BR112020010648A2 - detergent composition, laundry detergent composition, method to improve enzymatic cleaning in water and use of a protease enzyme - Google Patents

Abstract

  DETERGENT COMPOSITION, LAUNDRY DETERGENT COMPOSITION, METHOD FOR IMPROVING ENZYMATIC CLEANING IN WATER AND USE OF AN ENZYME PROTEASE. The present invention provides a detergent composition comprising: (i) from 1 to 60% by weight, preferably from 2 to 50%, more preferably from 4 to 50% by weight of a surfactant; (ii) from 0.0005 to 1% by weight, preferably from 0.005 to 0.6% by weight of a protease enzyme having at least 90% sequence identity with SEQ ID NO: 1; and a method and use to improve enzymatic cleaning in water using said protease.

Description

Invention Patent Descriptive Report DETERGENT COMPOSITION, LAUNDRY DETERGENT COMPOSITION, METHOD FOR IMPROVING ENZYMATIC CLEANING IN

WATER AND USE OF A PROTEASE ENZYME Field of the Invention

[0001] The present invention relates to a detergent composition comprising a surfactant that incorporates a new protease enzyme.

Background of the Invention

[0002] Water can be a scarce resource. Consumers who wish to use detergent compositions on a substrate, particularly laundry detergents on fabrics, may only be able to use water that is not ideal for cleaning. An example of this is that salt water (water with a significant sodium chloride content) as sea water is sometimes used.

[0003] Proteases are common ingredients in cleaning compositions. A problem with commercial proteases is that they work poorly in salt water conditions.

Summary of the Invention

[0004] It has been found that the incorporation of the new protease enzyme as defined in the present invention into detergent compositions shows improved cleanliness.

[0005] In one aspect, the present invention provides a detergent composition comprising: (i) from 1 to 60% by weight, preferably from 2 to 50%, more preferably from 4 to 50% by weight of surfactant; (ii) from 0.0005 to 1% by weight, preferably from 0.005 to 0.6% by weight of a protease enzyme having at least 90% sequence identity with SEQ ID NO: 1.

[0006] Preferably the protease enzyme has at least 95%, more preferably 97% sequence identity with SEQ ID NO: 1. More preferably, the protease enzyme has 100% sequence identity with SEQ ID NO: 1.

[0007] A preferred detergent composition is a laundry detergent composition. Preferably, the laundry detergent composition is a liquid or a powder, more preferably the detergent is a liquid detergent.

[0008] Preferably, the laundry detergent composition comprises anionic and / or non-ionic surfactant, more preferably the laundry detergent composition comprises both anionic and nonionic surfactants.

[0009] The laundry detergent preferably comprises an alkoxylated polyamine.

[0010] The laundry detergent preferably comprises a dirt-releasing polymer, more preferably a polyester-based dirt-releasing polymer.

[0011] Preferably, the laundry detergent comprises phosphonic acid chelating agent (or salt thereof) at a level that is less than 0.1% by weight, more preferably less than 0.01% by weight, even more preferably, the composition is free of phosphonic acid chelating agent (or salt thereof).

Preferred detergent compositions, particularly laundry detergent compositions additionally comprise an additional enzyme selected from the group consisting of: lipases, cellulases, alpha-amylases, peroxidases / oxidases, pectate lyases, mannases, and / or additional proteases.

[0013] In another aspect, the present invention provides a method for enhancing enzymatic cleaning in water having a sodium chloride content of 0.1 to 4%, preferably 0.25 to 3% at 20 ° C, said method comprising incorporating a protease enzyme having at least 90% sequence identity with SEQ ID NO: 1 in a detergent composition comprising from 1 to 60% by weight of a surfactant; and subsequent treatment of a substrate, preferably fabrics, with said composition.

In another aspect, the present invention provides for the use of a protease enzyme having at least 90%, preferably 95%, more preferably 97%, even more preferably 100% sequence identity with SEQ ID NO: 1 for improve enzymatic cleaning in water having a sodium chloride content of 0.1 to 4%, at a temperature of 15 ° C to 45 ° C.

Detailed Description of the Invention

[0015] The indefinite article "one" or "one" and its corresponding definite article "o" or "a" as used here means at least one, or one or more, unless otherwise specified.

[0016] The detergent composition can take any suitable form, for example liquid, solid (including powder) or gel.

[0017] The detergent composition can be applied to any suitable substrate. Particularly preferred substrates are fabrics. Particularly preferred detergent compositions are laundry detergent compositions.

[0018] Laundry detergent compositions can take any suitable form. Preferred forms are liquid or powder, with liquid being the most preferred. Surfactant

[0019] The detergent composition comprises surfactant (which includes 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 4 to 50% by weight of surfactant. Even more preferred levels of surfactant are 6 to 30% by weight, more preferably 8 to 20% by weight.

[0020] The detergent composition (preferably a laundry detergent composition) comprises anionic and / or nonionic surfactants, preferably comprising both anionic and nonionic surfactants.

[0021] Suitable anionic detergent compounds that can be used are generally water-soluble alkali metal salts of organic sulfates and sulfonates having alkyl radicals containing about 8 to about 22 carbon atoms, the term "alkyl" being used to include the alkyl portion of higher alkyl radicals.

[0022] Examples of suitable synthetic anionic detergent compounds are sodium and potassium alkyl sulphates, especially those obtained when sulphating higher C8 to C18 alcohols, produced for example from tallow or coconut oil, C9 to C20 alkyl sulfonates benzene of sodium and potassium, particularly linear secondary C10 to C15 benzene sulfonates of sodium; and alkyl glyceryl sodium ether sulfates, especially those higher alcohol ethers derived from tallow or coconut oil and synthetic petroleum alcohols.

[0023] The anionic surfactant is preferably selected from: linear alkyl benzene sulfonate; alkyl sulfates; alkyl ether sulfates; soaps; alkyl sulfonates (preferably methyl) ester and mixtures thereof.

[0024] 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 a C12-C14 n-alkyl sulfate with an average of 1 to 3 units of EO (ethoxylate).

[0025] Sodium lauryl ether sulfate (SLES) is particularly preferred. Preferably the linear alkyl benzene sulfonate is a C11-C15 alkyl sulfonate sodium benzene. Preferably the alkyl sulfate is a straight or branched C12-C18 alkyl sulfate. Sodium dodecyl sulfate is particularly preferred (SDS, also known as primary alkyl sulfate).

[0026] In liquid formulations preferably two or more surfactants are present, for example linear alkyl benzene sulfonate together with an alkyl ether sulfate.

[0027] In liquid formulations, preferably the laundry composition, in addition to the anionic surfactant, comprises non-ethoxylated alkyl surfactant

ionic, preferably from 2 to 8% by weight of nonionic alkyl ethoxylated surfactant.

[0028] Suitable non-ionic detergent compounds that can 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 non-ionic detergent compounds are the condensation products of linear or branched primary or secondary aliphatic alcohols C8 to C18 with ethylene oxide.

[0029] More preferably, the nonionic detergent compound is the nonionic alkyl ethoxylated surfactant which is a primary alcohol C8 to C18 with an average ethoxylation of 7 to 9 units of EO.

[0030] Preferably, the surfactants used are saturated. Protease

[0031] Protease has been found to perform well in salt water conditions. The protease can then be considered to be halotolerant. This means that it can work in an environment with a high salt content.

[0032] It has also been found that protease can perform well at a low temperature of 20 ° C.

[0033] Protease outperforms commercial protease enzymes in saltwater environments at both a high temperature of 40 ° C and a low temperature of 20 ° C.

[0034] The protease is present at a level of 0.0005 to 1% by weight, preferably from 0.005 to 0.6% by weight.

[0035] The protease enzyme has at least 90%, preferably 95%, or even 97% sequence identity to SEQ ID NO: 1. The most preferable protease enzyme can have 100% sequence identity to SEQ ID NO:

1. Alkoxylated polyamine

[0036] When the detergent composition is in the form of a laundry composition, it is preferred that an alkoxylated polyamine is included.

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.

[0038] The alkoxylated polyamine can be straight or branched. It can be branched to the extent that it is a dendrimer. Alkoxylation can typically be ethoxylation or propoxylation, or mixtures thereof. Where a nitrogen atom is alkoxylated, a preferred average degree of alkoxylation is 10 to 30, preferably 15 to 25.

A preferred material is alkoxylated polyethyleneimine, more preferably ethoxylated polyethyleneimine, with an average degree of ethoxylation being 10 to 30, preferably 15 to 25, where a nitrogen atom is ethoxylated. Dirt Release Polymer

[0040] When the detergent composition is in the form of a laundry composition, it is preferred that a dirt-releasing polymer is included.

Preferred levels of dirt release polymer are in the range of 0.1 to 10% by weight, preferably from 0.2 to 8% by weight, more preferably from 0.25 to 7% by weight, even more preferably from 0.5 to 6% by weight.

[0042] Polyester based dirt release polymers are described in WO 2014/029479 and WO 2016/005338. Additional enzymes

[0043] Additional enzymes, other than the specified proteases, may be present in the detergent composition. It is preferred that additional enzymes are present in the preferred laundry detergent composition.

[0044] 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.

[0045] Levels of enzyme present in the composition preferably relate to the level of enzyme as pure protein.

[0046] Preferred additional enzymes include those in the group consisting of:

additional lipases, cellulases, alpha-amylases, peroxidases / oxidases, pectate lyases, mannanases and / or proteases. Said preferred additional enzymes include a mixture of two or more of these enzymes.

[0047] Preferably the additional enzyme is selected from: lipases, cellulases, alpha-amylases and / or additional protease.

[0048] Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include Humicola lipases (Thermomyces synonym), for example H. lanuginosa (T. lanuginosus) as described in EP 258068 and EP 305216 or from H. insolens as described in WO 96/13580, a Pseudomonas lipase, for example example of P. alcaligenes or P. pseudoalalkigenes (EP 218272), P. cepacia (EP 331376), P. stutzeri (GB 1372034), P. fluorescens, strain of Pseudomonas sp. SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO 96/12012), a lipase from Bacillus, for example 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 variants of lipase 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.

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

[0050] 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 in relation to phospholipids.

[0051] Phospholipids, such as lecithin or phosphatidylcholine, consist of glycerol esterified with two fatty acids in the external position (sn-1) and in the middle (sn-2) and esterified with phosphoric acid in 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 A1 and A2 that hydrolyze a fatty acyl group (at position sn-1 and sn-2, respectively) to form lysophospholipid; and lysophospholipase (or phospholipase B) which can hydrolyze the remaining fatty acyl group to the lysophospholipid. Phospholipase C and phospholipase D (phosphodiesterases) release diacyl glycerol and phosphatidic acid respectively.

[0052] 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. Serine proteases of the subtilase type 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, which are the Subtilisin family, the Termitase family, the Proteinase K family, the Lantibiotic peptidase family, the Kexina family and the Pyrolysin family.

[0053] 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, substilisina Novo, subtilisina 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 can be those described in WO 92/175177, WO 01/016285, WO 02/026024 and WO 02/016547. Examples of trypsin-type 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 chymotrypsin proteases derived from Cellumonas described in WO 05/052161 and WO 05/052146.

[0054] Most preferably the protease is a subtilisin (EC 3.4.21.62).

[0055] 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, substilisina Novo, subtilisina 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. Preferably, 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, subtilisin is derived from Bacillus gibsonii or Bacillus Lentus.

[0056] 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®, all can be sold as Ultra® or Evity® (Novozymes A / S).

[0057] 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.

[0058] 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 more detail in GB 1296839 or the strain 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.).

[0059] 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, for example fungal cellulase produced by Humicola insolens, Thielavia terrestris, Myceliophthora thermophila and Fusarium oxysporum disclosed in US 4435307, US 5648267, US 56975, 776, 7676 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.

[0060] Suitable peroxidases / oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include Coprinus peroxidases, example of 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).

[0061] Additional enzymes suitable for use are discussed in WO 2009/087524, WO 2009/090576, WO 2009/107091, WO 2009/111258 and WO 2009/148983.

[0062] 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

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

[0064] Chelating agents may be present or absent in detergent compositions.

[0065] Preferably the laundry detergent comprises phosphonic acid chelating agent (or salt thereof) at a level that is less than 0.1% by weight, more preferably less than 0.01% by weight, even more preferably the composition it is free of phosphonic acid chelating agent (or salt thereof).

[0066] Examples of phosphonic acid chelating agents (or salt thereof) are: 1-hydroxyethylidene-1,1-diphosphonic acid (HEDP); diethylenetriaminopenta (methylenephosphonic) acid (DTPMP); hexamethylenediaminetetra (methylenephosphonic) acid (HDTMP); aminotris (methylenephosphonic) (ATMP); ethylenediaminetetra (methylenephosphonic) acid (EDTMP); tetramethylenediaminetetra (methylenephosphonic) acid (TDTMP); and phosphonobutanotricarboxylic acid (PBTC). Additional materials

[0067] Optional but preferred additional materials that can be included in detergent compositions (preferably laundry detergent compositions) include fluorescent agent, perfume, shade dye and polymers. Fluorescent agent

[0068] The composition preferably comprises a fluorescent agent

(optical brightener). Fluorescent agents are well known and many of said fluorescent agents are commercially available. Generally, these fluorescent agents are supplied and used in the form of their alkali metal salts, for example, sodium salts.

[0069] The total amount of fluorescent agent or agents used in the composition is generally from 0.0001 to 0.5% by weight, preferably 0.005 to 2% by weight, more preferably 0.01 to 0.1% by weight.

[0070] Preferred classes of fluorescent agent are: biphenyl di-styryl compounds, Tinopal®, CBS-X, di-amino stilbenesulfonic acid compounds, eg Tinopal DMS pure Xtra and Blankophor® HRH, and pyrazoline compounds, eg Blankophor SN.

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.

[0072] Most preferred fluorescent agents are: 2- (4-styryl-3-sulfophenyl) -2H-naphthol [1,2-d] sodium triazole, 4,4'-bis {[(4-anilino-6- (N-methyl-N-2-hydroxyethyl) amino 1,3,5-triazin-2-yl)] amino} di-sodium stylbene-2-2'-disulfonate, 4,4'-bis {[(4 -anilino-6-morpholino-1,3,5-triazin-2-yl)] amino} di-sodium stylbene-2,2'-disulfonate and 4,4'-bis (2-sulfo-styrene) di-biphenyl sodium.

[0073] 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

[0074] The composition preferably comprises a perfume. Many suitable examples of perfume are provided in the 1992 CTFA (Cosmetics, Toiletries and Fragrances Association) international buyer guide, published by the CFTA Publications and the 80th annual edition of the OPD Chemicals Buyers Directory of

1993, published by Schnell Publishing Co.

[0075] Preferably the perfume comprises at least one note (compound) of: alpha-isomethyl ionone; benzyl salicylate; citronelol; coumarin; hexyl cinnamal; 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-damascona; beta-ionone; verdil acetate; dodecanal; cinnamic hexyl aldehyde; cyclopentadecanolide; benzenoacetic acid, 2-phenylethyl ester; amyl salicylate; beta-karyophylene; ethyl undecylenate; geranyl anthranylate; alpha-irone; beta-phenyl ethyl benzoate; alpha-santalol; cedrol; cedril acetate; cedril shape; cyclohexyl salicylate; gamma-dodecalactone; and beta-phenylethyl phenyl acetate.

[0076] Useful components of perfume include materials with both natural and synthetic origin. They include compounds alone or mixtures. Specific examples of such components can be found in the current literature, for example, Fenaroli Guide to Aroma Ingredients, 1975, CRC Press; Adjuntos de Comida Sintéticos, 1947 by M.B. Jacobs, edited by Van Nostrand; or Perfume and Aroma Chemicals by S. Arctander 1969, Montclair, N.J. (USA).

[0077] 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.

[0078] In perfume mixtures, preferably 15 to 25% by weight are head notes. Head notes are defined by Poucher (Jornal da Sociedade de Químicos Cosméticos 6 (2): 80 [1955]). Preferred top notes are selected from citrus oils, linalool, linalyl acetate, lavender, dihydromyrcenol, rose oxide and cis-3-hexanol.

[0079] The International Fragrance Association published a list of fragrance ingredients (perfumes) in 2011 (http://ifraorg.org/en-

us / ingredients # .U7Z4hPldWzk).

[0080] The Fragrance Materials Research Institute provides a database of perfumes (fragrances) with reliable information.

[0081] Head notes of perfume can be used to suggest whiteness and brightness benefit of the invention.

[0082] Some or all of the 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 ° C. It is also advantageous to encapsulate perfume components that have a low CLog P (which are those that have a greater tendency to be partitioned in water), preferably with a CLog P of less than 3.0. These relatively low boiling point and relatively low CLog P materials have been called “prolonged flowering” 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 iso valerate, benzyl propionate, beta gamma hexenol, camphor gum, laevo-carvona, d-carvone, cinnamic alcohol, cinnamic format, cis-jasmine, cis-3-hexenyl acetate, cuminic alcohol, cyclal C, 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, flower acetate (decenyl tricycle acetate), fructene (decenyl tricycle propionate), geraniol, hexenol, hexenyl acetate, hexyl acetate, hexyl format, alkylene hydratropic oil, hydroxycitroneal, indone, isoamyl alcohol, iso mentone, isopulegyl acetate, isoquinolone, ligustral, linalool, linalool oxide, linalyl format, menthol, menthol acetophenone, methyl amyl ketone, methyl anthranylate, methyl benzoate, benzoate methyl benyl, methyl eugenol, methyl heptenone, methyl heptine carbonate, methyl heptyl ketone, methyl hexyl ketone, methyl phenyl carbinyl acetate, methyl salicylate, methyl-n-methyl anthranylate, 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 borne, pulegone, oxide rose, 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 perfume components other than the list given above of long-blooming perfumes present in the formulation.

[0083] 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 components of essential oils such as sage clarea, eucalyptus, geranium, lavender, apple extract, neroli, nutmeg, mint, violet and valerian fennel.

[0084] It is preferred that the laundry treatment composition does not contain a peroxygen bleach, for example sodium percarbonate, sodium perborate and peracid. Shading dye

[0085] Preferably the composition is a laundry detergent composition, so it comprises a shading dye. Preferably, the shading dye is present from 0.0001 to 0.1% by weight of the composition.

[0086] Dyes are described in Synthesis of Color Chemistry, Properties and Applications of dyes and organic pigments (H. Zollinger, Wiley VCH, Zürich, 2003) and, Chemistry of Industrial Dyes, Properties and Applications (K Hunger (ed), Wiley-VCH Weinheim 2003).

[0087] Shading 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.

[0088] Preferred shading dye chromophores are azo, azine, anthraquinone and triphenylmethane.

[0089] Azo, anthraquinone, phthalocyanine and triphenylmethane dyes preferably carry an anionic liquid charge or are uncharged. Azine preferably carries an anionic or cationic liquid charge. Blue or violet shading dyes deposit on the fabric during the washing or rinsing step of the washing process providing a visible tone to the fabric. In this context, the dye gives a white or blue color to a white fabric with a pitch angle of 240 to 345, more preferably 250 to 320, more preferably 250 to 280. The white fabric used in this test was a non-mercerized cotton sheet targeted.

[0090] Shading 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).

[0091] Mono-azo dyes preferably contain a heterocyclic ring and are more preferably thiophene dyes. The 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,

[0092] Bis-azo dyes are preferably sulfonated bis-azo dyes. Preferred examples of sulphonated bis-azo dyes are direct violet 7, direct violet 9, direct violet 11, direct violet 26, direct violet 35, direct violet 40, direct violet 41, direct violet 51, direct violet 66, violet Direct 99 and alkoxylated versions of them. Alkoxylated bis-azo dyes are described in WO 2012/054058 and WO 2010/151906.

[0093] An example of an alkoxylated bis-azo dye is:

[0094] Thiophene dyes are available from Milliken under the brand name Liquitint Violet DD and Liquitint Violet ION.

[0095] Azine dye 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: where: X3 is selected from: -H; -F; -CH3; -C2H5; -OCH3; and –OC2H5; 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.

[0096] The shading 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 shading dye, there are preferred ranges depending on the effectiveness of the shading dye which is dependent on the class and the particular effectiveness within any particular class. As mentioned above, the shading dye is a blue or violet shading dye.

[0097] A mixture of shading dyes can be used.

[0098] The shading 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 in the polyethylene imine are substituted as isopropyl alcohol groups by propoxylation. Preferably polyethylene imine prior to reaction with the dye and propoxylation has a molecular weight of 600 to 1800.

[0099] An example of the structure of a reactive anthraquinone covalently linked to a propoxylated imine polyethylene is: (Structure I) Polymers

[0100] The composition can comprise one or more additional polymers. Examples are carboxymethylcellulose, poly (ethylene glycol), poly (vinyl alcohol),

polycarboxylates such as polyacrylates, maleic acid / acrylic copolymers and lauryl methacrylate / acrylic acid copolymers. EXAMPLES

[0101] The invention will be demonstrated by the following non-limiting examples. Isolation and cultivation of a microorganism that produces protease

[0102] Strains of the protease-producing microorganism were isolated from samples of marine sediment collected from Jiaozhou Bay (China Yellow Sea 36 ° 09'N, 120 ° 32'E) by selective screening on agar plates with skimmed milk containing ( g / L of seawater): tryptone 5, yeast extract 2, skimmed milk powder 40, agar powder 16. The plates were incubated at 20 ° C for 48-72h to obtain bacterial colonies. Colonies with clear casein hydrolysis circles in milk were evaluated as protease producers. The proteolytic strain LA-05 exhibiting the largest hydrolysis circle was selected for further experiments. The growth and fermentation medium used for culture of the LA-05 strain consisted of 5g of tryptone, 2g of yeast extract and 1L of sea water, pH 7.0. The medium was autoclaved at 121 ° C for 20 min. First, the LA-05 strain alone was inoculated with 2% (v / v) growth culture and grown in an orbital shaking incubator at 20 ° C and 150 RPM for 12h. Then the culture was transferred to 2L conical flasks containing 400 mL of fermentation medium and incubated for 78h at 15-30 ° C and 150 RPM.

[0103] To obtain maximum enzyme yield, growth kinetics and enzyme production were measured every 6 hours during the incubation period (78h). The cell density was monitored by measuring the absorbance at 600nm. The cell-free supernatant was recovered by centrifugation at 12000 RPM for 20 min at 4 ° C, and then used as a crude enzyme preparation to determine protease activity. Strain identification and phylogenetic analysis

[0104] Strip tests to determine analytical profile index (API) and sequencing with the 16S rRNA gene were performed to identify the LA-05 strain gene. API strips were used to investigate the physiological and biochemical characteristics of the LA-05 strain according to the manufacturer's instructions.

[0105] The 16S rRNA sequence was amplified by PCR using a sense primer (27F, 5'-AGAGTTTGATCMTGGCTCAG-3 ') and reverse primer (1492R, 5'-TACGGYTACCTTGTTACGACTT-3'). The genomic DNA of the LA-05 strain was purified with a TIANamp Bacterium DNA kit (TIANGEN, Beijing, China) and then used as a model for PCR amplification including 30 cycles (the cycle parameters: denaturation at 94 ° C for 50s, annealing ring at 58 ° C for 50s, extension at 72 ° C for 100s).

[0106] The amplified product was cloned into vector pMD18-T (Takara, Dalian, China), and the recombinant plasmid pMD-16S was constructed. Then, the recombinant plasmid was transformed into the competent cells of Escherichia coli DH5a. LB culture medium containing ampicillin (60 µg / mL) was applied to recombinant E. coli DH5a culture clones. The DNA fragment of 16S rRNA bound in the recombinant plasmid was confirmed by commercial DNA sequencing (Sangon Biotech Co., Ltd., Shanghai, China). The multiple sequence alignment was done using an identification program using the EzTaxon database. Type culture strains with paired similarity above 97% were selected and subjected to evolutionary phylogenetic and molecular analyzes using Molecular Evolutionary Genetic Analysis (MEGA) software (version 7.0.9) by method of grouping neighbors. Statistical evaluation of the tree topology was calculated by bootstrap analysis with 1000 replications. Protease purification

[0107] All purification steps were carried out at 4 ° C unless stated otherwise. Concentration by ultrafiltration

[0108] Four hundred milliliters of cultures with 30h were centrifuged at 12000 RPM for 20min at 4 ° C. The supernatant was filtered through

0.22 µm to remove bacteria cells and debris from the medium completely, and recovered as a crude protease preparation. The prepared supernatant was concentrated by Millipore's Amicon Ultra-4 centrifugal filter devices with a molecular weight cut of 3 KDa. After washing three times with ultrapure water, the crude protease buffer was replaced with 50 mM Tris-HCl buffer containing 0.1 M NaCl (pH 8.0). Purification

[0109] Two milliliters of concentrated solution was loaded onto a gel filtration column (HiLoad 16/600 Superdex 200 pg, GE Healthcare) which was equilibrated and eluted with 50 mM Tris-HCl buffer containing 0.1 M NaCl (pH 8 , 0). The column was eluted with 180 ml of buffer at a flow rate of 1 ml / min until the optical density of the eluent at 280 nm is zero. Elution fractions of 3 mL each were collected and the protease activity was analyzed. Fractions showing protease activity were pooled and then applied to a HiTrap Q FF ion exchange column (1 mL) equilibrated with 50 mM Tris-HCl buffer containing 0.1 M NaCl (pH 8.0).

[0110] Subsequently, the column was washed with the same buffer and the bound proteins were eluted with a linear gradient of NaCl in the range of 0.1-1 M at a flow rate of 1mL / min. Elution fractions of 1 mL each were collected and protease activity was analyzed. Fractions containing protease activity were pooled and stored at -80 ° C for further studies. Determination of protease activity

[0111] Protein concentration was determined with a BCA protein test kit (Sangon Biotech, Shanghai, China) using bovine serum albumin (BSA) as a reference. Protease activity was determined according to the modified method (Lagzian and Asoodeh, 2012). In summary, a 0.25 mL aliquot of purified protease was incubated with 0.75 mL of 50 mM Tris-HCl buffer (pH 8.0) containing 1% (w / v) casein at 45 ° C for 10 min. The reaction was terminated by the addition of 0.5 ml of 20% (w / v) 20% trichloroacetic acid (TCA). The mixture was mixed with a laboratory shaker and placed at room temperature for 20 min. Subsequently, the precipitate was removed by centrifugation at 12000 RPM for 20 min and the absorbance of the supernatant was measured at 280 nm. A unit (U) of protease activity was defined as the amount of enzyme that hydrolyzes casein to release 1 µg of tyrosine per minute under experimental conditions. Units of protease activity were calculated using tyrosine (0-100 µg / mL) as standard. Electrophoresis, mass spectrometry, zymography and isoelectric focusing Molecular mass

[0112] The molecular mass of the purified protease was determined by electrophoresis on sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) using (5%, w / v) stacking gel and (12% w / v) according to standard protocols with Bio-Rad Mini-PROTEIN equipment (Farhadian et al., 2015). The gel was stained with Bright Blue Coomassie R-250 and decolorized with methanol / acetic acid / water (5/1/4, v / v / v). The relative molecular weight of the purified enzyme was estimated by comparing its mobility with a standard protein marker.

[0113] Meanwhile, the precise molecular mass of purified protease was analyzed, in positive linear mode, by the ionization and laser desorption mass spectrometry method assisted by laser-time-of-flight matrix (MALDI-TOF / MS) using Instrument FlashDetector (Bruker microflex LRF, Bruker Daltonics, USA). Data were collected with FlexControl 3.4 and analyzed with FlexAnalysis 3.4 software. Zymography

[0114] Zymography was performed to visualize the activity of the enzyme with a modified method (Machado et al., 2016). Briefly, samples were mixed with β-mercaptoethanol-free gel loading buffer and loaded to the electrophoresis gel without heating. Electrophoresis was performed in ice water. Subsequently, the gel was immersed in 50 mM Tris-HCl buffer

(pH 8.0) containing 2.5% Triton X-100 at 4 ° C and stirred lightly for 1h to remove the SDS. The gel was washed twice with 50 mM Tris-HCl buffer (pH 8.0) at 4 ° C for 20 min to extract residual Triton X-100, and then incubated with 1% (w / v) casein in 50 mM Tris -HCl (pH 8.0) at 25 ° C for 1h. The gel was soaked in 20% (w / v) trichloroacetic acid (TCA) to end the protease reaction, stained with bright blue Coomassie R-250 for 2h, and bleached with methanol / acetic acid / water (5/1 / 4, v / v / v) for one night to reveal the protease hydrolysis bands. Isoelectric focusing

[0115] Purified protease isoelectric focusing was performed on gel strips with immobilized pH gradients (IPG) from 3 to 10 (Bio-Rad, USA) using Multiphor II electrophoresis system (GE Healthcare). Briefly, the purified protease was desalted and washed with 1% glycine buffer by ultrafiltration. Pre-electrophoresis started at 700 V for 20 min at 15 ° C after fixing the IPG strip. Then, the sample and IEF standards were subjected to electrophoresis at 2000 V for 90 min at 15 ° C. Finally, the band was fixed by treatment with TCA buffer for 30 min, stained with bright blue Coomassie R-250 and bleached with a methanol / acetic acid / water mixture. The value for the purified protease was evaluated by ImageQuant TL software version 7.0. Determination of the N-terminal amino acid sequence

[0116] The protease purified by SDS-PAGE was transferred to a polyvinylidene difluoride (PVDF) membrane in CAPS buffer according to the Matsudaira protocol (Matsudaira, 1987). The PVDF membrane was lightly stained with bright blue Coomassie R-250 and the band containing the enzyme was cut. Subsequently, the N-terminal amino acid sequence was analyzed by Edman's automated degradation method using a PPSQ-21A protein sequencer (Shimadzu). The first 20 amino acid residues were aligned with those in the UniProtKB / Swiss-Prot database and Protein Data Bank protein database using the BLAST homology search (NCBI, USA).

Identification of gene encoding potential protease and modeling of three-dimensional structure

[0117] The strip containing the protease obtained by the denatured SDS-PAGE was carefully cut and the proteins were analyzed by tandem mass spectrometry (MALDI MS / MS) as described in the published protocol (Marchand et al., 2009). All spectra acquired from the samples were processed using TOF / TOF Explorer software (AB SCIEX) in standard mode. The identification of the fingerprint of the peptide mass (PMF) was sought using GPS Explorer (V 3.6) with a MASCOT search engine (2.3) compared to the NCBI database (non-redundant protein sequences). Proteins with protein score (C.I.) confidence intervals above 95% were considered reliable identifications.

[0118] Candidate proteins were selected based on MASCOT search results, proteolytic activity and secretion mechanism. To amplify the protease coding sequences by PCR, the alignment of multiple sequences was performed on the candidate coding sequences in DNAMAN software. A pair of primers, 5'-ATGAACCAACAACGTCAACTACAGCTG-3 'and 5'-CGGGTCAATCTAAACGCAACG-3', was designed according to the conserved regions of the upstream and downstream coding sequences. The coding sequence was amplified and cloned into pMD18-T vector using Escherichia coli DH5a as the host strain by Sanger sequencing. Eventually, the nucleotide sequence obtained was translated into the amino acid sequence, the mature protease with 321 amino acid residues. Traces of metals in the purified protease were determined by flame atomic absorption spectrometry. Washing studies in mini bottles

[0119] Cotton samples stained with blood / milk / ink on E116 cotton (Test Materials Center - Netherlands) together with cotton cover were used for washing in mini flasks. Dyes were applied in triplicate between the wash bottles. Using water prepared for FH26, containing 1g / L of the laundry formulation (two types, labeled F1 and F2) protease (control reference or halotolerant) was added at a concentration of 5mg / L in a total volume of 100 mL. This level of enzyme is equivalent to a level of 0.5% by weight of protease in the formulation. The control reference used was the leading commercial protease material (Carnival Evity from Novozymes). To replicate salt water conditions, 2% final salt concentration (NaCl) was also used.

[0120] Washings were carried out at 20 ° C and 40 ° C, with 250 RPM agitation for 1h. Washing at 20 ° C demonstrated the benefits of the invention even in low temperature conditions.

[0121] After washing, the stains were separated from the washing liquid and rinsed twice in a beaker containing 1L of FH26 water, before taking off to dry overnight. After drying, the stain plates were digitally scanned and their deltaE were measured. This value expresses the cleaning effect and is defined as the color difference between the white fabric and the stained fabric after being washed.

[0122] Mathematically, the definition of deltaE is: 𝑑𝑒𝑙𝑡𝑎𝐸 = √ [(𝛥𝐿) 2 + (𝛥𝑎) 2 + (𝛥𝑏) 2] where ΔL is measured by the difference in darkness between the washed fabric and the white one; Δa and Δb are measured by the difference in redness and yellowing respectively between both tissues. From this equation, it is clear that the lower the deltaE value, the whiter the fabric will be. In relation to this color measurement technique, reference is made to the International Commission de l'Eclairage (CIE); Recommendation in uniform color spaces, color difference equations, psychometric color terms, supplement No. 2 to CIE publication, No. 15, Colorimetry, CIE Central Office, Paris, 1978.

[0123] Here the cleaning effect is expressed in the form of stain removal index (SRI):

SRI = 100 - deltaE

[0124] The higher the SRI, the cleaner the fabric, SRI = 100 (white).

[0125] Formulations used Table 1 Formulation 1 (F1)% by weight Demineralized water Up to 100 Non-ionic surfactant (25-7) 4,365 Tinopal 5BMGX 0,200 Acusol WR 0,700 TEA 8,820 EU acid LAS 5,820 Glycerol 2,000 Prifac 5908 0,860 Dequest 2010 1,500 EU SLES 4,365 BIT - Proxel 0.040 Citric acid 1,000 Table 2 Formulation 2 (F2)% by weight Demineralized water Up to 100 Tinopal CBS-X 0.090 NaOH 0.457 TEA 1,500 Citric acid 0.400 LAS acid (Indiatuba) 4,500 EPEI 2,325 SLES 3EO (Texapon N70 LST 13,500) EPEI 0.775

BIT 0.0200 MIT 0.0095 Soladona LA059 - used 2012 0.9800 NaCl 1.5000 CAPB 1.5000

[0126] The two enzymes tested were added in formulations 1 and 2 to give an effective enzyme level in the formulation of 0.5% by weight. The proteases were: Protease 1 (comparison) = Carnival Evity, an enzyme commercially available from Novozymes; Protease 2 (according to the present invention) = a protease with 100% according to SEQ ID NO: 1.

[0127] The results are shown in table 3: Table 3 20 ° C F1 F1 + F1 + F2 F2 + F2 + protease 1 protease 2 protease 1 protease 2 SRI 59.90 67.54 70.26 54.88 57.99 72.57 Deviation 0.84 1.29 1.82 0.95 0.25 0.57 standard 40 ° C F1 F1 + F1 + F2 F2 + F2 + protease 1 protease 2 protease 1 protease 2 SRI 54.13 67, 05 69.20 52.39 57.45 63.75 Deviation 1.42 0.48 0.90 0.68 1.14 1.01 standard

[0128] The higher the SRI (stain removal index), the better the cleaning.

[0129] The results of the washing studies show that, as expected, both proteases give a cleaning benefit over the formulation with only control in all washing conditions (both at 20 ° C and 40 ° C, as well as in different formulations). The 20 ° C wash study shows the added benefit of improved cleaning even in a low temperature (20 ° C) wash condition.

[0130] In FH26 water containing 2% salt (NaCl) the benefit performance of the halotolerant protease is clearly apparent. At both temperatures 20 ° C and 40 ° C, the halotolerant protease outperforms the commercial protease control in formulations 1 and 2. The cleaning benefit (over reference protease 1) from the halotolerant protease is even more notable in the laundry formulation 2 which does not contain the chelating agent, which is present in formulation 1. Surprisingly, the performance of the benefit is immensely improved at a washing temperature of 20 ° C, which further highlights the potential applicability of this technology to improve the cleaning of stains in sea water conditions.

Claims (14)

Claims 1. Detergent composition characterized by comprising: (i) from 1 to 60% by weight, preferably from 2 to 50% by weight, more preferably 4 to 50% by weight of surfactant; (ii) from 0.0005 to 1% by weight, preferably from 0.005 to 0.6% by weight of a protease enzyme having at least 90% sequence identity with SEQ ID NO: 1. Detergent composition according to claim 1, characterized in that the protease enzyme has at least 95%, more preferably 97% sequence identity with SEQ ID NO: 1. Detergent composition according to claim 1 or 2, characterized in that the protease enzyme has 100% sequence identity with SEQ ID NO: 1. Detergent composition according to any one of claims 1 to 3, characterized in that the detergent composition is a laundry detergent composition. Laundry detergent composition as defined in claim 4, characterized in that the laundry detergent composition is liquid or powder, preferably liquid detergent. 6. Laundry detergent composition as defined in claim 4 or according to claim 5 characterized by the laundry detergent composition comprising anionic and / or non-ionic surfactant, preferably comprising both anionic and nonionic surfactants. Laundry detergent composition according to any one of claims 4 to 6, characterized in that the laundry detergent composition comprises an alkoxylated polyamine, preferably at a level of 0.1 to 8% by weight, more preferably from 0.2 to 6% by weight, more preferably from 0.5 to 5% by weight. Laundry detergent composition according to any one of claims 4 to 7, characterized in that the laundry detergent composition comprises a dirt-releasing polymer, preferably a polyester-based dirt-releasing polymer. Laundry detergent composition according to any one of claims 4 to 8, characterized in that the level of phosphonic acid chelating agent (or salt thereof) is less than 0.1% by weight, preferably 0.01% by weight, more preferably the composition is free of phosphonic acid chelating agent (or salt thereof). Detergent composition according to any one of claims 1 to 9, characterized in that it additionally comprises an additional enzyme selected from the group consisting of: lipases, cellulases, alpha-amylases, peroxidases / oxidases, pectate lyases, mannases and / or additional proteases. 11. Method for improving enzymatic cleaning in water characterized by having a sodium chloride content of 0.1 to 4%, preferably 0.25 to 3% by weight at 20 ° C, said method comprising incorporation of a protease enzyme having at least 90% sequence identity to SEQ ID NO: 1 in a detergent composition comprising from 1 to 60% by weight of a surfactant; and subsequent treatment of a substrate, preferably fabrics, with said composition. Method according to claim 11, characterized in that the protease enzyme has at least 95%, more preferably 97% sequence identity with SEQ ID NO: 1; even more preferably, the protease enzyme has 100% sequence identity with SEQ ID NO: 1. Method according to claim 11 or 12, characterized in that the composition treating the substrate is a composition as defined in any one of claims 4 to 10. 14. Use of a protease enzyme characterized by having at least 90%, preferably 95%, more preferably 97%, even more preferably 100% sequence identity with SEQ ID NO: 1 to enhance enzymatic cleaning in water having a content of 0.1 to 4% sodium chloride at a temperature of 15 ° C to 45 ° C.