Liquid dishwashing detergent composition

Abstract

The present invention provides liquid detergent compositions suitable for hand dishwashing, wherein the compositions comprise a protease enzyme and provide hand skin care benefits.

Description

[0001] References to sequence listings

[0002] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference. Technical Field

[0003] The present invention relates to liquid detergent compositions suitable for hand dishwashing wherein the compositions comprise a protease enzyme and provide hand skin care benefits. Background Art

[0004] Hand dishwashing using conventional liquid detergent compositions can often damage the skin and / or lead to skin irritation and dryness. Although many attempts have been made to develop hand dishwashing detergents that are mild to the skin, there remains a need for detergent compositions having improved skin mildness while maintaining optimal cleaning performance.

[0005] Proteases designed for improved protein stain removal are standard ingredients in detergent compositions for laundry and automatic dishwashing machines and are found in a variety of different types of compositions such as powders for machine use, liquids, sachets, tablets, bars, etc. Proteases, on the other hand, have not been widely used in hand dishwashing liquids.

[0006] Liquid detergent compositions for hand dishwashing containing proteases have been described in the patent literature, for example in WO 95 / 07971, WO 97 / 25397, WO 2010 / 088161, WO 2010 / 088163, WO 2010 / 088164 and WO2012 / 015852. In many cases, such compositions include ingredients such as emollients, polymers, wetting agents, and other components designed to provide a pleasant skin feel or to reduce skin irritation and dryness that may be associated with hand dishwashing.

[0007] Summary of the Invention The present invention provides liquid detergent compositions containing protease enzymes which have been found to provide substantial skin care benefits even in the absence of specific skin care or moisturizing ingredients. Summary of the invention

[0008] The present invention generally relates to liquid detergent compositions suitable for hand dishwashing and comprising at least one protease enzyme.

[0009] In one aspect, the present invention relates to liquid detergent compositions comprising at least one protease, wherein use of these compositions, e.g., in hand dishwashing, provides at least one hand skin care benefit selected from keratinocyte removal, reduction of transepidermal water loss, improvement of hand skin barrier, and improvement of hand skin water retention.

[0010] In another aspect, the present invention relates to the use of a protease for the preparation of a liquid detergent composition suitable for hand dishwashing, wherein the composition provides at least one hand skin care benefit selected from the group consisting of keratinocyte removal, reduction of transepidermal water loss, improvement of hand skin barrier, and improvement of hand skin water retention.

[0011] In a further aspect, the present invention is directed to a process for preparing a hand dishwashing detergent composition having skin care benefits, the process comprising adding to the composition at least one protease enzyme.

[0012] In a further aspect, the present invention relates to the use of a composition comprising a protease as disclosed herein in a cleaning process, such as for use in hand dishwashing.

[0013] In a further aspect, the invention relates to a method of cleaning, e.g., for hand washing dishes or cleaning hard surfaces, the method comprising contacting the dishes or hard surfaces to be cleaned with a detergent composition comprising a protease as described herein under conditions suitable for cleaning the dishes or surfaces.

[0014] Sequence review

[0015] SEQ ID NO: 1 is from Bacillus lentus Sequence of protease polypeptide.

[0016] SEQ ID NO: 2 is the sequence of the BPN' protease polypeptide from Bacillus amyloliquefaciens.

[0017] SEQ ID NO: 3 is the sequence of TY145 protease from Bacillus sp.

[0018] SEQ ID NO: 4 is the sequence of the protease disclosed in GENESEQP under accession number BER84782.

[0019] SEQ ID NO:5 is from Bacillus licheniformis Protease (also known as Carlsberg subtilisin). BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is an alignment of the amino acid sequences of subtilisin 309 (SEQ ID NO: 1) and subtilisin BPN' (SEQ ID NO: 2).

[0021] definition

[0022] Subtilase / Protease: The terms "subtilase" and "protease" are used interchangeably herein and refer to enzymes that hydrolyze peptide bonds in proteins. This includes any enzyme belonging to the EC 3.4 enzyme group (including each of its 13 subclasses), and in particular endopeptidases (EC 3.4.21). EC numbers refer to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego, California, including Supplements 1-5 as published in Eur. J. Biochem. 1994, 223, 1-5; Eur. J. Biochem. 1995, 232, 1-6; Eur. J. Biochem. 1996, 237, 1-5; Eur. J. Biochem. 1997, 250, 1-6; and Eur. J. Biochem. 1999, 264, 610-650, respectively.

[0023] Protease activity: The term "protease activity" means proteolytic activity (EC 3.4), in particular endopeptidase activity (EC 3.4.21). There are several types of protease activity, the three main types of activity being: trypsin-like (wherein there is cleavage of an amide substrate after Arg or Lys at P1), chymotrypsin-like (wherein cleavage occurs after a hydrophobic amino acid at P1), and elastase-like (cleavage after Ala at P1). Protease activity can be determined according to the procedure described in WO2016 / 087619.

[0024] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

[0025] For purposes of the present invention, use the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. [J.Molecular Biology] 48:443-453) implemented in the Needle program of EMBOSS package (EMBOSS:The European Molecular Biology Open Software Suite [EMBOSS:European Molecular Biology Open Software Suite], Rice et al., 2000, Trends Genet. [Genetics Trend] 16:276-277) (preferred 5.0.0 version or updated version) to determine the sequence identity between two amino acid sequences. The parameters used are gap opening penalty 10, gap extension penalty 0.5 and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output (obtained using non-simplified options) of Needle, which is labeled as "longest identity", is used as identity percentage and is calculated as follows:

[0026] (number of identical residues x 100) / (alignment length - total number of gaps in the alignment)

[0027] For the purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) implemented in the Needle program of the EMBOSS package (EMBOSS:The European Molecular Biology Open Software Suite [EMBOSS:European Molecular Biology Open Software Suite], Rice et al., 2000, supra) (preferably 5.0.0 version or updated version). The parameters used are gap opening penalty 10, gap extension penalty 0.5, and EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle, labeled as "longest identity" (obtained using the non-simplified option), is used as identity percentage and is calculated as follows:

[0028] (number of identical deoxyribonucleotides x 100) / (alignment length - total number of gaps in the alignment)

[0029] Variant: The term "variant" means a polypeptide having protease activity comprising an alteration (i.e., substitution, insertion, and / or deletion) at one or more positions. Substitution means replacing the amino acid occupying a position with a different amino acid, deletion means removing the amino acid occupying a position, and insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position.

[0030] Fragment: The term "fragment" means a polypeptide having one or more amino acids missing from the amino and / or carboxyl terminus of a mature polypeptide, wherein the fragment has subtilase activity. Such a fragment preferably contains at least 85%, at least 90% or at least 95% of the number of amino acids in SEQ ID NO: 1.

[0031] Variant naming conventions

[0032] For the purposes of the present invention, the polypeptide of SEQ ID NO: 2 is used to determine the corresponding amino acid residue numbering in the variant of SEQ ID NO: 1. The amino acid sequence of the variant of SEQ ID NO: 1 is aligned with SEQ ID NO: 2, and the amino acid position number corresponding to any amino acid residue in the polypeptide of SEQ ID NO: 1 is determined based on the alignment. For further information, see the paragraph "Numbering of amino acid positions / residues" below.

[0033] For SEQ ID NO:3, amino acid position numbering is according to SEQ ID NO:3.

[0034] The identification of the corresponding amino acid residue in another subtilase can be determined by aligning the multiple polypeptide sequences using several computer programs using their corresponding default parameters, including but not limited to MUSCLE (Multiple Sequence Comparison by Log-Expectation; Version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32:1792-1797), MAFFT (Version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30:3059-3066; Katoh et al., 2005, Nucleic Acids Research 33:511-518; Katoh and Toh, 2007, Bioinformatics 23:372-374; Katoh et al., 2009, Methods in Molecular Biology 537:39- 64 ; Katoh and Toh, 2010, Bioinformatics 26:1899-1900 ), and EMBOSS EMMA using ClustalW (version 1.83 or later; Thompson et al., 1994, Nucleic Acids Research 22:4673-4680).

[0035] In describing variants of the present invention, the nomenclature described below has been adjusted for ease of reference. Accepted IUPAC single-letter or three-letter abbreviations for amino acids are used. The terms "alteration" or "mutation" are used interchangeably herein to refer to substitutions, insertions, and deletions.

[0036] replace . For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. For example, the threonine at position 220 is replaced by alanine as "Thr220Ala" or "T220A". Multiple substitutions can be separated by a plus sign ("+"), for example, "Thr220Ala+Gly229Val" or "T220A+G229V" represents that the threonine (T) and glycine (G) at positions 220 and 229 are replaced by alanine (A) and valine (V), respectively. Alternatively, multiple substitutions can be listed with separate mutations separated by spaces or commas. Alternative substitutions in specific positions can be represented by a slash (" / "). For example, the threonine in position 220 is replaced by alanine, valine or leucine and can be represented as "T220A / V / L".

[0037] Missing . For amino acid deletions, the following nomenclature is used: original amino acid, position, *. Accordingly, the deletion of threonine at position 220 is represented as "Thr220*" or "T220*". Multiple deletions can be separated by a plus sign ("+"), for example, "Thr220*+Gly229*" or "T220*+G229*", or alternatively can be separated by spaces or commas. The "X" used before the position number is used for substitution as described above, for example "X131*" means that the amino acid residue at position 131 is deleted.

[0038] insert . For amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Accordingly, the insertion of lysine after threonine at position 220 is represented as "Thr220ThrLys" or "T220TK". Insertions of multiple amino acids are represented as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2; etc.]. For example, the insertion of lysine and alanine after threonine at position 220 is represented as "Thr220ThrLysAla" or "T220TKA".

[0039] In such cases, the inserted amino acid residue(s) are numbered by adding lowercase letters to the position number of the amino acid residue preceding the inserted amino acid residue(s). In the above example, the sequence would therefore be:

[0040] <![CDATA[ Parents: ]]> <![CDATA[ Variants: ]]> 220 220 220a 220b T TKA

[0041] Multiple changes Variants containing multiple changes are separated by plus signs ("+"), for example, "Arg170Tyr+Gly195Glu" or "R170Y+G195E" represent substitutions of arginine and glycine at positions 170 and 195 by tyrosine and glutamic acid, respectively. Alternatively, multiple changes can be listed as separate mutations separated by spaces or commas.

[0042] For example, a combination of substitution and insertion may be represented as follows: S99AD, which represents that the serine residue at position 99 is substituted with an alanine residue and an aspartic acid residue is inserted.

[0043] Different changes When different changes can be introduced at one position, these different changes can be separated by commas, for example, "Arg170Tyr,Glu" represents that the arginine at position 170 is replaced by tyrosine or glutamic acid. Therefore, "Tyr167Gly,Ala+Arg170Gly,Ala" represents the following variants:

[0044] "Tyr167Gly+Arg170Gly", "Tyr167Gly+Arg170Ala", "Tyr167Ala+Arg170Gly", and "Tyr167Ala+Arg170Ala".

[0045] Different changes at one position can also be indicated by a slash (" / "), such as "T220A / V / L", as explained above. Alternatively, different changes can be indicated by brackets, such as Arg170[Tyr,Gly] or a one-letter code R170[Y,G].

[0046] The number of the amino acid position / residue. As used herein, the amino acid position numbering is based on the numbering of the BPN' polypeptide of SEQ ID NO: 2. Thus, the amino acid positions of a parent protease polypeptide having, for example, SEQ ID NO: 1 are those of the corresponding positions of SEQ ID NO: 2. This numbering system is conventional in the art, with position numbering for subtilisins in the patent literature typically being based on the corresponding position numbering of BPN'.

[0047] Specifically, the numbering is based on the alignment in Table 1 of WO 89 / 06279, which shows the mature polypeptide comprising the subtilisin BPN' (BASBPN) sequence (sequence c in that table) and subtilisin 309 from Bacillus lentus (also known as Alignment of five proteases of the mature polypeptide of )(BLSAVI)(sequence a in the table).

[0048] For reference purposes only Figure 1 , and Table 1 based on WO 89 / 06279 shows an alignment between SEQ ID NO: 1 and SEQ ID NO: 2, from which the position numbers corresponding to the positions of SEQ ID NO: 2 can be easily determined.

[0049] For a parent protease other than SEQ ID NO:1, the amino acid sequence of another protease can be aligned similarly to SEQ ID NO:2 to determine the amino acid position numbers that correspond to those of SEQ ID NO:2.

[0050] For SEQ ID NO:3, position numbering is based on SEQ ID NO:3. DETAILED DESCRIPTION

[0051] As explained above, the present invention relates to liquid detergent compositions suitable for hand dishwashing, wherein these compositions comprise at least one protease and can provide one or more skin care benefits.

[0052] In a particular aspect, the present invention relates to a liquid detergent composition suitable for hand dishwashing, the composition comprising at least one protease, wherein the protease is a variant of the polypeptide of SEQ ID NO: 1 comprising two or more substitutions selected from the group consisting of S9E, N43R, N76D, Q206L, Y209W, S259D and L262E, wherein the variant has protease activity and has at least 80%, at least 85%, at least 90% or at least 95% but less than 100% sequence identity to SEQ ID NO: 1, and wherein the position numbering is based on the numbering of SEQ ID NO: 2.

[0053] In one embodiment of this aspect, the protease is a variant of a polypeptide of SEQ ID NO: 1 comprising 3, 4, 5, 6 or all substitutions selected from the group consisting of S9E, N43R, N76D, Q206L, Y209W, S259D and L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, such as a variant having at least 80%, at least 85%, at least 90% or at least 95% sequence identity with SEQ ID NO: 1. In one embodiment, the protease comprises or consists of a polypeptide of SEQ ID NO: 1 having 3, 4, 5, 6 or all of said substitutions.

[0054] In another embodiment, the protease is a variant of a polypeptide of SEQ ID NO: 1 comprising 3, 4, 5, 6, 7, 8 or all substitutions selected from the group consisting of S9E, N43R, N76D, V205I, Q206L, Y209W, S259D, N261W and L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, such as a variant having at least 80%, at least 85%, at least 90% or at least 95% sequence identity with SEQ ID NO: 1. In one embodiment, the protease comprises or consists of a polypeptide of SEQ ID NO: 1 having 3, 4, 5, 6, 7, 8 or all of the substitutions.

[0055] In another embodiment, the protease variant comprises or consists of SEQ ID NO: 1 with substitutions S9E, N43R, N76D, V205I, Q206L, Y209W, S259D, N261W and L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, for example, a variant having at least 80%, at least 85%, at least 90% or at least 95% sequence identity with SEQ ID NO: 1. In a preferred embodiment, the protease comprises or consists of a polypeptide of SEQ ID NO: 1 with substitutions S9E, N43R, N76D, V205I, Q206L, Y209W, S259D, N261W and L262E.

[0056] In another embodiment, the protease is a variant of a polypeptide of SEQ ID NO: 1 comprising 3, 4, 5, 6, 7, 8, 9, 10 or all substitutions selected from the group consisting of S9E, N43R, N76D, N185E, S188E, Q191N, A194P, Q206L, Y209W, S259D and L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, such as a variant having at least 80%, at least 85%, at least 90% or at least 95% sequence identity with SEQ ID NO: 1. In one embodiment, the protease comprises or consists of a polypeptide of SEQ ID NO: 1 having 3, 4, 5, 6, 7, 8, 9, 10 or all of the substitutions.

[0057] In another embodiment, the protease comprises or consists of SEQ ID NO: 1 with substitutions S9E, N43R, N76D, N185E, S188E, Q191N, A194P, Q206L, Y209W, S259D and L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, such as a variant having at least 80%, at least 85%, at least 90% or at least 95% sequence identity with SEQ ID NO: 1. In one embodiment, the protease comprises or consists of a polypeptide of SEQ ID NO: 1 with substitutions S9E, N43R, N76D, N185E, S188E, Q191N, A194P, Q206L, Y209W, S259D and L262E.

[0058] In another aspect, the present invention relates to a liquid detergent composition suitable for hand dishwashing, the composition comprising at least one protease, wherein the protease is selected from the group consisting of:

[0059] (a) a polypeptide of SEQ ID NO: 1;

[0060] (b) a variant of the polypeptide of SEQ ID NO: 1 having the mutation S99SE (i.e. insertion of E after position 99), wherein the position numbering is based on the numbering of SEQ ID NO: 2;

[0061] (c) a variant of the polypeptide of SEQ ID NO: 1 having the mutation S99AD (i.e. substitution of S99 with A followed by insertion of D), wherein the position numbering is based on the numbering of SEQ ID NO: 2;

[0062] (d) a variant of the polypeptide of SEQ ID NO: 1 having the substitutions Y167A+R170S+A194P, wherein the position numbering is based on the numbering of SEQ ID NO: 2;

[0063] (e) a variant of the polypeptide of SEQ ID NO: 1 having the substitutions S99D+S101R / E+S103A+V104I+G160S; for example a variant of SEQ ID NO: 1 having the substitutions S3T+V4I+S99D+S101E+S103A+V104I+G160S+V205I, wherein the position numbering is based on the numbering of SEQ ID NO: 2;

[0064] (f) a polypeptide of SEQ ID NO: 3 or a variant thereof, e.g., a variant of SEQ ID NO: 3 having the substitutions S27K+N109K+S111E+S171E+S173P+G174K+S175P+F180Y+G182A+L184F+Q198E+N199K+T297P, wherein the position numbering is based on the numbering of SEQ ID NO: 3; and

[0065] (g) The polypeptide of SEQ ID NO:4.

[0066] In one embodiment, use of a composition comprising the above protease in hand dishwashing provides at least one hand skin care benefit selected from keratinocyte removal, reduction of transepidermal water loss, improvement of hand skin barrier, and improvement of hand skin water retention.

[0067] Another aspect of the present invention relates to the use of a protease for the preparation of a liquid detergent composition suitable for hand dishwashing, wherein the protease provides at least one hand skin care benefit selected from keratinocyte removal, reduced transepidermal water loss, improved hand skin barrier, and improved hand skin water retention. In a preferred embodiment, the protease of this aspect is a variant of the polypeptide of SEQ ID NO: 1 comprising two or more substitutions selected from the group consisting of S9E, N43R, N76D, Q206L, Y209W, S259D and L262E, wherein the variant has protease activity and has at least 80%, at least 85%, at least 90% or at least 95% but less than 100% sequence identity with SEQ ID NO: 1, and wherein the position numbering is based on the numbering of SEQ ID NO: 2. Thus, the protease of this aspect may, for example, comprise or consist of a polypeptide of SEQ ID NO: 1 having substitutions S9E, N43R, N76D, V205I, Q206L, Y209W, S259D, N261W and L262E; or the protease may comprise or consist of a polypeptide of SEQ ID NO: 1 having substitutions S9E, N43R, N76D, N185E, S188E, Q191N, A194P, Q206L, Y209W, S259D and L262E.

[0068] A preferred protease for use in this aspect of the invention is a protease comprising, or consisting of, the polypeptide of SEQ ID NO: 1 having substitutions S9E, N43R, N76D, V205I, Q206L, Y209W, S259D, N261W and L262E.

[0069] In another embodiment of this aspect of the invention, the protease may be selected from the group consisting of:

[0070] (a) a polypeptide of SEQ ID NO: 1;

[0071] (b) a variant of the polypeptide of SEQ ID NO: 1 having the mutation S99SE, wherein the position numbering is based on the numbering of SEQ ID NO: 2;

[0072] (c) a variant of the polypeptide of SEQ ID NO: 1 having the mutation S99AD, wherein the position numbering is based on the numbering of SEQ ID NO: 2;

[0073] (d) a variant of the polypeptide of SEQ ID NO: 1 having the substitutions Y167A+R170S+A194P, wherein the position numbering is based on the numbering of SEQ ID NO: 2;

[0074] (e) a variant of the polypeptide of SEQ ID NO: 1 having the substitutions S99D+S101R / E+S103A+V104I+G160S; for example a variant of SEQ ID NO: 1 having the substitutions S3T+V4I+S99D+S101E+S103A+V104I+G160S+V205I, wherein the position numbering is based on the numbering of SEQ ID NO: 2;

[0075] (f) a polypeptide of SEQ ID NO: 3 or a variant thereof, e.g., a variant of SEQ ID NO: 3 having the substitutions S27K+N109K+S111E+S171E+S173P+G174K+S175P+F180Y+G182A+L184F+Q198E+N199K+T297P, wherein the position numbering is based on the numbering of SEQ ID NO: 3; and

[0076] (g) The polypeptide of SEQ ID NO:4.

[0077] As mentioned above, it has been found that the liquid detergent compositions of the present invention containing proteases can provide substantial skin care benefits even in the absence of specific skin care or moisturizing ingredients. Thus, in one embodiment, the compositions of the present invention may optionally be free of, for example, emollients, humectants, polymers, wetting agents, and other ingredients used for hand dishwashing to provide skin care benefits or, for example, to reduce skin irritation and dryness that may be associated with hand dishwashing.

[0078] Another aspect of the present invention relates to a method for preparing a hand dishwashing detergent composition having skin care benefits, or for providing skin care benefits to a hand dishwashing detergent composition, comprising adding at least one protease to the composition. The skin care benefits preferably comprise at least one hand skin care benefit selected from keratinocyte removal, reduced transepidermal water loss, improved hand skin barrier, and improved hand skin water retention. In a preferred embodiment, the protease of this aspect is a variant of the polypeptide of SEQ ID NO: 1 comprising two or more substitutions selected from the group consisting of S9E, N43R, N76D, Q206L, Y209W, S259D and L262E, wherein the variant has protease activity and has at least 80%, at least 85%, at least 90% or at least 95% but less than 100% sequence identity with SEQ ID NO: 1, and wherein the position numbering is based on the numbering of SEQ ID NO: 2. Thus, the protease of this aspect may, for example, comprise or consist of a polypeptide of SEQ ID NO: 1 having substitutions S9E, N43R, N76D, V205I, Q206L, Y209W, S259D, N261W and L262E; or the protease may comprise or consist of a polypeptide of SEQ ID NO: 1 having substitutions S9E, N43R, N76D, N185E, S188E, Q191N, A194P, Q206L, Y209W, S259D and L262E.

[0079] A preferred protease for use in this aspect of the invention is a protease comprising, or consisting of, the polypeptide of SEQ ID NO: 1 having substitutions S9E, N43R, N76D, V205I, Q206L, Y209W, S259D, N261W and L262E.

[0080] In another embodiment of this aspect of the invention, the protease may be selected from the group consisting of:

[0081] (a) a polypeptide of SEQ ID NO: 1;

[0082] (b) a variant of the polypeptide of SEQ ID NO: 1 having the mutation S99SE, wherein the position numbering is based on the numbering of SEQ ID NO: 2;

[0083] (c) a variant of the polypeptide of SEQ ID NO: 1 having the mutation S99AD, wherein the position numbering is based on the numbering of SEQ ID NO: 2;

[0084] (d) a variant of the polypeptide of SEQ ID NO: 1 having the substitutions Y167A+R170S+A194P, wherein the position numbering is based on the numbering of SEQ ID NO: 2;

[0085] (e) a variant of the polypeptide of SEQ ID NO: 1 having the substitutions S99D+S101R / E+S103A+V104I+G160S; for example a variant of SEQ ID NO: 1 having the substitutions S3T+V4I+S99D+S101E+S103A+V104I+G160S+V205I, wherein the position numbering is based on the numbering of SEQ ID NO: 2;

[0086] (f) a polypeptide of SEQ ID NO: 3 or a variant thereof, e.g., a variant of SEQ ID NO: 3 having the substitutions S27K+N109K+S111E+S171E+S173P+G174K+S175P+F180Y+G182A+L184F+Q198E+N199K+T297P, wherein the position numbering is based on the numbering of SEQ ID NO: 3; and

[0087] (g) The polypeptide of SEQ ID NO:4.

[0088] Another aspect of the invention relates to the use of a liquid detergent composition as described herein comprising at least one protease in a cleaning process. In this aspect, the cleaning process is typically hand dishwashing, although other uses are possible, particularly other manual cleaning processes, including hard surface cleaning and manual laundry washing. In a preferred embodiment, the protease of this aspect is a variant of a polypeptide of SEQ ID NO: 1 comprising two or more substitutions selected from the group consisting of S9E, N43R, N76D, Q206L, Y209W, S259D and L262E, wherein the variant has protease activity and has at least 80%, at least 85%, at least 90% or at least 95% but less than 100% sequence identity with SEQ ID NO: 1, and wherein the position numbering is based on the numbering of SEQ ID NO: 2. Thus, the protease of this aspect may, for example, comprise or consist of a polypeptide of SEQ ID NO: 1 having substitutions S9E, N43R, N76D, V205I, Q206L, Y209W, S259D, N261W and L262E; or the protease may comprise or consist of a polypeptide of SEQ ID NO: 1 having substitutions S9E, N43R, N76D, N185E, S188E, Q191N, A194P, Q206L, Y209W, S259D and L262E.

[0089] A preferred protease for use in this aspect of the invention is a protease comprising, or consisting of, the polypeptide of SEQ ID NO: 1 having substitutions S9E, N43R, N76D, V205I, Q206L, Y209W, S259D, N261W and L262E.

[0090] In another embodiment of this aspect of the invention, the protease may be selected from the group consisting of:

[0091] (a) a polypeptide of SEQ ID NO: 1;

[0092] (b) a variant of the polypeptide of SEQ ID NO: 1 having the mutation S99SE, wherein the position numbering is based on the numbering of SEQ ID NO: 2;

[0093] (c) a variant of the polypeptide of SEQ ID NO: 1 having the mutation S99AD, wherein the position numbering is based on the numbering of SEQ ID NO: 2;

[0094] (d) a variant of the polypeptide of SEQ ID NO: 1 having the substitutions Y167A+R170S+A194P, wherein the position numbering is based on the numbering of SEQ ID NO: 2;

[0095] (e) a variant of the polypeptide of SEQ ID NO: 1 having the substitutions S99D+S101R / E+S103A+V104I+G160S; for example a variant of SEQ ID NO: 1 having the substitutions S3T+V4I+S99D+S101E+S103A+V104I+G160S+V205I, wherein the position numbering is based on the numbering of SEQ ID NO: 2;

[0096] (f) a polypeptide of SEQ ID NO: 3 or a variant thereof, e.g., a variant of SEQ ID NO: 3 having the substitutions S27K+N109K+S111E+S171E+S173P+G174K+S175P+F180Y+G182A+L184F+Q198E+N199K+T297P, wherein the position numbering is based on the numbering of SEQ ID NO: 3; and

[0097] (g) The polypeptide of SEQ ID NO:4.

[0098] Another aspect of the invention relates to a method of cleaning, such as a method for hand washing dishes or cleaning hard surfaces, comprising contacting the dishes or hard surfaces to be cleaned with a detergent composition comprising at least one protease as described herein under conditions suitable for cleaning the dishes or surfaces. In a preferred embodiment, the protease of this aspect is a variant of the polypeptide of SEQ ID NO: 1 comprising two or more substitutions selected from the group consisting of S9E, N43R, N76D, Q206L, Y209W, S259D and L262E, wherein the variant has protease activity and has at least 80%, at least 85%, at least 90% or at least 95% but less than 100% sequence identity with SEQ ID NO: 1, and wherein the position numbering is based on the numbering of SEQ ID NO: 2. Thus, the protease of this aspect may, for example, comprise or consist of a polypeptide of SEQ ID NO: 1 having substitutions S9E, N43R, N76D, V205I, Q206L, Y209W, S259D, N261W and L262E; or the protease may comprise or consist of a polypeptide of SEQ ID NO: 1 having substitutions S9E, N43R, N76D, N185E, S188E, Q191N, A194P, Q206L, Y209W, S259D and L262E.

[0099] A preferred protease for use in this aspect of the invention is a protease comprising, or consisting of, the polypeptide of SEQ ID NO: 1 having substitutions S9E, N43R, N76D, V205I, Q206L, Y209W, S259D, N261W and L262E.

[0100] In another embodiment of this aspect of the invention, the protease may be selected from the group consisting of:

[0101] (a) a polypeptide of SEQ ID NO: 1;

[0102] (b) a variant of the polypeptide of SEQ ID NO: 1 having the mutation S99SE, wherein the position numbering is based on the numbering of SEQ ID NO: 2;

[0103] (c) a variant of the polypeptide of SEQ ID NO: 1 having the mutation S99AD, wherein the position numbering is based on the numbering of SEQ ID NO: 2;

[0104] (d) a variant of the polypeptide of SEQ ID NO: 1 having the substitutions Y167A+R170S+A194P, wherein the position numbering is based on the numbering of SEQ ID NO: 2;

[0105] (e) a variant of the polypeptide of SEQ ID NO: 1 having the substitutions S99D+S101R / E+S103A+V104I+G160S; for example a variant of SEQ ID NO: 1 having the substitutions S3T+V4I+S99D+S101E+S103A+V104I+G160S+V205I, wherein the position numbering is based on the numbering of SEQ ID NO: 2;

[0106] (f) a polypeptide of SEQ ID NO: 3 or a variant thereof, e.g., a variant of SEQ ID NO: 3 having the substitutions S27K+N109K+S111E+S171E+S173P+G174K+S175P+F180Y+G182A+L184F+Q198E+N199K+T297P, wherein the position numbering is based on the numbering of SEQ ID NO: 3; and

[0107] (g) The polypeptide of SEQ ID NO:4.

[0108] Obviously, in any aspect of the present invention described herein, including but not limited to the use of a protease for preparing a liquid detergent composition suitable for hand dishwashing, a method of preparing a hand dishwashing detergent composition having skin care benefits or providing skin care benefits to a hand dishwashing detergent composition by adding at least one protease to the composition, the use of a liquid detergent composition comprising at least one protease in a cleaning process, and a method of cleaning using the liquid detergent composition, the protease can be any protease or variant described in detail in the specification and claims.

[0109] In one embodiment of any aspect disclosed herein, the hand skin care benefit comprises enhanced keratinocyte removal.

[0110] In one embodiment of any aspect disclosed herein, the hand skin care benefit comprises reduced transepidermal water loss and / or improved hand skin barrier.

[0111] In one embodiment of any aspect disclosed herein, the hand skin care benefit comprises improved water retention of the hand skin.

[0112] Liquid detergent compositions of the present invention suitable for hand dishwashing typically comprise protease in an amount of from about 0.001% w / w to about 4% w / w, such as from about 0.01% w / w to about 2% w / w, such as from about 0.05% w / w to about 1% w / w, such as from about 0.1% w / w to about 0.5% w / w, wherein the percentages are enzyme protein by weight of the composition.

[0113] In preferred embodiments, the liquid detergent compositions of the present invention provide not only hand skin care benefits as described herein, but also provide good cleaning performance, meaning that their cleaning performance is at least as good as, or preferably better than, that of a corresponding liquid detergent composition without a protease. Proteases are well known for their protein stain removal properties in, for example, laundry and automatic dishwashing detergents, and therefore, it is expected that the compositions of the present invention will generally provide cleaning benefits as well as hand skin care benefits.

[0114] In another embodiment, the protease is highly stable, providing good storage stability to the composition. Non-limiting examples of proteases that have been found to have good stability and are therefore suitable for use in liquid detergent compositions of the present invention that are intended to have good shelf life include: variants of SEQ ID NO: 1 having substitutions S9E, N43R, N76D, V205I, Q206L, Y209W, S259D, N261W and L262E, and variants of SEQ ID NO: 1 having substitutions S9E, N43R, N76D, N185E, S188E, Q191N, A194P, Q206L, Y209W, S259D and L262E.

[0115] In addition to the amino acid changes specifically disclosed herein, the protease variants in the compositions of the present invention may include additional changes at one or more other positions. These additional changes may be minor, i.e., typically conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein and do not change the net formal charge as described herein; typically small deletions of 1-10 or 1-5 amino acids; or small amino-terminal extensions or carboxyl-terminal extensions. Another possible change is the truncation of the N-terminus and / or C-terminus, such as truncation of 1-5 amino acids at one or both ends, and the resulting protease variant fragments retain protease activity.

[0116] Examples of conservative substitutions are within the following groups: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not alter specific activity are known in the art and are described, for example, by H. Neurath and RL Hill, 1979 in The Proteins, Academic Press, New York. Common conservative substitution groups include, but are not limited to: G=A=S, I=V=L=M, D=E, Y=F, and N=Q (where, for example, "G=A=S" means that these three amino acids can be substituted for each other).

[0117] Alternatively, these amino acid changes have such a property that the physicochemical properties of the polypeptide are changed. For example, the amino acid changes can increase the thermal stability of the polypeptide, change the substrate specificity, change the optimal pH, and the like.

[0118] Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resulting mutant molecules are tested for protease activity to identify amino acid residues that are critical for the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. Active sites of enzymes or other biological interactions can also be determined by physical analysis of the structure, such as by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, together with mutations to putative contact site amino acids. See, e.g., de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identities of essential amino acids can also be inferred from alignments with related polypeptides.

[0119] Detergent composition

[0120] For the purposes of the present invention, the detergent composition is a liquid, particularly a liquid suitable for hand dishwashing, although it is envisaged that the compositions of the present invention may also be used for other purposes such as hard surface cleaning or hand laundry washing.

[0121] Liquid composition

[0122] The liquid detergent compositions of the present invention are typically formulated as liquid dishwashing detergent compositions suitable for hand dishwashing as is well known in the art.

[0123] In addition to the protease, these compositions will typically contain 30% to 90% by weight of an aqueous liquid carrier in which the other components of the composition are dissolved, dispersed or suspended. Preferably, the aqueous liquid carrier will contain 45% to 70%, more preferably 45% to 65% by weight of the composition. In a preferred embodiment, the aqueous liquid carrier is water. Alternatively, the aqueous liquid carrier may contain other materials that are liquid or soluble in the liquid carrier at room temperature (about 20° C.-25° C.), and these materials may provide other functions in addition to the function of an inert filler. Such materials may include, for example, hydrotropes and solvents, as discussed in more detail below.

[0124] The liquid dishwashing composition may have any suitable pH value. Preferably, the pH of the composition is adjusted to between about 6 and 10, preferably between about 7 and 9, such as about 7 or about 8. The pH of the composition may be adjusted using pH adjusting ingredients known in the art.

[0125] Surfactants

[0126] The composition of the present invention preferably comprises at least one surfactant selected from nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, zwitterionic surfactants, semi-polar nonionic surfactants, and mixtures thereof. The amount of the surfactant included can be from about 1% to about 50% by weight of the liquid detergent composition, preferably from about 5% to about 40% by weight, more preferably from about 5% to about 30% by weight, such as from about 10% to about 20% by weight. Non-limiting examples of suitable surfactants are discussed below.

[0127] In one embodiment, the highly effective yet mild surfactant system may comprise from about 4% to about 40%, preferably from about 6% to about 32%, more preferably from about 11% to about 25%, and most preferably from about 11% to about 18% of anionic surfactant, and optionally no more than about 15%, preferably no more than about 10%, more preferably no more than about 5%, by weight of the total composition, of a sulfonate surfactant.

[0128] Suitable anionic surfactants for use in the compositions and methods of the present invention include sulfates, sulfosuccinates, sulfonates, and / or alkyl ethoxy sulfates, more preferably a combination of alkyl sulfates and / or alkyl ethoxy sulfates having a combined degree of ethoxylation of less than about 5, preferably less than about 3, more preferably less than about 2.

[0129] In alternative embodiments, the surfactant system can be based on high levels of nonionic surfactants (such as from about 10% to about 45%, preferably from about 15% to about 40%, more preferably from about 20% to about 35% by weight of the total composition), preferably in combination with amphoteric surfactants, and more preferably in combination with low levels of anionic surfactants (such as less than 20%, preferably less than 10%, more preferably less than about 5% by weight of the total composition).

[0130] Sulfate surfactants

[0131] Suitable sulfate surfactants for use in the compositions herein include C 10 -C 14 Water soluble salts or acids of alkyl or hydroxyalkyl sulfates and / or ether sulfates. Suitable counterions include hydrogen, alkali metal cations or ammonium or substituted ammonium, preferably sodium.

[0132] When the hydrocarbyl chain is branched, it preferably comprises C 14 Alkyl branching units. The average branching percentage of the sulfate surfactant is preferably greater than 30% of the total hydrocarbyl chain, more preferably from 35% to 80%, most preferably from 40% to 60%.

[0133] Sulfate surfactants can be selected from C8-C 20 Primary, branched and random alkyl sulfates (AS); C 10 -C 18 Secondary (2,3) alkyl sulfate; C 10 -C 18 Alkyl alkoxy sulfate (AExS), wherein x is preferably from 1 to 30; C 10 -C 18 Alkyl alkoxy carboxylates, preferably containing 1 to 5 ethoxy units; mid-chain branched alkyl sulfates as discussed in US 6,020,303 and US 6,060,443; and mid-chain branched alkyl alkoxy sulfates as discussed in US 6,008,181 and US 6,020,303.

[0134] Alkyl sulfosuccinate-sulfoacetate

[0135] Other suitable anionic surfactants are alkyl (preferably dialkyl) sulfosuccinates and / or sulfoacetates. The dialkyl sulfosuccinates may be C 6-15 A linear or branched dialkyl sulfosuccinate. The alkyl moieties may be symmetrical (ie, identical alkyl moieties) or asymmetrical (ie, different alkyl moieties). Preferably, the alkyl moieties are symmetrical.

[0136] Sulfonate surfactants

[0137] The compositions of the present invention will preferably comprise no more than 15%, preferably no more than 10%, even more preferably no more than 5% by weight of the total composition of a sulfonate surfactant. These surfactants include C 10 -C 14 Water-soluble salts or acids of alkyl or hydroxyalkyl sulfonates; as discussed in WO 99 / 05243, WO 99 / 05242, WO 99 / 05244, WO 99 / 05082, WO 99 / 05084, WO 99 / 05241, WO 99 / 07656, WO 00 / 23549 and WO 00 / 23548; 11 -C 18 Alkylbenzene sulfonate (LAS), modified alkylbenzene sulfonate (MLAS); methyl sulfonate (MES); and alpha-olefin sulfonate (AOS). These surfactants also include paraffin sulfonates, which can be monosulfonates and / or disulfonates, obtained by sulfonating paraffins of 10 to 20 carbon atoms. Sulfonate surfactants also include alkyl glyceryl sulfonate surfactants.

[0138] Amphoteric and zwitterionic surfactants

[0139] Amphoteric and zwitterionic surfactants may be included in the composition at levels of from 0.01% to 20%, preferably from 0.2% to 15%, more preferably from 0.5% to 12% by weight. Suitable amphoteric and zwitterionic surfactants are amine oxides and betaines.

[0140] Most preferred are amine oxides, especially cocodimethylamine oxide or cocamidopropyl dimethylamine oxide. The amine oxides may have a straight chain or intermediate branched alkyl portion. Typical straight chain amine oxides include those of formula R 1 -N(R 2 )(R 3 )→O water-soluble amine oxide, where R 1 It is C 8-18 Alkyl part; R 2 and R 3 Independently selected from C 1-3 Alkyl groups and C 1-3The group consisting of hydroxyalkyl groups preferably includes methyl, ethyl, propyl, isopropyl, 2-hydroxyethyl, 2-hydroxypropyl and 3-hydroxypropyl. These linear amine oxide surfactants particularly include linear C 10 -C 18 Alkyl dimethyl amine oxide and linear C8-C 12 Alkoxyethyl dihydroxyethyl amine oxides. Preferred amine oxides include linear C 10 , Straight chain C 10 -C 12 , and straight chain C 12 -C 14 Alkyl dimethyl amine oxides. As used herein, "intermediate branching" means that the amine oxide has an alkyl portion having n1 carbon atoms and an alkyl branch on an alkyl portion having n2 carbon atoms. The alkyl branch is located on the alpha carbon of the nitrogen atom from the alkyl portion. This type of branching of amine oxides is also referred to as internal amine oxides in the art. The sum of n1 and n2 is 10 to 24 carbon atoms, preferably 12 to 20, and more preferably 10 to 16. The number of carbon atoms of an alkyl portion (n1) should be approximately the same as the number of carbon atoms of an alkyl branch (n2), so that the one alkyl portion and the one alkyl branch are symmetrical. As used herein, "symmetrical" means that in at least 50 wt%, more preferably at least 75 wt% to 100 wt% of the intermediate branched amine oxides used herein, n1-n2 is less than or equal to 5, preferably 4, and most preferably 0 to 4 carbon atoms.

[0141] The amine oxide further comprises two moieties independently selected from C 1-3 Alkyl, C 1-3 A hydroxyalkyl group, or a polyethylene oxide group containing an average of about 1 to about 3 ethylene oxide groups. Preferably, the two moieties are selected from C 1-3 Alkyl, more preferably both are selected to be C1 alkyl.

[0142] Other suitable surfactants include betaines, such as alkyl betaines, alkyl amido betaines, amidazolinium betaine, sulfobetaines (INCI Sultaines) and phosphobetaines.

[0143] A preferred surfactant system is a mixture of anionic surfactant and amphoteric or zwitterionic surfactant in a ratio ranging from 1:1 to 5:1, preferably 1:1 to 3.5:1.

[0144] For further information on amphoteric and zwitterionic surfactants, see WO 2012 / 015852.

[0145] Nonionic surfactants

[0146] When present as a co-surfactant, a typical amount of nonionic surfactant may be 0.1% to 20%, preferably 0.5% to 15%, more preferably 0.5% to 10% by weight of the liquid detergent composition. When present as a primary surfactant, a typical amount may be 0.1% to 45%, preferably 15% to 40%, more preferably 20% to 35% by weight of the total composition. Suitable nonionic surfactants include condensation products of fatty alcohols with 1 to 25 moles of ethylene oxide. The alkyl chain of the fatty alcohol may be straight or branched, primary or secondary, and typically contains 8 to 22 carbon atoms. Particularly preferred are condensation products of alcohols having alkyl groups containing 10 to 18 carbon atoms (preferably 10 to 15 carbon atoms) with 2 to 18 moles (preferably 2 to 15 moles, more preferably 5 to 12 moles) of ethylene oxide per mole of alcohol.

[0147] Also suitable are alkyl polyglycosides, alkyl glyceryl ethers and sorbitan esters, and fatty acid amide surfactants, as described, for example, in WO 2012 / 015852.

[0148] Cationic surfactants

[0149] When present in the composition, the cationic surfactant is present in an effective amount, for example, from 0.1% to 20% by weight of the liquid detergent composition. Suitable cationic surfactants are quaternary ammonium surfactants. Suitable quaternary ammonium surfactants are selected from mono-C6-C 16 , preferably C6-C 10 The group consisting of N-alkyl or alkenyl ammonium surfactants, wherein the remaining N positions are substituted by methyl, hydroxyethyl or hydroxypropyl groups. Another preferred cationic surfactant is a C6-C 18 Alkyl or alkenyl esters, such as quaternary chloroesters.

[0150] Cationic polymers

[0151] In one embodiment, the liquid hand dishwashing composition herein may include at least one cationic polymer to further enhance the skin benefits. The cationic polymer may be present in an amount of 0.001% to 10%, preferably 0.01% to 5%, more preferably 0.05% to 1% by weight of the total composition. Suitable cationic polymers contain cationic nitrogen-containing moieties (such as quaternary ammonium) or cationic protonated amino moieties and may have an average molecular weight of from about 5000 to about 10 million, preferably at least about 100,000, more preferably at least about 200,000, but preferably no more than about 3,000,000. For further information on the use of cationic polymers in liquid dishwashing compositions, see, for example, WO 2012 / 015852.

[0152] Wetting Agents

[0153] The liquid dishwashing composition may further comprise one or more wetting agents to provide additional hand skin mildness benefits. Alternatively, the composition may be free of wetting agents.

[0154] When present, the wetting agent may be used in an amount of 0.1% to 50%, preferably 1% to 20%, more preferably 1% to 10%, even more preferably 1% to 6%, and most preferably 2% to 5% by weight of the total composition.

[0155] Suitable wetting agents include those materials that show affinity to water and help enhance the absorption of water on the substrate (preferably skin). Specific non-limiting examples of particularly suitable wetting agents include glycerol, diglycerol, polyethylene glycol (PEG-4), propylene glycol, hexylene glycol, butylene glycol, (di) propylene glycol, triacetin, polyalkylene glycol, and mixtures thereof. Others may be polyethylene glycol ethers of methyl glucose, pyrrolidone carboxylic acid (PCA) and its salts, pyridoxal and salts (such as sodium pidolate), polyols (like sorbitol, xylitol and maltitol), or polymeric polyols (like polydextrose) or natural extracts (like soap bark (quillaia), or lactic acid or urea). Also included are alkyl polysaccharides, polybetaine polysiloxanes, and mixtures thereof. Other suitable wetting agents are polymer wetting agents of water-soluble and / or swellable polysaccharide families, such as hyaluronic acid, chitosan and / or fructose-rich polysaccharides, such as provided by SOLABIA S (CAS-No.178463-23-5).

[0156] Wetting agents containing oxygen atoms are preferred over wetting agents containing nitrogen or sulfur atoms. More preferred wetting agents are polyols or carboxyl groups, such as glycerol, diglycerol, sorbitol, propylene glycol, polyethylene glycol, butylene glycol; and / or pidolic acid and its salts, and most preferred are wetting agents selected from the group consisting of glycerol, sorbitol, sodium lactate and urea, or mixtures thereof.

[0157] Cleaning polymers

[0158] The liquid hand dishwashing composition herein may optionally further comprise an alkoxylated polyethyleneimine polymer. The composition may comprise from 0.01% to 10%, preferably from 0.01% to 2%, more preferably from 0.1% to 1.5%, even more preferably from 0.2% to 1.5% of an alkoxylated polyethyleneimine polymer, such as described in WO2007 / 135645, by weight of the total composition.

[0159] The alkoxylated polyethyleneimine polymer may have a polyethyleneimine backbone having a weight average molecular weight of 400 to 10,000, preferably 400 to 7,000, alternatively 3,000 to 7,000.

[0160] Alkoxylation of the polyethyleneimine backbone includes: (1) one or two alkoxylation modifications per nitrogen atom (depending on whether the modification occurs at an internal nitrogen atom or a terminal nitrogen atom of the polyethyleneimine backbone), the alkoxylation modification consisting of replacement of hydrogen atoms with a polyalkylene chain having an average of about 1 to about 40 alkoxy moieties per modification, wherein the terminal alkoxy moiety of the alkoxylation modification is terminated with hydrogen, C1-C4 alkyl, or a mixture thereof; (2) substitution of one C1-C4 alkyl moiety or benzyl moiety and one or two alkoxylation modifications per nitrogen atom (depending on whether the substitution occurs at an internal nitrogen atom or a terminal nitrogen atom of the polyethyleneimine backbone), the alkoxylation modification consisting of replacement of hydrogen atoms with a polyalkyleneoxy chain having an average of about 1 to about 40 alkoxy moieties per modification, wherein the terminal alkoxy moiety is terminated with hydrogen, C1-C4 alkyl, or a mixture thereof; or (3) a combination thereof.

[0161] The composition may further comprise an amphiphilic graft polymer based on a water-soluble polyalkylene oxide (A) as graft base and side chains formed by polymerization of a vinyl ester component (B), said polymer having an average of not more than 1 grafting site per 50 alkylene oxide units and an average molar mass Mw of 3,000 to 100,000, as described in WO 2007 / 138053.

[0162] Magnesium ion

[0163] The optional presence of magnesium ions may be used in detergent compositions when the compositions are used in softened water containing few divalent ions. When used, magnesium ions are preferably added to the compositions of the present invention as hydroxide, chloride, acetate, sulfate, formates, oxides or nitrates. When included, magnesium ions are present at active levels of 0.01% to 1.5%, preferably 0.015% to 1%, more preferably 0.025% to 0.5% by weight of the liquid detergent composition.

[0164] Solvents

[0165] The present composition may optionally contain a solvent. Suitable solvents include C 4-14 Ethers and diethers, glycols, alkoxylated glycols, C6-C 16 Glycol ethers, alkoxylated aromatic alcohols, aromatic alcohols, aliphatic branched alcohols, alkoxylated aliphatic branched alcohols, alkoxylated linear C1-C5 alcohols, linear C1-C5 alcohols, amines, C8-C 14 Alkyl and cycloalkyl hydrocarbons and halogenated hydrocarbons, and mixtures thereof. When present, the liquid detergent composition will contain from 0.01% to 20%, preferably from 0.5% to 20%, more preferably from 1% to 10% of the solvent by weight of the liquid detergent composition. These solvents may be used in combination with an aqueous liquid carrier (such as water), or they may be used in the absence of any aqueous liquid carrier.

[0166] Water-soluble additives

[0167] The liquid detergent compositions of the present invention may optionally contain an effective amount of a hydrotrope to render the liquid detergent composition suitably compatible in water. Suitable hydrotropes for use herein include anionic hydrotropes, particularly sodium, potassium and ammonium xylene sulfonate, sodium, potassium and ammonium toluene sulfonate, sodium, potassium and ammonium cumene sulfonate, and mixtures thereof, and related compounds. The liquid detergent compositions of the present invention may contain from 0% to 15% of a hydrotrope, or mixtures thereof, by weight of the total liquid detergent composition, preferably from 1% to 10%, most preferably from 3% to 10%, by weight of the total liquid hand dishwashing composition.

[0168] Polymer foam stabilizers

[0169] The compositions of the present invention may optionally contain polymeric foam stabilizers. These polymeric foam stabilizers provide increased foam volume and foam duration to liquid detergent compositions. These polymeric foam stabilizers may be selected from homopolymers of (N,N-dialkylamino)alkyl esters and (N,N-dialkylamino)alkyl acrylates.

[0170] The weight average molecular weight of the polymer foam booster determined via conventional gel permeation chromatography is 1000 to 2000000, preferably 5000 to 1000000, more preferably 10000 to 750000, more preferably 20000 to 500000, even more preferably 35000 to 200000. The polymer foam stabilizer may optionally be present in the form of a salt (inorganic or organic).

[0171] A preferred polymeric foam stabilizer is (N,N-dimethylamino)alkyl acrylate.Other preferred foam boosting polymers are copolymers of hydroxypropyl acrylate / dimethylaminoethyl methacrylate (copolymers of HPA / DMAM).

[0172] When present in the composition, the polymeric suds boosters / stabilisers may be present at from 0.01% to 15%, preferably from 0.05% to 10%, more preferably from 0.1% to 5% by weight of the liquid detergent composition.

[0173] Another preferred class of polymeric foam booster polymers is a hydrophobically modified cellulose polymer having a number average molecular weight (Mw) of less than 45,000, preferably between 10,000 and 40,000, more preferably between 13,000 and 25,000. The hydrophobically modified cellulose polymer includes water-soluble cellulose ether derivatives, such as nonionic cellulose derivatives and cationic cellulose derivatives. Preferred cellulose derivatives include methylcellulose, hydroxypropylmethylcellulose, hydroxyethylmethylcellulose, and mixtures thereof.

[0174] Diamine

[0175] Another optional ingredient of the compositions of the present invention is a diamine. Since liquid detergent compositions exhibit considerable variation, the composition may contain from 0% to 15%, preferably from 0.1% to 15%, preferably from 0.2% to 10%, more preferably from 0.25% to 6%, more preferably from 0.5% to 1.5% by weight of the composition of at least one diamine.

[0176] Preferred organic diamines are those in which pK1 and pK2 are in the range of 8.0 to 11.5, preferably in the range of 8.4 to 11, even more preferably in the range of 8.6 to 10.75. Preferred materials include 1,3-bis(methylamino)-cyclohexane (pKa=10 to 10.5), 1,3-propylenediamine (pK1=10.5; pK2=8.8), 1,6-hexanediamine (pK1=11; pK2=10), 1,3-pentane ... (pK1 = 10.5; pK2 = 8.9), 2-methyl-1,5-pentanediamine (pK1 = 11.2; pK2 = 10.0) Other preferred materials include primary / primary diamines with alkylene spacers ranging from C4 to C5.

[0177] carboxylic acid

[0178] The liquid detergent composition of the present invention may contain a linear or cyclic carboxylic acid or its salt to improve the rinse feel of the composition. The presence of anionic surfactants (especially when present in higher amounts ranging from 15% to 35% by weight of the composition) results in the composition imparting a slippery feel to the user's hands and dishes. This slippery feel is reduced when a carboxylic acid as defined herein is used.

[0179] Carboxylic acids useful herein include C 1-6 A straight chain acid or a cyclic acid containing at least 3 carbons. The straight or cyclic carbon-containing chain of the carboxylic acid or its salt may be substituted with a substituent selected from the group consisting of hydroxyl groups, esters, ethers, aliphatic groups having 1 to 6 carbon atoms, more preferably 1 to 4 carbon atoms, and mixtures thereof.

[0180] Preferred carboxylic acids are those selected from the group consisting of salicylic acid, maleic acid, acetylsalicylic acid, 3-methylsalicylic acid, 4-hydroxyisophthalic acid, dihydroxyfumaric acid, 1,2,4-benzenetricarboxylic acid, valeric acid and salts thereof and mixtures thereof. When the carboxylic acid is in salt form, the cation of the salt is preferably selected from alkali metals, alkaline earth metals, monoethanolamine, diethanolamine or triethanolamine and mixtures thereof.

[0181] When present, the carboxylic acid or salt thereof is preferably present at a level of from 0.1% to 5%, more preferably from 0.2% to 1% and most preferably from 0.25% to 0.5% by weight of the total composition.

[0182] Other components

[0183] The liquid detergent compositions herein may further comprise various other optional ingredients suitable for use in liquid detergent compositions such as perfumes, dyes, sunscreens, other enzymes, chelating agents, pH buffers and rheology modifiers, including those of the polyacrylate, polysaccharide or polysaccharide derivative type and / or combinations of solvents with polycarboxylate polymers.

[0184] thickness

[0185] The liquid hand dishwashing compositions herein are typically thickened and preferably at 20 s-1and 20° C. having a viscosity of 50 to 5000 centipoise (50-5000 mPa*s), more preferably 100 to 4000 centipoise (100-4000 mPa*s), even more preferably 500-3500 centipoise (500-3500 mPa*s), and most preferably 800 to 3000 centipoise (800-3000 mPa*s). The viscosity can be determined by conventional methods known in the art, for example, using an AR 550 rheometer from TA Instruments using a steel plate spindle with a diameter of 40 mm and a gap size of 500 μm. s-1 High shear viscosity and 0.05 s-1 The low shear viscosity can be increased from 0.1 to s-1 To 25 s-1 The preferred rheology can be achieved using internal existing detergent ingredient structuring or by using external rheology modifiers and / or crystalline structurants, which provide the composition with pseudoplastic or shear thinning rheology and time-dependent recovery of viscosity after shear (thixotropy).

[0186] Crystal structure agent

[0187] The composition of the present invention may further comprise one or more crystalline structurants, which are materials that form a linear structural system and / or an insoluble particle network in the matrix of the entire composition. The crystalline structurant may be crystallized in situ in the aqueous liquid matrix of the composition or in a premix for forming such an aqueous liquid matrix. It has been found that the network created by the crystalline wax structurant prevents the hydrophobic emollient droplets from coalescing and phase separation in the product, thereby providing excellent hand dishwashing liquid composition stability.

[0188] When present, the crystalline structurant is typically included at a level of 0.02% to 5%, preferably 0.025% to 3%, more preferably 0.05% to 2%, most preferably 0.1% to 1.5% by weight of the total composition. Preferred crystalline structurants are: hydroxyl-containing crystalline structurants, such as hydroxyl-containing fatty acids, fatty esters or fatty soap waxy materials, etc., such as those described in US 6,080,707.

[0189] Other suitable crystal structurants include C 10-22 Ethylene glycol fatty acid ester. 10-22Ethylene glycol fatty acid esters can be used alone or in combination with another crystalline structurant such as hydrogenated castor oil. Typical examples are monoesters and / or diesters of ethylene glycol, propylene glycol, diethylene glycol, dipropylene glycol, triethylene glycol or tetraethylene glycol with fatty acids containing from about 6 to about 22 carbon atoms (preferably from about 12 to about 18 carbon atoms), such as caproic acid, caprylic acid, 2-ethylhexanoic acid, capric acid, lauric acid, isotridecanoic acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, isostearic acid, oleic acid, elaidic acid, petroleic acid, linoleic acid, linolenic acid, arachidic acid, olefinic acid, behenic acid, erucic acid, and mixtures thereof.

[0190] For further information on suitable crystal structurants, see WO 2012 / 015852.

[0191] Additional enzymes

[0192] The detergent composition may comprise one or more additional enzymes, such as an amylase, an arabinase, a carbohydrase, a cellulase (e.g., an endoglucanase), a cutinase, a deoxyribonuclease, a galactanase, a haloperoxidase, a lipase, a mannanase, an oxidase (e.g., a laccase and / or a peroxidase), a pectinase, a pectin lyase, an additional protease, a xylanase, a xanthanase, a xyloglucanase, or an oxidoreductase.

[0193] When the composition comprises one or more additional enzymes, the additional enzyme is preferably an amylase and / or a lipase, in particular an amylase.

[0194] The properties of the enzyme or enzymes selected should be compatible with the selected detergent (eg, pH optimum, compatibility with other enzyme or non-enzyme ingredients, etc.).

[0195] Protease

[0196] In addition to the proteases disclosed herein, the composition may include one or more additional proteases, including those of bacteria, fungi, plants, viruses, or animal origin. Proteases of microbial origin are preferred. Proteases may be alkaline proteases, such as serine proteases or metalloproteases. Serine proteases may be, for example, serine proteases of the S1 family (such as trypsin) or the S8 family (such as subtilisin). Metalloproteases may be, for example, thermolysin or another metalloprotease from, for example, the M4 family, such as those metalloproteases from the M5, M7, or M8 families.

[0197] Examples of metalloproteases are neutral metalloproteases as described in WO 2007 / 044993 (Genencor Int.), such as those derived from Bacillus amyloliquefaciens.

[0198] Suitable commercially available proteases include those sold under the following trade names: Duralase Tm 、Durazym Tm , Ultra, Ultra, Ultra, Ultra, Blaze 100T, Blaze 125T, Blaze 150T, Uno, and Excel (Novozymes A / S), those proteases sold under the following trade names: Ox, OxP, Purafect Excellenz P1000 TM 、Excellenz P1250 TM , P100, P110, Effectenz P1000 TM 、Effectenz P1050 TM 、Effectenz P2000 TM , and (Danisco / DuPont), Axapem TM (Gist-Brocases NV), BLAP (sequence shown in Figure 29 of US 5352604) and its variants (Henkel AG), and KAP (Bacillus alkalophilus subtilisin) from Kao Corporation.

[0199] Lipase and cutinase

[0200] Suitable lipases and cutinases include those of bacterial or fungal origin. Chemically modified mutant enzymes or protein engineered mutant enzymes are included. Examples include lipases from Thermomyces, e.g., from T. lanuginosus (formerly named Humicola lanuginosa) as described in EP 258068 and EP 305216; cutinases from Humicola, e.g., H. insolens (WO 96 / 13580); lipases from strains of Pseudomonas (some of these are now renamed Burkholderia), e.g., P. alcaligenes or P. pseudoalcaligenes (EP 218272), P. cepacia (EP 331376), Pseudomonas species strain SD705 (WO 96 / 13580); 95 / 06720 and WO 96 / 27002), P. wisconsinensis (WO 96 / 12012); GDSL-type Streptomyces lipase (WO 2010 / 065455); cutinase from Magnaporthe grisea (WO 2010 / 107560); cutinase from Pseudomonas mendocina (US 5,389,536); lipase from Thermobifida fusca (WO 2011 / 084412); Geobacillus stearothermophilus lipase (WO 2011 / 084417); lipase from Bacillus subtilis (WO 2011 / 084417); subtilis (WO 2011 / 084599); and lipases from Streptomyces griseus (WO 2011 / 150157) and S. pristinae spiralis (WO 2012 / 137147).

[0201] Other examples are lipase variants such as those described in EP 407225, WO 92 / 05249, WO 94 / 01541, WO 94 / 25578, WO 95 / 14783, WO 95 / 30744, WO 95 / 35381, WO 95 / 22615, WO 96 / 00292, WO 97 / 04079, WO 97 / 07202, WO 00 / 34450, WO 00 / 60063, WO 01 / 92502, WO 2007 / 87508 and WO 2009 / 109500.

[0202] Preferred commercial lipase products include Lipolase TM 、Lipex TM 、Lipolex TM and Lipoclean TM (Novozymes A / S), Lumafast (from Genencor), and Lipomax (from Gist-Brocades).

[0203] Still other examples are lipases sometimes referred to as acyltransferases or perhydrolases, e.g., acyltransferases with homology to Candida antarctica lipase A (WO 2010 / 111143), acyltransferases from Mycobacterium smegmatis (WO 2005 / 056782), perhydrolases from the CE 7 family (WO 2009 / 067279), and variants of the Mycobacterium smegmatis perhydrolase, in particular the S54V variant used in the commercial product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd (WO 2010 / 100028).

[0204] Amylase

[0205] Suitable amylases that can be used with the protease may be alpha-amylases or glucoamylases and may be of bacterial or fungal origin. Chemically modified mutants or protein engineered mutants are included. Amylases include, for example, alpha-amylases obtained from Bacillus, such as a special strain of Bacillus licheniformis (described in more detail in GB 1,296,839).

[0206] Suitable amylases include those having SEQ ID NO: 2 in WO 95 / 10603 or variants thereof having 90% sequence identity to SEQ ID NO: 3. Preferred variants are described in WO 94 / 02597, WO 94 / 18314, WO 97 / 43424 and in SEQ ID NO: 4 of WO 99 / 19467, such as variants having substitutions at one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408 and 444.

[0207] Various suitable amylases include those having SEQ ID NO: 6 in WO 02 / 10355 or variants thereof having 90% sequence identity to SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are those having deletions in positions 181 and 182 and a substitution in position 193.

[0208] Other suitable amylases are hybrid alpha-amylases comprising residues 1-33 of an alpha-amylase derived from Bacillus amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006 / 066594 and residues 36-483 of a Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 4 of WO 2006 / 066594, or variants thereof having 90% sequence identity. Preferred variants of this hybrid alpha-amylase are those having a substitution, deletion or insertion in one or more of the following positions: G48, T49, G107, H156, A181, N190, M197, 1201, A209 and Q264. Most preferred variants of the hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase derived from Bacillus amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006 / 066594 and residues 36-483 of SEQ ID NO: 4 are those having the following substitutions:

[0209] M197T;

[0210] H156Y+A181T+N190F+A209V+Q264S; or

[0211] G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.

[0212] Other suitable amylases are amylases having the sequence of SEQ ID NO:6 in WO 99 / 19467 or variants thereof having 90% sequence identity with SEQ ID NO:6. Preferred variants of SEQ ID NO:6 are those having substitutions, deletions or insertions in one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216 and K269. Particularly preferred amylases are those having deletions in positions R181 and G182, or positions H183 and G184.

[0213] Additional amylases that can be used are those having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 7 of WO 96 / 23873, or variants thereof having 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 7. Preferred variants of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 7 are those having substitutions, deletions or insertions in one or more of the following positions: 140, 181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304, and 476 (using SEQ ID 2 of WO 96 / 23873 for numbering). More preferred variants are those with deletions in two positions selected from 181, 182, 183 and 184, such as 181 and 182, 182 and 183, or positions 183 and 184. The most preferred amylase variants of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 7 are those with deletions in positions 183 and 184 and substitutions in one or more of positions 140, 195, 206, 243, 260, 304 and 476.

[0214] Other amylases that can be used are those having SEQ ID NO: 2 of WO 2008 / 153815, SEQ ID NO: 10 in WO 01 / 66712, or variants thereof having 90% sequence identity to SEQ ID NO: 2 of WO 2008 / 153815, or variants thereof having 90% sequence identity to SEQ ID NO: 10 in WO 01 / 66712. Preferred variants of SEQ ID NO: 10 in WO 01 / 66712 are those having substitutions, deletions or insertions in one or more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211 and 264.

[0215] Further suitable amylases are those having SEQ ID NO: 2 of WO 2009 / 061380 or variants thereof having 90% sequence identity to SEQ ID NO: 2. Preferred variants of SEQ ID NO: 2 are those having C-terminal truncations and / or substitutions, deletions or insertions in one or more of the following positions: Q87, Q98, S125, N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444, and G475. More preferred variants of SEQ ID NO: 2 are those with substitutions in one or more of the following positions: Q87E, R, Q98R, S125A, N128C, T131I, T165I, K178L, T182G, M201L, F202Y, N225E, R, N272E, R, S243Q, A, E, D, Y305R, R309A, Q320R, Q359E, K444E, and G475K, and / or those with deletions in positions R180 and / or S181 or T182 and / or G183. The most preferred amylase variants of SEQ ID NO: 2 are those with the following substitutions:

[0216] N128C+K178L+T182G+Y305R+G475K;

[0217] N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;

[0218] S125A+N128C+K178L+T182G+Y305R+G475K; or

[0219] S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K,

[0220] wherein the variant is C-terminally truncated and optionally further comprises a substitution at position 243 and / or a deletion at position 180 and / or position 181.

[0221] Further suitable amylases are those having SEQ ID NO: 1 of WO 2013 / 184577 or variants thereof having 90% sequence identity to SEQ ID NO: 1. Preferred variants of SEQ ID NO: 1 are those having substitutions, deletions or insertions in one or more of the following positions: K176, R178, G179, T180, G181, E187, N192, M199, 1203, S241, R458, T459, D460, G476 and G477. More preferred variants of SEQ ID NO: 1 are those having substitutions in one or more of the following positions: K176L, E187P, N192FYH, M199L, I203YF, S241QADN, R458N, T459S, D460T, G476K and G477K, and / or those having deletions in positions R178 and / or S179 or T180 and / or G181. The most preferred amylase variants of SEQ ID NO: 1 comprise the following substitutions:

[0222] E187P+I203Y+G476K

[0223] E187P+I203Y+R458N+T459S+D460T+G476K

[0224] and optionally further comprises a substitution at position 241 and / or a deletion at position 178 and / or position 179.

[0225] Further suitable amylases are those having SEQ ID NO: 1 of WO 2010 / 104675 or variants thereof having 90% sequence identity to SEQ ID NO: 1. Preferred variants of SEQ ID NO: 1 are those having substitutions, deletions or insertions in one or more of the following positions: N21, D97, V128, K177, R179, S180, I181, G182, M200, L204, E242, G477 and G478.

[0226] More preferred variants of SEQ ID NO: 1 are those having substitutions in one or more of the following positions: N21D, D97N, V128I, K177L, M200L, L204YF, E242QA, G477K and G478K, and / or those having deletions in positions R179 and / or S180 or I181 and / or G182. The most preferred amylase variants of SEQ ID NO: 1 comprise substitutions N21D+D97N+V128I, and optionally further comprise substitutions at position 200 and / or deletions at position 180 and / or position 181.

[0227] Other suitable amylases are alpha-amylases having SEQ ID NO: 12 in WO 01 / 66712 or variants having at least 90% sequence identity to SEQ ID NO: 12. Preferred amylase variants are those having substitutions, deletions or insertions in one or more of the following positions of SEQ ID NO: 12 in WO 01 / 66712: R28, R118, N174; R181, G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484. Particularly preferred amylases include variants having a deletion of D183 and G184 and having substitutions R118K, N195F, R320K and R458K, and variants additionally having substitutions at one or more positions selected from the group consisting of: M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345, and A339, most preferably variants additionally having substitutions in all of these positions.

[0228] Other examples are amylase variants such as those described in WO 2011 / 098531, WO 2013 / 001078 and WO 2013 / 001087. Commercially available amylases include Duramyl TM Termamyl TM 、Fungamyl TM 、Stainzyme TM 、Stainzyme Plus TM 、Natalase TM 、Liquozyme X、BAN TM , and Prime (from Novozymes A / S), and Rapidase TM 、Purastar TM / Effect TM , Powerase, Preferenz S1000, Preferenz S100 and Preferenz S110 (from Genencor International Inc. / DuPont.

[0229] A preferred amylase is a variant of the amylase having SEQ ID NO: 13 in WO 2016 / 180748 having changes H1*+N54S+V56T+K72R+G109A+F113Q+R116Q+W167F+Q172G+A174S+G182*+D183*+G184T+N195F+V206L+K391A+P473R+G476K.

[0230] Another preferred amylase is a variant of the amylase having SEQ ID NO: 1 in WO 2013 / 001078 having changes D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+G304R+G476K.

[0231] Another preferred amylase is a variant of an amylase having SEQ ID NO: 1 in WO 2018 / 141707 having changes H1*+G7A+G109A+W140Y+G182*+D183*+N195F+V206Y+Y243F+E260G+N280S+G304R+E391A+G476K.

[0232] Further preferred amylases are variants of the amylase having SEQ ID NO: 1 in WO 2017 / 191160 having the changes L202M+T246V.

[0233] Deoxyribonuclease (DNase)

[0234] Suitable deoxyribonuclease (DNA enzyme) is any enzyme that catalyzes the hydrolysis and cleavage of the phosphodiester bond in the DNA backbone to degrade DNA. DNA enzymes available from bacteria are preferred, and in particular, DNA enzymes available from Bacillus species are preferred; in particular, DNA enzymes available from Bacillus subtilis or Bacillus licheniformis are preferred. Examples of such DNA enzymes are described in WO 2011 / 098579 and WO 2014 / 087011.

[0235] Oxidoreductase

[0236] In one embodiment, the composition may comprise an oxidoreductase, which is an enzyme that catalyzes a reduction-oxidation reaction. A preferred oxidoreductase is superoxide dismutase.

[0237] Usage and cleaning methods

[0238] The present invention also relates to the use of the liquid detergent composition for cleaning, in particular for hard surface cleaning such as dishwashing, in particular for hand dishwashing.

[0239] This aspect further relates to a cleaning method, especially a method for cleaning hard surfaces or for dishwashing, which comprises contacting the hard surface or dishware with a liquid detergent composition under conditions suitable for cleaning the surface or dishware. In a specific embodiment, the cleaning method is for hand washing dishware and comprises contacting the dishware with a detergent composition comprising a protease as described herein under conditions suitable for cleaning the dishware.

[0240] In one embodiment, the present invention relates to the use of a protease for the preparation of a liquid detergent composition suitable for hand dishwashing, wherein the composition provides hand skin care benefits, wherein the protease is a variant of the polypeptide of SEQ ID NO: 1 comprising two or more substitutions selected from the group consisting of S9E, N43R, N76D, Q206L, Y209W, S259D and L262E, wherein the variant has protease activity and has at least 80%, at least 85%, at least 90% or at least 95% but less than 100% sequence identity to SEQ ID NO: 1, and wherein the position numbering is based on the numbering of SEQ ID NO: 2. In this embodiment, the protease can be any of those described in more detail above.

[0241] In one embodiment, the present invention relates to the use of a protease as disclosed herein for the preparation of a liquid detergent composition suitable for hand dishwashing, wherein the composition provides a hand skin care benefit, and wherein the use of the composition in hand dishwashing provides at least one hand skin care benefit selected from keratinocyte removal, reduction of transepidermal water loss, improvement of hand skin barrier, and improvement of hand skin water retention.

[0242] It should be understood that the protease used in these uses and cleaning methods can be any of the proteases described in more detail elsewhere herein.

[0243] The present invention is further described by the following examples, which should not be construed as limiting the scope of the present invention.

[0244] Examples

[0245] Example 1 - Skin Care Benefits of Compositions of the Invention

[0246] A study was conducted to evaluate the skin care benefits of compositions of the present invention comprising a protease.

[0247] These studies were: 1) a randomized, single-blind, single-use, within-group and between-group controlled evaluation of the skin care benefits of the test product on skin texture (dead cell removal and improvement in skin smoothness); 2) a randomized, single-blind, single-use, between-group controlled evaluation of the hand skin moisturizing benefits obtained using the test product.

[0248] These studies compared a commercial hand dishwashing detergent (Fairy hand dishwashing detergent) alone (no enzyme) or with 0.2% by weight of a protease, wherein the protease is a variant of SEQ ID NO: 1 having substitutions S9E, N43R, N76D, V205I, Q206L, Y209W, S259D, N261W and L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2. The protease was obtained from Novozymes A / S, Denmark, and added to the commercial detergent in liquid form.

[0249] Testing Procedure

[0250] Thirty subjects were recruited and completed the study in Shanghai, China. All subjects were provided with a thorough explanation of the study in accordance with local laws and regulations, and all subjects gave verbal and written informed consent. Participation was voluntary, and subjects were free to leave the study at any time. All 30 participants completed the study.

[0251] The recruited subjects were not allowed to use any cosmetics, topical medications, or other hand care products for at least three days before the test. The subjects were not allowed to use hand cream products on the morning of the test or wash their hands one hour before the test.

[0252] To establish a baseline, subjects were asked to sit in a defined environment for 30 minutes without any beverages allowed. The forearms were then exposed and placed under the test conditions, remaining relaxed. Two test areas were selected and marked on the back of each hand. The texture of the dorsal hand was evaluated using the VC98 instrument (Courage & Khazaka, Germany). The water loss rate of hand skin was measured by the instrument (Courage & Khazaka, Germany).

[0253] Product Application: Two researchers helped each subject soak and wash their hands with the product at the same time. Hands were treated with different products (with and without protease). Subjects' hands were soaked in the product solution for 5 minutes, and then staff applied 10g of product foam (squeezed from the foaming bottle) with a sponge to wash the subject's hands for 15 minutes (20 times / minute). Afterwards, the subject washed his hands with clean water (temperature 30℃-35℃) and gently removed excess water from his hands with another dry sponge. Then use The instrument measures the water loss value for 180 seconds to calculate the water retention time immediately after drying.

[0254] The subjects' hands were kept dry at room temperature for 60 minutes and then used The instrument evaluates the hand surface texture and smoothness and uses The instrument tested the hydration rate for water loss. After the instrumental evaluation, the subjects were asked to fill in a self-assessment questionnaire.

[0255] Measured the following parameters:

[0256] (1) Skin texture: Before and after product use (60 minutes later), use The instrument tested two areas on the back of the hand. The average of the two areas of each hand was calculated. Keratinocyte parameters (keratinocyte area ratio) were analyzed using Image-Pro Plus software (Media Cybernetics, Inc.).

[0257] (2) Transepidermal water loss: Before and after application (after 60 minutes), two areas on the back of the hand were tested using VapoMeter (Delfin, Finland). The average of the two areas on each hand was calculated.

[0258] (3) Skin moisture retention time: Use The instrument was used for 180 seconds to determine the water retention time. The average of the two areas of each hand was calculated.

[0259] For each parameter, the delta value was determined for the test product hand (ie detergent + protease) and the control hand (ie detergent alone).

[0260] result

[0261] Corneocyte area ratio:

[0262] The delta value (unitless) for corneocyte area was -0.0757 for detergent with 0.2% protease compared to -0.0277 for detergent alone (P = 0.002).

[0263] A larger corneocyte area ratio means that there are more corneocytes on the skin. Therefore, a larger Δ value of the corneocyte area ratio indicates better corneocyte removal performance. Therefore, it has been demonstrated that the addition of proteases has a positive effect on corneocyte removal.

[0264] Transepidermal water loss (TEWL):

[0265] The Δ value of TEWL (g / m 2 h) was -7.5 (P<0.001).

[0266] The TEWL delta value for the control hand (detergent alone) was 3.8 g / m 2h, which indicates water loss due to poor skin barrier function after washing with detergent. In comparison, the TEWL delta value for the test product hands (detergent + protease) was -7.5 g / m 2 h, which represents an increase in moisture, suggesting that despite the damaging effects of detergents on the hand skin barrier, this can be converted into a significant barrier protection benefit by the addition of proteases.

[0267] Skin moisture retention time:

[0268] The delta value (seconds) for skin moisture retention time of the detergent with 0.2% protease was 120.2 compared to 83.6 for the detergent alone (P=0.021).

[0269] Longer skin moisture retention time indicates better hand skin moisturizing effect. Compared with the hand washed with detergent only, the skin moisture retention time of the other hand washed with detergent + protease was longer, indicating that the addition of 0.2% protease has a positive effect on hand skin moisturizing.

[0270] Questionnaire Results:

[0271] For each of the following questions, more participants chose the “product area” (hands washed with detergent + protease) compared to the “control area” (detergent alone):

[0272] After washing with this product, which hand is smoother than before?

[0273] After washing, which hand is moisturized and more hydrated than before?

[0274] After washing with this product, which hand is softer than before?

[0275] After washing with this product, which hand has better skin tone than before?

[0276] After washing with the product, which hand has less dead or cracked skin than before?

[0277] Which product made your skin feel softer and less irritated than before?

[0278] Which product exfoliates more naturally than before?

[0279] For the following questions, more participants chose the “control area” (detergent alone) compared to the “product area” (hands washed with detergent + protease):

[0280] Which product made your hand skin feel tighter after use?

[0281] in conclusion

[0282] Under the conditions of the study with 30 subjects, it was observed that:

[0283] The hand washed with detergent + protease had significantly fewer keratinocytes compared to the hand washed with detergent alone (P=0.002), indicating that the addition of 0.2% protease had a positive effect on keratinocyte removal.

[0284] The transepidermal water loss of hands washed with detergent + protease was significantly reduced compared with hands washed with detergent alone (P<0.001), indicating that the addition of 0.2% protease has a positive effect on the hand skin barrier.

[0285] Compared with the hand washed with detergent only, the water retention time of the skin of the other hand washed with detergent + protease was significantly prolonged (P=0.021), indicating that adding 0.2% protease can improve the water retention time of the hand skin.

[0286] Further, based on the responses to the questionnaire, it was found that more subjects experienced improved moisturizing and hydration after washing with a detergent having 0.2% protease compared to a hand washed with detergent alone. It was also found that more subjects indicated that the skin of the hand washed with a detergent having 0.2% protease felt less tight compared to the other hand washed with detergent alone.

[0287] Example 2 - Keratinocyte Removal by Compositions of the Invention

[0288] A study was conducted to evaluate the keratinocyte removal of the hand dishwashing compositions of the present invention.

[0289] Testing Procedure

[0290] A solution of commercial hand dishwashing detergent (Blue Moon Green Tea Hand Dishwashing Detergent; 10 g dishwashing detergent / liter tap water) was prepared in advance. Five subjects with visibly chapped hands were recruited for testing. One hand was treated with detergent alone, while the other hand was treated with a detergent containing one of five different proteases. The proteases used were as follows (for proteases 1-4, position numbering is based on that of SEQ ID NO:2):

[0291] 1. SEQ ID NO: 1 with substitutions S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E

[0292] 2. SEQ ID NO: 1

[0293] 3. SEQ ID NO: 1 with substitutions S9E+N43R+N76D+N185E+S188E+Q191N+A194P+Q206L+Y209W+S259D+L262E

[0294] 4. SEQ ID NO: 1 with change S99AD (i.e. substitution of S99A and insertion of D)

[0295] 5. SEQ ID NO:5

[0296] The protease concentration in the dishwashing liquid was 0.3% by weight.

[0297] Clear Scotch tape was used to obtain corneocytes from the left and right hands. The corneocytes were found to be well distributed on both hands. Four replicate tapes were used for each hand for testing.

[0298] The tape with keratinocytes was incubated with 500 ml detergent alone or detergent + 0.3% protease solution for 10 minutes at 40° C. The keratinocytes remaining on the tape were stained with 1 mg / mL toluidine blue O for 15 min at room temperature and then rinsed with tap water after staining.

[0299] Residual corneocytes were measured at R460 nm on the DigiEye system. The benefit of adding protease was expressed as the enhancement of corneocyte removal calculated as:

[0300] Corneocyte removal benefit (%) = [residual corneocytes (with protease) - residual corneocytes (detergent only)] / corneocytes before washing

[0301] Table 1: Keratinocyte removal by 0.3% protease in hand dishwashing detergent

[0302]

[0303] As can be seen from the table above, all the proteases tested provided keratinocyte removal benefits, with Protease 2 The benefits provided were smaller than those of the other proteases, whereas Protease 1 provided significantly better keratinocyte removal than the other proteases tested. Sequence Listing <110> Novozymes A / S <120> Liquid dishwashing detergent composition <130> 15018-WO-PCT[2] <160> 5 <170> PatentIn Version 3.5 <210> 1 <211> 269 <212> PRT <213> Bacillus lentus <400> 1 Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala 1 5 10 15 His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp 20 25 30 Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser 35 40 45 Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr 50 55 60 His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu 65 70 75 80 Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala 85 90 95 Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala 100 105 110 Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser 115 120 125 Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly 130 135 140 Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser 145 150 155 160 Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln 165 170 175 Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile 180 185 190 Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr 195 200 205 Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala 210 215 220 Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile 225 230 235 240 Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu 245 250 255 Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg 260 265 <210> 2 <211> 275 <212> PRT <213> Bacillus amyloliquefaciens <400> 2 Ala Gln Ser Val Pro Tyr Gly Val Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala 35 40 45 Ser Met Val Pro Ser Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His 50 55 60 Gly Thr His Val Ala Gly Thr Val Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly Val Ala Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Gly Ala Asp Gly Ser Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala Ile Ala Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro Ser Gly Ser Ala Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala 130 135 140 Ser Gly Val Val Val Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly 145 150 155 160 Ser Ser Ser Thr Val Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala 165 170 175 Val Gly Ala Val Asp Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Val 180 185 190 Gly Pro Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly Asn Lys Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Ser 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn 225 230 235 240 Trp Thr Asn Thr Gln Val Arg Ser Ser Leu Glu Asn Thr Thr Thr Lys 245 250 255 Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 <210> 3 <211> 311 <212> PRT <213> Bacillus species <400> 3 Ala Val Pro Ser Thr Gln Thr Pro Trp Gly Ile Lys Ser Ile Tyr Asn 1 5 10 15 Asp Gln Ser Ile Thr Lys Thr Thr Gly Gly Ser Gly Ile Lys Val Ala 20 25 30 Val Leu Asp Thr Gly Val Tyr Thr Ser His Leu Asp Leu Ala Gly Ser 35 40 45 Ala Glu Gln Cys Lys Asp Phe Thr Gln Ser Asn Pro Leu Val Asp Gly 50 55 60 Ser Cys Thr Asp Arg Gln Gly His Gly Thr His Val Ala Gly Thr Val 65 70 75 80 Leu Ala His Gly Gly Ser Asn Gly Gln Gly Val Tyr Gly Val Ala Pro 85 90 95 Gln Ala Lys Leu Trp Ala Tyr Lys Val Leu Gly Asp Asn Gly Ser Gly 100 105 110 Tyr Ser Asp Asp Ile Ala Ala Ala Ile Arg His Val Ala Asp Glu Ala 115 120 125 Ser Arg Thr Gly Ser Lys Val Val Ile Asn Met Ser Leu Gly Ser Ser 130 135 140 Ala Lys Asp Ser Leu Ile Ala Ser Ala Val Asp Tyr Ala Tyr Gly Lys 145 150 155 160 Gly Val Leu Ile Val Ala Ala Ala Gly Asn Ser Gly Ser Gly Ser Asn 165 170 175 Thr Ile Gly Phe Pro Gly Gly Leu Val Asn Ala Val Ala Val Ala Ala 180 185 190 Leu Glu Asn Val Gln Gln Asn Gly Thr Tyr Arg Val Ala Asp Phe Ser 195 200 205 Ser Arg Gly Asn Pro Ala Thr Ala Gly Asp Tyr Ile Ile Gln Glu Arg 210 215 220 Asp Ile Glu Val Ser Ala Pro Gly Ala Ser Val Glu Ser Thr Trp Tyr 225 230 235 240 Thr Gly Gly Tyr Asn Thr Ile Ser Gly Thr Ser Met Ala Thr Pro His 245 250 255 Val Ala Gly Leu Ala Ala Lys Ile Trp Ser Ala Asn Thr Ser Leu Ser 260 265 270 His Ser Gln Leu Arg Thr Glu Leu Gln Asn Arg Ala Lys Val Tyr Asp 275 280 285 Ile Lys Gly Gly Ile Gly Ala Gly Thr Gly Asp Asp Tyr Ala Ser Gly 290 295 300 Phe Gly Tyr Pro Arg Val Lys 305 310 <210> 4 <211> 274 <212> PRT <213> Artificial Sequence <220> <223> Bacillus licheniformis variant <400> 4 Ala Gln Thr Val Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val 1 5 10 15 Gln Ala Gln Gly Phe Lys Gly Ala Asn Val Lys Val Ala Val Leu Asp 20 25 30 Thr Gly Ile Gln Ala Ser His Pro Asp Leu Asn Val Val Gly Gly Ala 35 40 45 Ser Phe Val Ala Gly Glu Ala Tyr Asn Thr Asp Gly Asn Gly His Gly 50 55 60 Thr His Val Ser Gly Thr Val Ala Ala Leu Asp Asn Asn Ile Gly Val 65 70 75 80 Leu Gly Val Ala Pro Ser Val Ser Leu Tyr Ala Val Lys Val Leu Asn 85 90 95 Ser Ser Gly Ser Gly Ser Tyr Ser Gly Ile Val Ser Gly Ile Glu Trp 100 105 110 Ala Thr Thr Asn Gly Met Asp Val Ile Asn Met Ser Leu Gly Ser Pro 115 120 125 Ser Gly Ser Thr Ala Met Lys Gln Ala Val Asp Asn Ala Tyr Ala Arg 130 135 140 Gly Val Val Val Val Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly Asn 145 150 155 160 Thr Asn Thr Ile Gln Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val 165 170 175 Gly Ala Val Asp Ser Asn Ser Gln Arg Ala Ser Phe Ser Ser Val Gly 180 185 190 Ala Glu Leu Glu Val Met Ala Pro Gly Val Gly Val Tyr Ser Thr Tyr 195 200 205 Pro Thr Asn Thr Tyr Ala Thr Leu Ser Gly Thr Ser Met Ala Ser Pro 210 215 220 His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Leu 225 230 235 240 Ser Ala Ser Gln Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Tyr Leu 245 250 255 Gly Pro Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala 260 265 270 Ala Gln <210> 5 <211> 274 <212> PRT <213> Bacillus licheniformis <400> 5 Ala Gln Thr Val Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val 1 5 10 15 Gln Ala Gln Gly Phe Lys Gly Ala Asn Val Lys Val Ala Val Leu Asp 20 25 30 Thr Gly Ile Gln Ala Ser His Pro Asp Leu Asn Val Val Gly Gly Ala 35 40 45 Ser Phe Val Ala Gly Glu Ala Tyr Asn Thr Asp Gly Asn Gly His Gly 50 55 60 Thr His Val Ala Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val 65 70 75 80 Leu Gly Val Ala Pro Ser Val Ser Leu Tyr Ala Val Lys Val Leu Asn 85 90 95 Ser Ser Gly Ser Gly Ser Tyr Ser Gly Ile Val Ser Gly Ile Glu Trp 100 105 110 Ala Thr Thr Asn Gly Met Asp Val Ile Asn Met Ser Leu Gly Gly Ala 115 120 125 Ser Gly Ser Thr Ala Met Lys Gln Ala Val Asp Asn Ala Tyr Ala Arg 130 135 140 Gly Val Val Val Val Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly Asn 145 150 155 160 Thr Asn Thr Ile Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val 165 170 175 Gly Ala Val Asp Ser Asn Ser Asn Arg Ala Ser Phe Ser Ser Val Gly 180 185 190 Ala Glu Leu Glu Val Met Ala Pro Gly Ala Gly Val Tyr Ser Thr Tyr 195 200 205 Pro Thr Asn Thr Tyr Ala Thr Leu Asn Gly Thr Ser Met Ala Ser Pro 210 215 220 His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Leu 225 230 235 240 Ser Ala Ser Gln Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Tyr Leu 245 250 255 Gly Ser Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala 260 265 270 Ala Gln

Claims

1. A method for providing skin care benefits to a hand dishwashing detergent composition, the method comprising adding to the composition at least one protease, wherein the protease is a variant of the polypeptide of SEQ ID NO: 1 consisting of the following substitutions S9E, N43R, N76D, V205I, Q206L, Y209W, S259D, N261W and L262E, wherein the position numbering is based on the numbering of SEQ ID NO:

2.

2. The method of claim 1, wherein the composition comprises protease in an amount of 0.001% w / w to 4% w / w, wherein the percentages are enzyme protein by weight of the composition.

3. The method of claim 2, wherein the composition comprises protease in an amount of 0.01% w / w to 2% w / w, wherein the percentages are enzyme protein by weight of the composition.

4. The method of any one of claims 1 to 3, wherein the composition is a liquid detergent composition and has a pH value between 6 and 10.

5. The method of claim 4, wherein the pH of the composition is between 7 and 9.

6. The method of claim 1, wherein the skin care benefit is selected from the group consisting of keratinocyte removal, reduction in transepidermal water loss, improved hand skin barrier, and improved hand skin water retention.

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