATE AND USE OF AN ESTERASE ENZYME Field of the Invention

[0001] The present invention relates to a detergent composition, more specifically a laundry detergent composition, said composition comprising a novel esterase enzyme.

Background of the Invention

[0002] Sebum is an oily dirt that remains a stain that is difficult to remove from used clothes. With an incentive to encourage consumers to wash at low temperatures, the challenge of effective sebum removal remains. Tallow consists of a variety of fats and esters including wax esters, cholesteryl esters, squalene and many free fatty acids/alcohols. Sebum is liquid at body temperature but solid at room temperature.

[0003] These properties are particularly important for collar/cuff dirt removal because it is easier to remove liquid body oil than solids from clothing. Current laundry enzymes are not able to degrade all of the sebum components which makes removal from the fabric difficult.

[0004] There is a problem with sebum removal where detergents including current commercial enzymes do not properly remove sebum.

Summary of the Invention

[0005] Incorporation of a novel esterase enzyme into detergent compositions has been found to improve sebum removal from fabrics.

[0006] In one aspect the present invention provides a detergent composition comprising:

(i) from 1 to 60% by weight, preferably from 2 to 50% by weight, more preferably from 3 to 45%, even more preferably from 5 to 40% by weight, even more preferably from 6 to 40% by weight of a surfactant; and, (ii) from 0.0005 to 5% by weight, preferably from 0.005 to 2.5% by weight, more preferably from 0.01 to 1% by weight of an esterase enzyme of the EC 3.1.1.1 enzyme class, having at least 60% sequence identity to any one of SEQ ID NOs: 1 to 4.

[0007] Preferably the esterase enzyme is at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 97%, at least 98% or even at least 99% sequence identity to any one of SEQ ID NOs: 1 to 4.

[0008] Even more preferably the esterase enzyme has 100% sequence identity to any one of SEQ ID NOs: 1 to 4.

[0009] Preferably the detergent composition comprises from 0.1 to 10% by weight, preferably from 0.2 to 9% by weight, more preferably from 0.25 to 8, even more preferably 0.5 to 6% by weight, even more preferably 1 to 5% by weight of soil release polymer, more preferably a polyester based soil release polymer.

[0010] Preferably the polyester soil release polymer is a polyethylene and/or polypropylene terephthalate based soil release polymer, preferably a polypropylene terephthalate based soil release polymer.

[0011] Preferably the detergent composition comprises an alkoxylated polyamine, preferably at a level of from 0.1 to 8% by weight, more preferably from 0.2 to 6% by weight, even more preferably from 0.5 to 5 % by weight.

[0012] Preferably the detergent composition is a laundry detergent composition. Preferably the laundry detergent composition is a liquid or a powder, even more preferably a liquid detergent.

[0013] Preferably the surfactant in the detergent composition comprises anionic and/or non-ionic surfactant, in one case comprising both anionic and non-ionic surfactants.

[0014] Preferred detergent compositions, particularly laundry detergent compositions, further comprise an additional enzyme selected from the group consisting of: lipases, proteases, cellulases, alpha-amylases, peroxides/oxidases, pectate lyases and/or mannanases.

[0015] Preferred detergent compositions, particularly laundry detergent compositions, further comprise an additional ingredient selected from fluorescent agent, perfume, toning dyes and polymers, and mixtures thereof.

[0016] In another aspect the present invention provides a method of treating a fabric substrate with a stain of sebum, said method comprising incorporating an esterase enzyme of the EC 3.1.1.1 enzyme class, having at least 60%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 97%, at least 98% or even at least 99% sequence identity, even more preferably 100% sequence identity to any one of SEQ ID NOs: 1 to 4 in a detergent composition comprising from 1 to 60% by weight of a surfactant ; and subsequently treating a fabric substrate with a sebum stain with said composition.

[0017] In another aspect the present invention provides the use of an esterase enzyme of the EC 3.1.1.1 enzyme class, having at least 60%, preferably at least 70%, more preferably at least 75%, most preferably at least 80% , more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 97%, at least 98% or even at least 99% sequence identity, even more preferably 100% sequence identity to any of SEQ ID NOs: 1 to 4 to improve cleaning of sebum stains on fabric.

Detailed Description of the Invention

[0018] The indefinite article “a” or “an” and its corresponding definite article “the” or “the” as used herein means at least one, or one or more, unless otherwise specified.

[0019] All % levels of ingredients in compositions (formulations) listed here are in % by weight based on total formulation unless otherwise noted.

[0020] It is understood that any reference to a preferred ingredient of the detergent composition is considered to be combinable matter with any other preferred ingredient of the detergent composition disclosed herein.

[0021] The detergent composition may take any suitable form, for example liquids, solids (including powders) or gels.

[0022] The detergent composition can be applied to any suitable substrate. Particularly preferred substrates are fabrics. Particularly preferred detergent compositions are laundry detergent compositions.

[0023] Laundry detergent compositions may take any suitable form. Preferred forms are liquid or powder, with liquid being most preferred. Sequence information

[0024] The sequences disclosed herein are SEQ ID NOs 1 to 4.

[0025] The sequence SEQ ID 1 is from Thermogutta terrifontis

[0026] The sequence SEQ ID 2 is from Thermogutta terrifontis

[0027] The sequence is a mutant of SEQ ID 1 and differs at position 37.

[0028] The sequence SEQ ID 3 is from Thermogutta terrifontis

[0029] This sequence is a mutant of SEQ ID 1 and differs at position 251.

[0030] Sequence SEQ ID 4 is from Archaeoglobus fulgidus Enzyme esterase

[0031] The esterase enzyme has at least 60% sequence identity to any one of SEQ ID NOs: 1 to 4.

[0032] Preferably the esterase enzyme is at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 97%, at least 98% or even at least 99% sequence identity to any one of SEQ ID NOs: 1 to 4.

[0033] Even more preferably the esterase enzyme has 100% sequence identity to any one of SEQ ID NOs: 1 to 4.

[0034] Esterase can be described as being of the EC enzyme class

3.1.1.1, known as carboxylesterase.

[0035] Preferred esterases are from Thermogutta terrifontis or Archaeoglobus fulgidus. Surfactant

[0036] The detergent composition comprises surfactant (which may include a single surfactant or a mixture of two or more surfactants). The composition comprises from 1 to 60% by weight, preferably from 2 to 50% by weight, more preferably from 3 to 45% by weight of surfactant, even more preferably from 5 to 40% by weight, even more preferably from 6 to 40 % by weight of surfactant.

[0037] The detergent composition (preferably a laundry detergent composition) comprises anionic and/or non-ionic surfactants, preferably comprising both anionic and non-ionic surfactants.

[0038] Suitable anionic detergent compounds that may be used are generally water-soluble alkali metal salts of organic sulfates and sulfonates having alkyl radicals containing from about 8 to about 22 carbon atoms, the term alkyl being used to include the moiety alkyl of higher alkyl radicals.

[0039] Examples of suitable synthetic anionic detergent compounds are sodium and potassium alkyl sulfates, especially those obtained by sulfating higher C 8 to C 18 alcohols, produced for example from tallow or coconut oil, C 9 to C 18 alkyl sulfonates sodium and potassium C 20 benzene, particularly sodium linear secondary C 10 to C 15 alkyl benzene sulfonates; and sodium alkyl glyceryl ether sulfates, especially those ethers of higher alcohols derived from tallow or coconut oil and synthetic alcohols derived from petroleum.

[0040] The anionic surfactant is preferably selected from: linear alkyl benzene sulfonate; alkyl sulfates; alkyl ether sulfates; soaps; alkyl (preferably methyl) ester sulfonates and mixtures thereof.

[0041] The most preferred anionic surfactants are selected from: linear alkyl benzene sulfonate; alkyl sulfate; alkyl ether sulfate and mixtures thereof. Preferably the alkyl ether sulfate is an n-C 12 -C 14 alkyl ether sulfate with an average of 1 to 3 EO (ethoxylate) units.

[0042] Sodium lauryl ether sulfate (SLES) is particularly preferred. Preferably the linear alkyl benzene sulfonate is a sodium C 11 -C 15 alkyl benzene sulfonate. Preferably the alkyl sulfate is a linear or branched sodium C 12 -C 18 alkyl sulfate. Sodium dodecyl sulfate is particularly preferred (SDS, also known as primary alkyl sulfate).

[0043] In liquid formulations preferably two or more anionic surfactants are present, for example linear alkyl benzene sulfonate together with an alkyl ether sulfate.

[0044] In liquid formulations, preferably the laundry composition, in addition to the anionic surfactant, comprises alkyl ethoxylated non-ionic surfactant, preferably from 2 to 8% by weight of alkyl ethoxylated non-ionic surfactant.

[0045] Suitable nonionic detergent compounds that 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 C 8 to C 18 aliphatic linear or branched primary or secondary alcohols with ethylene oxide.

[0046] More preferably, the non-ionic detergent compound is the alkyl ethoxylated non-ionic surfactant which is a C 8 to C 18 primary alcohol with an average ethoxylation of 7 to 9 EO units.

[0047] Preferably, the surfactants used are saturated. dirt release polymer

[0048] Dirt releasing polymer is present at a level of from 0.1 to 10% by weight. Preferred inclusion levels of the dirt releasing polymer are preferably from 0.2 to 9% by weight, more preferably from 0.25 to 8% by weight, even more preferably from 0.5 to 6% by weight, even more preferably from 1 to 5% by weight.

[0049] Dirt Release Polymer is a polyester based dirt release polymer. More preferably the polyester soil release polymer is a polyethylene and/or polypropylene terephthalate based soil release polymer, even more preferably a polypropylene terephthalate based soil release polymer.

[0050] Suitable polyester-based soil release polymers are described in WO 2014/029479 and WO 2016/005338 . alkoxylated polyamine

[0051] The detergent composition preferably comprises an alkoxylated polyamine. Especially when the detergent composition is in the form of a laundry composition, it is preferred that an alkoxylated polyamine is included.

[0052] Preferred levels of alkoxylated polyamine are in the range of 0.1 to 8% by weight, preferably 0.2 to 6% by weight, more preferably 0.5 to 5% by weight. Another preferred level is 1 to 4% by weight.

[0053] The alkoxylated polyamine can be linear or branched. It may be branched to the extent that it is a dendrimer. Alkoxylation can typically be ethoxylation or propoxylation, or a mixture of both. Where a nitrogen atom is alkoxylated, a preferred average degree of alkoxylation is from 10 to 30, preferably from 15 to 25.

[0054] A preferred material is alkoxylated polyethyleneimine, more preferably ethoxylated polyethyleneimine, with an average degree of ethoxylation being from 10 to 30, preferably from 15 to 25, where a nitrogen atom is ethoxylated. additional enzymes

[0055] Additional enzymes other than the specified lipase may be present in the detergent composition. It is preferred that additional enzymes are present in the preferred laundry detergent composition.

[0056] If present, then the level of each enzyme in the laundry composition of the invention is from 0.0001% by weight to 0.1% by weight.

[0057] Enzyme levels present in the composition preferably refer to the enzyme level as pure protein.

Additional preferred enzymes include those in the group consisting of: lipases, proteases, cellulases, alpha-amylases, peroxidases/oxidases, pectate lyases, and/or mannanases. Said preferred additional enzymes include a mixture of two or more of these enzymes.

[0059] Preferably the additional enzyme is selected from: lipases, proteases, cellulases and/or alpha-amylases.

[0060] Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein-engineered mutants are included. Examples of useful lipases include Humicola lipases (synonymous

Thermomyces), eg H. lanuginosa (T. lanuginosus) as described in EP 258068 and EP 305216 or from H. insolens as described in WO 96/13580, a lipase from Pseudomonas, eg from P. alcaligenes or P. pseudoalcaligenes (EP 218272 ), P. cepacia (EP 331376), P. stutzeri (GB 1372034), P. fluorescens, Pseudomonas sp. SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO 96/12012), a lipase from Bacillus, eg from B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1131, 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422). Other examples are lipase variants such as those described in WO 92/05249, WO 94/01541, EP 407225, EP 260105, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783 , WO 95/22615, WO 97/04079 and WO97/07202, WO 00/60063.

[0061] Preferred commercially available lipase enzymes include Lipolase®, and Lipolase Ultra®, Lipex® and Lipoclean® (Novozymes A/S).

[0062] The method of the invention can be carried out in the presence of phospholipase classified as EC 3.1.1.4 and/or EC 3.1.1.32. In the context of the present invention, the term "phospholipase" is to be understood as an enzyme that has activity towards phospholipids.

[0063] Phospholipids, such as lecithin or phosphatidylcholine, consist of glycerol esterified with two fatty acids at an outer (sn-1) and middle (sn-2) position and esterified with phosphoric acid at the third position; phosphoric acid, in turn, can be esterified to an amino alcohol. Phospholipases are enzymes that participate in the hydrolysis of phospholipids. Several types of phospholipase activity can be distinguished, including phospholipases A 1 and A 2 which hydrolyze a fatty acyl group (at the sn-1 and sn-2 position, respectively) to form lysophospholipid; and lysophospholipase (or phospholipase B) which can hydrolyze the remaining fatty acyl group into lysophospholipid. Phospholipase C and phospholipase D (phosphodiesterases) release diacyl glycerol and phosphatidic acid respectively.

[0064] Protease enzymes hydrolyze bonds between peptides and proteins, in the context of laundry, this leads to improved removal of stains containing proteins or peptides. Examples of suitable protease families include aspartic proteases; cysteine proteases; glutamic proteases; asparagine peptide lyases; serine proteases and threonine proteases. Such protease families are described in the MEROPS peptidase database (http://merops.sanger.ac.uk/). Serine proteases are preferred. Subtilase-type serine protease are more preferred. The term "subtilase" refers to a subgroup of serine protease according to Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523. Serine proteases are a subgroup of proteases defined by having a serine at the active site, which form a covalent adduct with the substrate. Subtilases can be divided into 6 subdivisions, i.e., the Subtilisin family, the Thermitase family, the Proteinase K family, the Lantibiotic peptidase family, the Kexin family, and the Pyrolysin family.

[0065] Examples of subtilases are those derived from Bacillus such as Bacillus lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii described in US 7262042 and WO 09/021867, and subtilisin lentus, subtilisin Novo, subtilisin Carlsberg, Bacillus licheniformis, subtilisin BPN', subtilisin 309, subtilisin 147 and subtilisin 168 described in WO 89/06279 and protease PD 138 described in WO 93/18140. Other useful proteases may be those described in WO 92/175177, WO 01/016285, WO 02/026024 and WO 02/016547. Examples of trypsin-like proteases are trypsin (eg of porcine or bovine origin) and the Fusarium protease described in WO 89/06270, WO 94/25583 and WO 05/040372 and Cellumonas-derived chymotrypsin proteases described in WO 05/052161 and WO 05/052146.

[0066] More preferably the protease is a subtilisin (EC 3.4.21.62).

[0067] Examples of subtilases are those derived from Bacillus such as Bacillus lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii described in US 7262042 and WO 09/021867, and subtilisin lentus, substilisin Novo, subtilisin Carlsberg, Bacillus licheniformis, subtilisin BPN', subtilisin 309, subtilisin 147 and subtilisin 168 described in

WO 89/06279 and PD 138 protease described in WO 93/18140. Preferably, the subtilisin is derived from Bacillus, preferably Bacillus lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii as described in US 6312936 B1, US 5679630, US 4760025, US 7262042 and WO 09/021867 . More preferably the subtilisin is derived from Bacillus gibsonii or Bacillus Lentus.

[0068] Suitable commercially available protease enzymes include those sold under the brand names Alcalase®, Blaze®, Duralase®, Duranzym®, Relase®, Relase Ultra®, Savinase®, Savinase Ultra®, Primase®, Polarzyme®, Kannase ®, Liquanase®, Liquanase Ultra®, Ovozyme®, Coronase®, Coronase® Ultra, Neutrase®, Everlase® and Esperase® can all be sold as Ultra® or Evity® (Novozymes A/S).

[0069] The composition may use cutinase, classified in EC 3.1.1.74. The cutinase used according to the invention can be of any origin. Preferably the cutinases are of microbial origin, in particular of bacterial, fungal or yeast origin.

[0070] 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. licheniformis, described in greater detail in GB 1296839 or the strains of Bacillus sp. disclosed in WO 95/026397 or WO 00/060060. Commercially available amylases are Duramyl®, Termamyl®, Termamyl Ultra®, Natalase®, Stainzyme®, Amplify®, Fungamyl® and BAN® (Novozymes A/S), Rapidase® and Purastar® (from Genencor International Inc.).

[0071] Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein-engineered mutants are included. Suitable cellulases include cellulases of the genus Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, eg fungal cellulase produced by Humicola insolens, Thielavia terrestris, Myceliophthora thermophila and Fusarium oxysporum disclosed in US 4435307, US 5648263, US 5691178, US 5776757, WO 89 /09259, WO 96/029397 and WO 98/012307. Commercially available cellulases include Celluzyme®, Carezyme®, Celluclean®, Endolase®, Renozyme® (Novozyme A/S), Clazinase® and Puradax HA® (Genencor International Inc.), and KAC-500(B)®, (Kao Corporation ). Celluclean® is preferred.

[0072] Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein-engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g. from C. cinereus and variants thereof such as those described in WO 93/24618, WO 95/10602 and WO 98/15257. Commercially available peroxidases include Guardzyme ® and Novozym ® 51004 (Novozymes A/S).

[0073] Additional enzymes suitable for use are discussed in WO 2009/087524 , WO 2009/090576 , WO 2009/107091 , WO 2009/111258 and WO 2009/148983 .

[0074] The aqueous solution used in the method preferably has an enzyme present. The enzyme is preferably present in the aqueous solution used in the method at a concentration in the range of 0.01 to 10 ppm, preferably 0.05 to 1 ppm. Enzyme Stabilizers

[0075] Any enzyme present in the composition can be stabilized using conventional stabilizing agents, eg a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid or boric acid derivative, eg an aromatic borate ester , or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition can be formulated as described in eg WO 92/19709 and WO 92/19708. additional materials

[0076] Additional optional, but preferred materials that may be included in detergent compositions (preferably laundry detergent compositions) include fluorescent agent, perfume, toning dye, polymers and chelating agents. fluorescent agent

[0077] The composition preferably comprises a fluorescent agent (optical brightener). Fluorescent agents are well known and many such fluorescent agents are commercially available. Generally, these fluorescing agents are supplied and used in the form of their alkali metal salts, for example sodium salts.

[0078] The total amount of fluorescent agent or agents used in the composition is generally 0.0001 to 0.5% by weight, preferably 0.005 to 2% by weight, more preferably 0.01 to 0.1% by weight.

[0079] Preferred classes of fluorescing agent are: di-styryl biphenyl compounds, Tinopal®, CBS-X, di-amino stilbene disulfonic acid compounds, eg Tinopal DMS pure Xtra and Blankophor® HRH, and pyrazoline compounds, eg Blankophor SN.

[0080] Preferred fluorescent agents are fluorescent agents with CAS No. 3426-43-5; CAS No. 35632-99-6; CAS No. 24565-13-7; CAS No. 12224-16-7; CAS No. 13863-31-5; CAS No. 4193-55-9; CAS No. 16090-02-1; CAS No. 133-66-4; CAS No. 68444-86-0; CAS No. 27344-41-8.

[0081] More preferred fluorescent agents are: Sodium 2-(4-styryl-3-sulfophenyl)-2H-naphthol[1,2-d]triazole, 4,4'-bis{[(4-anilino-6- Disodium (N-methyl-N-2-hydroxyethyl)amino 1,3,5-triazin-2-yl)]amino}stilbene-2-2'-disulfonate, 4,4'-bis{[(4 disodium-anilino-6-morpholino-1,3,5-triazin-2-yl)]amino}stilbene-2,2'-disulfonate and di-di-4,4'-bis(2-sulfostyryl)biphenyl sodium.

[0082] The aqueous solution used in the method has a fluorescent agent present. The fluorescent agent is present in the aqueous solution used in the method preferably in the range of 0.0001 g/L to 0.1 g/L, more preferably 0.001 to 0.02 g/L. perfume

[0083] The composition preferably comprises a perfume. Many suitable perfume examples are provided in the 1992 CTFA (Cosmetics, Toiletries and Fragrance Association) international buyers guide, published by CFTA Publications and 80th annual edition of the OPD Chemicals Buyers Directory, 1993, published by Schnell Publishing Co.

[0084] Preferably the perfume comprises at least one note (compound) of: alpha-isomethyl ionone; benzyl salicylate; citronellol; coumarin; cinnamal hexyl; linalool; pentanoic acid, 2-methyl-, ethyl ester; octanal; benzyl acetate; 1,6-octadien-3-ol, 3,7-dimethyl-, 3-acetate; cyclohexanol, 2-(1,1-dimethylethyl)-1-acetate; delta-damascone; beta-ionone; verdel acetate; dodecanal; cinnamic hexyl aldehyde; cyclopentadecanolide; benzeneacetic acid, 2-phenylethyl ester; amyl salicylate; beta-caryophyllene; ethyl undecylenate; geranyl anthranilate; alpha-irone; beta-phenyl ethyl benzoate; alpha-santalol; cedrol; cedryl acetate; cedilla shape; cyclohexyl salicylate; gamma-dodecalactone; and beta-phenylethyl phenyl acetate.

[0085] Useful components of perfume include materials of both natural and synthetic origin. They include compounds alone and mixtures. Specific examples of such components can be found in the current literature, e.g. Fenaroli Guide to Flavor Ingredients, 1975, CRC Press; Synthetic Food Adjuncts, 1947 by M.B. Jacobs, edited by Van Nostrand; or Perfume and Flavor Chemicals by S. Arctander 1969, Montclair, N.J. (USA).

[0086] It is common practice for a plurality of perfume components to be present in a formulation. In the compositions of the present invention it is envisaged that there are four or more, preferably five or more, more preferably six or more or even more preferably seven or more different perfume components.

[0087] In perfume mixtures preferably 15 to 25% by weight are top notes. Headnotes 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.

[0088] The International Fragrance Association published a list of fragrance ingredients (perfumes) in 2011 (http://ifraorg.org/en-us/ingredients#.U7Z4hPldWzk).

[0089] The Fragrance Materials Research Institute provides a perfume (fragrance) database with safety information.

[0090] Perfume head notes may be used to suggest whiteness and brilliance benefit of the invention.

[0091] Some or all perfumes can be encapsulated, typical perfume components where it is advantageous to encapsulate include those with a relatively low boiling point, preferably those with a boiling point of less than 300, preferably 100-250° Celsius. It is also advantageous to encapsulate perfume components that have a low CLog P (i.e., those that have a greater tendency to partition in water), preferably with a CLog P of less than 3.0. These relatively low boiling, relatively low CLog P materials have been called "extended bloom" perfume ingredients and include one or more of the following materials: allyl caproate, amyl acetate, amyl propionate, anisic aldehyde, anisole, benzaldehyde, benzyl acetate, benzyl acetone, benzyl alcohol, benzyl formate, benzyl isovalrate, benzyl propionate, beta gamma hexenol, camphor gum, laevo-carvone, d-carvone, cinnamic alcohol, cinnamyl formate, cis-jasmone, cis-3-hexenyl acetate, cuminic alcohol, C-cyclal, dimethyl benzyl carbinol, dimethyl benzyl carbinol acetate, ethyl acetate, ethyl acetate acetate, ethyl amyl ketone, ethyl benzoate, ethyl butyrate, ethyl hexyl ketone, ethyl phenyl acetate, eucalyptol, eugenol, fenchyl acetate, fluor acetate (tricycle decenyl acetate), fructene (tricycle decenyl propionate), geraniol, hexenol, hexenyl acetate, hexyl acetate, hexyl formate, alk hydrotropic oil, hydroxycitronellal, indone, isoamyl alcohol, isomenthone, isopulegyl acetate, isoquinolone, ligustral, linalool,

linalool oxide, linalyl formate, menthone, menthyl acetophenone, methyl amyl ketone, methyl anthranilate, methyl benzoate, methyl benyl acetate, methyl eugenol, methyl heptenone, methyl heptin carbonate, methyl heptyl ketone, methyl hexyl ketone, acetate of methyl phenyl carbinyl, methyl salicylate, methyl-n-methyl anthranilate, nerol, octalactone, octyl alcohol, p-cresol, p-cresol methyl ether, p-methoxy acetophenone, p-methyl acetophenone, phenoxy ethanol, phenyl acetaldehyde , phenyl ethyl acetate, phenyl ethyl alcohol, phenyl ethyl dimethyl carbinol, prenyl acetate, propyl bornate, pulegone, rose oxide, safrole, 4-terpinelol, alpha-terpinelol and/or viridine. It is common practice for a plurality of perfume components to be present in a formulation. In the compositions of the present invention it is envisaged that there are four or more, preferably five or more, more preferably six or more or even more preferably seven or more different perfume components from the above given list of long flowering perfumes to be present in the perfume.

[0092] Another group of perfumes with which the present invention can be applied are the so-called 'aromatherapy' materials. These include various components also used in perfumery, including essential oil components such as clary sage, eucalyptus, geranium, lavender, apple extract, neroli, nutmeg, mint, violet fennel and valerian.

[0093] It is preferred that the laundry treatment composition does not contain a peroxygen bleach, e.g. sodium percarbonate, sodium perborate and peracid. toning dye

[0094] Preferably when the composition is a laundry detergent composition, then it comprises a toning dye. Preferably, the toning dye is present from 0.0001 to 0.1% by weight of the composition.

[0095] Dyes are described in Synthesis of Color Chemistry, Properties and Applications of Organic Dyes and Pigments (H. Zollinger, Wiley VCH, Zürich, 2003) and, Chemical of Industrial Dyes, Properties and Applications (K

Hunger (ed), Wiley-VCH Weinheim 2003).

[0096] Toning dyes for use in laundry compositions preferably have an extinction coefficient at maximum absorption in the visible range (400 to 700nm) of more than 5000 L mol-1cm-1, preferably more than 10000 L mol-1cm -1. The dyes are blue or violet in color.

[0097] Preferred toning dye chromophores are azo, azine, anthraquinone and triphenylmethane.

[0098] Azo, anthraquinone, phthalocyanine and triphenylmethane dyes preferably carry a net anionic charge or are uncharged. Azine preferably carries a net anionic or cationic charge. Blue or violet toning dyes deposit on the fabric during the wash or rinse step of the washing process providing a visible tone to the fabric. In this context, the dye imparts a blue or violet color to a white fabric with a pitch angle of 240 to 345, more preferably 250 to 320, most preferably 250 to 280. The white fabric used in this test was a non-woven woven cotton sheet. mercerized targeted.

[0099] Toning dyes are described in WO 2005/003274, WO 2006/032327 (Unilever), WO 2006/032397 (Unilever), WO 2006/045275 (Unilever), WO 2006/027086 (Unilever), WO 2008/017570 ( Unilever), WO 2008/141880 (Unilever), WO 2009/132870 (Unilever), WO 2009/141173 (Unilever), WO 2010/099997 (Unilever), WO 2010/102861 (Unilever), WO 2010/148624 (Unilever) , WO 2008/087497 (P&G), WO 2011/011799 (P&G), WO 2012/054820 (P&G), WO 2013/142495 (P&G) and WO 2013/151970 (P&G).

[0100] Mono-azo dyes preferably contain a heterocyclic ring and are more preferably thiophene dyes. Mono-azo dyes are preferably alkoxylated and are preferably uncharged or anionically charged at pH=7. Alkoxylated thiophene dyes are described in WO 2013/142495 and WO 2008/087497. Preferred examples of thiophene dyes are shown below:

and,

[0101] Bis-azo dyes are preferably sulfonated bis-azo dyes. Preferred examples of sulfonated bis-azo compounds are direct violet 7, direct violet 9, direct violet 11, direct violet 26, direct violet 31, direct violet 35, direct violet 40, direct violet 41, direct violet 51, direct violet 66, violet direct 99 and alkoxylated versions thereof. Bis-azo alkoxylated dyes are described in WO 2012/054058 and WO 2010/151906.

[0102] An example of a bis-azo alkoxylated dye is:

[0103] Thiophene dyes are available from Milliken under the brand names Liquitint Violet DD and Liquitint Violet ION.

[0104] Azine dyes are preferably selected from sulfonated 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 phenazine dye selected from: wherein: X3 is selected from: -H; -F; -CH3; -C 2H5; -OCH3; and -OC 2H5; X4 is selected from: -H; -CH3; -C2H5; -OCH3; and -OC2H5; Y2 is selected from: -OH; -OCH2CH2OH; -CH(OH)CH2OH; -OC(O)CH3; and C(O)OCH3.

[0105] The toning dye is present in the composition in the range of 0.0001 to 0.5% by weight, preferably 0.001 to 0.1% by weight. Depending on the nature of the toning dye, there are preferred ranges depending on the effectiveness of the toning dye which is class dependent and particular effectiveness within any particular class. As mentioned above, the toning dye is a blue or violet toning dye.

[0106] A mixture of toning dyes can be used.

[0107] The toning dye is even more preferably a reactive blue anthraquinone dye covalently linked to an alkoxylated polyethyleneimine. Alkoxylation is preferably selected from ethoxylation and propoxylation, more preferably propoxylation. Preferably 80 to 95 mol% of the N-H groups on the polyethylene imine are substituted as isopropyl alcohol groups by propoxylation. Preferably the polyethylene imine prior to dye reaction and propoxylation has a molecular weight of 600 to 1800.

[0108] An example of the structure of a preferred reactive anthraquinone covalently linked to a propoxylated polyethylene imine is: (Structure I) Polymers

[0109] The composition may comprise one or more additional polymers. Examples are carboxymethylcellulose, poly(ethylene glycol), poly(vinyl alcohol), polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers. chelating agent

[0110] Chelating agents may be present or absent from detergent compositions.

[0111] If present, then the chelating agent is present at a level of from 0.01 to 5% by weight.

[0112] Examples of phosphonic acid (or salt thereof) chelating agents are: 1-hydroxyethylidene-1,1-diphosphonic acid (HEDP); diethylenetriaminepenta(methylenephosphonic) acid (DTPMP); hexamethylenediaminetetra(methylenephosphonic) acid (HDTMP); aminotris(methylenephosphonic) acid (ATMP); ethylenediaminetetra(methylenephosphonic) acid (EDTMP); tetramethylenediaminetetra(methylenephosphonic) acid (TDTMP); and phosphonobutanetricarboxylic acid (PBTC).

Examples

[0113] The invention will be demonstrated by the following non-limiting examples. Cloning & expression including TtEst Thermogutta terrifontis sequence information:

[0114] The DNA sequence encoding a protein with putative hydrolytic activity was identified using the ANASTASIA galaxy data line from the HotZyme database. The gene was amplified from the genomic DNA and cloning was performed using the aLICator LIC cloning and expression kit to an N-terminal His6 tag (pLATE31, E. coli, ArcticExpress (DE3) RIL was transformed (heat shock) and used as an expression strain for protein production.

[0115] TtEst L37A and L251A mutants were prepared using the QuikChange Lightining site-directed mutagenesis kit according to the manufacturer's instructions. The mutant constructs were overexpressed in the same way as the native protein. AfEst2: Archaeoglobus fulgidus Esterase:

[0116] The gene encoding AF-Est2 (site tag: AF1537; Uniprot accession number: O28735) was amplified by PCR, without its stop codon, using A. fulgidus chromosomal DNA as a template and respectively introducing a site of Ncol and Xhol restriction. The generated PCR product was digested by Ncol and Xhol and the product was purified and ligated into the protein expression vector pET24d digested with the same restriction enzymes, resulting in plasmid pWUR365 for the expression of the 6x-His tagged AF-Est2 protein C-terminal. E. coli strain BL21-CodonPlus (DE3)-RIPL was transformed with the expression plasmid. Fermentation (collection) & purification TtEst Thermogutta terrifontis:

[0117] Protein production was performed in 2L Erlenmeyer flasks with 1L of LB medium and the appropriate antibiotic for plasmid selection (Ampicillin, 100 µg/mL, Gentamicin 20 µg/mL). LB medium containing only ampicillin was inoculated with 1-3% (v/v) pre-culture and incubated at 37°C and 180 rpm until reaching OD 600 = 0.4. Subsequently, the incubation temperature was lowered to 12 °C for 1 h. Gene expression was induced by the addition of IPTG to 1 mM final and performed by 2d at 12 °C and 180 rpm. Cells were collected by centrifugation (4750 x g, 20 min, 4 °C) and stored at -80 °C.

[0118] Cell lysis was performed by resuspension of the equilibrium cell slurry buffer (25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 20 mM imidazole, 10 mL buffer to 1 g of net cell weight) and sonication on ice to break down the cells. Protein purification was performed using a 1 ml HisTrap FF column (GE Healthcare) and an AKTA purification system (GE Healthcare) for affinity chromatography via poly histidine labeling. Protein elution was performed by a linear gradient for 30 min using buffer with increased imidazole concentration (25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 500 mM imidazole). Elution fractions were identified by absorbance (280 nm) and applied to an SDS-PAGE. Fractions containing the protein of interest were pooled and dialyzed overnight against 5 L of imidazole-free buffer (25 mM Tris-HCl, pH 8.0, 500 mM NaCl). The dialyzed protein was supplemented with 0.005% (v/v) sodium azide and 10% (v/v) glycerol for freezing and storage at -80°C.

AfEst2: Archaeoglobus fulgidus Esterase:

[0119] Protein production was performed in Luria-Bertani (LB) medium containing 50 µg/mL each of kanamycin, chloramphenicol and streptomycin. The LB medium containing kanamycin and chloramphenicol with 1-3% (v/v) pre-culture is incubated at 37 °C and 180 rpm until reaching OD 600 = 0.6. Gene expression was induced by adding IPTG to 1 mM final and performed overnight at 30 °C and 180 rpm. Cells were collected by centrifugation (4750 x g, 20 min, 4 °C) and stored at -80 °C.

[0120] Cell lysis was performed by resuspension of cell paste equilibration buffer (25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 20 mM imidazole, 10 mL of buffer to 1 g of net cell weight) and sonication on ice to break down the cells. Protein purification was performed using a 1 ml HisTrap FF column using an AKTA purifier for affinity chromatography via poly histidine labeling. Protein elution was performed by a linear gradient for 30 min using buffer with increased imidazole concentration (25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 500 mM imidazole). Elution fractions were identified by absorbance (280 nm) and applied to an SDS-PAGE. Fractions containing the protein of interest were pooled and dialyzed overnight against 5 L of imidazole-free buffer (25 mM Tris-HCl, pH 8.0, 500 mM NaCl). The dialyzed protein was supplemented with 0.005% (v/v) sodium azide and 10% (v/v) glycerol for freezing and storage at -80°C. Bioanalytical Determination of protein concentration

[0121] The total amount of protein from the enzyme samples was estimated using Sigma-Aldrich's BCA assay kit (bicinchoninic acid) and working reagent was prepared as instructed in the user manual. The BCA reagent was prepared by mixing solution A [1% (w/v) bicinchoninic acid as sodium salt, 2% (w/v) sodium carbonate, 0.16% (w/v) tartrate of sodium, 0.4% (w/v) of sodium hydroxide, 0.95% (w/v) of sodium hydrogen carbonate, pH 11.5] with solution B [4% (w/v) of sodium sulfate copper] in ratio (v/v) 50:1. A serial dilution of bovine serum albumin (2 mg/mL) was performed in deionized water to create a 7-point standard curve. To run the assay, BCA reagent (200 µL) was added into 96-well plate wells, followed by sample protein dilutions (20 µL). Microtiter plates (MTP) were sealed and incubated at 37 °C for 30 min. After incubation, the absorbance at 540 nm was measured in a spectrophotometer. Determination of esterase purity

[0122] Protein samples containing esterase (20 µL) were prepared with SDS-PAGE sample buffer and heated at 70 °C for 10 min before running on 4-12% Bis-Tris NuPage gels with MOPS buffer at 170V. PageRulerPlus molecular weight marker was run along with the samples for molecular mass determination. Each gel was then stained using GelCode Blue Safe protein dye following the manufacturer's protocol. Biochemical determination of esterase activity

[0123] Esterase activity was determined by a colorimetric method using 4-nitrophenyl-valerate (C5) and 4-nitrophenyl-dodecanoate (C12) as substrates. 4-Nitrophenyl-dodecanoate (25mg) or 4-nitrophenyl-valerate (18mg) were dissolved in 10 mL of solvent (methanol) to prepare 8mM stock solutions. Before performing the assay, 1 mL of stock solution was added to 7 mL of acidified water (pH 4.5), to give a final concentration of 1 mM. In 96-well microtiter plates, 60 µL dH2O, 115 µL Tris-HCl buffer (pH 8.5, 50 mM), 5 µL diluted enzyme solution and 20 µL substrate (multi-channel at the end) were added . For blanks, enzyme solution was replaced with dH2O. Following the addition of reagents, the release of product (4-nitrophenol) was monitored at 405nm for 15 min at room temperature.

application test

Human-like sebum model composition and application to tissue

[0124] Table 1A shows the composition of human-like sebum to be used in washing studies, and which is comparable to human sebum analyzed in the literature (Table 1B). Macrolex violet dye (0.4% w/w) was added to the model tallow, and then 100 µL was applied to a 10x10 cm sample of polycotton which was preheated to 60 °C. Stain drainage was facilitated by leaving the stain to dry overnight at 60°C. Uniformity of staining was confirmed by colorimetric determination of SRI values across the sample which was subsequently cut into circles smaller than 30 mm in diameter, allowing for adaptation into 6-well microtiter plates for subsequent wash tests.

[0125] Table 1: (A) Human-like sebum composition tested. Shown in comparison (B) is the composition of human sebum as proposed by Nikkari 1974, in Ro 2005, Stefaniak 2010. Human-like sebum model was designed to mimic the literature description.

Table 1A (A) Tested human-like sebum model Ingredient Type % Inclusion Oleic acid Fatty acids 8 Isostearic acid (12%) 4 Tricaprin Triglycerides 1.8 Triolein (39.2%) 28.2 Triisostearin 9.2 oleyl Wax esters 11.9 Myristyl myristate (29.8%) 11.9 Isostearyl isostearate 6 Squalene Squalene (13.8%) 13.8 Cholesterol oleate Cholesterol (esters) 3.4 Cholesterol (5.1% ) 1.7 Total 99.9

Table 1B (B) Human tallow (literature) Type % median inclusion (range) Fatty acids 28.3 (2.3 - 38.3) Triglycerides 32.5 (14.8 - 44) Wax esters 25 (10 - 26) Squalene 10.6 (3.3 – 20) Cholesterol (esters) 6 (1 – 9.5) Washing studies for human-like sebum enzymatic wash performance

[0126] Prewash readings were taken for the 30 mm diameter sebum stains to measure stain intensity. Washing studies were conducted either in a 5 mL volume (inside a 6-well plate, at 40 °C for 1 hour at 100 rpm) or in 100 mL (inside glass vials, at 40 °C for 1 hour at 100 rpm). Enzymes were present at 25 mg/L within 2 g/L of a 7.5% surfactant formulation. The stains were then rinsed three times after washing to completely remove the washing liquid and any residual enzyme. After drying, the stain plates were digitally scanned and their deltaE was measured. This value is used to express cleaning effect and is defined as the color difference between a white garment and that of a garment stained after being washed.

[0127] Mathematically, the definition of deltaE is: 𝑑𝑒𝑙𝑡𝑎𝐸 = √[(𝛥𝐿)2 + (𝛥𝑎)2 + (𝛥𝑏)2 ] where ΔL is measured by the difference in darkness between washed and white fabric; Δa and Δb are measured by the difference in redness and yellowness respectively between both tissues. From this equation, it is clear that the lower the value of deltaE, the whiter the fabric will be. In relation to this color measurement technique, reference is made to the Commission Internationale de l'Eclairage (CIE); Recommendation on Uniform Color Spaces, Color Difference Equations, Psychometric Color Terms, Supplement No.

CIE, nº 15, Colorimetry, Central Office of the CIE, Paris, 1978.

[0128] Here the cleaning effect is expressed in the form of stain removal index (SRI): SRI = 100 - deltaE

[0129] The higher the SRI, the cleaner the fabric, SRI = 100 (white). Human-like enzymatic cleaning performance against sebum

[0130] Washing studies in a wash volume of 5 mL identified that the esterase enzymes of SEQ ID 1 to 4, the enzymes named TtEst, TtEst37, TtEst151, AfEst2, all showed improved performance in relation to human-like sebum removal against the control samples that includes the clothes washing esterase reference (Cutinase) and the clothes washing lipase reference (Lipex Evity). The 3-5 SRI increase units for the experimental enzymes shown is a whitening cleanse enhancement visualized above that of the cutinase control enzyme and the laundry wash lipase reference (Lipase Evity). The test was performed in triplicate at 40 °C for 1 h. The applied formulation contains 7.5% of total surfactant.

[0131] The >3 SRI increase units for the lipase enzyme of the invention is a clearly visualized cleaning improvement compared to Cutinase and Lipex Evity (Table 2).

Table 2 Wash Sample Performance (SRI) Negative control (water) 68.5 ± 1.03 Positive control (formulation + Lipex Evity) 70.6 ± 0.6 Positive control (formulation + Cutinase) 73.9 ± 0.98 Invention (formulation + TtEst esterase of SEQ ID 1) 76.1 ± 1.1 Invention (formulation + TtEst37 esterase of SEQ ID 2) 76.2 ± 1.2 Invention (formulation + TtEst251 esterase of SEQ ID 3) 78.8 ± 2.3 Invention (formulation + AfEst2 esterase from SEQ ID 4) 77.2 ± 1.0

[0132] Table 2: Cleaning performance of the esterase enzymes of SEQ ID 1 to 4 (relative to human-like sebum model) shown in comparison to wash controls in either: water, or commercial esterase plus reference formulation (Cutinase ) or more reference formulation of commercial laundry lipase (Lipex Evity).

[0133] The Stain Removal Index (SRI) indicating washing performance was measured. The ± statistics refer to the 95% confidence level. The test shows that the esterases of SEQ ID 1 to 4 perform much better against sebum than the commercial enzyme esterase (Cutinase) and lipase (Lipex Evity).

Human-like enzymatic cleaning performance against sebum

[0134] Washing studies in a volume of 100 mL confirm that the lipase enzyme of SEQ ID 4 showed improved performance regarding human-like sebum removal versus control samples that include the current commercial esterase enzyme (Cutinase) and lipase (Lipex Evity) (Table 3). The test was performed in triplicate at 40 °C for 1 h. The applied formulation contains 7.5% of total surfactant.

Table 3 Sample Washing performance (SRI) Negative control (water) 72.6 ± 1.2 Positive control (formulation + Lipex Evity) 79.7 ± 1.28 Positive control (formulation + Cutinase) 81.4 ± 1.65 Invention (formulation + AfEst2 esterase of SEQ ID 4) 84.2 ± 0.12

[0135] Table 3: Cleaning performance of the esterase enzyme of SEQ ID 4 (relative to human-like sebum model) shown in comparison to wash controls in either: water, or commercial esterase plus reference formulation (Cutinase) or most reference formulation of commercial clothing washing lipase (Lipex Evity).

Claims (12)

claims 1. Detergent composition characterized in that it comprises: (i) from 1 to 60% by weight, preferably from 2 to 50% by weight, more preferably from 3 to 45%, even more preferably from 5 to 40% by weight, even more preferably from 6 to 40% by weight of a surfactant; and, (ii) from 0.0005 to 5% by weight, preferably from 0.005 to 2.5% by weight, more preferably from 0.01 to 1% by weight of an esterase enzyme of the EC 3.1.1.1 enzyme class, having at least 60% sequence identity to any one of SEQ ID NOs: 1 to 4. A detergent composition according to claim 1 characterized in that the esterase enzyme is at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, still more preferably at least 95%, even more preferably at least 97%, at least 98% or even at least 99% sequence identity to any one of SEQ ID NOs: 1 to 4. A detergent composition according to claim 1 or 2 characterized in that the esterase enzyme has 100% sequence identity to any one of SEQ ID NOs: 1 to 4. Detergent composition according to any one of claims 1 to 3 characterized in that it comprises 0.1 to 10% by weight, preferably from 0.2 to 9% by weight, more preferably from 0.25 to 8, even more preferably 0 .5 to 6% by weight, even more preferably 1 to 5% by weight of soil release polymer, more preferably a polyester based soil release polymer. Detergent composition according to claim 4, characterized in that the polyester soil release polymer is a polyethylene and/or polypropylene terephthalate based soil release polymer, preferably a polypropylene terephthalate based soil release polymer. Detergent composition according to any one of claims 1 to 5, characterized in that the detergent composition comprises an alkoxylated polyamine, preferably at a level of from 0.1 to 8% by weight, more preferably from 0.2 to 6% by weight. weight, even more preferably from 0.5 to 5% by weight. A detergent composition according to any one of claims 1 to 6 characterized in that the detergent composition is a laundry detergent composition, preferably the laundry detergent composition is a liquid or a powder, even more preferably a liquid detergent. A laundry detergent composition according to claim 7, characterized in that the surfactant comprises anionic and/or non-ionic surfactant, preferably comprising both anionic and non-ionic surfactants. A detergent composition according to any one of claims 1 to 8, characterized in that it is preferably a laundry detergent composition, further comprising an additional enzyme selected from the group consisting of: lipases, proteases, cellulases, alpha-amylases, peroxides /oxidases, pectate lyases and/or mannanases. A detergent composition according to any one of claims 1 to 9, characterized in that it is preferably a laundry detergent composition, further comprising an additional ingredient selected from fluorescent agent, perfume, toning dyes and polymers, and mixtures thereof. 11. Method of treating a fabric substrate characterized by being with a tallow stain, said method comprising incorporating an esterase enzyme of the EC 3.1.1.1 enzyme class, having at least 60%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 97%, at least 98% or even at least at least 99% sequence identity, even more preferably 100% sequence identity to any one of SEQ ID NOs: 1 to 4 in a detergent composition comprising from 1 to 60% by weight of a surfactant; and subsequently treating a fabric substrate with a sebum stain with said composition. 12. Use of an esterase enzyme, characterized in that it is of the EC 3.1.1.1 enzyme class, having at least 60%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, most preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 97%, at least 98% or even at least 99% sequence identity, most preferably 100% sequence identity with any of SEQ ID NOs: 1 to 4 to improve cleaning of sebum stains on fabric.