WO2013188344A2 - Laundry soap bars - Google Patents

Laundry soap bars Download PDF

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Publication number
WO2013188344A2
WO2013188344A2 PCT/US2013/045091 US2013045091W WO2013188344A2 WO 2013188344 A2 WO2013188344 A2 WO 2013188344A2 US 2013045091 W US2013045091 W US 2013045091W WO 2013188344 A2 WO2013188344 A2 WO 2013188344A2
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WIPO (PCT)
Prior art keywords
ala
gly
phe
val
leu
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PCT/US2013/045091
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French (fr)
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WO2013188344A3 (en
Inventor
Nelson Eduardo Prieto
Lise Munch MIKKELSON
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Novozymes A/S
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Application filed by Novozymes A/S filed Critical Novozymes A/S
Priority to BR112014030846A priority Critical patent/BR112014030846A2/en
Priority to AP2014008122A priority patent/AP3789A/en
Priority to MX2014015186A priority patent/MX2014015186A/en
Priority to CN201380031099.5A priority patent/CN104583382B/en
Publication of WO2013188344A2 publication Critical patent/WO2013188344A2/en
Publication of WO2013188344A3 publication Critical patent/WO2013188344A3/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D17/00—Detergent materials or soaps characterised by their shape or physical properties
    • C11D17/0047—Detergents in the form of bars or tablets
    • C11D17/0065—Solid detergents containing builders
    • C11D17/0069—Laundry bars
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/20—Organic compounds containing oxygen
    • C11D3/2072—Aldehydes-ketones
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38609—Protease or amylase in solid compositions only
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38681—Chemically modified or immobilised enzymes

Definitions

  • the present invention relates to laundry soap bars with improved enzyme stability and to a process for preparing said laundry soap bars and to uses of the laundry soap bars.
  • Protease stability can also be improved by adding one or more stabilizers to the laundry soap bar.
  • boron containing compounds are used as stabilizers.
  • GB 2186883 de-scribes laundry soap bars with a water content of 10-33% and containing proteases in which the proteolytic enzyme is stabilized by a mixture of a boron compound, a polyol, an organic acid or its alkali metal salt, and an alkali metal salt of an inorganic acid which is not a boron compound.
  • WO 98/54285 describes high-moisture protease containing laundry soap bars with improved protease stability.
  • the improved stability is obtained by adding a stabilizing agent made of a bo- rate compound in conjunction with a polyol, a carboxylate salt, a carboxylic acid, or mixtures thereof.
  • a stabilizing agent made of a bo- rate compound in conjunction with a polyol, a carboxylate salt, a carboxylic acid, or mixtures thereof.
  • Various boron compounds are well known as stabilizers for subtilisins in liquid detergents, e.g. WO 96/41859.
  • WO 98/18897 describes a process for incorporating enzymes into laundry detergent bar compositions that minimizes the loss of enzyme stability during the manufacturing process of the bar. This is achieved by a process in which the enzyme prills are added after milling and cooling and then plodding the mixture.
  • a-MAPI, antipain, GE20372A and chymostatin A, B and C are described as peptide aldehydes with activity as protease inhibitors: R.J. Broadbridge and M. Akhtar, Chem. Commun., (1998), 1449-1450; E. Sarubbi, P.F. Seneci, M.R. Angelastro, N.P. Peet, M. Denaro, K. Islam, FEBS Letters, (1993), 319 (3), 253-256 and I.J. Galpin, A.H. Wilby, A.G. Place, R.J. Beynon, Int. J. Peptide Protein Res., (1984), 23, 477-486.
  • a laundry soap bar comprising:
  • protease inhibitors of which at least one is a peptide aldehyde, a hydrosulfite adduct or a hemiacetal adduct thereof;
  • the invention also relates to the use of laundry soap bars in cleaning laundry and for washing laundry by hand. Furthermore the invention relates to the preparation of laundry soap bars and the use of premixes in the preparation thereof.
  • alkyl in the present context represents a linear or branched hydrocarbon radical having 1 -6 carbon atoms. Representative examples include methyl, ethyl, n-propyl, iso- propyl, n-butyl, / ' so-butyl, sec-butyl, ie f-butyl, 1-ethylpropyl, 2-methylbutyl, pentyl, 3,3- dimethylbutyl, hexyl, etc.
  • Preferred "alkyl” groups are methyl, ethyl, n-propyl, / ' so-propyl, n-butyl, / ' so-butyl and sec-butyl.
  • aryl in the present context represents a carbocyclic aromatic ring having 6- 10 carbon atoms.
  • aryl also represents fused ring systems in which 2 neighbouring atoms are used to fuse the 2 aromatic rings together.
  • Representative examples of “aryl” rings are phenyl and naphthalenyl.
  • arylalkyl in the present context represents a carbocyclic aromatic ring having 6-10 carbon atoms attached to a alkyl group of 1 -6 carbon atoms.
  • Representative examples of “arylalkyl” rings are benzyl, phenylethyl and phenylpropyl.
  • halogen in the present context represents fluorine, chlorine, bromine and iodine.
  • amino acid covers amino acids with L- or D-configuration unless otherwise specified.
  • the invention relates to laundry soap bars for hand washing laundry.
  • laundry soap bar includes laundry bars, soap bars, combo bars, syndet bars and detergent bars.
  • the types of bar usually differ in the type of surfactant they contain, and the term laundry soap bar includes those containing soaps from fatty acids and/or synthetic soaps.
  • the laundry soap bar has a physical form which is solid and not a liquid, gel or a powder at room temperature.
  • solid is defined as a physical form which does not significantly change over time, i.e. if a solid object (e.g. laundry soap bar) is placed inside a container, the solid object does not change to fill the container it is placed in.
  • the bar is a solid typically in bar form but can be in other solid shapes such as round or oval.
  • Sequence identity The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity”.
  • the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 or later. Version 6.1.0 was used.
  • the optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
  • the output of Needle labelled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
  • substantially pure polypeptide means a preparation that contains at most 10%, at most 8%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1 %, and at most 0.5% by weight of other polypeptide material with which it is natively or recombi- nantly associated.
  • the polypeptide is at least 92% pure, e.g., at least 94% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99%, at least 99.5% pure, and 100% pure by weight of the total polypeptide material present in the preparation.
  • the polypeptides of the present invention are preferably in a substantially pure form. This can be accomplished, for example, by preparing the polypeptide by well-known recombinant methods or by classical purification methods.
  • variant means a polypeptide having protease activity comprising an altera- tion, i.e., a substitution, insertion, and/or deletion of one or more (several) amino acid residues at one or more (several) positions.
  • a substitution means a replacement of an amino acid occupying a position with a different amino acid;
  • a deletion means removal of an amino acid occupying a position; and
  • an insertion means adding 1-3 amino acids adjacent to an amino acid occupying a position.
  • One object of the present invention is to provide enzyme containing laundry soap bars with improved storage stability.
  • a further object of the present invention is to provide a process for manufacturing a solid formulation such as laundry soap bars wherein the enzyme can be added in the initial phase of the laundry soap bar manufacturing process and still maintain a significant enzyme activity after manufacturing.
  • a peptide aldehyde (or hydrosulfite adduct or hemiacetal adduct) protease inhibitor with the salt of a monovalent cation and an organic anion has a stabilizing effect in laundry soap bars comprising a protease, such as a subtilisin, and optionally one or more additional enzymes.
  • a laundry soap bar comprising:
  • protease inhibitors of which at least one is a peptide aldehyde, a hydrosulfite adduct or a hemiacetal adduct thereof;
  • An embodiment of the invention is the laundry soap bar which contains, for example, less than 0.1 % w/w, e.g. less than 0.05% w/w, e.g. less than 0.02% w/w, e.g. less than 0.01 % w/w, e.g. less than 0.001 % w/w or 0% w/w by weight of the total composition of the laundry soap bar of one or more boron containing compounds.
  • boron contain- ing compounds are boric acid, borate, borax, phenylboronic acid and phenylboronic acid derivatives such as 4-formylphenylboronic acid.
  • An objective of the invention is to provide a laundry soap bar which has good physical properties, such as wet cracking properties, washdown feel, foam height, bar hardness and/or mush factor.
  • An embodiment of the invention is to provide a laundry soap bar with a mush factor less than 0.30, preferably less than 0.27, more preferably less than 0.24, e.g. between 0.10 and 0.30, preferably between 0.15 and 0.25.
  • An additional embodiment of the invention is to provide a laundry soap bar with wet cracking properties of between 1 and 4, preferably between 1 and 3, more preferably between 1 and 2, when assessed 2 hours after the laundry soap bar has been immersed in water for 1 hour.
  • a further embodiment of the invention is to provide a laundry soap bar with foam height of between 10 and 20 cm, preferably between 10.5 and 17 cm, more preferably between 1 1 and 15 cm, both initially and after 5 minutes.
  • An embodiment of the invention is to provide a laundry soap bar with wet bar (washdown) feel between 0 and 2, preferably between 0 and 1 .
  • An additional embodiment of the invention is to provide a laundry soap bar with hardness of between 2500 and 6000 g, preferably between 2750 and 5500 g, more preferably between 3000 and 5000 g, even more preferably between 3250 and 5000 g, even more preferably between 3500 and 5000 g.
  • the protease inhibitor of the laundry soap bar is a peptide aldehyde of the formula P-(A) y -L-(B) x -B°-H or a hydrosulfite adduct or hemiacetal adduct thereof, wherein:
  • i. H is hydrogen
  • iii. x is 1 , 2 or 3 for (B) x , and B is independently a single amino acid connected to B° via the C-terminal of the B amino acid
  • v. y is 0, 1 or 2 for (A) y , and A is independently a single amino acid residue connected to L via the /V-terminal of the A amino acid, with the proviso that if L is absent then A is absent;
  • P is selected from the group consisting of hydrogen and an /V-terminal protection group, with the proviso that if L is absent then P is an /V-terminal protection group;
  • R is independently selected from the group consisting of C 1-6 alkyl, C 6- io aryl or C 7-10 arylalkyl optionally substituted with one or more, identical or different, sub- stituent's R';
  • R" is a C 1-6 alkyl group.
  • the protease inhibitor of the laundry soap bar is a hydrosulfite adduct of a peptide aldehyde is of the formula P-(A) y -L-(B) x -N(H)-CHR-CH(OH)- S0 3 M, wherein
  • i. M is hydrogen or an alkali metal
  • ii. x is 1 , 2 or 3 for (B) x , and B is independently a single amino acid connected to B° via the C-terminal of the B amino acid
  • iv. y is 0, 1 or 2 for (A) y , and A is independently a single amino acid residue connected to L via the /V-terminal of the A amino acid, with the proviso that if L is ab- sent then A is absent;
  • v. P is selected from the group consisting of hydrogen and an /V-terminal protection group, with the proviso that if L is absent then P is an /V-terminal protection group;
  • R" is a C 1-6 alkyl group.
  • M is Na or K.
  • R is a C 7-10 ar- ylalkyl substituted with -OH, preferably C 7 arylalkyl substituted with -OH.
  • B° is selected from the group consisting of D- or L-form of arginine (Arg), 3,4-dihydroxyphenylalanine, isoleucine (lie), leucine (Leu), methionine (Met), norleucine (NIe), norvaline (Nva), phenylalanine (Phe), m-tyrosine, p-tyrosine (Tyr) and valine (Val).
  • B is an amino acid with L-configuration.
  • B 1 may be selected from the group consisting of alanine (Ala), cysteine (Cys), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (NIe), norvaline (Nva), proline (Pro), serine (Ser), threonine (Thr) and valine (Val).
  • B 2 may be selected from the group consisting of alanine (Ala), arginine (Arg), capreo- mycidine (Cpd), cysteine (Cys), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (NIe), norvaline (Nva), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), and valine (Val).
  • B 3 may be selected from the group consisting of isoleucine (lie), leucine (Leu), norleu- cine (NIe), norvaline (Nva), phenylalanine (Phe), phenylglycine, tyrosine (Tyr), tryptophan (Trp) and valine (Val).
  • x is 1 , 2 or 3.
  • a 1 may be selected from the group consisting of alanine (Ala), arginine (Arg), capreo- mycidine (Cpd), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (NIe), norvaline (Nva), phenylalanine (Phe), threonine (Thr), tyrosine (Tyr), tryptophan (Trp) and valine (Val),
  • a 1 is preferably phenylalanine or tyrosine
  • a 2 may be selected from the group consisting of arginine (Arg), isoleucine (lie), leucine (Leu), norleucine (NIe), norvaline (Nva), phenylalanine (Phe), phenylglycine, Tyrosine (Tyr), tryptophan (Trp) and valine (Val).
  • P is hydrogen
  • L is absent. In one embodiment, L is absent and A is absent.
  • P may be selected from the group consisting of formyl, acetyl (Ac), benzoyl (Bz), trifluoroacetyl, methoxysuccinyl, fluorenylmethyloxycarbonyl (Fmoc), methox- ycarbonyl (MEO-CO), (fluoromethoxy)carbonyl, benzyloxycarbonyl (Cbz), t-butyloxycarbonyl
  • P is benzyloxycarbonyl (Cbz).
  • the peptide aldehyde adduct is L-Alaninamide, N- [(phenylmethoxy)carbonyl]glycyl-/V-[2-hydroxy-1-[(4-hydroxyphenyl)methyl]-2-sulfoethyl]-, sodium salt (1 :1 ).
  • the protease inhibitor is one of the following peptide aldehydes or a hydrosulfite adduct thereof: Cbz-Arg-Ala-Tyr-H, Ac-Gly-Ala-Tyr-H, Cbz-Gly-Ala-Tyr-H, Cbz-Gly-Ala-Leu-H, Cbz-Val-Ala-Leu-H, Cbz-Gly-Ala-Phe-H, Cbz-Gly-Ala-Val-H, Cbz-Gly- Gly-Tyr-H, Cbz-Gly-Gly-Phe-H, Cbz-Arg-Val-Tyr-H, Cbz-Leu-Val-Tyr-H, Ac-Leu-Gly-Ala-Tyr- H, Ac-Phe-Gly-Ala-Tyr-H, Ac-Tyr-Gly-Ala-Tyr-H, Ac-Phe-G
  • the molar ratio of the peptide aldehyde or hydrosulfite adduct or hemiacetal adduct to the protease is between 1 :1 and 1000:1 .
  • the peptide aldehyde or hydrosulfite adduct or hemiacetal adduct is present in an amount of 0.001 ppm to 0.4% by weight of the total composition of the laundry soap bar.
  • the protease is present in an amount of 0.0001 % to 5.0 % by weight calculated as the percentage of enzyme protein to the total composition of the laundry soap bar.
  • preferred monovalent cations are sodium, potassium or ammonium. Most preferred cation is sodium.
  • organic ani- on formate acetate, citrate or lactate are preferred.
  • One preferred salt of a monovalent cation and an organic anion is sodium formate.
  • the salt of a monovalent cation and an organic anion is present in an amount of 0.1 % to 10% by weight of the total composition.
  • the laundry soap bar of the invention may comprise one or more additional enzymes selected from the list consisting of amylase, lipase, cellulase, mannanase, pectate lyase, carbohydrase and protease.
  • the protease may be obtained from Bacillus subtilis; Bacillus li- cheniformis; Bacillus lentus and any mixtures thereof.
  • the soap of the laundry soap bar is from an animal and/or vegetable source.
  • the invention further concerns a process of preparing the laundry soap bars as described above comprising the steps of:
  • the process is further described under preparation of laundry soap bars.
  • the process includes preparation of a premix, where the protease inhibitor which is a peptide aldehyde or a hydrosulfite adduct or hemiacetal adduct thereof; and a salt of a monovalent cation and an organic anion is mixed together prior to step (a).
  • the premix compres a protease; a protease inhibitor which is a peptide aldehyde or a hydrosulfite adduct or hemiacetal adduct thereof; a salt of a monovalent cation and/or a monovalent organic anion; a polyol and a pH controlling compound is prepared prior to step (a).
  • the invention further concerns a premix to be used in the above process, comprising a protease; a protease inhibitor which is a peptide aldehyde or a hydrosulfite adduct or hemiacetal adduct thereof; and a salt of a monovalent cation and/or a monovalent organic anion.
  • a laundry soap bar for washing a textile is also part of the invention.
  • the laundry soap bar is preferably for hand washing.
  • the protease may be of animal, vegetable or microbial origin, including chemically or genetically modified mutants. It may be a serine protease, and in particular a subtilase.
  • subtilases refers to a sub-group of serine protease according to Siezen et al., Protein Engng. 4 (1991 ) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523.
  • Serine proteases or serine peptidases is a subgroup of proteases characterised by having a serine in the active site, which forms a covalent adduct with the substrate.
  • subtilases (and the serine proteases) are characterised by having two active site amino acid residues apart from the serine, namely a histidine and an aspartic acid residue.
  • Subtilases are defined by homology analysis of more than 170 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases.
  • the subtilases may be divided into 6 sub-divisions, i.e. the Subtilisin family, the Thermitase family, the Proteinase K family, the Lantibiotic peptidase family, the Kexin family and the Pyrolysin family.
  • subtilisin family (EC 3.4.21.62) may be further divided into 3 sub-groups, i.e. I-S1 ("true" subtilisins), I-S2 (highly alkaline proteases) and intracellular subtilisins. Definitions or grouping of enzymes may vary or change, however, in the context of the present invention the above division of subtilases into sub-division or sub-groups shall be understood as those described by Siezen et al., Protein Engng. 4 (1991 ) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523.
  • subtilisins are those derived from Bacillus such as subtilisin lentus, Bacillus lentus, subtilisin Novo, subtilisin Carlsberg, Bacillus licheniformis, subtilisin BPN', subtilisin 309, subtilisin 147 and subtilisin 168 described in WO 89/06279 and protease PD138 (WO 93/18140). Additional examples are described in WO 98/0201 15, WO 01/44452, WO 01/58275, WO 01/58276, WO 03/006602 and WO 04/099401 .
  • trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270 and WO 94/25583.
  • subtilisins examples include KannaseTM, EverlaseTM, Pri- maseTM, DuralaseTM, EsperaseTM, AlcalaseTM, DurazymTM, SavinaseTM, OvozymeTM, Liqua- naseTM, CoronaseTM, PolarzymeTM, PyraseTM, Pancreatic Trypsin NOVO (PTN), Bio-FeedTM Pro and Clear-LensTM Pro; Blaze (Novozymes A/S).
  • proteases include RonozymeTM Pro, MaxataseTM, MaxacalTM, MaxapemTM, OpticleanTM, ProperaseTM, PurafectTM, Purafect OxTM, Purafact PrimeTM, ExcellaseTM, FN2TM, FN 3TM and FN4TM (available from Genen- cor International Inc.).
  • An embodiment of the invention is a protease which has at least 90%, e.g. , at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 1 .
  • a further of the invention is a protease which has at least 90%, e.g., at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 2.
  • An additional embodiment of the invention is a protease which has at least 90%, e.g., at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 3.
  • the laundry soap bar composition may optionally comprise of one or more additional enzymes such as 5, 4, 3, 2 or 1 additional enzyme(s) and the additional enzymes may be an additional protease, a lipase, a cutinase, an amylase, a carbohy- drase, a cellulase, a pectinase, a pectate lyase, a mannanase, an arabinase, a galactanase, a xylanase, an oxidase, a laccase, and/or peroxidase.
  • additional enzymes such as 5, 4, 3, 2 or 1 additional enzyme(s) and the additional enzymes may be an additional protease, a lipase, a cutinase, an amylase, a carbohy- drase, a cellulase, a pectinase, a pectate lyase, a
  • Suitable lipases and cutinases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples include lipase from Thermomy- ces, e.g., from T. lanuginosus (previously named Humicola lanuginosa) as described in EP 258068 and EP 305216, cutinase from Humicola, e.g. H. insolens as described in WO 96/13580, a Pseudomonas lipase, e.g., from P. alcaligenes or P. pseudoalcaligenes (EP 218272), P. cepacia (EP 331376), P. stutzeri (GB 1372034), P.
  • Thermomy- ces e.g., from T. lanuginosus (previously named Humicola lanuginosa) as described in EP 258068 and EP 305216
  • cutinase from Humicola e.g
  • lipase variants such as those described in WO 92/05249, WO
  • LipolaseTM Lipolase UltraTM, and LipexTM
  • LecitaseTM LipolexTM
  • LipocleanTM LipoprimeTM
  • Other commercially available lipases include Lumafast (Genencor Int Inc); Lipomax (Gist-Brocades/Genencor Int Inc) and Bacillus sp lipase from Solvay.
  • a carbohydrase is a general term for enzymes that cleave carbohydrates.
  • carbohydrases are named after the substrates they act on, for example amylases act on amylase and cellulases act on cellulose.
  • Many carbohydrases have found use in cleaning and laundry applications, such as amylase, cellulase, pectinase, pectate lyase, mannanase, arabinase, galactanase and xylanase, and all these can be applied in the laundry soap bar of the present invention.
  • Suitable amylases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, a-amylases obtained from Bacillus, e.g., a special strain of Bacillus lichen iformis, described in more detail in GB 1 ,296,839.
  • Examples of useful amylases are the variants described in WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181 , 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391 , 408, and 444.
  • amylases are Stainzyme; Stainzyme Plus; DuramylTM, Terma- mylTM, Termamyl Ultra; Natalase, FungamylTM and BANTM (Novozymes A/S), RapidaseTM and PurastarTM (from Genencor International Inc.). Lyases
  • the lyase may be a pectate lyase derived from Bacillus, particularly B. lichniformis or B. agaradhaerens, or a variant derived of any of these, e.g. as described in US 6124127, WO 99/027083, WO 99/027084, WO 02/006442, WO 02/092741 , WO 03/095638,
  • a commercially available pectate lyase is XPect; Pectawash and Pectaway (Novozymes A/S).
  • the mannanase may be an alkaline mannanase of Family 5 or 26. It may be a wild- type from Bacillus or Humicola, particularly B. agaradhaerens, B. licheniformis, B. halodurans, B. clausii, or H. insolens. Suitable mannanases are described in WO 99/064619. A commercially available mannanase is Mannaway (Novozymes A/S).
  • Suitable cellulases may be of bacterial or fungal origin. Chemically or genetically modified mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in US 4,435,307, US 5,648,263, US 5,691 ,178, US 5,776,757 and WO 89/09259.
  • cellulases are the alkaline or neutral cellulases having color care benefits.
  • Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/1 1262, WO 96/29397, WO 98/08940.
  • Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471 , WO 98/12307 and PCT/DK98/00299.
  • cellulases include CarezymeTM, CelluzymeTM, CellucleanTM, CelluclastTM, and EndolaseTM ; Renozyme; Whitezyme (Novozymes A/S), ClazinaseTM, Pura- dax, Puradax HA, and Puradax EG (available from Genencor) and KAC-500(B)TM (Kao Corporation).
  • the protease inhibitor maybe any compound which stabilises or inhibits the protease so that the protease or other enzyme(s) in the laundry soap bar are not degraded.
  • protease inhibitors are aprotinin, bestatin, calpain inhibitor I and II, chymostatin, leupeptin, pep- statin, phenylmethanesulfonyl fluoride (PMSF), boric acid, borate, borax, boronic acids, phenyl- boronic acids such as 4-formylphenylboronic acid (4-FPBA), peptide aldehydes or hydrosulfite adducts or hemiacetal adducts thereof and peptide trifluromethyl ketones.
  • protease inhibitors such as 5,4,3,2 or 1 inhibitor(s) of which at least one is a peptide aldehyde, a hydrosulfite adduct or a hemiacetal adduct thereof.
  • the peptide aldehyde may have the formula P-(A) y -L-(B) x -B°-H or a hydrosulfite adduct or hemiacetal adduct thereof, wherein:
  • i. H is hydrogen
  • iii. x is 1 , 2 or 3 for (B) x , and B is independently a single amino acid connected to B° via the C-terminal of the B amino acid
  • v. y is 0, 1 or 2 for (A) y , and A is independently a single amino acid residue connected to L via the /V-terminal of the A amino acid, with the proviso that if L is absent then A is absent;
  • P is selected from the group consisting of hydrogen and an /V-terminal protection group, with the proviso that if L is absent then P is an /V-terminal protection group;
  • R is independently selected from the group consisting of C 1-6 alkyl, C 6- io aryl or C 7- io arylalkyl optionally substituted with one or more, identical or different, sub- stituent's R';
  • R' is independently selected from the group consisting of halogen, -OH, -OR", - SH, -SR", -NH 2 , -NHR", -NR" 2 , -C0 2 H, -CONH 2 , -CONHR", -CONR” 2 , -
  • R" is a C 1-6 alkyl group.
  • x may be 1 , 2 or 3 and therefore B may be 1 , 2 or 3 amino acid residues respectively.
  • B may represent B 1 , B 2 -B 1 or B 3 -B 2 -B 1 , where B 3 , B 2 and B 1 each represent one amino acid residue, y may be 0, 1 or 2 and therefore A may be absent, or 1 or 2 amino acid residues respectively having the formula A 1 or A 2 -A 1 wherein A 2 and A 1 each represent one amino acid residue.
  • B° may be a single amino acid residue with L- or D-configuration, which is connected to H via the C-terminal of the amino acid, wherein R is a C 1-6 alkyl, C 6- io aryl or C 7-10 arylalkyl side chain, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, phenyl or benzyl, and wherein R may be optionally substituted with one or more, identical or different, substituent's R'.
  • R is a C 1-6 alkyl, C 6- io aryl or C 7-10 arylalkyl side chain, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, phenyl or benzyl, and wherein R may be optionally substituted with one or more, identical or different, substituent's R'.
  • arginine Arg
  • 3,4-dihydroxyphenylalanine isoleucine (lie), leucine (Leu), methionine (Met), norleucine (NIe), norvaline (Nva), phenylalanine (Phe), m- tyrosine, p-tyrosine (Tyr) and valine (Val).
  • B° is leucine, methionine, phenylalanine, p-tyrosine and valine.
  • B 1 which is connected to B° via the C-terminal of the B 1 amino acid, may be an aliphatic, hydrophobic and/or neutral amino acid.
  • B 1 are alanine (Ala), cysteine (Cys), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (NIe), norvaline (Nva), proline (Pro), serine (Ser), threonine (Thr) and valine (Val).
  • B 1 are alanine, glycine, isoleucine, leucine and valine.
  • a particular embodiment is when B 1 is alanine, glycine or valine.
  • B 2 which is connected to B 1 via the C-terminal of the B 2 amino acid, may be an aliphatic, hydrophobic, neutral and/or polar amino acid.
  • B 2 are alanine (Ala), arginine (Arg), capreomycidine (Cpd), cysteine (Cys), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (NIe), norvaline (Nva), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), and valine (Val).
  • B 2 are alanine, arginine, capreomycidine, glycine, isoleucine, leucine, phenylalanine and valine.
  • a particular embodiment is when B 2 is arginine, glycine, leucine, phenylalanine or valine.
  • B 3 which if present is connected to B 2 via the C-terminal of the B 3 amino acid, may be a large, aliphatic, aromatic, hydrophobic and/or neutral amino acid.
  • B 3 isoleucine (lie), leucine (Leu), norleucine (NIe), norvaline (Nva), phenylalanine (Phe), phenylglycine, tyrosine (Tyr), tryptophan (Trp) and valine (Val).
  • B 3 are leucine, phenylalanine, tyrosine and tryptophan.
  • a 1 which if present is connected to L via the /V-terminal of the amino acid, may be an aliphatic, aromatic, hydrophobic, neutral and/or polar amino acid.
  • Examples of A 1 are alanine (Ala), arginine (Arg), capreomycidine (Cpd), glycine (Gly), isoleucine (lie), leucine (Leu), norleu- cine (NIe), norvaline (Nva), phenylalanine (Phe), threonine (Thr), tyrosine (Tyr), tryptophan (Trp) and valine (Val).
  • a 1 are alanine, arginine, glycine, leucine, phenylalanine, tyrosine, tryptophan and valine.
  • B 2 is leucine, phenylalanine, tyrosine or tryptophan.
  • the A 2 residue which if present is connected to A 1 via the /V-terminal of the amino acid, may be a large, aliphatic, aromatic, hydrophobic and/or neutral amino acid.
  • a 2 are arginine (Arg), isoleucine (lie), leucine (Leu), norleucine (NIe), norvaline (Nva), phenylalanine (Phe), phenylglycine, Tyrosine (Tyr), tryptophan (Trp) and valine (Val).
  • Particular examples of A 2 are phenylalanine and tyrosine.
  • the N-terminal protection group P may be selected from formyl, acetyl (Ac), benzoyl (Bz), trifluoroacetyl, methoxysuccinyl, aromatic and aliphatic urethane protecting groups such as fluorenylmethyloxycarbonyl (Fmoc), methoxycarbonyl, (fluoromethoxy)carbonyl, ben- zyloxycarbonyl (Cbz), t-butyloxycarbonyl (Boc) and adamantyloxycarbonyl; p-methoxybenzyl carbonyl (Moz), benzyl (Bn), p-methoxybenzyl (PMB), p-methoxyphenyl (PMP), methoxyacetyl, methylamino carbonyl, methylsulfonyl, ethylsulfonyl, benzylsulfonyl, methylphosphoramidyl (M
  • P is preferably acetyl, methoxycarbonyl, benzyloxycarbonyl, methylamino carbonyl, methylsulfonyl, benzylsulfonyl and benzylphosphoramidyl.
  • P is preferably acetyl, methoxycarbonyl, methylsulfonyl, ethylsulfonyl and methylphosphoramidyl.
  • Suitable peptide aldehydes are described in WO94/04651 , W095/25791 , W098/13458, W098/13459, WO98/13460, W098/13461 , W098/13462, WO07/141736, WO07/145963, WO09/1 18375, W010/055052 and W01 1/036153.
  • the peptide aldehyde may be Cbz-Arg-Ala-Tyr-H (L-Alaninamide, N2-[(phenylmethoxy)carbonyl]-L-arginyl-N-[(1 S)-1 - formyl-2-(4-hydroxyphenyl)ethyl]-),
  • MeO-CO-Phe-Gly-Ala-Leu-H (L-Alaninamide, N-(methoxycarbonyl)-L- phenylalanylglycyl-N-[(1 S)-1 -formyl-3-methylbutyl]-),
  • MeO-CO-Phe-Gly-Ala-Phe-H (L-Alaninamide, N-(methoxycarbonyl)-L- phenylalanylglycyl-N-[(1 S)-1 -formyl-2-phenylethyl]-),
  • MeS02-Phe-Gly-Ala-Leu-H (L-Alaninamide, N-(methylsulfonyl)-L-phenylalanylglycyl-N- [(1 S)-1 -formyl-3-methylbutyl]-),
  • PhCH2S02-Val-Ala-Leu-H (L-Alaninamide, N-[(phenylmethyl)sulfonyl]-L-valyl-N-[(1 S)- 1 -formyl-3-methylbutyl]-),
  • a preferred example is Cbz-Gly-Ala-Tyr-H.
  • a-MAPI (3,5,8,1 1 -Tetraazatridecanoic acid, 6-[3-[(aminoiminomethyl)amino]propyl]-12- formyl-9-(1-methylethyl)-4,7,10-trioxo-13-phenyl-2-(phenylmethyl)-, (2S,6S,9S,12S)-
  • L-Valinamide N2-[[(1 -carboxy-2-phenylethyl)amino]carbonyl]-L-arginyl-N-(1 -formyl-2- phenylethyl)-, [1 (S),2(S)]-; L-Valinamide, N2-[[[(1 S)-1 -carboxy-2-phenylethyl]amino]carbonyl]-L- arginyl-N-[(1 S)-1-formyl-2-phenylethyl]- (9CI); SP-Chymostatin B),
  • Chymostatin B (L-Valinamide, (2S)-2-[(4S)-2-amino-3,4,5,6-tetrahydro-4-pyrimidinyl]-N- [[[(1 S)-1 -carboxy-2-phenylethyl]amino]carbonyl]glycyl-N-(1 -formyl-2-phenylethyl)-
  • Chymostatin C (L-lsoleucinamide, (2S)-2-[(4S)-2-amino-3,4,5,6-tetrahydro-4- pyrimidinyl]-N-[[[(1 S)-1-carboxy-2-phenylethyl]amino]carbonyl]glycyl-N-(1 -formyl-2-phenylethyl)- L-lsoleucinamide, (2S)-2-[(4S)-2-amino-1 ,4,5,6-tetrahydro-4-pyrimidinyl]-N-[[[(1 S)-1- carboxy-2-phenylethyl]amino]carbonyl]glycyl-N-(1-formyl-2-phenylethyl)- (9CI); L-
  • Isoleucinamide L-2-(2-amino-1 ,4,5,6-tetrahydro-4-pyrimidinyl)-N-[[(1-carboxy-2- phenylethyl)amino]carbonyl]glycyl-N-(1 -formyl-2-phenylethyl)-, stereoisomer).
  • the protease inhibitor may be an adduct of a peptide aldehyde.
  • the adduct maybe a hydrosulfite adduct having the formula P-(A) y -L-(B) x -N(H)-CHR- CH(OH)-S0 3 M, wherein P, A, y, L, B, x and R are defined as above, and M is H or an alkali metal, preferably Na or K.
  • the adduct may be a hemiacetal having the formula P- (A) y -L-(B) x -N(H)-CHR-CH(OH)-OR, wherein P, A, y, L, B, x and R are defined as above.
  • the general formula of the hydrosulfite adduct of a peptide aldehyde may also be writ- ten: P-A 2 -A 1 -L-B 3 -B 2 -B 1 -N(H)-CHR-CH(OH)-S0 3 M, where P, A 2 , A 1 ,L, B 3 , B 2 , B 1 , R and M are as defined above.
  • the adduct of a peptide aldehyde can be Cbz-Gly-Ala-N(H)-CH(CH 2 -p- C 6 H 4 OH)-CH(OH)-S0 3 Na (Sodium (2S)-[(N- ⁇ N-[(benzyloxy)carbonyl]glycyl ⁇ -L-alaninyl)amino]-1- hydroxy-3-(4-hydroxyphenyl)propane-1 -sulfonate) or Cbz-Gly-Ala-N(H)-CH(CH2Ph)-CH(OH)- S0 3 Na (Sodium (2S)-[(N- ⁇ N-[(benzyloxy)carbonyl]glycyl ⁇ -L-alaninyl)amino]-1-hydroxy-3- (phenyl)propane-l -sulfonate) or "MeO-CO_Val-Ala-N(H)-CH(CH2CH(CH 3
  • MeO-CO-Phe-Gly-Ala-NHCH(CH 2 CH(CH 3 ) 2 ))C(OH)(S0 3 M)-H, where M Na,
  • PhCH 2 0(OH)(0)P-Val-Ala-NHCH(CH 2 CH(CH 3 ) 2 ))C(OH)(S0 3 M)-H, where M Na ,
  • PhCH 2 S0 2 -Val-Ala-NHCH(CH 2 CH(CH 3 ) 2 ))C(OH)(S0 3 M)-H, where M Na,
  • PhCH 2 0(OH)(0)P-Leu-Ala-NHCH(CH 2 CH(CH 3 ) 2 ))C(OH)(S0 3 M)-H, where M Na ,
  • PhCH 2 0(OH)(0)P-Phe-Ala-NHCH(CH 2 CH(CH 3 ) 2 ))C(OH)(S0 3 M)-H, where M Na ,
  • the salt used in the bar is a salt of a monovalent cation and an organic anion.
  • the monovalent cation may be for example Na + , K + or NH 4 + .
  • the organic anion may be for example formate, acetate, citrate or lactate.
  • a salt of a monovalent cation and an organic anion may be, for example, sodium formate, potassium formate, ammonium formate, sodium acetate, potassium acetate, ammonium acetate, sodium lactate, potassium lactate, ammonium lactate, mono-sodium citrate, di-sodium citrate, tri-sodium citrate, sodium potassium citrate, potassium citrate, ammonium citrate or the like.
  • a particular embodiment is sodium formate. COMPOSITION OF LAUNDRY SOAP BAR
  • the laundry soap bar of the invention comprises the premix and soap.
  • the laundry soap bar may further comprise complexing agents like EDTA and HEDP (1 -hydroxyethane 1 ,1- diphosphonic acid), perfumes and/or different type of fillers.
  • the laundry soap bar may comprise additional compounds. Said compounds are not limited to surfactants e.g.
  • anionic synthetic surfactants builders, polymeric soil release agents, detergent chelators, polyols such as glycerol, pH controlling compounds, stabilizing agents, fillers, dyes, colorants, dye transfer inhibitors, alkoxylated polycarbonates, suds suppressers, structurants, binders, leaching agents, bleaching activators, clay soil removal agents, anti-redeposition agents, polymeric dispersing agents, brighteners, fabric softeners, perfumes and/or other compounds known in the art.
  • polyols such as glycerol, pH controlling compounds, stabilizing agents, fillers, dyes, colorants, dye transfer inhibitors, alkoxylated polycarbonates, suds suppressers, structurants, binders, leaching agents, bleaching activators, clay soil removal agents, anti-redeposition agents, polymeric dispersing agents, brighteners, fabric softeners, perfumes and/or other compounds known in the art.
  • the protease and the optional additional enzyme(s) may each be present in the laundry soap bar in an amount in the range from 0.00001 % to 2.0% by weight calculated as the percentage of enzyme protein of the total composition. Typical amounts are in the range from 0.0001 % to 0.4%, preferably in the range 0.001 % to 0.1 %, most preferably in the range 0.004% to 0.08% by weight of the total composition of the laundry soap bar.
  • the molar ratio of the peptide aldehyde (or hydrosulfite adduct or hemiacetal adduct) to the protease may be at least 1 :1 or 1 .5:1 , and it may be less than 1000:1 , more preferred less than 500:1 , even more preferred less than 100:1.
  • the molar ratio of the peptide al- dehyde (or hydrosulfite adduct or hemiacetal adduct) to the protease may be from 1000:1 to 1 :1 , preferably from 200:1 to 1 :1 , more preferably from 50:1 to 1 :1 , even more preferably from 20:1 to 2:1 , or most preferably the molar ratio is from 10:1 to 2:1.
  • the peptide aldehyde (or hydrosulfite adduct or hemiacetal adduct) may be present in the laundry soap bar in an amount in the range from 0.001 ppm to 0.4% by weight of the total composition. Typical amounts are in the range from 0.01 ppm to 0.1 %, preferably in the range 0.1 ppm to 0.02%, most preferably in the range 1 ppm to 100 ppm by weight of the total composition of the laundry soap bar.
  • the salt of a monovalent cation and an organic anion may be present in the laundry soap bar in an amount of at least 0.1 % w/w, e.g. at least 1 .0%, at least 1 .5% or at least 2.0% by weight of the total composition of the laundry soap bar.
  • the amount of the salt is typically below 10% w/w, below 8% or below 6%.
  • the salt may be present in an amount of 0.1 % to 10% by weight of the total composition, preferably 1 % to 8% and more preferably 2% to 6%.
  • Polyols useful in the present invention are characterized by solubility in water, carbon backbones of length between C2 and C6 and multiple hydroxyl groups, preferably containing between 2 and 6 hydroxyl groups per molecule.
  • Polyols useful herein include, but are not limited to 1 ,2-butanediol, 3-chloro-1 ,2- propanediol, ethylenediol, 1 ,2-hexanediol, glycerol (glycerine), mannose, propylene glycol, sorbitol, sucrose and mixtures thereof.
  • the polyol level will usually be between 0.5% and 10%, and preferably between 1 % and 5%, and more preferably between 1.5% and 3% by weight of the final bar composition.
  • soaps can be described as either high glycerine or low glycerine soaps.
  • Low glycerine soaps comprises from 0 to 2% glycerine, if more glycerine is needed it may be added in the manufacturing of the soap while the soap is still liquid to ensure proper uptake on or to soap noodles.
  • the laundry soap bar comprises from 1 to 6 % glycerine.
  • the laundry soap bar comprises from 2% to 5% glycerine by weight of total laundry soap bar.
  • the laundry soap bar comprises about 4% glycerine.
  • the pH controlling agent may be any suitable compound which is able of controlling the pH.
  • the pH controlling agent is an acid.
  • the pH controlling compound may be selected from the group consisting of a fatty acid, Acetic acid, Aconitic acid, Adipic acid, Arachidic acid, Arachi- donic acid, Aspartic acid, Behenic acid, Butyric acid, Capric acid, Capronic acid, Cerotic acid,
  • Citric acid Formic acid, Fumaric acid, Glutamic acid, Glutaric acid, Glyceric acid, Glyceric acid- 3-phosphate, Glyoxylic acid, Isocitric acid, a-Ketoglutaric acid, Lactic acid, Why aicd, Linoleic acid, Linolenic acid, Maleic acid, Malic acid, Malonic acid, Myristic acid, Oleic acid, Oxalic acid, Oxaloacetic acid, Palmitic acid, Palmitoleic acid, Phosphatidic acid, Phosphoenolpyruvic acid, Pimelic acid, Propionic acid, Pyruvic acid, Stearic acid, Succinic acid, Tartraic acid and Valeric acid.
  • the pH controlling agent will usually be present at a level between 0.04% and about 8%, preferably between 0.1 % and 6%. And more preferably between 0.2% and about 2% by weight of the final bar composition.
  • the amount of pH controlling agent is in a particular embodiment 0.001 % to 5% w/w of the laundry soap bar. In a more particular embodiment the amount of the pH controlling agent is 0.01 % to 2% w/w of the laundry soap bar. In a most particular embodiment the amount of the pH controlling agent is 0.05% to 1 % w/w of the laundry soap bar. In a particular embodiment the amount of pH controlling agent is around 0.5% w/w of the final bar composition.
  • Moisture enhances the mixing of the premix ingredients. Moisture further provides the laundry soap bar with acceptable feel and other physical characteristics. Moisture e.g. water can be added to the premix by being included with an ingredient and/or as free water added to the premix.
  • the laundry soap bar contains less than 30% w/w water (moisture) content of the final laundry soap bar, or from 10% to 30%, preferably from 20% to 30% water (moisture) content of the final laundry soap bar.
  • the soap suitable for use of the present invention includes water soluble salts of higher fatty acids.
  • the soap may be from an animal and/or vegetable source. Soap can be made by direct saponification of fats and oils or by neutralisation of free fatty acids. Suitable soaps are sodium, potassium, ammonium, and alkyloammonium salts of higher fatty acids containing from about 8 to about 24 carbon atoms, such as from 12 to about 18 carbon atoms.
  • the soap is selected from sodium and potassium salts of mixtures of fatty acid derived from coconut oil and tallow, such as sodium or potassium tallow and palm oil.
  • nut oil such as coconut or palm kernel oil, may be added in amounts of 5-30% by weight of the final bar.
  • Synthetic anionic surfactants which are suitable for use in the present invention include the water soluble salts, preferably the alkali metal, ammonium and alkyl ammonium salts of organic sulphuric reaction products having in their molecular structure an alkyl group containing from about 10 to about 20 carbon atoms and a sulfonic acid or sulphuric acid ester group.
  • this group of synthetic surfactants are the sodium and potassium alkyi sulfates, especially those obtained by sulfating the higher alcohols (C8-18 carbon atoms) such as those produced by reducing the glycerides of tallow or coconut oil; and the sodium and potassium alkyi benzene sulfonates in which the alkyi group contains from about 9 to about 15 carbon atoms, in straight chain or branched chain configuration.
  • LAS linear straight chain alkyi benzene sulfonates
  • the alkali metal salts, particularly the sodium salts of these surfactants are preferred.
  • an anionic synthetic detergent suitable for use herein are the sodium alkyi glyceryl ether sulfonates (AES), especially those ethers of higher alcohols derived from tallow and coconut oil; sodium coconut oil fatty acid monoglyceride sulfonates and sulfates; and sodium or potassium salts of alkyi ethylene oxide ether sulfates containing about 1 to about 10 units ethylene oxide per molecule and wherein the alkyi group contains from about 10 to about 20 carbon atoms.
  • AES sodium alkyi glyceryl ether sulfonates
  • a suitable anionic synthetic detergent also includes the water soluble salts of ester of alpha- sulphonated fatty acids containing from about 6 to about 20 carbon atoms in the fatty acid group and from about 1 to about 10 carbon atoms in the ester group; water soluble salts of 2-acyloxyalkane-1 -sulfonic acids containing from about 2 to about 9 carbon atoms in the acyl group and from 9 to about 23 carbon atoms in the alkane moiety; water soluble salts of olefin and paraffin sulfonates containing from about 12 to about 20 carbon atoms; and beta- alkyloxy alkane sulfonates containing from about 1 to about 3 carbon atoms in the alkyi group and from about 8 to about 20 carbon atoms in the alkane moiety.
  • Preferred anionic synthetic surfactant examples are C10-18 alkyi sulfates (AS), C10-18 linear alkyi benzene sulfonates (LAS), C10-14 alkyi glyceryl ether sulfonates (AES), and mixtures thereof.
  • linear alkyi benzene sulfonate coco fatty alcohol sulphate, soda ash, calcium car- bonate, coco fatty alcohol, Ti0 2 , cellulase, diethylenetriamine (methylenephosphonic acid), coco monoethanolamide, fluorecent whitening agents, substituted methylcellulose, perfume, moisture.
  • the laundry soap bars of the present invention may be processed in conventional laundry soap bar making equipment such as but not limited to: mixers, plodders, e.g a two stage vacuum plodder, extruders, cutters, logo-stampers, cooling tunnels and wrappers.
  • the invention is not limited to preparing the laundry soap bars by any single method.
  • a preferred embodiment of the invention is to prepare a premix containing a soap, a protease and optionally a second enzyme, a protease inhibitor which is a peptide aldehyde or a hydrosulfite adduct or hemiacetal adduct thereof, and a salt of a monovalent cation and an organic anion and then plodding the mixture.
  • the process may further comprise the steps of milling, extruding, cutting, stamping, cooling and/or wrapping.
  • the premix of the invention may be added to the soap at different stages of the process.
  • the premix is added to the soap before the refining step.
  • An embodiment of the present invention is to add the enzyme in liquid form.
  • the protease and optionally a second enzyme is added at the same time as the protease inhibitor or hydrosulfite adduct or hemiacetal adduct thereof.
  • the mixing may take place in a mixer e.g. in a Double Sigma Amalgator type MSA-100 mixer.
  • a plodder e.g. a Duplex plodder, in which the plodding step is performed.
  • the plodder operates preferably at high vacuum, so that entrapped air/gas is removed.
  • the product is extruded and the extruded bar subsequently moves to the cutter where the bar is cut to the desired bar length.
  • the pieces are stamped into their final shape in stamping presses.
  • High-speed presses are normally used on high-volume production lines.
  • the form of the stamp is established by designers, taking into account legal considerations such as the minimum net weight for a laundry bar.
  • the bars are printed with the product brand name.
  • the bar can be cooled, e.g. in a cooling tunnel, before it following normal procedure is wrapped, cased and sent to storage.
  • the laundry soap bar of the invention is for use for cleaning laundry and for washing laundry by hand.
  • a laundry soap bar comprising:
  • protease inhibitors of which at least one is a peptide aldehyde, a hydrosulfite adduct or a hemiacetal adduct thereof;
  • H is hydrogen
  • B is independently a single amino acid connected to B° via the C-terminal of the amino acid
  • A is absent if L is absent or is independently a single amino acid residue connected to L via the /V-terminal of the amino acid;
  • P is selected from the group consisting of hydrogen or if L is absent an /V-terminal protection group
  • x is 1 , 2 or 3;
  • y 0, 1 , or 2
  • R is independently selected from the group consisting of C 1-6 alkyl, C 6- io aryl or C 7-10 arylalkyl optionally substituted with one or more, identical or different, substit- uent's R';
  • R" is a C 1-6 alkyl group.
  • B° is selected from the group consisting of D- or L-form of arginine (Arg), 3,4-dihydroxyphenylalanine, isoleucine (lie), leucine (Leu), methionine (Met), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), m-tyrosine, p- tyrosine (Tyr) and valine (Val).
  • B 1 is selected from the group consisting of alanine (Ala), cysteine (Cys), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (Nle), norvaline (Nva), proline (Pro), serine (Ser), threonine (Thr) and valine (Val).
  • Al alanine
  • cysteine Cys
  • Gly glycine
  • lie leucine
  • Leu norleucine
  • Nva norvaline
  • proline Pro
  • serine serine
  • Thr threonine
  • Thr threonine
  • valine val
  • B2 is selected from the group consisting of alanine (Ala), arginine (Arg), capreomycidine (Cpd), cysteine (Cys), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), and valine (Val).
  • alanine Al
  • arginine Arg
  • Cpd capreomycidine
  • cysteine Cys
  • Gly isoleucine
  • lie leucine
  • Leu norleucine
  • Nva norvaline
  • Phe proline
  • Pro serine
  • Ser serine
  • Thr threonine
  • valine val
  • B 3 is selected from the group consisting of isoleucine (lie), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), phenylglycine, tyrosine (Tyr), tryptophan (Trp) and valine (Val).
  • a 1 is selected from the group consisting of alanine (Ala), arginine (Arg), capreomycidine (Cpd), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), threonine (Thr), tyrosine (Tyr), tryptophan (Trp) and valine (Val).
  • a 1 is selected from the group consisting of alanine (Ala), arginine (Arg), capreomycidine (Cpd), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), threonine (Thr), tyrosine (Tyr), tryptophan (Trp) and valine (Val).
  • a 1 is selected from the group consisting of alanine (A
  • the laundry soap bar of paragraph 4a wherein the protease inhibitor is one of the following peptide aldehydes or a hydrosulfite adduct thereof: Cbz-Arg-Ala-Tyr-H, Ac-Gly-Ala- Tyr-H, Cbz-Gly-Ala-Tyr-H, Cbz-Gly-Ala-Leu-H, Cbz-Val-Ala-Leu-H, Cbz-Gly-Ala-Phe-H, Cbz-Gly-Ala-Val-H, Cbz-Gly-Gly-Tyr-H, Cbz-Gly-Gly-Phe-H, Cbz-Arg-Val-Tyr-H, Cbz-Leu- Val-Tyr-H, Ac-Leu-Gly-Ala-Tyr-H, Ac-Phe-Gly-Ala-Tyr-H, Ac-Tyr-Gly-Ala-Tyr-
  • the laundry soap bar of any of paragraphs 1 a to 18a comprising of one or more additional enzymes selected from the list consisting of amylase, lipase, cellulase, mannanase, pec- tate lyase, carbohydrase and protease.
  • the laundry soap bar of any of paragraphs 1 a to 19a wherein the protease is obtained from Bacillus subtilis; Bacillus licheniformis; Bacillus lentus and any mixtures thereof.
  • the laundry soap bar of any of paragraphs 1 a to 20a wherein the soap is from an animal and/or vegetable source.
  • a process of preparing the laundry soap bars according to any of paragraphs 1 a to 21 a comprising the steps of:
  • step (a) plodding the mixture from step (a).
  • a. The process of paragraph 22a, wherein the protease; the protease inhibitor which is a peptide aldehyde or a hydrosulfite adduct or hemiacetal adduct thereof; and a salt of a monovalent cation and an organic anion is mixed together prior to step (a).
  • a premix to be used in the process of any of paragraphs 22a to 24a comprising a protease; a protease inhibitor which is a peptide aldehyde or a hydrosulfite adduct or hemiace- tal adduct thereof; and a salt of a monovalent cation and/or a monovalent organic anion.
  • a laundry soap bar comprising:
  • protease inhibitors of which at least one is a peptide aldehyde, a hydrosulfite adduct or a hemiacetal adduct thereof;
  • the laundry soap bar of embodiment 1 which contains less than 0.1 % w/w of the total composition of the laundry soap bar of one or more boron containing compounds.
  • x. H is hydrogen
  • xii. x is 1 , 2 or 3 for (B) x , and B is independently a single amino acid connected to B° via the C-terminal of the B amino acid
  • xiv. y is 0, 1 or 2 for (A) y , and A is independently a single amino acid residue connected to L via the /V-terminal of the A amino acid, with the proviso that if L is absent then A is absent;
  • xv. P is selected from the group consisting of hydrogen and an /V-terminal protection group, with the proviso that if L is absent then P is an /V-terminal protection group;
  • R is independently selected from the group consisting of C 1-6 alkyl, C 6- io aryl or C 7-10 arylalkyl optionally substituted with one or more, identical or different, sub- stituent's R';
  • R" is a C 1-6 alkyl group.
  • x. x is 1 , 2 or 3 for (B) x , and B is independently a single amino acid connected to B° via the C-terminal of the B amino acid
  • xii. y is 0, 1 or 2 for (A) y , and A is independently a single amino acid residue connected to L via the /V-terminal of the A amino acid, with the proviso that if L is absent then A is absent;
  • xiii. P is selected from the group consisting of hydrogen and an /V-terminal protection group, with the proviso that if L is absent then P is an /V-terminal protection group;
  • R is independently selected from the group consisting of C 1-6 alkyl, C 6- io aryl or C 7-10 arylalkyl optionally substituted with one or more, identical or different, sub- stituent's R';
  • R' is independently selected from the group consisting of halogen, -OH, -OR", -
  • xvi. R" is a C 1-6 alkyl group. 7.
  • M is Na or K.
  • B° is selected from the group consisting of D- or L-form of arginine (Arg), 3,4-dihydroxyphenylalanine, isoleucine (lie), leucine (Leu), methionine (Met), norleucine (NIe), norvaline (Nva), phenylalanine
  • B is an amino acid with L- configuration.
  • B 1 is selected from the group consisting of alanine (Ala), cysteine (Cys), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (Nle), norvaline (Nva), proline (Pro), serine (Ser), threonine (Thr) and valine (Val).
  • B 2 is selected from the group consisting of alanine (Ala), arginine (Arg), capreomycidine (Cpd), cysteine (Cys), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), and valine (Val).
  • B 3 is selected from the group consisting of isoleucine (lie), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), phenylglycine, tyrosine (Tyr), tryptophan (Trp) and valine (Val).
  • x is 1 .
  • a 1 is selected from the group consisting of alanine (Ala), arginine (Arg), capreomycidine (Cpd), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), threonine (Thr), tyrosine (Tyr), tryptophan (Trp) and valine (Val).
  • a 1 is selected from the group consisting of alanine (Ala), arginine (Arg), capreomycidine (Cpd), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), threonine (Thr), tyrosine (Tyr), tryptophan (Trp) and valine (Val).
  • a 2 is selected from the group consisting of arginine (Arg), isoleucine (lie), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), phenylglycine, Tyrosine (Tyr), tryptophan (Trp) and valine (Val).
  • P is selected from the group consisting of formyl, acetyl (Ac), benzoyl (Bz), trifluoroacetyl, methoxy- succinyl, fluorenylmethyloxycarbonyl (Fmoc), methoxycarbonyl (MEO-CO), (fluorometh- oxy)carbonyl, benzyloxycarbonyl (Cbz), t-butyloxycarbonyl (Boc), adamantyloxycarbon- yl, p-methoxybenzyl carbonyl (Moz), benzyl (Bn), p-methoxybenzyl (PMB), p- methoxyphenyl (PMP), methoxyacetyl, methylamino carbonyl (MeNCO), methylsulfonyl
  • MeO-CO-Phe-Gly-Ala-NHCH(CH 2 CH(CH 3 ) 2 ))C(OH)(S0 3 M)-H, where M Na,
  • the laundry soap bar of any of embodiments 1 to 37 wherein the salt of a monovalent cation and an organic anion is present in an amount of 0.1 % to 10% by weight of the total composition.
  • the laundry soap bar of any of embodiments 1 to 38 comprising of one or more additional enzymes selected from the list consisting of amylase, lipase, cellulase, man- nanase, pectate lyase, carbohydrase and protease.
  • the laundry soap bar of any of embodiments 1 to 39 wherein the protease is obtained from Bacillus subtilis; Bacillus licheniformis; Bacillus lentus and any mixtures thereof.
  • the laundry soap bar of any of embodiments 1 to 40 wherein the soap is from an animal and/or vegetable source.
  • a process of preparing the laundry soap bars according to any of embodiments 1 to 41 comprising the steps of:
  • step (a) plodding the mixture from step (a).
  • the protease; the protease inhibitor which is a peptide aldehyde or a hydrosulfite adduct or hemiacetal adduct thereof; and a salt of a monovalent cation and an organic anion is mixed together prior to step (a).
  • a premix comprising a protease; a protease inhibitor which is a peptide aldehyde or a hydrosulfite adduct or hemiacetal adduct thereof; a salt of a monovalent cation and/or a monovalent organic anion; a polyol and a pH controlling compound is prepared prior to step (a). .
  • a premix to be used in the process of any of embodiments 42 to 44 comprising a protease; a protease inhibitor which is a peptide aldehyde or a hydrosulfite adduct or hemiacetal adduct thereof; and a salt of a monovalent cation and/or a monovalent organic anion.
  • TFM Total fatty matter (80/20 tallow/vegetable)
  • Citric acid is then added with stirring until dispersed.
  • the premix was mixed with the soap base using a Sunlab mixer model 7.5 and then processed using a Sunlab Merton sampler model U.
  • the bars were stamped using a Sunlab Stamper model LMK-1 .
  • Hardness, or firmness, of laundry soap bars is defined as the maximum peak force (in grams) during the first compression cycle for the probe to penetrate a set depth into the laundry soap bar.
  • Laundry soap bar hardness was measured at room temperature ( ⁇ 23°C) assimilating ASTM D937-92 (ASTM International, West Conshohocken, PA) method by using a Texture Analyzer (model TA-XT2) with a 45° conical probe (model TA-15) using 5kg load cell, compression test mode, and 1 mm/sec test speed.
  • the test bars are kept in a polythene bag or wrapper at room temperature until needed for testing to keep from drying.
  • pH assay pH is measured on a 1 %w solution using a pH meter. The solution is prepared with distilled water.
  • Mush is the description for the gelatinous hydrated soap formed when toilet bars are left in contact with water. Mush is measured by determining the amount of soap gel formed after exposure to moisture.
  • the weight and dimension of the dry soap is determined.
  • the toilet bar is skew- ered near to the top using a wire or rod so that it can hang inside a beaker with about half of the bar immersed.
  • the bar is suspended in the beaker that is filled with deionized water at 40° C to the point where about half of the bar is immersed and left for two hours to cool.
  • the bar is removed from the water, the water is drained, and the bar is allowed to drain suspended for 15 minutes, after which the bar is weighted.
  • the soft soap gel developed on the immersed part of the bar is scraped off using a steel spatula avoiding scraping off the harder soap underneath.
  • the surface area of the soap immersed is obtained by measuring the circumference of the soap at the point of immersion and the depth from the middle point of the immersion to the end of the soap. Record the surface ar- ea (circumference x depth + area of collar), total water adsorbed (Wet weight - Dry weight) and mush (weight of soft soap removed). These measurements are used to calculate the mushing factor where:
  • Mushing factor Weight of Mush (g) / Surface Area (sq. cm.).
  • the mushing factor is evaluated using the following scale:
  • Wet cracking is the term used to describe the cracks which can appear on the surface of a toilet bar after it has been used and allowed to dry for a specified period.
  • test laundry soap bars are conditioned for at least 15 hours at room temperature. The bars are then totally immersed in water (30°C initial) for a period of one hour. They are then cleansed of mush that has been formed, taking special care to remove mush from the ends of the soap, by rubbing between the hands under the water. They are then removed and placed on a wire mesh tray where they are allowed to dry at normal room temperature.
  • the degree of cracking is assessed by visually examining the bars after 2, 24, and/or 48 hours, and rating them using the photo scale shown in page 423 of the reference book "Soap Manufacturing Technology" mentioned above.
  • the wet cracking is evaluated using the following scale:
  • the main purpose of this test is to establish any negative effect on bar feel in use caused by poor processing or by additives.
  • the test is carried out by hand washing with the bar and grading the product on a scale ranging from smooth to very gritty.
  • a bowl is filled with about 2L of tap water at the desired start/use temperature (20 °C, 30 °C, and/or 40 °C).
  • the bar is immersed in the water for 30 seconds and is then rubbed between the hands under the water for at least 3 times of 10 rubs each to remove any rough edges or relief from the stamped design.
  • the bar is rubbed under the water for at least 30 seconds and the feel of the bar assessed on a smoothness / sandiness scale, i.e. the presence of hard particles which detract from a smooth feeling of the bar on the skin, using the following scale:
  • test is repeated at successively higher temperatures until the product is rated as satisfactory and the testing is discontinued at that point.
  • Foam height was measured according to ASTM D1 173 (ASTM International, West Conshohocken, PA) using 1 g soap/L tap water (-25 ppm water hardness) at initial time 0 and after 5 minutes.
  • the Tergo-To-Meter is a medium scale model wash system that can be applied to test different wash conditions simultaneously.
  • a TOM is basically a large temperature controlled water bath with open metal beakers submerged into it. Each beaker constitutes one small top loader style washing machine and during an experiment, each of them will contain a solution of a specific detergent/enzyme system and the soiled and unsoiled fabrics its performance is tested on. Mechanical stress is achieved by a rotating stirring arm, which stirs the liquid within each beaker. Because the TOM beakers may have no lid, it is possible to withdraw samples during a TOM experiment and assay for information on-line during wash.
  • the TOM model wash system is mainly used in benchtop scale testing of detergents and enzymes at e.g, US or LA AP wash conditions. In a TOM experiment, factors such as the ballast to soil ratio and the fabric to wash liquor ratio can be varied. The TOM provides the ability to conduct screenings at small scale before conducting the more time consuming full scale experiments in top loader washing machines.
  • the water bath has a steel beakers and 1 rotating arm per beaker with capacity of up to 1200 mL of detergent solution. Temperature ranges from 5 to 80°C.
  • the water bath has to be filled up with deionised water. Rotational speed can be set up to 120 rpm/min.
  • wash solution with desired amount of detergent, temperature and water hardness in a bucket. Let soap bar dissolve during magnet stirring for up to 10 min. Wash solution shall be used within 30 to 60 min after preparation.
  • Savinase 16L available from Novozymes A/S, containing the subtilisin Bacillus lentus (SEQ ID NO: 2) was used as the test protease.
  • the test material EMPA1 17 is blood/milk/ink on cotton/polyester and was obtained from EMPA Testmaterials AG, Movenstrasse 12, CH-9015 St. Gallen, Switzerland.
  • the storage stability test was done by incubating the laundry soap bar for eight weeks at 30°C and/or 37°C in closed vials. Following the incubation, the residual protease activity was determined the laundry soap bar base using a slightly modified method described in H. Lund, S.G. Kaasgaard, P. Skagerlind, L. Jorgensen, C.I. Jorgensen and M. van de Weert, J Surfact. Deterg., (2012), 15, 9-21.
  • acceptable enzyme stability for an enzyme is having at least 60% residual activity when stored for eight weeks at 30°C as determined by the hydrolysis of the substrate ⁇ /,/V-dimethyl casein.
  • Example 1 Properties of Laundry soap bar with and without Protease Inhibitor
  • the laundry soap bars were prepared according to the Preparation of Laundry Soap
  • the total protease concentration was 0.016% by weight calculated as the percentage of enzyme protein to the total composition of the laundry soap bar.
  • Table 2 Storage stability of Savinase in laundry soap bars after 4 and 8 weeks at 30°C and 37°C based on wash performance of EMPA 1 17 swatches in a Terg-O-tometer
  • Table 3 Storage stability of Savinase in laundry soap bars after 4 and 8 weeks at 30°C and 37°C as determined by the hydrolysis of /V,/V-dimethyl casein
  • Tables 2 and 3 show that the Savinase without any in-situ protease stabiliser present had no stability after just 4 weeks at 30°C as measured by the wash performance of blood/milk/ink on cotton/polyester swatches and by the percentage of residual activity as determined by the hydrolysis of ⁇ /,/V-dimethyl casein.
  • the addition of the protease inhibitor and especially together with the addition of sodium formate significantly increased storage stability even after 8 weeks at 37°C.
  • the mushing factor was determined according the mush assay described above. Table 4 shows that laundry soap bars containing sodium formate had improved mushing factor over bars without sodium formate. Table 5: Wet Cracking properties of the laundry soap bars
  • the wet cracking properties were determined according the wet cracking assay de- scribed above. Table 5 shows that all laundry soap bars had acceptable wet cracking properties after a single immersion of 1 hour, when assessed after 2, 24 and 48 hours.
  • Example 2 Properties of Laundry soap bar with varying amounts of Sodium Formate The laundry soap bars were prepared according to the Preparation of Laundry Soap
  • the total protease concentration used was 0.016% by weight calculated as the percentage of enzyme protein to the total composition of the laundry soap bar.
  • Table 9 Storage stability of Savinase in laundry soap bars after 4 and 8 weeks at 30°C and 37°C based on wash performance of EMPA 1 17 swatches in a Terg-O-tometer
  • Table 10 Storage stability of Savinase in laundry soap bars after 4 and 8 weeks at 30°C and 37°C as determined by the hydrolysis of /V,/V-dimethyl casein
  • Tables 9 and 10 show that Savinase in the presence of inhibitor but without any sodium formate present has reduced stability at 30°C and almost no activity at 37°C even after just 4 weeks of storage as measured by the wash performance of blood/milk/ink on cotton/polyester swatches.
  • the addition of both the protease inhibitor and sodium formate significantly increased storage stability even after 8 weeks at 37°C as measured by the wash perfor- mance of blood/milk/ink on cotton/polyester swatches and by the percentage of residual activity as determined by the hydrolysis of ⁇ /,/V-dimethyl casein.
  • the mushing factor was determined according the mush assay described above. Table

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Abstract

The present invention relates to laundry soap bars with improved enzyme stability and to a process for preparing said laundry soap bars and to uses of the laundry soap bars.

Description

LAUNDRY SOAP BARS
REFERENCE TO A SEQUENCE LISTING
This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference. FIELD OF THE INVENTION
The present invention relates to laundry soap bars with improved enzyme stability and to a process for preparing said laundry soap bars and to uses of the laundry soap bars.
BACKGROUND OF THE INVENTION
The addition of enzymes, and particularly proteases, to laundry soap bars is a well- known feature in order to improve the removal of protein soiling. In addition, when formulating a laundry soap bar a second, non-protease enzyme (such as an amylase or a lipase) may also be included to improve the detergency towards other soilings. However, the stability of enzymes during production and then long term storage of the laundry soap bars becomes an issue of significant importance. The activity of the added enzymes can be affected by many factors, and one factor is the water content of the laundry soap bar. Laundry soap bars with high water content generally can result in a reduction of enzyme activity during storage, and this is a particular issue for proteases.
Protease stability can also be improved by adding one or more stabilizers to the laundry soap bar. Often boron containing compounds are used as stabilizers. GB 2186883 de- scribes laundry soap bars with a water content of 10-33% and containing proteases in which the proteolytic enzyme is stabilized by a mixture of a boron compound, a polyol, an organic acid or its alkali metal salt, and an alkali metal salt of an inorganic acid which is not a boron compound. WO 98/54285 describes high-moisture protease containing laundry soap bars with improved protease stability. The improved stability is obtained by adding a stabilizing agent made of a bo- rate compound in conjunction with a polyol, a carboxylate salt, a carboxylic acid, or mixtures thereof. Various boron compounds are well known as stabilizers for subtilisins in liquid detergents, e.g. WO 96/41859.
The above cited prior art all relates to enzyme stability during storage and use. However, loss of enzyme activity during manufacturing the bars is also a significant problem when in- corporating enzymes in laundry detergent bar compositions. WO 98/18897 describes a process for incorporating enzymes into laundry detergent bar compositions that minimizes the loss of enzyme stability during the manufacturing process of the bar. This is achieved by a process in which the enzyme prills are added after milling and cooling and then plodding the mixture.
However, a trend towards boron-free detergents has begun after the recent EU REACH classification classed boric acid as reprotoxic chemical. It has been shown in liquid de- tergent compositions that a reversible peptide aldehyde protease inhibitor can result in improved enzyme stabilization, such as that described in EP 0583534, WO 94/04651 and WO 98/13458. In addition, WO 10/055052 describes the use of a peptide aldehyde to stabilize a protease in liquid detergents whilst WO 07/141736, WO 07/145963 and WO 09/1 18375 disclose that a peptide aldehyde can be used to stabilize the subtilisin and any second enzyme.
Finally, a-MAPI, antipain, GE20372A and chymostatin A, B and C are described as peptide aldehydes with activity as protease inhibitors: R.J. Broadbridge and M. Akhtar, Chem. Commun., (1998), 1449-1450; E. Sarubbi, P.F. Seneci, M.R. Angelastro, N.P. Peet, M. Denaro, K. Islam, FEBS Letters, (1993), 319 (3), 253-256 and I.J. Galpin, A.H. Wilby, A.G. Place, R.J. Beynon, Int. J. Peptide Protein Res., (1984), 23, 477-486.
SUMMARY OF THE INVENTION
We have surprisingly found that the combination of a peptide aldehyde protease inhibitor, a hydrosulfite adduct of a peptide aldehyde protease inhibitor or a hemiacetal adduct of a peptide aldehyde protease inhibitor in combination with a salt of a monovalent cation and an organic anion has a stabilizing effect in laundry soap bars comprising a protease, such as a subtilisin, and optionally one or more additional enzymes.
Accordingly, the invention provides a laundry soap bar comprising:
a) one or more soaps or synthetic surfactants or any mixture thereof; b) a protease and optionally one or more additional enzymes;
c) one or more protease inhibitors of which at least one is a peptide aldehyde, a hydrosulfite adduct or a hemiacetal adduct thereof; and
d) a salt of a monovalent cation and an organic anion.
The invention also relates to the use of laundry soap bars in cleaning laundry and for washing laundry by hand. Furthermore the invention relates to the preparation of laundry soap bars and the use of premixes in the preparation thereof. DEFINITIONS
All chemical names herein are the CAS (Chemical abstract service) registered names. CAS is a unique registry number system to identify chemical substances.
Alky I The term "alkyl" in the present context represents a linear or branched hydrocarbon radical having 1 -6 carbon atoms. Representative examples include methyl, ethyl, n-propyl, iso- propyl, n-butyl, /'so-butyl, sec-butyl, ie f-butyl, 1-ethylpropyl, 2-methylbutyl, pentyl, 3,3- dimethylbutyl, hexyl, etc. Preferred "alkyl" groups are methyl, ethyl, n-propyl, /'so-propyl, n-butyl, /'so-butyl and sec-butyl.
Aryl
The term "aryl" in the present context represents a carbocyclic aromatic ring having 6- 10 carbon atoms. The term "aryl" also represents fused ring systems in which 2 neighbouring atoms are used to fuse the 2 aromatic rings together. Representative examples of "aryl" rings are phenyl and naphthalenyl.
Aryl alkyl
The term "arylalkyl" in the present context represents a carbocyclic aromatic ring having 6-10 carbon atoms attached to a alkyl group of 1 -6 carbon atoms. Representative examples of "arylalkyl" rings are benzyl, phenylethyl and phenylpropyl.
Halogen
The term "halogen" in the present context represents fluorine, chlorine, bromine and iodine. Amino acid
The term "amino acid" covers amino acids with L- or D-configuration unless otherwise specified.
Laundry soap bar
The invention relates to laundry soap bars for hand washing laundry. The term laundry soap bar includes laundry bars, soap bars, combo bars, syndet bars and detergent bars. The types of bar usually differ in the type of surfactant they contain, and the term laundry soap bar includes those containing soaps from fatty acids and/or synthetic soaps. The laundry soap bar has a physical form which is solid and not a liquid, gel or a powder at room temperature. The term solid is defined as a physical form which does not significantly change over time, i.e. if a solid object (e.g. laundry soap bar) is placed inside a container, the solid object does not change to fill the container it is placed in. The bar is a solid typically in bar form but can be in other solid shapes such as round or oval.
Sequence Identity The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".
For purposes of the present invention, the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 or later. Version 6.1.0 was used. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labelled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment).
Substantially pure polypeptide
The term "substantially pure polypeptide" means a preparation that contains at most 10%, at most 8%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1 %, and at most 0.5% by weight of other polypeptide material with which it is natively or recombi- nantly associated. Preferably, the polypeptide is at least 92% pure, e.g., at least 94% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99%, at least 99.5% pure, and 100% pure by weight of the total polypeptide material present in the preparation. The polypeptides of the present invention are preferably in a substantially pure form. This can be accomplished, for example, by preparing the polypeptide by well-known recombinant methods or by classical purification methods.
Variant
The term "variant" means a polypeptide having protease activity comprising an altera- tion, i.e., a substitution, insertion, and/or deletion of one or more (several) amino acid residues at one or more (several) positions. A substitution means a replacement of an amino acid occupying a position with a different amino acid; a deletion means removal of an amino acid occupying a position; and an insertion means adding 1-3 amino acids adjacent to an amino acid occupying a position. DETAILED DESCRIPTION OF THE INVENTION
One object of the present invention is to provide enzyme containing laundry soap bars with improved storage stability. A further object of the present invention is to provide a process for manufacturing a solid formulation such as laundry soap bars wherein the enzyme can be added in the initial phase of the laundry soap bar manufacturing process and still maintain a significant enzyme activity after manufacturing. We have surprisingly found that the combination of a peptide aldehyde (or hydrosulfite adduct or hemiacetal adduct) protease inhibitor with the salt of a monovalent cation and an organic anion has a stabilizing effect in laundry soap bars comprising a protease, such as a subtilisin, and optionally one or more additional enzymes.
Accordingly, the invention provides a laundry soap bar comprising:
a) one or more soaps or synthetic surfactants or any mixture thereof; b) a protease and optionally one or more additional enzymes;
c) one or more protease inhibitors of which at least one is a peptide aldehyde, a hydrosulfite adduct or a hemiacetal adduct thereof; and
d) a salt of a monovalent cation and an organic anion.
An embodiment of the invention is the laundry soap bar which contains, for example, less than 0.1 % w/w, e.g. less than 0.05% w/w, e.g. less than 0.02% w/w, e.g. less than 0.01 % w/w, e.g. less than 0.001 % w/w or 0% w/w by weight of the total composition of the laundry soap bar of one or more boron containing compounds. Non limiting examples of boron contain- ing compounds are boric acid, borate, borax, phenylboronic acid and phenylboronic acid derivatives such as 4-formylphenylboronic acid.
An objective of the invention is to provide a laundry soap bar which has good physical properties, such as wet cracking properties, washdown feel, foam height, bar hardness and/or mush factor. An embodiment of the invention is to provide a laundry soap bar with a mush factor less than 0.30, preferably less than 0.27, more preferably less than 0.24, e.g. between 0.10 and 0.30, preferably between 0.15 and 0.25. An additional embodiment of the invention is to provide a laundry soap bar with wet cracking properties of between 1 and 4, preferably between 1 and 3, more preferably between 1 and 2, when assessed 2 hours after the laundry soap bar has been immersed in water for 1 hour.
A further embodiment of the invention is to provide a laundry soap bar with foam height of between 10 and 20 cm, preferably between 10.5 and 17 cm, more preferably between 1 1 and 15 cm, both initially and after 5 minutes. An embodiment of the invention is to provide a laundry soap bar with wet bar (washdown) feel between 0 and 2, preferably between 0 and 1 . An additional embodiment of the invention is to provide a laundry soap bar with hardness of between 2500 and 6000 g, preferably between 2750 and 5500 g, more preferably between 3000 and 5000 g, even more preferably between 3250 and 5000 g, even more preferably between 3500 and 5000 g.
In one embodiment of the invention the protease inhibitor of the laundry soap bar is a peptide aldehyde of the formula P-(A)y-L-(B)x-B°-H or a hydrosulfite adduct or hemiacetal adduct thereof, wherein:
i. H is hydrogen; ii. B° is a single amino acid residue with L- or D-configuration of the formula -NH- CH(R)-C(=0)-;
iii. x is 1 , 2 or 3 for (B)x, and B is independently a single amino acid connected to B° via the C-terminal of the B amino acid
iv. L is absent or L is independently a linker group of the formula -C(=0)-,
-C(=0)-C(=0)-, -C(=S)-, -C(=S)-C(=S)- or -C(=S)-C(=0)-;
v. y is 0, 1 or 2 for (A)y, and A is independently a single amino acid residue connected to L via the /V-terminal of the A amino acid, with the proviso that if L is absent then A is absent;
vi. P is selected from the group consisting of hydrogen and an /V-terminal protection group, with the proviso that if L is absent then P is an /V-terminal protection group;
vii. R is independently selected from the group consisting of C1-6 alkyl, C6-io aryl or C7-10 arylalkyl optionally substituted with one or more, identical or different, sub- stituent's R';
viii. R' is independently selected from the group consisting of halogen, -OH, -OR", - SH, -SR", -NH2, -NHR", -NR"2, -C02H, -CONH2, -CONHR", -CONR"2, - NHC(=N)NH2; and
ix. R" is a C1-6 alkyl group.
In one embodiment of the invention, the protease inhibitor of the laundry soap bar is a hydrosulfite adduct of a peptide aldehyde is of the formula P-(A)y-L-(B)x-N(H)-CHR-CH(OH)- S03M, wherein
i. M is hydrogen or an alkali metal;
ii. x is 1 , 2 or 3 for (B)x, and B is independently a single amino acid connected to B° via the C-terminal of the B amino acid
iii. L is absent or L is independently a linker group of the formula -C(=0)-,
-C(=0)-C(=0)-, -C(=S)-, -C(=S)-C(=S)- or -C(=S)-C(=0)-;
iv. y is 0, 1 or 2 for (A)y, and A is independently a single amino acid residue connected to L via the /V-terminal of the A amino acid, with the proviso that if L is ab- sent then A is absent;
v. P is selected from the group consisting of hydrogen and an /V-terminal protection group, with the proviso that if L is absent then P is an /V-terminal protection group;
vi. R is independently selected from the group consisting of C1-6 alkyl, C6-io aryl or C7-10 arylalkyl optionally substituted with one or more, identical or different, sub- stituent's R'; vii. R' is independently selected from the group consisting of halogen, -OH, -OR", - SH, -SR", -NH2, -NHR", -NR"2, -C02H, -CONH2, -CONHR", -CONR"2, - NHC(=N)NH2; and
viii. R" is a C1-6 alkyl group.
In one embodiment of the invention, M is Na or K. In one embodiment R is a C7-10 ar- ylalkyl substituted with -OH, preferably C7 arylalkyl substituted with -OH.
In one embodiment, B° is selected from the group consisting of D- or L-form of arginine (Arg), 3,4-dihydroxyphenylalanine, isoleucine (lie), leucine (Leu), methionine (Met), norleucine (NIe), norvaline (Nva), phenylalanine (Phe), m-tyrosine, p-tyrosine (Tyr) and valine (Val).
In one embodiment of the invention, B is an amino acid with L-configuration. B1 may be selected from the group consisting of alanine (Ala), cysteine (Cys), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (NIe), norvaline (Nva), proline (Pro), serine (Ser), threonine (Thr) and valine (Val).
B2 may be selected from the group consisting of alanine (Ala), arginine (Arg), capreo- mycidine (Cpd), cysteine (Cys), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (NIe), norvaline (Nva), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), and valine (Val).
B3 may be selected from the group consisting of isoleucine (lie), leucine (Leu), norleu- cine (NIe), norvaline (Nva), phenylalanine (Phe), phenylglycine, tyrosine (Tyr), tryptophan (Trp) and valine (Val).
In one embodiment, x is 1 , 2 or 3.
In one embodiment, L is -C(=0)- or -C(S) and y is 1 or 2.
A1 may be selected from the group consisting of alanine (Ala), arginine (Arg), capreo- mycidine (Cpd), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (NIe), norvaline (Nva), phenylalanine (Phe), threonine (Thr), tyrosine (Tyr), tryptophan (Trp) and valine (Val), A1 is preferably phenylalanine or tyrosine
A2 may be selected from the group consisting of arginine (Arg), isoleucine (lie), leucine (Leu), norleucine (NIe), norvaline (Nva), phenylalanine (Phe), phenylglycine, Tyrosine (Tyr), tryptophan (Trp) and valine (Val).
In one embodiment, P is hydrogen.
In one embodiment, L is absent. In one embodiment, L is absent and A is absent. For these embodiments, P may be selected from the group consisting of formyl, acetyl (Ac), benzoyl (Bz), trifluoroacetyl, methoxysuccinyl, fluorenylmethyloxycarbonyl (Fmoc), methox- ycarbonyl (MEO-CO), (fluoromethoxy)carbonyl, benzyloxycarbonyl (Cbz), t-butyloxycarbonyl
(Boc), adamantyloxycarbonyl, p-methoxybenzyl carbonyl (Moz), benzyl (Bn), p- methoxybenzyl (PMB), p-methoxyphenyl (PMP), methoxyacetyl, methylamino carbonyl (MeNCO), methylsulfonyl (MeS02), ethylsulfonyl (EtS02), benzylsulfonyl (PhCH2S02), methylphosphoramidyl (MeOP(OH)(=0)) and benzylphosphoramidyl (PhCH20-P(OH)(0)). P is preferably acetyl (Ac), methoxycarbonyl (MEO-CO), methylsulfonyl (MeS02), ethylsulfonyl (EtS02), benzyloxycarbonyl (Cbz) or methylphosphoramidyl (MeOP(OH)(=0)). In a most preferred embodiment P is benzyloxycarbonyl (Cbz).
In one embodiment, the peptide aldehyde adduct is L-Alaninamide, N- [(phenylmethoxy)carbonyl]glycyl-/V-[2-hydroxy-1-[(4-hydroxyphenyl)methyl]-2-sulfoethyl]-, sodium salt (1 :1 ).
In one embodiment, the protease inhibitor is one of the following peptide aldehydes or a hydrosulfite adduct thereof: Cbz-Arg-Ala-Tyr-H, Ac-Gly-Ala-Tyr-H, Cbz-Gly-Ala-Tyr-H, Cbz-Gly-Ala-Leu-H, Cbz-Val-Ala-Leu-H, Cbz-Gly-Ala-Phe-H, Cbz-Gly-Ala-Val-H, Cbz-Gly- Gly-Tyr-H, Cbz-Gly-Gly-Phe-H, Cbz-Arg-Val-Tyr-H, Cbz-Leu-Val-Tyr-H, Ac-Leu-Gly-Ala-Tyr- H, Ac-Phe-Gly-Ala-Tyr-H, Ac-Tyr-Gly-Ala-Tyr-H, Ac-Phe-Gly-Ala-Leu-H, Ac-Phe-Gly-Ala- Phe-H, Ac-Phe-Gly-Val-Tyr-H, Ac-Phe-Gly-Ala-Met-H, Ac-Trp-Leu-Val-Tyr-H, MeO-CO-Val-
Ala-Leu-H, MeNCO-Val-Ala-Leu-H, MeO-CO-Phe-Gly-Ala-Leu-H, MeO-CO-Phe-Gly-Ala- Phe-H, MeS02-Phe-Gly-Ala-Leu-H, MeS02-Val-Ala-Leu-H, PhCH20-P(OH)(0)-Val-Ala-Leu- H, EtS02-Phe-Gly-Ala-Leu-H, PhCH2S02-Val-Ala-Leu-H, PhCH20-P(OH)(0)-Leu-Ala-Leu-H, PhCH20-P(OH)(0)-Phe-Ala-Leu-H, or MeO-P(OH)(0)-Leu-Gly-Ala-Leu-H, α-ΜΑΡΙ, β-ΜΑΡΙ, Phe-C(=0)-Arg-Val-Tyr-H, Phe-C(=0)-Gly-Gly-Tyr-H, Phe-C(=0)-Gly-Ala-Phe-H, Phe-
C(=0)-Gly-Ala-Tyr-H, Phe-C(=0)-Gly-Ala-L-H, Phe-C(=0)-Gly-Ala-Nva-H, Phe-C(=0)-Gly- Ala-Nle-H, Tyr-C(=0)-Arg-Val-Tyr-H, Tyr-C(=0)-Gly-Ala-Tyr-H, Phe-C(=S)-Arg-Val-Phe-H, Phe-C(=S)-Arg-Val-Tyr-H, Phe-C(=S)-Gly-Ala-Tyr-H, Antipain, GE20372A, GE20372B, Chymostatin A, Chymostatin B, and Chymostatin C.
In one embodiment, the molar ratio of the peptide aldehyde or hydrosulfite adduct or hemiacetal adduct to the protease is between 1 :1 and 1000:1 .
In one embodiment, the peptide aldehyde or hydrosulfite adduct or hemiacetal adduct is present in an amount of 0.001 ppm to 0.4% by weight of the total composition of the laundry soap bar.
In one embodiment, the protease is present in an amount of 0.0001 % to 5.0 % by weight calculated as the percentage of enzyme protein to the total composition of the laundry soap bar.
For the salt of a monovalent cation and an organic anion, preferred monovalent cations are sodium, potassium or ammonium. Most preferred cation is sodium. For the organic ani- on formate, acetate, citrate or lactate are preferred. One preferred salt of a monovalent cation and an organic anion is sodium formate. The salt of a monovalent cation and an organic anion is present in an amount of 0.1 % to 10% by weight of the total composition.
The laundry soap bar of the invention may comprise one or more additional enzymes selected from the list consisting of amylase, lipase, cellulase, mannanase, pectate lyase, carbohydrase and protease. The protease may be obtained from Bacillus subtilis; Bacillus li- cheniformis; Bacillus lentus and any mixtures thereof.
The soap of the laundry soap bar is from an animal and/or vegetable source.
The invention further concerns a process of preparing the laundry soap bars as described above comprising the steps of:
a) mixing a soap; a protease; a protease inhibitor which is a peptide aldehyde or a hy- drosulfite adduct or hemiacetal adduct thereof; and a salt of a monovalent cation and an organic anion; and
b) plodding the mixture from step (a).
The process is further described under preparation of laundry soap bars. The process includes preparation of a premix, where the protease inhibitor which is a peptide aldehyde or a hydrosulfite adduct or hemiacetal adduct thereof; and a salt of a monovalent cation and an organic anion is mixed together prior to step (a).
The premix compres a protease; a protease inhibitor which is a peptide aldehyde or a hydrosulfite adduct or hemiacetal adduct thereof; a salt of a monovalent cation and/or a monovalent organic anion; a polyol and a pH controlling compound is prepared prior to step (a).
The invention further concerns a premix to be used in the above process, comprising a protease; a protease inhibitor which is a peptide aldehyde or a hydrosulfite adduct or hemiacetal adduct thereof; and a salt of a monovalent cation and/or a monovalent organic anion. The use of a laundry soap bar for washing a textile is also part of the invention. The laundry soap bar is preferably for hand washing.
The invention is further described in the below text.
Protease
The protease may be of animal, vegetable or microbial origin, including chemically or genetically modified mutants. It may be a serine protease, and in particular a subtilase. The term "subtilases" refers to a sub-group of serine protease according to Siezen et al., Protein Engng. 4 (1991 ) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523. Serine proteases or serine peptidases is a subgroup of proteases characterised by having a serine in the active site, which forms a covalent adduct with the substrate. Further the subtilases (and the serine proteases) are characterised by having two active site amino acid residues apart from the serine, namely a histidine and an aspartic acid residue.
Subtilases are defined by homology analysis of more than 170 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases. The subtilases may be divided into 6 sub-divisions, i.e. the Subtilisin family, the Thermitase family, the Proteinase K family, the Lantibiotic peptidase family, the Kexin family and the Pyrolysin family.
The Subtilisin family (EC 3.4.21.62) may be further divided into 3 sub-groups, i.e. I-S1 ("true" subtilisins), I-S2 (highly alkaline proteases) and intracellular subtilisins. Definitions or grouping of enzymes may vary or change, however, in the context of the present invention the above division of subtilases into sub-division or sub-groups shall be understood as those described by Siezen et al., Protein Engng. 4 (1991 ) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523.
Examples of subtilisins are those derived from Bacillus such as subtilisin lentus, Bacillus lentus, subtilisin Novo, subtilisin Carlsberg, Bacillus licheniformis, subtilisin BPN', subtilisin 309, subtilisin 147 and subtilisin 168 described in WO 89/06279 and protease PD138 (WO 93/18140). Additional examples are described in WO 98/0201 15, WO 01/44452, WO 01/58275, WO 01/58276, WO 03/006602 and WO 04/099401 . Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270 and WO 94/25583.
Examples of useful proteases are the variants described in WO 92/19729, WO
98/201 15, WO 98/201 16, and WO 98/34946, especially the variants with substitutions in one or more of the following positions: 27, 36, 57, 76, 87, 97, 101 , 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235, and 274.
Examples of commercially available subtilisins include Kannase™, Everlase™, Pri- mase™, Duralase™, Esperase™, Alcalase™, Durazym™, Savinase™, Ovozyme™, Liqua- nase™, Coronase™, Polarzyme™, Pyrase™, Pancreatic Trypsin NOVO (PTN), Bio-Feed™ Pro and Clear-Lens™ Pro; Blaze (Novozymes A/S). Other commercially available proteases include Ronozyme™ Pro, Maxatase™, Maxacal™, Maxapem™, Opticlean™, Properase™, Purafect™, Purafect Ox™, Purafact Prime™, Excellase™, FN2™, FN 3™ and FN4™ (available from Genen- cor International Inc.).
An embodiment of the invention is a protease which has at least 90%, e.g. , at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 1 . A further of the invention is a protease which has at least 90%, e.g., at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 2. An additional embodiment of the invention is a protease which has at least 90%, e.g., at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 3.
Additional enzyme
In addition to the protease, the laundry soap bar composition may optionally comprise of one or more additional enzymes such as 5, 4, 3, 2 or 1 additional enzyme(s) and the additional enzymes may be an additional protease, a lipase, a cutinase, an amylase, a carbohy- drase, a cellulase, a pectinase, a pectate lyase, a mannanase, an arabinase, a galactanase, a xylanase, an oxidase, a laccase, and/or peroxidase. Lipase and cutinase
Suitable lipases and cutinases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples include lipase from Thermomy- ces, e.g., from T. lanuginosus (previously named Humicola lanuginosa) as described in EP 258068 and EP 305216, cutinase from Humicola, e.g. H. insolens as described in WO 96/13580, a Pseudomonas lipase, e.g., from P. alcaligenes or P. pseudoalcaligenes (EP 218272), P. cepacia (EP 331376), P. stutzeri (GB 1372034), P. fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g., from B. subtilis (Dartois et al , Biochemica et Biophysica Acta, (1993), 1 131 , 253- 360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422).
Other examples are lipase variants such as those described in WO 92/05249, WO
94/01541 , EP 407225, EP 260105, WO 95/35381 , WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079, WO 97/07202, WO 00/060063, WO 07/087508 and WO 09/109500.
Preferred commercially available lipase enzymes include Lipolase™, Lipolase Ultra™, and Lipex™; Lecitase™, Lipolex™; Lipoclean™, Lipoprime™ (Novozymes A/S). Other commercially available lipases include Lumafast (Genencor Int Inc); Lipomax (Gist-Brocades/Genencor Int Inc) and Bacillus sp lipase from Solvay.
Carbohydrase
A carbohydrase is a general term for enzymes that cleave carbohydrates. In general carbohydrases are named after the substrates they act on, for example amylases act on amylase and cellulases act on cellulose. Many carbohydrases have found use in cleaning and laundry applications, such as amylase, cellulase, pectinase, pectate lyase, mannanase, arabinase, galactanase and xylanase, and all these can be applied in the laundry soap bar of the present invention.
Amylase
Suitable amylases (a and/or β) include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, a-amylases obtained from Bacillus, e.g., a special strain of Bacillus lichen iformis, described in more detail in GB 1 ,296,839.
Examples of useful amylases are the variants described in WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181 , 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391 , 408, and 444.
Commercially available amylases are Stainzyme; Stainzyme Plus; Duramyl™, Terma- myl™, Termamyl Ultra; Natalase, Fungamyl™ and BAN™ (Novozymes A/S), Rapidase™ and Purastar™ (from Genencor International Inc.). Lyases
The lyase may be a pectate lyase derived from Bacillus, particularly B. licherniformis or B. agaradhaerens, or a variant derived of any of these, e.g. as described in US 6124127, WO 99/027083, WO 99/027084, WO 02/006442, WO 02/092741 , WO 03/095638, A commercially available pectate lyase is XPect; Pectawash and Pectaway (Novozymes A/S).
Mannanase
The mannanase may be an alkaline mannanase of Family 5 or 26. It may be a wild- type from Bacillus or Humicola, particularly B. agaradhaerens, B. licheniformis, B. halodurans, B. clausii, or H. insolens. Suitable mannanases are described in WO 99/064619. A commercially available mannanase is Mannaway (Novozymes A/S).
Cellulase
Suitable cellulases may be of bacterial or fungal origin. Chemically or genetically modified mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in US 4,435,307, US 5,648,263, US 5,691 ,178, US 5,776,757 and WO 89/09259.
Especially suitable cellulases are the alkaline or neutral cellulases having color care benefits. Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/1 1262, WO 96/29397, WO 98/08940. Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471 , WO 98/12307 and PCT/DK98/00299.
Commercially available cellulases include Carezyme™, Celluzyme™, Celluclean™, Celluclast™, and Endolase™ ; Renozyme; Whitezyme (Novozymes A/S), Clazinase™, Pura- dax, Puradax HA, and Puradax EG (available from Genencor) and KAC-500(B)™ (Kao Corporation).
Protease inhibitor
The protease inhibitor maybe any compound which stabilises or inhibits the protease so that the protease or other enzyme(s) in the laundry soap bar are not degraded. Examples of protease inhibitors are aprotinin, bestatin, calpain inhibitor I and II, chymostatin, leupeptin, pep- statin, phenylmethanesulfonyl fluoride (PMSF), boric acid, borate, borax, boronic acids, phenyl- boronic acids such as 4-formylphenylboronic acid (4-FPBA), peptide aldehydes or hydrosulfite adducts or hemiacetal adducts thereof and peptide trifluromethyl ketones. There may be one or more protease inhibitors, such as 5,4,3,2 or 1 inhibitor(s) of which at least one is a peptide aldehyde, a hydrosulfite adduct or a hemiacetal adduct thereof.
Peptide aldehyde inhibitor
The peptide aldehyde may have the formula P-(A)y-L-(B)x-B°-H or a hydrosulfite adduct or hemiacetal adduct thereof, wherein:
i. H is hydrogen;
ii. B° is a single amino acid residue with L- or D-configuration of the formula -NH- CH(R)-C(=0)-;
iii. x is 1 , 2 or 3 for (B)x, and B is independently a single amino acid connected to B° via the C-terminal of the B amino acid
iv. L is absent or L is independently a linker group of the formula -C(=0)-,
-C(=0)-C(=0)-, -C(=S)-, -C(=S)-C(=S)- or -C(=S)-C(=0)-;
v. y is 0, 1 or 2 for (A)y, and A is independently a single amino acid residue connected to L via the /V-terminal of the A amino acid, with the proviso that if L is absent then A is absent;
vi. P is selected from the group consisting of hydrogen and an /V-terminal protection group, with the proviso that if L is absent then P is an /V-terminal protection group; vii. R is independently selected from the group consisting of C1-6 alkyl, C6-io aryl or C7-io arylalkyl optionally substituted with one or more, identical or different, sub- stituent's R';
viii. R' is independently selected from the group consisting of halogen, -OH, -OR", - SH, -SR", -NH2, -NHR", -NR"2, -C02H, -CONH2, -CONHR", -CONR"2, -
NHC(=N)NH2; and
ix. R" is a C1-6 alkyl group. x may be 1 , 2 or 3 and therefore B may be 1 , 2 or 3 amino acid residues respectively. Thus, B may represent B1, B2-B1 or B3-B2-B1, where B3, B2 and B1 each represent one amino acid residue, y may be 0, 1 or 2 and therefore A may be absent, or 1 or 2 amino acid residues respectively having the formula A1 or A2-A1 wherein A2 and A1 each represent one amino acid residue.
B° may be a single amino acid residue with L- or D-configuration, which is connected to H via the C-terminal of the amino acid, wherein R is a C1-6 alkyl, C6-io aryl or C7-10 arylalkyl side chain, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, phenyl or benzyl, and wherein R may be optionally substituted with one or more, identical or different, substituent's R'. Particular examples are the D- or L-form of arginine (Arg), 3,4-dihydroxyphenylalanine, isoleucine (lie), leucine (Leu), methionine (Met), norleucine (NIe), norvaline (Nva), phenylalanine (Phe), m- tyrosine, p-tyrosine (Tyr) and valine (Val). A particular embodiment is when B° is leucine, methionine, phenylalanine, p-tyrosine and valine.
B1, which is connected to B° via the C-terminal of the B1 amino acid, may be an aliphatic, hydrophobic and/or neutral amino acid. Examples of B1 are alanine (Ala), cysteine (Cys), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (NIe), norvaline (Nva), proline (Pro), serine (Ser), threonine (Thr) and valine (Val). Particular examples of B1 are alanine, glycine, isoleucine, leucine and valine. A particular embodiment is when B1 is alanine, glycine or valine.
If present, B2, which is connected to B1 via the C-terminal of the B2 amino acid, may be an aliphatic, hydrophobic, neutral and/or polar amino acid. Examples of B2 are alanine (Ala), arginine (Arg), capreomycidine (Cpd), cysteine (Cys), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (NIe), norvaline (Nva), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), and valine (Val). Particular examples of B2 are alanine, arginine, capreomycidine, glycine, isoleucine, leucine, phenylalanine and valine. A particular embodiment is when B2 is arginine, glycine, leucine, phenylalanine or valine.
B3, which if present is connected to B2 via the C-terminal of the B3 amino acid, may be a large, aliphatic, aromatic, hydrophobic and/or neutral amino acid. Examples of B3 are isoleucine (lie), leucine (Leu), norleucine (NIe), norvaline (Nva), phenylalanine (Phe), phenylglycine, tyrosine (Tyr), tryptophan (Trp) and valine (Val). Particular examples of B3 are leucine, phenylalanine, tyrosine and tryptophan.
The linker group L may be absent or selected from the group consisting of-C(=0)-, - C(=0)-C(=0)-, -C(=S)-, -C(=S)-C(=S)- or -C(=S)-C(=0)-. Particular embodiments of the inven- tion are when L is absent or L is a carbonyl group -C(=0)-.
A1, which if present is connected to L via the /V-terminal of the amino acid, may be an aliphatic, aromatic, hydrophobic, neutral and/or polar amino acid. Examples of A1 are alanine (Ala), arginine (Arg), capreomycidine (Cpd), glycine (Gly), isoleucine (lie), leucine (Leu), norleu- cine (NIe), norvaline (Nva), phenylalanine (Phe), threonine (Thr), tyrosine (Tyr), tryptophan (Trp) and valine (Val). Particular examples of A1 are alanine, arginine, glycine, leucine, phenylalanine, tyrosine, tryptophan and valine. A particular embodiment is when B2 is leucine, phenylalanine, tyrosine or tryptophan.
The A2 residue, which if present is connected to A1 via the /V-terminal of the amino acid, may be a large, aliphatic, aromatic, hydrophobic and/or neutral amino acid. Examples of A2 are arginine (Arg), isoleucine (lie), leucine (Leu), norleucine (NIe), norvaline (Nva), phenylalanine (Phe), phenylglycine, Tyrosine (Tyr), tryptophan (Trp) and valine (Val). Particular examples of A2 are phenylalanine and tyrosine.
The N-terminal protection group P (if present) may be selected from formyl, acetyl (Ac), benzoyl (Bz), trifluoroacetyl, methoxysuccinyl, aromatic and aliphatic urethane protecting groups such as fluorenylmethyloxycarbonyl (Fmoc), methoxycarbonyl, (fluoromethoxy)carbonyl, ben- zyloxycarbonyl (Cbz), t-butyloxycarbonyl (Boc) and adamantyloxycarbonyl; p-methoxybenzyl carbonyl (Moz), benzyl (Bn), p-methoxybenzyl (PMB), p-methoxyphenyl (PMP), methoxyacetyl, methylamino carbonyl, methylsulfonyl, ethylsulfonyl, benzylsulfonyl, methylphosphoramidyl (MeOP(OH)(=0)) and benzylphosphoramidyl (PhCH2OP(OH)(=0)).
The general formula of the peptide aldehyde may also be written: P-A2- A1-L- B3- B2 B1-
B°-H, where P, A2, A1,L, B3, B2, B1 and B° are as defined above.
In the case of a tripeptide aldehyde with a protection group (i.e. x=2, L is absent and A is absent), P is preferably acetyl, methoxycarbonyl, benzyloxycarbonyl, methylamino carbonyl, methylsulfonyl, benzylsulfonyl and benzylphosphoramidyl. In the case of a tetrapeptide alde- hyde with a protection group (i.e. x=3, L is absent and A is absent), P is preferably acetyl, methoxycarbonyl, methylsulfonyl, ethylsulfonyl and methylphosphoramidyl.
Suitable peptide aldehydes are described in WO94/04651 , W095/25791 , W098/13458, W098/13459, WO98/13460, W098/13461 , W098/13462, WO07/141736, WO07/145963, WO09/1 18375, W010/055052 and W01 1/036153.
More particularly, the peptide aldehyde may be Cbz-Arg-Ala-Tyr-H (L-Alaninamide, N2-[(phenylmethoxy)carbonyl]-L-arginyl-N-[(1 S)-1 - formyl-2-(4-hydroxyphenyl)ethyl]-),
Ac-Gly-Ala-Tyr-H (L-Alaninamide, N-acetylglycyl-N-[(1 S)-1-formyl-2-(4- hydroxyphenyl)ethyl]-)
Cbz-Gly-Ala-Tyr-H (L-Alaninamide, N-[(phenylmethoxy)carbonyl]glycyl-N-[(1 S)-1- formyl-2-(4-hydroxyphenyl)ethyl]-),
Cbz-Gly-Ala-Leu-H (L-Alaninamide, N-[(phenylmethoxy)carbonyl]glycyl-N-[(1 S)-1- formyl-3-methylbutyl]-),
Cbz-Val-Ala-Leu-H (L-Alaninamide, N-[(phenylmethoxy)carbonyl]-L-valyl-N-[(1 S)-1 - formyl-3-methylbutyl]-),
Cbz-Gly-Ala-Phe-H (L-Alaninamide, N-[(phenylmethoxy)carbonyl]glycyl-N-[(1 S)-1- formyl-2-phenylethyl]-),
Cbz-Gly-Ala-Val-H (L-Alaninamide, N-[(phenylmethoxy)carbonyl]glycyl-N-[(1 S)-1- formyl-2-methylpropyl]-),
Cbz-Gly-Gly-Tyr-H (Glycinamide, N-[(phenylmethoxy)carbonyl]glycyl-N-[(1 S)-1 -formyl-
2-(4-hydroxyphenyl)ethyl]-),
Cbz-Gly-Gly-Phe-H (Glycinamide, N-[(phenylmethoxy)carbonyl]glycyl-N-[(1 S)-1 -formyl- 2-phenylethyl]-),
Cbz-Arg-Val-Tyr-H (L-Valinamide, N2-[(phenylmethoxy)carbonyl]-L-arginyl-N-[(1 S)-1- formyl-2-(4-hydroxyphenyl)ethyl]-),
Cbz-Leu-Val-Tyr-H (L-Valinamide, N-[(phenylmethoxy)carbonyl]-L-leucyl-N-[(1 S)-1- formyl-2-(4-hydroxyphenyl)ethyl]-)
Ac-Leu-Gly-Ala-Tyr-H (L-Alaninamide, N-acetyl-L-leucylglycyl-N-[(1 S)-1-formyl-2-(4- hydroxyphenyl)ethyl]-),
Ac-Phe-Gly-Ala-Tyr-H (L-Alaninamide, N-acetyl-L-phenylalanylglycyl-N-[(1 S)-1-formyl-
2-(4-hydroxyphenyl)ethyl]-),
Ac-Tyr-Gly-Ala-Tyr-H (L-Alaninamide, N-acetyl-L-tyrosylglycyl-N-[(1 S)-1 -formyl-2-(4- hydroxyphenyl)ethyl]-),
Ac-Phe-Gly-Ala-Leu-H (L-Alaninamide, N-acetyl-L-phenylalanylglycyl-N-[(1 S)-1 -formyl- 3-methylbutyl]-),
Ac-Phe-Gly-Ala-Phe-H (L-Alaninamide, N-acetyl-L-phenylalanylglycyl-N-[(1 S)-1 -formyl-
2- phenylethyl]-)
Ac-Phe-Gly-Val-Tyr-H (L-Valinamide, N-acetyl-L-phenylalanylglycyl-N-[(1 S)-1 -formyl-2- (4-hydroxyphenyl)ethyl]-),
Ac-Phe-Gly-Ala-Met-H (L-Alaninamide, N-acetyl-L-phenylalanylglycyl-N-[(1 S)-1-formyl-
3- (methylthio)propyl]-), Ac-Trp-Leu-Val-Tyr-H (L-Valinamide, N-acetyl-L-tryptophyl-L-leucyl-N-[(1 S)-1 -formyl-2- (4-hydroxyphenyl)ethyl]-),
MeO-CO-Val-Ala-Leu-H (L-Alaninamide, N-(methoxycarbonyl)-L-valyl-N-[(1 S)-1-formyl- 3-methylbutyl]-)
MeNHCO-Val-Ala-Leu-H (L-Alaninamide, N-(aminomethylcarbonyl)-L-valyl-N-[(1 S)-1- formyl-3-methylbutyl]-),
MeO-CO-Phe-Gly-Ala-Leu-H (L-Alaninamide, N-(methoxycarbonyl)-L- phenylalanylglycyl-N-[(1 S)-1 -formyl-3-methylbutyl]-),
MeO-CO-Phe-Gly-Ala-Phe-H (L-Alaninamide, N-(methoxycarbonyl)-L- phenylalanylglycyl-N-[(1 S)-1 -formyl-2-phenylethyl]-),
MeS02-Phe-Gly-Ala-Leu-H (L-Alaninamide, N-(methylsulfonyl)-L-phenylalanylglycyl-N- [(1 S)-1 -formyl-3-methylbutyl]-),
MeS02-Val-Ala-Leu-H (L-Alaninamide, N-(methylsulfonyl)-L-valyl-N-[(1 S)-1 -formyl-3- methylbutyl]-),
PhCH20-P(OH)(0)-Val-Ala-Leu-H (L-Alaninamide, N-
[hydroxy(phenylmethoxy)phosphinyl]-L-valyl-N-[(1 S)-1-formyl-3-methylbutyl]-),
EtS02-Phe-Gly-Ala-Leu-H (L-Alaninamide, N-(ethylsulfonyl)-L-phenylalanylglycyl-N- [(1 S)-1 -formyl-3-methylbutyl]-),
PhCH2S02-Val-Ala-Leu-H (L-Alaninamide, N-[(phenylmethyl)sulfonyl]-L-valyl-N-[(1 S)- 1 -formyl-3-methylbutyl]-),
PhCH20-P(OH)(0)-Leu-Ala-Leu-H (L-Alaninamide, N-
[hydroxy(phenylmethoxy)phosphinyl]-L-leucyl-N-[(1 S)-1-formyl-3-methylbutyl]-),
PhCH20-P(OH)(0)-Phe-Ala-Leu-H (L-Alaninamide, N-
[hydroxy(phenylmethoxy)phosphinyl]-L-phenylalanyl-N-[(1 S)-1-formyl-3-methylbutyl]-), or
MeO-P(OH)(0)-Leu-Gly-Ala-Leu-H; (L-Alaninamide, N-(hydroxymethoxyphosphinyl)-L- leucylglycyl-N-[(1 S)-1-formyl-3-methylbutyl]-).
A preferred example is Cbz-Gly-Ala-Tyr-H.
Further examples of such peptide aldehydes include
a-MAPI (3,5,8,1 1 -Tetraazatridecanoic acid, 6-[3-[(aminoiminomethyl)amino]propyl]-12- formyl-9-(1-methylethyl)-4,7,10-trioxo-13-phenyl-2-(phenylmethyl)-, (2S,6S,9S,12S)-
L-Valinamide, N2-[[(1 -carboxy-2-phenylethyl)amino]carbonyl]-L-arginyl-N-(1 -formyl-2- phenylethyl)-, [1 (S),2(S)]-; L-Valinamide, N2-[[[(1 S)-1 -carboxy-2-phenylethyl]amino]carbonyl]-L- arginyl-N-[(1 S)-1-formyl-2-phenylethyl]- (9CI); SP-Chymostatin B),
β-ΜΑΡΙ (L-Valinamide, N2-[[[(1 S)-1-carboxy-2-phenylethyl]amino]carbonyl]-L-arginyl-N- [(1 R)-1-formyl-2-phenylethyl]-L-Valinamide, N2-[[(1-carboxy-2-phenylethyl)amino]carbonyl]-L- arginyl-N-(1 -formyl-2-phenylethyl)-, [1 (S),2(R)]-), Phe-C(=0)-Arg-Val-Tyr-H (L-Valinamide, N2-[[[(1 S)-1-carboxy-2- phenylethyl]amino]carbonyl]-L-arginyl-N-[(1 S)-1 -formyl-2-(4-hydroxyphenyl)ethyl]- (9CI)),
Phe-C(=0)-Gly-Gly-Tyr-H, (3,5,8,1 1 -Tetraazatridecanoic acid, 12-formyl-13-(4- hydroxyphenyl)-4,7,10-trioxo-2-(phenylmethyl)-, (2S,12S)-),
Phe-C(=0)-Gly-Ala-Phe-H, (3,5,8,1 1-Tetraazatridecanoic acid, 12-formyl-9-methyl-
4,7,10-trioxo-13-phenyl-2-(phenylmethyl)-, (2S,9S,12S)-),
Phe-C(=0)-Gly-Ala-Tyr-H (3,5,8,1 1 -Tetraazatridecanoic acid, 12-formyl-13-(4- hydroxyphenyl)-9-methyl-4,7,10-trioxo-2-(phenylmethyl)-, (2S,9S,12S)-),
Phe-C(=0)-Gly-Ala-Leu-H, (3,5,8,1 1 -Tetraazapentadecanoic acid, 12-formyl-9,14- dimethyl-4,7,10-trioxo-2-(phenylmethyl)-, (2S,9S,12S)-),
Phe-C(=0)-Gly-Ala-Nva-H, (3,5,8,1 1 -Tetraazapentadecanoic acid, 12-formyl-9-methyl- 4,7,10-trioxo-2-(phenylmethyl)-, (2S,9S,12S)-),
Phe-C(=0)-Gly-Ala-Nle-H (3,5,8, 1 1-Tetraazahexadecanoic acid, 12-formyl-9-methyl- 4,7,10-trioxo-2-(phenylmethyl)-, (2S,9S,12S)-),
Tyr-C(=0)-Arg-Val-Tyr-H (L-Valinamide, N2-[[[(1 S)-1-carboxy-2-(4- hydroxyphenyl)ethyl]amino]carbonyl]-L-arginyl-N-[(1 S)-1-formyl-2-(4-hydroxyphenyl)ethyl]- (9CI))
Tyr-C(=0)-Gly-Ala-Tyr-H (3,5,8,1 1 -Tetraazatridecanoic acid, 12-formyl-13-(4- hydroxyphenyl)-2-[(4-hydroxyphenyl)methyl]-9-methyl-4,7,10-trioxo-, (2S,9S,12S)-)
Phe-C(=S)-Arg-Val-Phe-H, (3,5,8,1 1-Tetraazatridecanoic acid, 6-[3-
[(aminoiminomethyl)amino]propyl]-12-formyl-9-(1-methylethyl)-7,10-dioxo-13-phenyl-2- (phenylmethyl)-4-thioxo-, (2S,6S,9S,12S)-),
Phe-C(=S)-Arg-Val-Tyr-H, (3,5,8,1 1-Tetraazatridecanoic acid, 6-[3-
[(aminoiminomethyl)amino]propyl]-12-formyl-13-(4-hydroxyphenyl)-9-(1-methylethyl)-7,10-di 2-(phenylmethyl)-4-thioxo-, (2S,6S,9S,12S)-),
Phe-C(=S)-Gly-Ala-Tyr-H, (3,5,8,1 1 -Tetraazatridecanoic acid, 12-formyl-13-(4- hydroxyphenyl)-9-methyl-7,10-dioxo-2-(phenylmethyl)-4-thioxo-, (2S,9S,12S)-),
Antipain (L-Valinamide, N2-[[(1-carboxy-2-phenylethyl)amino]carbonyl]-L-arginyl-N-[4- [(aminoiminomethyl)amino]-1-formylbutyl]- ),
GE20372A (L-Valinamide, N2-[[[(1 S)-1-carboxy-2-(4- hydroxyphenyl)ethyl]amino]carbonyl]-L-arginyl-N-[(1 S)-1-formyl-2-phenylethyl]-
L-Valinamide, N2-[[[1-carboxy-2-(4-hydroxyphenyl)ethyl]amino]carbonyl]-L-arginyl-N- (1-formyl-2-phenylethyl)-, [1 (S),2(S)]-) ,
GE20372B (L-Valinamide, N2-[[[(1 S)-1-carboxy-2-(4- hydroxyphenyl)ethyl]amino]carbonyl]-L-arginyl-N-[(1 R)-1 -formyl-2-phenylethyl]-
L-Valinamide, N2-[[[1-carboxy-2-(4-hydroxyphenyl)ethyl]amino]carbonyl]-L-arginyl-N- (1 -formyl-2-phenylethyl)-, [1 (S),2(R)]-), Chymostatin A (L-Leucinamide, (2S)-2-[(4S)-2-amino-3,4,5,6-tetrahydro-4-pyrimidinyl]- N-[[[(1 S)-1-carboxy-2-phenylethyl]amino]carbonyl]glycyl-N-(1-formyl-2-phenylethyl)-
L-Leucinamide, (2S)-2-[(4S)-2-amino-1 !4!5,6-tetrahydro-4-pyrimidinyl]-N-[[[(1 S)-1- carboxy-2-phenylethyl]amino]carbonyl]glycyl-N-(1 -formyl-2-phenylethyl)- (9CI); L-Leucinamide, L-2-(2-amino-1 ,4,5,6-tetrahydro-4-pyrimidinyl)-N-[[(1-carboxy-2- phenylethyl)amino]carbonyl]glycyl-N-(1 -formyl-2-phenylethyl)-, stereoisomer),
Chymostatin B (L-Valinamide, (2S)-2-[(4S)-2-amino-3,4,5,6-tetrahydro-4-pyrimidinyl]-N- [[[(1 S)-1 -carboxy-2-phenylethyl]amino]carbonyl]glycyl-N-(1 -formyl-2-phenylethyl)-
L-Valinamide, (2S)-2-[(4S)-2-amino-1 ,4,5,6-tetrahydro-4-pyrimidinyl]-N-[[[(1 S)-1- carboxy-2-phenylethyl]amino]carbonyl]glycyl-N-(1 -formyl-2-phenylethyl)- (9CI); L-Valinamide, L- 2-(2-amino-1 ,4,5,6-tetrahydro-4-pyrimidinyl)-N-[[(1-carboxy-2-phenylethyl)amino]carbonyl]glycyl- N-(1 -formyl-2-phenylethyl)-, stereoisomer), and
Chymostatin C (L-lsoleucinamide, (2S)-2-[(4S)-2-amino-3,4,5,6-tetrahydro-4- pyrimidinyl]-N-[[[(1 S)-1-carboxy-2-phenylethyl]amino]carbonyl]glycyl-N-(1 -formyl-2-phenylethyl)- L-lsoleucinamide, (2S)-2-[(4S)-2-amino-1 ,4,5,6-tetrahydro-4-pyrimidinyl]-N-[[[(1 S)-1- carboxy-2-phenylethyl]amino]carbonyl]glycyl-N-(1-formyl-2-phenylethyl)- (9CI); L-
Isoleucinamide, L-2-(2-amino-1 ,4,5,6-tetrahydro-4-pyrimidinyl)-N-[[(1-carboxy-2- phenylethyl)amino]carbonyl]glycyl-N-(1 -formyl-2-phenylethyl)-, stereoisomer).
Peptide aldehyde adducts
Instead of a peptide aldehyde, the protease inhibitor may be an adduct of a peptide aldehyde. The adduct maybe a hydrosulfite adduct having the formula P-(A)y-L-(B)x-N(H)-CHR- CH(OH)-S03M, wherein P, A, y, L, B, x and R are defined as above, and M is H or an alkali metal, preferably Na or K. Alternatively, the adduct may be a hemiacetal having the formula P- (A)y-L-(B)x-N(H)-CHR-CH(OH)-OR, wherein P, A, y, L, B, x and R are defined as above. A pre- ferred embodiment is a hydrosulfite adduct wherein P=Cbz, B2=Gly; B1= Ala; B° = Tyr (so R=PhCH2, R'=OH), x=2, y=0, L=A=absent and M=Na (Cbz-Gly-Ala-N(H)-CH(CH2-p-C6H4OH)- CH(OH)-S03Na, L-Alaninamide, /V-[(phenylmethoxy)carbonyl]glycyl-/V-[2-hydroxy-1 -[(4- hydroxyphenyl)methyl]-2-sulfoethyl]-, sodium salt (1 :1 )).
The general formula of the hydrosulfite adduct of a peptide aldehyde may also be writ- ten: P-A2-A1-L-B3-B2-B1-N(H)-CHR-CH(OH)-S03M, where P, A2, A1,L, B3, B2, B1, R and M are as defined above.
Alternatively, the adduct of a peptide aldehyde can be Cbz-Gly-Ala-N(H)-CH(CH2-p- C6H4OH)-CH(OH)-S03Na (Sodium (2S)-[(N-{N-[(benzyloxy)carbonyl]glycyl}-L-alaninyl)amino]-1- hydroxy-3-(4-hydroxyphenyl)propane-1 -sulfonate) or Cbz-Gly-Ala-N(H)-CH(CH2Ph)-CH(OH)- S03Na (Sodium (2S)-[(N-{N-[(benzyloxy)carbonyl]glycyl}-L-alaninyl)amino]-1-hydroxy-3- (phenyl)propane-l -sulfonate) or "MeO-CO_Val-Ala-N(H)-CH(CH2CH(CH3)2)-CH(OH)-S03Na ( Sodium (2S)-[(N-{N-[(benzyloxy)carbonyl]glycyl}-L-alaninyl)amino]-1-hydroxy-3-(2- propanyl)propane-1 -sulfonate).
Other preferred peptide aldehyde bisulfites are
Cbz-Arg-Ala-NHCH(CH2C6H4OH)C(OH)(S03M)-H where M=Na,
Ac-Gly-Ala-NHCH(CH2C6H4OH)C(OH)(S03M)-H! where M=Na,
Cbz-Gly-Ala-NHCH(CH2C6H4OH)C(OH)(S03M)-H! where M=Na (L-Alaninamide, N- [(phenylmethoxy)carbonyl]glycyl-N-[2-hydroxy-1-[(4-hydroxyphenyl)methyl]-2-sulfoethyl]-, sodium salt (1 :1 )),
Cbz-Gly-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H, where M=Na,
Cbz-Val-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H! where M=Na,
Cbz-Gly-Ala-NHCH(CH2Ph)C(OH)(S03M)-H, where M=Na,
Cbz-Gly-Ala-NHCH(CH(CH3)2)C(OH)(S03M)-H, where M=Na,
Cbz-Gly-Gly-NHCH(CH2C6H40H)C(OH)(S03M)-H, where M=Na,
Cbz-Gly-Gly-NHCH(CH2Ph)C(OH)(S03M)-H, where M=Na,
Cbz-Arg-Val-NHCH(CH2C6H40H)C(OH)(S03M)-H, where M=Na,
Cbz-Leu-Val-NHCH(CH2C6H40H)C(OH)(S03M)-H, where M=Na,
Ac-Leu-Gly-Ala-NHCH(CH2C6H40H)C(OH)(S03M)-H, where M=Na,
Ac-Phe-Gly-Ala-NHCH(CH2C6H40H)C(OH)(S03M)-H, where M=Na,
Ac-Tyr-Gly-Ala-NHCH(CH2C6H40H)C(OH)(S03M)-H, where M=Na,
Ac-Phe-Gly-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H, where M=Na,
Ac-Phe-Gly-Ala-NHCH(CH2Ph)C(OH)(S03M)-H, where M=Na,
Ac-Phe-Gly-Val-NHCH(CH2C6H40H)C(OH)(S03M)-H, where M=Na ,
Ac-Phe-Gly-Ala-NHCH(CH2CH2SCH3)(S03M)-H, where M=Na,
Ac-Trp-Leu-Val-NHCH(CH2C6H40H)C(OH)(S03M)-H, where M=Na,
MeO-CO-Val-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H, where M=Na,
MeNCO-Val-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H, where M=Na,
MeO-CO-Phe-Gly-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H, where M=Na,
MeO-CO-Phe-Gly-Ala-NHCH(CH2Ph)C(OH)(S03M)-H, where M=Na,
MeS02-Phe-Gly-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H, where M=Na,
MeS02-Val-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H, where M=Na,
PhCH20(OH)(0)P-Val-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H, where M=Na ,
EtS02-Phe-Gly-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H, where M=Na,
PhCH2S02-Val-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H, where M=Na,
PhCH20(OH)(0)P-Leu-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H, where M=Na ,
PhCH20(OH)(0)P-Phe-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H, where M=Na ,
MeO(OH)(0)P-Leu-Gly-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H, where M=Na,and Phe-urea-Arg-Val-NHCH(CH2C6H4OH)C(OH)(S03M)-H where M=Na.
Salt
The salt used in the bar is a salt of a monovalent cation and an organic anion. The monovalent cation may be for example Na+, K+ or NH4 +. The organic anion may be for example formate, acetate, citrate or lactate. Thus a salt of a monovalent cation and an organic anion may be, for example, sodium formate, potassium formate, ammonium formate, sodium acetate, potassium acetate, ammonium acetate, sodium lactate, potassium lactate, ammonium lactate, mono-sodium citrate, di-sodium citrate, tri-sodium citrate, sodium potassium citrate, potassium citrate, ammonium citrate or the like. A particular embodiment is sodium formate. COMPOSITION OF LAUNDRY SOAP BAR
Laundry soap bar ingredients
The laundry soap bar of the invention comprises the premix and soap. The laundry soap bar may further comprise complexing agents like EDTA and HEDP (1 -hydroxyethane 1 ,1- diphosphonic acid), perfumes and/or different type of fillers. The laundry soap bar may comprise additional compounds. Said compounds are not limited to surfactants e.g. anionic synthetic surfactants, builders, polymeric soil release agents, detergent chelators, polyols such as glycerol, pH controlling compounds, stabilizing agents, fillers, dyes, colorants, dye transfer inhibitors, alkoxylated polycarbonates, suds suppressers, structurants, binders, leaching agents, bleaching activators, clay soil removal agents, anti-redeposition agents, polymeric dispersing agents, brighteners, fabric softeners, perfumes and/or other compounds known in the art.
Amounts
The protease and the optional additional enzyme(s) may each be present in the laundry soap bar in an amount in the range from 0.00001 % to 2.0% by weight calculated as the percentage of enzyme protein of the total composition. Typical amounts are in the range from 0.0001 % to 0.4%, preferably in the range 0.001 % to 0.1 %, most preferably in the range 0.004% to 0.08% by weight of the total composition of the laundry soap bar.
The molar ratio of the peptide aldehyde (or hydrosulfite adduct or hemiacetal adduct) to the protease may be at least 1 :1 or 1 .5:1 , and it may be less than 1000:1 , more preferred less than 500:1 , even more preferred less than 100:1. Alternatively the molar ratio of the peptide al- dehyde (or hydrosulfite adduct or hemiacetal adduct) to the protease may be from 1000:1 to 1 :1 , preferably from 200:1 to 1 :1 , more preferably from 50:1 to 1 :1 , even more preferably from 20:1 to 2:1 , or most preferably the molar ratio is from 10:1 to 2:1. The peptide aldehyde (or hydrosulfite adduct or hemiacetal adduct) may be present in the laundry soap bar in an amount in the range from 0.001 ppm to 0.4% by weight of the total composition. Typical amounts are in the range from 0.01 ppm to 0.1 %, preferably in the range 0.1 ppm to 0.02%, most preferably in the range 1 ppm to 100 ppm by weight of the total composition of the laundry soap bar.
The salt of a monovalent cation and an organic anion may be present in the laundry soap bar in an amount of at least 0.1 % w/w, e.g. at least 1 .0%, at least 1 .5% or at least 2.0% by weight of the total composition of the laundry soap bar. The amount of the salt is typically below 10% w/w, below 8% or below 6%. Alternatively the salt may be present in an amount of 0.1 % to 10% by weight of the total composition, preferably 1 % to 8% and more preferably 2% to 6%.
Polyols
Polyols useful in the present invention are characterized by solubility in water, carbon backbones of length between C2 and C6 and multiple hydroxyl groups, preferably containing between 2 and 6 hydroxyl groups per molecule.
Polyols useful herein include, but are not limited to 1 ,2-butanediol, 3-chloro-1 ,2- propanediol, ethylenediol, 1 ,2-hexanediol, glycerol (glycerine), mannose, propylene glycol, sorbitol, sucrose and mixtures thereof. The polyol level will usually be between 0.5% and 10%, and preferably between 1 % and 5%, and more preferably between 1.5% and 3% by weight of the final bar composition.
Glycerine
During known soap manufacturing glycerine is either being removed due to commercial reasons or it is left in the soap. Therefore soaps can be described as either high glycerine or low glycerine soaps. Low glycerine soaps comprises from 0 to 2% glycerine, if more glycerine is needed it may be added in the manufacturing of the soap while the soap is still liquid to ensure proper uptake on or to soap noodles. In a particular embodiment the laundry soap bar comprises from 1 to 6 % glycerine. In a more particular embodiment of the present invention the laundry soap bar comprises from 2% to 5% glycerine by weight of total laundry soap bar. In a most preferred embodiment the laundry soap bar comprises about 4% glycerine. pH controlling compound
It has been found that it may be necessary to lower the pH to improve stability of the enzyme. The pH controlling agent may be any suitable compound which is able of controlling the pH. In a particular embodiment the pH controlling agent is an acid.
In a particular embodiment the pH controlling compound may be selected from the group consisting of a fatty acid, Acetic acid, Aconitic acid, Adipic acid, Arachidic acid, Arachi- donic acid, Aspartic acid, Behenic acid, Butyric acid, Capric acid, Capronic acid, Cerotic acid,
Citric acid, Formic acid, Fumaric acid, Glutamic acid, Glutaric acid, Glyceric acid, Glyceric acid- 3-phosphate, Glyoxylic acid, Isocitric acid, a-Ketoglutaric acid, Lactic acid, Laurie aicd, Linoleic acid, Linolenic acid, Maleic acid, Malic acid, Malonic acid, Myristic acid, Oleic acid, Oxalic acid, Oxaloacetic acid, Palmitic acid, Palmitoleic acid, Phosphatidic acid, Phosphoenolpyruvic acid, Pimelic acid, Propionic acid, Pyruvic acid, Stearic acid, Succinic acid, Tartraic acid and Valeric acid.
The pH controlling agent will usually be present at a level between 0.04% and about 8%, preferably between 0.1 % and 6%. And more preferably between 0.2% and about 2% by weight of the final bar composition. The amount of pH controlling agent is in a particular embodiment 0.001 % to 5% w/w of the laundry soap bar. In a more particular embodiment the amount of the pH controlling agent is 0.01 % to 2% w/w of the laundry soap bar. In a most particular embodiment the amount of the pH controlling agent is 0.05% to 1 % w/w of the laundry soap bar. In a particular embodiment the amount of pH controlling agent is around 0.5% w/w of the final bar composition.
Moisture content
Moisture enhances the mixing of the premix ingredients. Moisture further provides the laundry soap bar with acceptable feel and other physical characteristics. Moisture e.g. water can be added to the premix by being included with an ingredient and/or as free water added to the premix. The laundry soap bar contains less than 30% w/w water (moisture) content of the final laundry soap bar, or from 10% to 30%, preferably from 20% to 30% water (moisture) content of the final laundry soap bar.
Soap
The soap suitable for use of the present invention includes water soluble salts of higher fatty acids. The soap may be from an animal and/or vegetable source. Soap can be made by direct saponification of fats and oils or by neutralisation of free fatty acids. Suitable soaps are sodium, potassium, ammonium, and alkyloammonium salts of higher fatty acids containing from about 8 to about 24 carbon atoms, such as from 12 to about 18 carbon atoms. In a particular embodiment the soap is selected from sodium and potassium salts of mixtures of fatty acid derived from coconut oil and tallow, such as sodium or potassium tallow and palm oil. Furthermore nut oil, such as coconut or palm kernel oil, may be added in amounts of 5-30% by weight of the final bar.
Synthetic surfactant
Synthetic anionic surfactants which are suitable for use in the present invention include the water soluble salts, preferably the alkali metal, ammonium and alkyl ammonium salts of organic sulphuric reaction products having in their molecular structure an alkyl group containing from about 10 to about 20 carbon atoms and a sulfonic acid or sulphuric acid ester group. Examples of this group of synthetic surfactants are the sodium and potassium alkyi sulfates, especially those obtained by sulfating the higher alcohols (C8-18 carbon atoms) such as those produced by reducing the glycerides of tallow or coconut oil; and the sodium and potassium alkyi benzene sulfonates in which the alkyi group contains from about 9 to about 15 carbon atoms, in straight chain or branched chain configuration. Especially valuable are the linear straight chain alkyi benzene sulfonates (LAS) in which the average number of carbon atoms in the alkyi group is from about 1 1-13, abbreviated as C1 1 -13 LAS. The alkali metal salts, particularly the sodium salts of these surfactants are preferred.
Other examples of an anionic synthetic detergent suitable for use herein are the sodium alkyi glyceryl ether sulfonates (AES), especially those ethers of higher alcohols derived from tallow and coconut oil; sodium coconut oil fatty acid monoglyceride sulfonates and sulfates; and sodium or potassium salts of alkyi ethylene oxide ether sulfates containing about 1 to about 10 units ethylene oxide per molecule and wherein the alkyi group contains from about 10 to about 20 carbon atoms.
In addition, a suitable anionic synthetic detergent also includes the water soluble salts of ester of alpha- sulphonated fatty acids containing from about 6 to about 20 carbon atoms in the fatty acid group and from about 1 to about 10 carbon atoms in the ester group; water soluble salts of 2-acyloxyalkane-1 -sulfonic acids containing from about 2 to about 9 carbon atoms in the acyl group and from 9 to about 23 carbon atoms in the alkane moiety; water soluble salts of olefin and paraffin sulfonates containing from about 12 to about 20 carbon atoms; and beta- alkyloxy alkane sulfonates containing from about 1 to about 3 carbon atoms in the alkyi group and from about 8 to about 20 carbon atoms in the alkane moiety.
Preferred anionic synthetic surfactant examples are C10-18 alkyi sulfates (AS), C10-18 linear alkyi benzene sulfonates (LAS), C10-14 alkyi glyceryl ether sulfonates (AES), and mixtures thereof.
An example of ingredients comprised in a conventional laundry soap bar (syndet bar) is:
linear alkyi benzene sulfonate, coco fatty alcohol sulphate, soda ash, calcium car- bonate, coco fatty alcohol, Ti02, cellulase, diethylenetriamine (methylenephosphonic acid), coco monoethanolamide, fluorecent whitening agents, substituted methylcellulose, perfume, moisture.
An example of detergent ingredients comprised in a conventional laundry soap bar (combo bar) is:
LAS, soap, cellulase, protease, borax, sodium silicate, citric acid, sodium carbonate, glycerol, propylene glycerol, sugar (sorbitol), MgS04, soda ash, talc, moisture. Preparation of the laundry soap bar
The laundry soap bars of the present invention may be processed in conventional laundry soap bar making equipment such as but not limited to: mixers, plodders, e.g a two stage vacuum plodder, extruders, cutters, logo-stampers, cooling tunnels and wrappers. The invention is not limited to preparing the laundry soap bars by any single method. A preferred embodiment of the invention is to prepare a premix containing a soap, a protease and optionally a second enzyme, a protease inhibitor which is a peptide aldehyde or a hydrosulfite adduct or hemiacetal adduct thereof, and a salt of a monovalent cation and an organic anion and then plodding the mixture.
Besides the mixing step and the plodding step the process may further comprise the steps of milling, extruding, cutting, stamping, cooling and/or wrapping.
The premix of the invention may be added to the soap at different stages of the process. In a particular embodiment of the present invention the premix is added to the soap before the refining step. An embodiment of the present invention is to add the enzyme in liquid form. In a particular embodiment of the present invention the protease and optionally a second enzyme is added at the same time as the protease inhibitor or hydrosulfite adduct or hemiacetal adduct thereof.
The mixing may take place in a mixer e.g. in a Double Sigma Amalgator type MSA-100 mixer. After mixing the mixture is transported into a plodder e.g. a Duplex plodder, in which the plodding step is performed. The plodder operates preferably at high vacuum, so that entrapped air/gas is removed.
The product is extruded and the extruded bar subsequently moves to the cutter where the bar is cut to the desired bar length. The pieces are stamped into their final shape in stamping presses. High-speed presses are normally used on high-volume production lines. The form of the stamp is established by designers, taking into account legal considerations such as the minimum net weight for a laundry bar. Optionally the bars are printed with the product brand name. The bar can be cooled, e.g. in a cooling tunnel, before it following normal procedure is wrapped, cased and sent to storage.
Use of laundry soap bars
The laundry soap bar of the invention is for use for cleaning laundry and for washing laundry by hand.
The invention is further summarized in the following paragraphs:
1 a. A laundry soap bar comprising:
a) one or more soaps or synthetic surfactants or any mixture thereof; b) a protease and optionally one or more additional enzymes;
c) one or more protease inhibitors of which at least one is a peptide aldehyde, a hydrosulfite adduct or a hemiacetal adduct thereof; and
d) a salt of a monovalent cation and an organic anion. a. The laundry soap bar of paragraph 1 a which contains less than 0.1 % w/w of the total composition of the laundry soap bar of one or more boron containing compounds. a. The laundry soap bar of any of paragraphs 1 a or 2a which contains less than 30% w/w water (moisture) content of the final laundry soap bar. a. The laundry soap bar of any of paragraphs 1 a to 3a wherein the protease inhibitor is a peptide aldehyde of the formula P-(A)y-L-(B)x-B°-H or a hydrosulfite adduct or hemiacetal adduct thereof, wherein:
H is hydrogen;
B° is a single amino acid residue with L- or D-configuration of the formula -NH- CH(R)-C(=0)-;
B is independently a single amino acid connected to B° via the C-terminal of the amino acid;
L is absent or independently a linker group of the formula -C(=0)-, -C(=0)-C(=0)-, -
C(=S)-, -C(=S)-C(=S)- or -C(=S)-C(=0)-;
A is absent if L is absent or is independently a single amino acid residue connected to L via the /V-terminal of the amino acid;
P is selected from the group consisting of hydrogen or if L is absent an /V-terminal protection group;
x is 1 , 2 or 3;
y is 0, 1 , or 2,
R is independently selected from the group consisting of C1-6 alkyl, C6-io aryl or C7-10 arylalkyl optionally substituted with one or more, identical or different, substit- uent's R';
R' is independently selected from the group consisting of halogen, -OH, -OR", -SH, - SR", -NH2, -NHR", -NR"2, -C02H, -CONH2, -CONHR", -CONR"2, - NHC(=N)NH2; and
R" is a C1-6 alkyl group. a. The laundry soap bar of paragraph 4a wherein B° is selected from the group consisting of D- or L-form of arginine (Arg), 3,4-dihydroxyphenylalanine, isoleucine (lie), leucine (Leu), methionine (Met), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), m-tyrosine, p- tyrosine (Tyr) and valine (Val). a. The laundry soap bar of any of paragraphs 4a to 5a wherein B1 is selected from the group consisting of alanine (Ala), cysteine (Cys), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (Nle), norvaline (Nva), proline (Pro), serine (Ser), threonine (Thr) and valine (Val). a. The laundry soap bar of any of paragraphs 4a to 6a wherein B2 is selected from the group consisting of alanine (Ala), arginine (Arg), capreomycidine (Cpd), cysteine (Cys), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), and valine (Val). a. The laundry soap bar of any of paragraphs 4a to 7a wherein B3 is selected from the group consisting of isoleucine (lie), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), phenylglycine, tyrosine (Tyr), tryptophan (Trp) and valine (Val). a. The laundry soap bar of any of paragraphs 4a to 8a wherein L is -C(=0)- and y is 1 or 2. 0a. The laundry soap bar paragraph 9a wherein A1 is selected from the group consisting of alanine (Ala), arginine (Arg), capreomycidine (Cpd), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), threonine (Thr), tyrosine (Tyr), tryptophan (Trp) and valine (Val). 1 a. The laundry soap bar of paragraph 4a wherein the protease inhibitor is one of the following peptide aldehydes or a hydrosulfite adduct thereof: Cbz-Arg-Ala-Tyr-H, Ac-Gly-Ala- Tyr-H, Cbz-Gly-Ala-Tyr-H, Cbz-Gly-Ala-Leu-H, Cbz-Val-Ala-Leu-H, Cbz-Gly-Ala-Phe-H, Cbz-Gly-Ala-Val-H, Cbz-Gly-Gly-Tyr-H, Cbz-Gly-Gly-Phe-H, Cbz-Arg-Val-Tyr-H, Cbz-Leu- Val-Tyr-H, Ac-Leu-Gly-Ala-Tyr-H, Ac-Phe-Gly-Ala-Tyr-H, Ac-Tyr-Gly-Ala-Tyr-H, Ac-Phe- Gly-Ala-Leu-H, Ac-Phe-Gly-Ala-Phe-H, Ac-Phe-Gly-Val-Tyr-H, Ac-Phe-Gly-Ala-Met-H, Ac- Trp-Leu-Val-Tyr-H, MeO-CO-Val-Ala-Leu-H, MeNCO-Val-Ala-Leu-H, MeO-CO-Phe-Gly- Ala-Leu-H, MeO-CO-Phe-Gly-Ala-Phe-H, MeS02-Phe-Gly-Ala-Leu-H, MeS02-Val-Ala- Leu-H, PhCH20-P(OH)(0)-Val-Ala-Leu-H, EtS02-Phe-Gly-Ala-Leu-H, PhCH2S02-Val-Ala- Leu-H, PhCH20-P(OH)(0)-Leu-Ala-Leu-H, PhCH20-P(OH)(0)-Phe-Ala-Leu-H, or MeO- P(OH)(0)-Leu-Gly-Ala-Leu-H, α-ΜΑΡΙ, β-ΜΑΡΙ, Phe-C(=0)-Arg-Val-Tyr-H, Phe-C(=0)- Gly-Gly-Tyr-H, Phe-C(=0)-Gly-Ala-Phe-H, Phe-C(=0)-Gly-Ala-Tyr-H, Phe-C(=0)-Gly-Ala- L-H, Phe-C(=0)-Gly-Ala-Nva-H, Phe-C(=0)-Gly-Ala-Nle-H, Tyr-C(=0)-Arg-Val-Tyr-H, Tyr- C(=0)-Gly-Ala-Tyr-H, Phe-C(=S)-Arg-Val-Phe-H, Phe-C(=S)-Arg-Val-Tyr-H, Phe-C(=S)- Gly-Ala-Tyr-H, Antipain, GE20372A, GE20372B, Chymostatin A, Chymostatin B, and
Chymostatin C.
12a. The laundry soap bar of any of paragraphs 1 a to 1 1 a wherein the molar ratio of the peptide aldehyde (or hydrosulfite adduct or hemiacetal adduct) to the protease is between 1 :1 and 1000:1.
13a. The laundry soap bar of any of paragraphs 1 a to 12a wherein the peptide aldehyde (or hydrosulfite adduct or hemiacetal adduct) is present in an amount of 0.001 ppm to 0.4% by weight of the total composition of the laundry soap bar.
14a. The laundry soap bar of any of paragraphs 1 a to 13a, wherein the protease is present in an amount of 0.0001 % to 5.0 % by weight calculated as the percentage of enzyme protein to the total composition of the laundry soap bar. 15a. The laundry soap bar of any of paragraphs 1 a to 14a wherein monovalent cation is sodium, potassium or ammonium.
16a. The laundry soap bar of any of paragraphs 1 a to 15a wherein the monovalent organic anion is formate, acetate, citrate or lactate.
17a. The laundry soap bar of any of paragraphs 1 a to 16a wherein the salt of a monovalent cation and an organic anion is sodium formate.
18a. The laundry soap bar of any of paragraphs 1 a to 17a wherein the salt of a monovalent cation and an organic anion is present in an amount of 0.1 % to 10% by weight of the total composition.
19a. The laundry soap bar of any of paragraphs 1 a to 18a comprising of one or more additional enzymes selected from the list consisting of amylase, lipase, cellulase, mannanase, pec- tate lyase, carbohydrase and protease. a. The laundry soap bar of any of paragraphs 1 a to 19a, wherein the protease is obtained from Bacillus subtilis; Bacillus licheniformis; Bacillus lentus and any mixtures thereof. a. The laundry soap bar of any of paragraphs 1 a to 20a, wherein the soap is from an animal and/or vegetable source. a. A process of preparing the laundry soap bars according to any of paragraphs 1 a to 21 a comprising the steps of:
a) mixing a soap; a protease; a protease inhibitor which is a peptide aldehyde or a hy- drosulfite adduct or hemiacetal adduct thereof; and a salt of a monovalent cation and an organic anion; and
b) plodding the mixture from step (a). a. The process of paragraph 22a, wherein the protease; the protease inhibitor which is a peptide aldehyde or a hydrosulfite adduct or hemiacetal adduct thereof; and a salt of a monovalent cation and an organic anion is mixed together prior to step (a). a. The process of any of paragraphs 22a to 23a, wherein a premix comprising a protease; a protease inhibitor which is a peptide aldehyde or a hydrosulfite adduct or hemiacetal ad- duct thereof; a salt of a monovalent cation and/or a monovalent organic anion; a polyol and a pH controlling compound is prepared prior to step (a). a. A premix to be used in the process of any of paragraphs 22a to 24a, comprising a protease; a protease inhibitor which is a peptide aldehyde or a hydrosulfite adduct or hemiace- tal adduct thereof; and a salt of a monovalent cation and/or a monovalent organic anion.
The invention is further summarised in the below embodiments:
1 . A laundry soap bar comprising:
a) one or more soaps or synthetic surfactants or any mixture thereof;
b) a protease and optionally one or more additional enzymes;
c) one or more protease inhibitors of which at least one is a peptide aldehyde, a hydrosulfite adduct or a hemiacetal adduct thereof; and
d) a salt of a monovalent cation and an organic anion. The laundry soap bar of embodiment 1 which contains less than 0.1 % w/w of the total composition of the laundry soap bar of one or more boron containing compounds.
The laundry soap bar of any of embodiments 1 or 2, which contains less than 30% w/w water (moisture) content of the final laundry soap bar.
The laundry soap bar of any of embodiments 1 to 3, wherein the protease inhibitor is a peptide aldehyde of the formula P-(A)y-L-(B)x-B°-H or a hydrosulfite adduct or hemiacetal adduct thereof, wherein:
x. H is hydrogen;
xi. B° is a single amino acid residue with L- or D-configuration of the formula -NH- CH(R)-C(=0)-;
xii. x is 1 , 2 or 3 for (B)x, and B is independently a single amino acid connected to B° via the C-terminal of the B amino acid
xiii. L is absent or L is independently a linker group of the formula -C(=0)-,
-C(=0)-C(=0)-, -C(=S)-, -C(=S)-C(=S)- or -C(=S)-C(=0)-;
xiv. y is 0, 1 or 2 for (A)y, and A is independently a single amino acid residue connected to L via the /V-terminal of the A amino acid, with the proviso that if L is absent then A is absent;
xv. P is selected from the group consisting of hydrogen and an /V-terminal protection group, with the proviso that if L is absent then P is an /V-terminal protection group;
xvi. R is independently selected from the group consisting of C1-6 alkyl, C6-io aryl or C7-10 arylalkyl optionally substituted with one or more, identical or different, sub- stituent's R';
xvii. R' is independently selected from the group consisting of halogen, -OH, -OR", - SH, -SR", -NH2, -NHR", -NR"2, -C02H, -CONH2, -CONHR", -CONR"2, - NHC(=N)NH2; and
xviii. R" is a C1-6 alkyl group.
The laundry soap bar of any of the preceding embodiments, wherein the protease inhibitor is a hydrosulfite adduct of a peptide aldehyde or hemiacetal adduct of a peptide aldehyde.
The laundry soap of any of the preceding embodiments, wherein the hydrosulfite adduct of a peptide aldehyde is of the formula P-(A)y-L-(B)x-N(H)-CHR-CH(OH)-S03M, wherein ix. M is hydrogen or an alkali metal;
x. x is 1 , 2 or 3 for (B)x, and B is independently a single amino acid connected to B° via the C-terminal of the B amino acid
xi. L is absent or L is independently a linker group of the formula -C(=0)-,
-C(=0)-C(=0)-, -C(=S)-, -C(=S)-C(=S)- or -C(=S)-C(=0)-;
xii. y is 0, 1 or 2 for (A)y, and A is independently a single amino acid residue connected to L via the /V-terminal of the A amino acid, with the proviso that if L is absent then A is absent;
xiii. P is selected from the group consisting of hydrogen and an /V-terminal protection group, with the proviso that if L is absent then P is an /V-terminal protection group;
xiv. R is independently selected from the group consisting of C1-6 alkyl, C6-io aryl or C7-10 arylalkyl optionally substituted with one or more, identical or different, sub- stituent's R';
xv. R' is independently selected from the group consisting of halogen, -OH, -OR", -
SH, -SR", -NH2, -NHR", -NR"2, -C02H, -CONH2, -CONHR", -CONR"2, - NHC(=N)NH2; and
xvi. R" is a C1-6 alkyl group. 7. The laundry soap bar of embodiment 6, wherein M is Na or K.
8. The laundry soap bar of any of embodiments 4 to 7, wherein R is a C7-10 arylalkyl substituted with -OH. 9. The laundry soap bar of any of embodiments 4 to 8, wherein R is a C7 arylalkyl substituted with -OH.
10. The laundry soap bar of any of embodiments 4 to 7, wherein B° is selected from the group consisting of D- or L-form of arginine (Arg), 3,4-dihydroxyphenylalanine, isoleucine (lie), leucine (Leu), methionine (Met), norleucine (NIe), norvaline (Nva), phenylalanine
(Phe), m-tyrosine, p-tyrosine (Tyr) and valine (Val).
1 1. The laundry bar of any of embodiments 4 to 10, wherein B is an amino acid with L- configuration. 12. The laundry soap bar of any of embodiments 4 to 1 1 , wherein B1 is selected from the group consisting of alanine (Ala), cysteine (Cys), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (Nle), norvaline (Nva), proline (Pro), serine (Ser), threonine (Thr) and valine (Val).
13. The laundry soap bar of any of embodiments 4 to 12, wherein B2 is selected from the group consisting of alanine (Ala), arginine (Arg), capreomycidine (Cpd), cysteine (Cys), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), and valine (Val).
14. The laundry soap bar of any of embodiments 4 to 13, wherein B3 is selected from the group consisting of isoleucine (lie), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), phenylglycine, tyrosine (Tyr), tryptophan (Trp) and valine (Val). 15. The laundry soap bar of any of embodiments 4 to 12, wherein x is 1 .
16. The laundry soap bar of any of embodiments 4 to 13, wherein x is 2.
17. The laundry soap bar of any of embodiments 4 to 14, wherein x is 3
18. The laundry soap bar of any of embodiments 4 to 17, wherein L is -C(=0)- or -C(S) and y is 1 or 2
19. The laundry soap bar of any of embodiments 4 to 18, wherein A1 is selected from the group consisting of alanine (Ala), arginine (Arg), capreomycidine (Cpd), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), threonine (Thr), tyrosine (Tyr), tryptophan (Trp) and valine (Val).
20. The laundry soap bar of any of embodiments 4 to 19, wherein A2 is selected from the group consisting of arginine (Arg), isoleucine (lie), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), phenylglycine, Tyrosine (Tyr), tryptophan (Trp) and valine (Val).
21. The laundry soap of any of embodiments 4 to 20, wherein A1 is phenylalanine or tyro- sine.
22. The laundry soap bar of any of embodiments 4 to 21 , wherein P is hydrogen. 23. The laundry soap bar of any of embodiments 4 to 17, wherein L is absent.
24. The laundry soap bar of any of embodiments 4 to 17 and 23, wherein A is absent. 25. The laundry soap bar of any of embodiments 4 to 17 and 23-24, wherein P is selected from the group consisting of formyl, acetyl (Ac), benzoyl (Bz), trifluoroacetyl, methoxy- succinyl, fluorenylmethyloxycarbonyl (Fmoc), methoxycarbonyl (MEO-CO), (fluorometh- oxy)carbonyl, benzyloxycarbonyl (Cbz), t-butyloxycarbonyl (Boc), adamantyloxycarbon- yl, p-methoxybenzyl carbonyl (Moz), benzyl (Bn), p-methoxybenzyl (PMB), p- methoxyphenyl (PMP), methoxyacetyl, methylamino carbonyl (MeNCO), methylsulfonyl
(MeS02), ethylsulfonyl (EtS02), benzylsulfonyl (PhCH2S02), methylphosphoramidyl (MeOP(OH)(=0)) and benzylphosphoramidyl (PhCH20-P(OH)(0)).
26. The laundry soap bar of any of embodiments 4 to 17 and 23-25, wherein P is selected from the group consisting of acetyl (Ac), methoxycarbonyl (MEO-CO), methylsulfonyl (MeS02), ethylsulfonyl (EtS02), benzyloxycarbonyl (Cbz) and methylphosphoramidyl (MeOP(OH)(=0)).
27. The laundry soap bar of any of embodiments 4 to 17 and 23-26, wherein P is benzyloxycarbonyl (Cbz).
28. The laundry soap bar of any of embodiments 4 to 17 and 23-27, wherein the peptide aldehyde adduct is L-Alaninamide, /V-[(phenylmethoxy)carbonyl]glycyl-/V-[2-hydroxy-1-[(4- hydroxyphenyl)methyl]-2-sulfoethyl]-, sodium salt (1 :1 ).
29. The laundry soap bar of any of embodiments 4 to 27, wherein the protease inhibitor is one of the following peptide aldehydes or a hydrosulfite adduct thereof: Cbz-Arg-Ala-Tyr- H, Ac-Gly-Ala-Tyr-H, Cbz-Gly-Ala-Tyr-H, Cbz-Gly-Ala-Leu-H, Cbz-Val-Ala-Leu-H, Cbz- Gly-Ala-Phe-H, Cbz-Gly-Ala-Val-H, Cbz-Gly-Gly-Tyr-H, Cbz-Gly-Gly-Phe-H, Cbz-Arg- Val-Tyr-H, Cbz-Leu-Val-Tyr-H, Ac-Leu-Gly-Ala-Tyr-H, Ac-Phe-Gly-Ala-Tyr-H, Ac-Tyr- Gly-Ala-Tyr-H, Ac-Phe-Gly-Ala-Leu-H, Ac-Phe-Gly-Ala-Phe-H, Ac-Phe-Gly-Val-Tyr-H, Ac-Phe-Gly-Ala-Met-H, Ac-Trp-Leu-Val-Tyr-H, MeO-CO-Val-Ala-Leu-H, MeNCO-Val- Ala-Leu-H, MeO-CO-Phe-Gly-Ala-Leu-H, MeO-CO-Phe-Gly-Ala-Phe-H, MeS02-Phe- Gly-Ala-Leu-H, MeS02-Val-Ala-Leu-H, PhCH20-P(OH)(0)-Val-Ala-Leu-H, EtS02-Phe- Gly-Ala-Leu-H, PhCH2S02-Val-Ala-Leu-H, PhCH20-P(OH)(0)-Leu-Ala-Leu-H, PhCH20- P(OH)(0)-Phe-Ala-Leu-H, or MeO-P(OH)(0)-Leu-Gly-Ala-Leu-H, α-ΜΑΡΙ, β-ΜΑΡΙ , Phe- C(=0)-Arg-Val-Tyr-H, Phe-C(=0)-Gly-Gly-Tyr-H, Phe-C(=0)-Gly-Ala-Phe-H, Phe-C(=0)- Gly-Ala-Tyr-H, Phe-C(=0)-Gly-Ala-L-H, Phe-C(=0)-Gly-Ala-Nva-H, Phe-C(=0)-Gly-Ala- Nle-H, Tyr-C(=0)-Arg-Val-Tyr-H, Tyr-C(=0)-Gly-Ala-Tyr-H, Phe-C(=S)-Arg-Val-Phe-H, Phe-C(=S)-Arg-Val-Tyr-H, Phe-C(=S)-Gly-Ala-Tyr-H, Antipain, GE20372A, GE20372B, Chymostatin A, Chymostatin B, and Chymostatin C.
30. The laundry soap bar of embodiments 6-27, wherein the wherein the adduct of a peptide aldehyde protease inhibitor is selected from the group consisting of:
Cbz-Gly-Ala-N(H)-CH(CH2-p-C6H4OH)-CH(OH)-S03Na,
Cbz-Gly-Ala-N(H)-CH(CH2Ph)-CH(OH)-S03Na,
MeO-CO_Val-Ala-N(H)-CH(CH2CH(CH3)2)-CH(OH)-S03Na,
Cbz-Arg-Ala-NHCH(CH2C6H4OH)C(OH)(S03M)-H where M=Na,
Ac-Gly-Ala-NHCH(CH2C6H4OH)C(OH)(S03M)-H, where M=Na,
Cbz-Gly-Ala-NHCH(CH2C6H4OH)C(OH)(S03M)-H, where M=Na,
Cbz-Gly-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H, where M=Na,
Cbz-Val-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H, where M=Na,
Cbz-Gly-Ala-NHCH(CH2Ph)C(OH)(S03M)-H, where M=Na,
Cbz-Gly-Ala-NHCH(CH(CH3)2)C(OH)(S03M)-H, where M=Na,
Cbz-Gly-Gly-NHCH(CH2C6H4OH)C(OH)(S03M)-H, where M=Na,
Cbz-Gly-Gly-NHCH(CH2Ph)C(OH)(S03M)-H, where M=Na,
Cbz-Arg-Val-NHCH(CH2C6H4OH)C(OH)(S03M)-H, where M=Na,
Cbz-Leu-Val-NHCH(CH2C6H4OH)C(OH)(S03M)-H, where M=Na,
Ac-Leu-Gly-Ala-NHCH(CH2C6H4OH)C(OH)(S03M)-H, where M=Na,
Ac-Phe-Gly-Ala-NHCH(CH2C6H4OH)C(OH)(S03M)-H, where M=Na,
Ac-Tyr-Gly-Ala-NHCH(CH2C6H4OH)C(OH)(S03M)-H, where M=Na,
Ac-Phe-Gly-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H, where M=Na,
Ac-Phe-Gly-Ala-NHCH(CH2Ph)C(OH)(S03M)-H, where M=Na,
Ac-Phe-Gly-Val-NHCH(CH2C6H4OH)C(OH)(S03M)-H, where M=Na ,
Ac-Phe-Gly-Ala-NHCH(CH2CH2SCH3)(S03M)-H, where M=Na,
Ac-Trp-Leu-Val-NHCH(CH2C6H4OH)C(OH)(S03M)-H, where M=Na,
MeO-CO-Val-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H, where M=Na,
MeNCO-Val-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H, where M=Na,
MeO-CO-Phe-Gly-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H, where M=Na,
MeO-CO-Phe-Gly-Ala-NHCH(CH2Ph)C(OH)(S03M)-H, where M=Na,
MeS02-Phe-Gly-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H, where M=Na,
MeS02-Val-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H, where M=Na, PhCH20(OH)(0)P-Val-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H! where M=Na , EtS02-Phe-Gly-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H! where M=Na,
PhCH2S02-Val-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H! where M=Na,
PhCH20(OH)(0)P-Leu-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H! where M=Na, PhCH20(OH)(0)P-Phe-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H! where M=Na,
MeO(OH)(0)P-Leu-Gly-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H! where M=Na, and Phe-urea-Arg-Val-NHCH(CH2C6H40H)C(OH)(S03M)-H where M=Na.
31. The laundry soap bar of any of embodiments 1 to 30, wherein the molar ratio of the pep- tide aldehyde or hydrosulfite adduct or hemiacetal adduct to the protease is between 1 :1 and 1000:1.
32. The laundry soap bar of any of embodiments 1 to 31 , wherein the peptide aldehyde or hydrosulfite adduct or hemiacetal adduct is present in an amount of 0.001 ppm to 0.4% by weight of the total composition of the laundry soap bar.
33. The laundry soap bar of any of embodiments 1 to 32, wherein the protease is present in an amount of 0.0001 % to 5.0 % by weight calculated as the percentage of enzyme protein to the total composition of the laundry soap bar.
34. The laundry soap bar of any of embodiments 1 to 33, wherein monovalent cation is sodium, potassium or ammonium.
35. The laundry soap bar of any of embodiments 1 to 34, wherein the monovalent cation is sodium.
36. The laundry soap bar of any of embodiments 1 to 35, wherein the monovalent organic anion is formate, acetate, citrate or lactate. 37. The laundry soap bar of any of embodiments 1 to 36, wherein the salt of a monovalent cation and an organic anion is sodium formate.
38. The laundry soap bar of any of embodiments 1 to 37, wherein the salt of a monovalent cation and an organic anion is present in an amount of 0.1 % to 10% by weight of the total composition. . The laundry soap bar of any of embodiments 1 to 38 comprising of one or more additional enzymes selected from the list consisting of amylase, lipase, cellulase, man- nanase, pectate lyase, carbohydrase and protease. . The laundry soap bar of any of embodiments 1 to 39, wherein the protease is obtained from Bacillus subtilis; Bacillus licheniformis; Bacillus lentus and any mixtures thereof. . The laundry soap bar of any of embodiments 1 to 40, wherein the soap is from an animal and/or vegetable source. . A process of preparing the laundry soap bars according to any of embodiments 1 to 41 comprising the steps of:
a) mixing a soap; a protease; a protease inhibitor which is a peptide aldehyde or a hy- drosulfite adduct or hemiacetal adduct thereof; and a salt of a monovalent cation and an organic anion; and
b) plodding the mixture from step (a). . The process of embodiment 42, wherein the protease; the protease inhibitor which is a peptide aldehyde or a hydrosulfite adduct or hemiacetal adduct thereof; and a salt of a monovalent cation and an organic anion is mixed together prior to step (a). . The process of any of embodiments 42 to 43, wherein a premix comprising a protease; a protease inhibitor which is a peptide aldehyde or a hydrosulfite adduct or hemiacetal adduct thereof; a salt of a monovalent cation and/or a monovalent organic anion; a polyol and a pH controlling compound is prepared prior to step (a). . A premix to be used in the process of any of embodiments 42 to 44, comprising a protease; a protease inhibitor which is a peptide aldehyde or a hydrosulfite adduct or hemiacetal adduct thereof; and a salt of a monovalent cation and/or a monovalent organic anion. . Use of a laundry soap bar of any of embodiments 1 to 41 for washing a textile. . Use of embodiment 46, wherein the washing is done by hand. Preparation of Laundry Soap Bars
Description of base used
The base properties included:
TFM (Total fatty matter) (80/20 tallow/vegetable)
Free alkalinity (NaOH) < 0.2%
Total alkalinity (NaOH) < 0.3%
Moisture -24% (measured using a moisture analyzer balance). Preparation of premix
1. Glycerine and water (if any) were mixed in a beaker while stirring at room temperature. Mix order is not important for this step.
2. Sodium formate (if any) is then added to the glycerine solution and mixed until dispersed.
3. Citric acid is then added with stirring until dispersed.
4. The enzyme(s) with/without inhibitor are added last with continued stirring.
5. The slurry is allowed to continue stirring for at least 20 minutes until fully dispersed. Addition of premix to soap base and preparation of bars
The premix was mixed with the soap base using a Sunlab mixer model 7.5 and then processed using a Sunlab Merton sampler model U. The bars were stamped using a Sunlab Stamper model LMK-1 .
Physical property Assays
Measurements were done, with modifications noted below, as described in L. Spitz,
"Soap Manufacturing Technology", AOCS Press, Urbana, IL, 2009, Chapter 15 (ISBN-13: 978- 1893997615).
Hardness assay
Hardness, or firmness, of laundry soap bars is defined as the maximum peak force (in grams) during the first compression cycle for the probe to penetrate a set depth into the laundry soap bar. Laundry soap bar hardness was measured at room temperature (~23°C) assimilating ASTM D937-92 (ASTM International, West Conshohocken, PA) method by using a Texture Analyzer (model TA-XT2) with a 45° conical probe (model TA-15) using 5kg load cell, compression test mode, and 1 mm/sec test speed. The test bars are kept in a polythene bag or wrapper at room temperature until needed for testing to keep from drying. pH assay pH is measured on a 1 %w solution using a pH meter. The solution is prepared with distilled water.
Colour assay
Colour is described visually in terms of its colour and/or translucency. Mush assay
Mush is the description for the gelatinous hydrated soap formed when toilet bars are left in contact with water. Mush is measured by determining the amount of soap gel formed after exposure to moisture.
First, the weight and dimension of the dry soap is determined. The toilet bar is skew- ered near to the top using a wire or rod so that it can hang inside a beaker with about half of the bar immersed. The bar is suspended in the beaker that is filled with deionized water at 40° C to the point where about half of the bar is immersed and left for two hours to cool. The bar is removed from the water, the water is drained, and the bar is allowed to drain suspended for 15 minutes, after which the bar is weighted. The soft soap gel developed on the immersed part of the bar is scraped off using a steel spatula avoiding scraping off the harder soap underneath. Weigh the soft soap removed directly or weigh the bar after removal of the soft soap and subtract from the weight of wet soap to obtain an indirect result. The surface area of the soap immersed is obtained by measuring the circumference of the soap at the point of immersion and the depth from the middle point of the immersion to the end of the soap. Record the surface ar- ea (circumference x depth + area of collar), total water adsorbed (Wet weight - Dry weight) and mush (weight of soft soap removed). These measurements are used to calculate the mushing factor where:
Mushing factor (gm/sq.cm) = Weight of Mush (g) / Surface Area (sq. cm.).
The mushing factor is evaluated using the following scale:
< 0.2 good;
> 0.2 - 0.3 satisfactory;
> 0.3 not satisfactory.
Wet cracking assay
Wet cracking is the term used to describe the cracks which can appear on the surface of a toilet bar after it has been used and allowed to dry for a specified period. First, test laundry soap bars are conditioned for at least 15 hours at room temperature. The bars are then totally immersed in water (30°C initial) for a period of one hour. They are then cleansed of mush that has been formed, taking special care to remove mush from the ends of the soap, by rubbing between the hands under the water. They are then removed and placed on a wire mesh tray where they are allowed to dry at normal room temperature. The degree of cracking is assessed by visually examining the bars after 2, 24, and/or 48 hours, and rating them using the photo scale shown in page 423 of the reference book "Soap Manufacturing Technology" mentioned above.
The wet cracking is evaluated using the following scale:
1-2 good;
3 satisfactory;
4 less satisfactory;
≥5 not satisfactory.
Wet bar (washdown) feel assay
The main purpose of this test is to establish any negative effect on bar feel in use caused by poor processing or by additives. The test is carried out by hand washing with the bar and grading the product on a scale ranging from smooth to very gritty. A bowl is filled with about 2L of tap water at the desired start/use temperature (20 °C, 30 °C, and/or 40 °C). The bar is immersed in the water for 30 seconds and is then rubbed between the hands under the water for at least 3 times of 10 rubs each to remove any rough edges or relief from the stamped design. The bar is rubbed under the water for at least 30 seconds and the feel of the bar assessed on a smoothness / sandiness scale, i.e. the presence of hard particles which detract from a smooth feeling of the bar on the skin, using the following scale:
0 - Smooth/no roughness (satisfactory);
1 - Slightly sandy (satisfactory);
2 - Sandy (less satisfactory);
3 - Very sandy/gritty (not satisfactory);
≥4 - Very gritty (not satisfactory).
If the result is not rated satisfactory, then the test is repeated at successively higher temperatures until the product is rated as satisfactory and the testing is discontinued at that point.
Foam assay
Foam height was measured according to ASTM D1 173 (ASTM International, West Conshohocken, PA) using 1 g soap/L tap water (-25 ppm water hardness) at initial time 0 and after 5 minutes. Terg-O-tometer (TOM) wash assay
The Tergo-To-Meter (TOM) is a medium scale model wash system that can be applied to test different wash conditions simultaneously. A TOM is basically a large temperature controlled water bath with open metal beakers submerged into it. Each beaker constitutes one small top loader style washing machine and during an experiment, each of them will contain a solution of a specific detergent/enzyme system and the soiled and unsoiled fabrics its performance is tested on. Mechanical stress is achieved by a rotating stirring arm, which stirs the liquid within each beaker. Because the TOM beakers may have no lid, it is possible to withdraw samples during a TOM experiment and assay for information on-line during wash.
The TOM model wash system is mainly used in benchtop scale testing of detergents and enzymes at e.g, US or LA AP wash conditions. In a TOM experiment, factors such as the ballast to soil ratio and the fabric to wash liquor ratio can be varied. The TOM provides the ability to conduct screenings at small scale before conducting the more time consuming full scale experiments in top loader washing machines.
Equipment: The water bath has a steel beakers and 1 rotating arm per beaker with capacity of up to 1200 mL of detergent solution. Temperature ranges from 5 to 80°C. The water bath has to be filled up with deionised water. Rotational speed can be set up to 120 rpm/min.
1. Set temperature in the Terg-O-Tometer and start the rotation in the water bath. Wait for the temperature to adjust (tolerance is +/- 0,5 °C)
2. All beakers shall be clean and without traces of prior test material.
3. Prepare wash solution with desired amount of detergent, temperature and water hardness in a bucket. Let soap bar dissolve during magnet stirring for up to 10 min. Wash solution shall be used within 30 to 60 min after preparation.
4. Add 1000 ml wash solution into a TOM beaker
5. Start agitation at 100 rpm.
6. Sprinkle the swatches into the beaker and then the ballast load.
7. Time measurement start when the swatches and ballast are added to the beaker.
8. Wash for 20 minutes
9. Stop agitation
10. Transfer the wash load from TOM beaker to a sieve and rinse with cold tap water
1 1. Separate the soil swatches from the ballast load. The soil swatches are transferred to a 5 L beaker with cold tap water under running water. Keep the ballast load separately for the coming inactivation. 12. Set the timer to 5 minutes.
13. Press gently the water out by hand and place the test swatches on a tray covered with a paper. Add another paper on top of the swatches.
14. Let the swatches dry overnight and then measure using a colorimeter as described be- low.
Test protease
Savinase 16L, available from Novozymes A/S, containing the subtilisin Bacillus lentus (SEQ ID NO: 2) was used as the test protease.
Test Material
The test material EMPA1 17 is blood/milk/ink on cotton/polyester and was obtained from EMPA Testmaterials AG, Movenstrasse 12, CH-9015 St. Gallen, Switzerland.
Cleaning and Stain Removal assay
Wash performance was measured at laboratory scale using a method similar to ASTM D3050 (ASTM International, West Conshohocken, PA) with the modifications mentioned here. Soiled test swatches (EMPA1 17) were washed in a Terg-O-tometer (see description above) using 3 gr/L laundry soap bar at 100 rpm. The swatches were washed at 30°C using 150 ppm water hardness for 20 minutes then rinsed with tap water for 5 minutes. After drying, the cleanliness of the swatches was determined by light remission using a colorimeter measurement of 460 nm. Enzyme stability/Activity assay
The storage stability test was done by incubating the laundry soap bar for eight weeks at 30°C and/or 37°C in closed vials. Following the incubation, the residual protease activity was determined the laundry soap bar base using a slightly modified method described in H. Lund, S.G. Kaasgaard, P. Skagerlind, L. Jorgensen, C.I. Jorgensen and M. van de Weert, J Surfact. Deterg., (2012), 15, 9-21.
Laundry soap bars containing enzymes were crumbled and incubated in an oven at 30 or 37°C in closed vials for the desired time intervals. Following the incubation, the residual activity was determined. Unless otherwise stated, residual activity was calculated relative to the activity of the sample at time T=0 days. Protease activity was measured on a Konelab 30 analyzer (Thermo clinical Labsystems, Espoo, Finland). The sample was diluted in 50 mM borate, 150 mM KCI, pH 9.0 containing 2% (w/v) Na2S04 (160 mM) and 0.0025% (w/v) alcohol ethoxylate. Hydrolysis of the substrate Λ/,/V-di methyl casein (0.32% (w/v) was monitored at 40°C, pH 8.3 at wavelength 405 nm after 8 min incubation time over a period of 2 min. For the following examples, acceptable enzyme stability for an enzyme is having at least 60% residual activity when stored for eight weeks at 30°C as determined by the hydrolysis of the substrate Λ/,/V-dimethyl casein.
Example 1 : Properties of Laundry soap bar with and without Protease Inhibitor
The laundry soap bars were prepared according to the Preparation of Laundry Soap
Bars method described above and the composition is given in table 1. In this experiment, laundry soap bars with and without protease inhibitor and with and without sodium formate were prepared. The laundry soap bars were tested using the Cleaning and Stain Removal and Enzyme Stability/Activity assays and the results are shown in tables 2 and 3. The physical proper- ties of the laundry soap bars were also assessed and the results are reported in tables 4-7.
Table 1 : Formulation of laundry soap bars
The total protease concentration was 0.016% by weight calculated as the percentage of enzyme protein to the total composition of the laundry soap bar. The protease inhibitor, if used, was where: P=Cbz, B2=Gly; B1= Ala; B° = Tyr (so R=PhCH2, R -OH), x=2, y=0, L=A=absent and M=Na, L-Alaninamide, /V-[(phenylmethoxy)carbonyl]glycyl-/V-[2-hydroxy-1 -[(4- hydroxyphenyl)methyl]-2-sulfoethyl]-, sodium salt (1 :1 )).
Figure imgf000043_0001
The mentioned amounts of ingredients are referred to in %w.
Table 2: Storage stability of Savinase in laundry soap bars after 4 and 8 weeks at 30°C and 37°C based on wash performance of EMPA 1 17 swatches in a Terg-O-tometer
Figure imgf000043_0002
2 55.3 54.6 53.7 54.6 0.1 -0.6 -1.5 -0.6
3 67.3 65.1 57.3 55.3 12.1 9.9 2.1 0.0
4 69.0 68.9 68.1 67.6 13.8 13.6 12.9 12.3 aValues compared to bar only (no enzyme) value of 55.2. Stabilised control (Savinase + 4-formylphenylboronic acid): 69.3.
Table 3: Storage stability of Savinase in laundry soap bars after 4 and 8 weeks at 30°C and 37°C as determined by the hydrolysis of /V,/V-dimethyl casein
Figure imgf000044_0001
Tables 2 and 3 show that the Savinase without any in-situ protease stabiliser present had no stability after just 4 weeks at 30°C as measured by the wash performance of blood/milk/ink on cotton/polyester swatches and by the percentage of residual activity as determined by the hydrolysis of Λ/,/V-dimethyl casein. In comparison, the addition of the protease inhibitor and especially together with the addition of sodium formate significantly increased storage stability even after 8 weeks at 37°C.
Table 4: Mushing Factor of the laundry soap bars
Figure imgf000044_0002
The mushing factor was determined according the mush assay described above. Table 4 shows that laundry soap bars containing sodium formate had improved mushing factor over bars without sodium formate. Table 5: Wet Cracking properties of the laundry soap bars
Figure imgf000045_0001
The wet cracking properties were determined according the wet cracking assay de- scribed above. Table 5 shows that all laundry soap bars had acceptable wet cracking properties after a single immersion of 1 hour, when assessed after 2, 24 and 48 hours.
Table 6: Foaming of the laundry soap bars
Figure imgf000045_0002
loose 1 1.0 10.5
The foaming of the laundry soap bar was determined according the foam assay described above. Table 6 shows that all soap bars initially gave good foam quality and height and this was also the case after 5 minutes indicating good foam stability. Table 7: pH, bar hardness, bar colour and wet bar (wash down) feel of the laundry soap bars
Figure imgf000046_0001
The pH, colour, hardness and wet bar (wash down) feel were determined according the pH, colour hardness and wet bar (wash down) assays respectively described above. Table 7 shows that pH was consistent indicating that pH was independent of the addition of stabiliser and/or sodium formate. Laundry soap bars containing sodium formate had a higher hardness which may be beneficial since a harder bar could last longer. All laundry soap bars had a good wet bar feel.
Example 2: Properties of Laundry soap bar with varying amounts of Sodium Formate The laundry soap bars were prepared according to the Preparation of Laundry Soap
Bars method described above and the composition is given in table 8. In this experiment, the amounts of glycerine, citric acid and sodium formate were varied and the properties of the laundry soap bar investigated. The laundry soap bars were tested using the Cleaning and Stain Removal and Enzyme Stability/Activity assays and the results are shown in tables 9 and 10. The physical properties of the laundry soap bars were also assessed and the results are reported in tables 1 1 -14.
Table 8: Formulation of laundry soap bars
The total protease concentration used was 0.016% by weight calculated as the percentage of enzyme protein to the total composition of the laundry soap bar. The protease inhibi- tor used was where: P=Cbz, B2=Gly; B1= Ala; B° = Tyr (so R=PhCH2, R -OH), x=2, y=0, L=A=absent and M=Na, L-Alaninamide, /V-[(phenylmethoxy)carbonyl]glycyl-/V-[2-hydroxy-1 -[(4- hydroxyphenyl)methyl]-2-sulfoethyl]-, sodium salt (1 :1 ), at a concentration of 9 ppm.
Figure imgf000047_0001
The mentioned amounts of ingredients are referred to in %w.
Table 9: Storage stability of Savinase in laundry soap bars after 4 and 8 weeks at 30°C and 37°C based on wash performance of EMPA 1 17 swatches in a Terg-O-tometer
Figure imgf000047_0002
8 56.3 55.7 54.8 50.8 24.5 23.9 23.0 19.0
9 54.3 50.8 36.3 33.7 22.5 19.0 4.6 1.9
10 57.5 56.3 54.2 49.4 25.7 24.5 22.5 17.6
1 1 57.2 56.1 56.2 53.9 25.4 24.3 24.4 22.1
12 55.5 55.1 54.4 52.5 23.7 23.4 22.6 20.7
14 58.7 56.3 56.0 54.4 26.9 24.5 24.2 22.6
16 57.2 56.8 56.5 54.9 25.4 25.0 24.8 23.1
17 57.3 56.5 55.9 55.8 25.5 24.7 24.1 24.0
18 56.1 55.2 54.2 54.2 24.3 23.4 22.4 22.4 aValues compared to buffer value of 31.78.
Table 10: Storage stability of Savinase in laundry soap bars after 4 and 8 weeks at 30°C and 37°C as determined by the hydrolysis of /V,/V-dimethyl casein
Figure imgf000048_0001
Tables 9 and 10 show that Savinase in the presence of inhibitor but without any sodium formate present has reduced stability at 30°C and almost no activity at 37°C even after just 4 weeks of storage as measured by the wash performance of blood/milk/ink on cotton/polyester swatches. In comparison, the addition of both the protease inhibitor and sodium formate significantly increased storage stability even after 8 weeks at 37°C as measured by the wash perfor- mance of blood/milk/ink on cotton/polyester swatches and by the percentage of residual activity as determined by the hydrolysis of Λ/,/V-dimethyl casein.
Table 1 1 : Mushing Factor of the laundry soap bars
Figure imgf000049_0001
The mushing factor was determined according the mush assay described above. Table
1 1 shows that laundry soap bars containing sodium formate had improved mushing factor over bars without sodium formate.
Table 12: Wet Cracking properties of the laundry soap bars
Figure imgf000049_0002
1 1 2 2 4 3
12 3 2 4 4
16 2 1 4 4
17 2 1 4 3
18 2 1 4 3
The wet cracking properties were determined according the wet cracking assay described above. Table 12 shows that laundry soap bars containing sodium formate had acceptable wet cracking properties after a single immersion of 1 hour, when assessed after 2 hours. Table 13: Foaming of the laundry soap bars
Figure imgf000050_0001
12.5 12.5
12.0 13.5
16 12.5 12.5 12.5 13.0
13.0 13.0
13.0 13.0
17 13.0 13.2 13.0 13.2
13.5 13.5
13.5 13.0
18 13.0 13.2 13.0 13.0
13.0 13.0
Table 14: pH, bar hardness, bar colour and wet bar (wash down) feel of the laundry soap bars
Figure imgf000051_0001
The pH, colour, hardness and wet bar (wash down) feel were determined according the pH, colour hardness and wet bar (wash down) assays respectively described above. Table 7 shows that pH was consistent indicating that pH was independent of the composition of the laundry soap bar. Laundry soap bars containing 2% sodium formate had a higher hardness than bars with no sodium formate or 4% sodium formate. All laundry soap bars had a good wet bar feel.

Claims

1 . A laundry soap bar comprising:
a) one or more soaps or synthetic surfactants or any mixture thereof; b) a protease and optionally one or more additional enzymes;
c) one or more protease inhibitors of which at least one is a peptide aldehyde, a hydrosulfite adduct or a hemiacetal adduct thereof; and
d) a salt of a monovalent cation and an organic anion.
2. The laundry soap bar of claim 1 , wherein the protease inhibitor is a peptide aldehyde of the formula P-(A)y-L-(B)x-B°-H or a hydrosulfite adduct or hemiacetal adduct thereof, wherein:
xix. H is hydrogen;
xx. B° is a single amino acid residue with L- or D-configuration of the formula -NH- CH(R)-C(=0)-;
xxi. x is 1 , 2 or 3 for (B)x, and B is independently a single amino acid connected to B° via the C-terminal of the (B)x amino acid
xxii. L is absent or L is independently a linker group of the formula -C(=0)-,
-C(=0)-C(=0)-, -C(=S)-, -C(=S)-C(=S)- or -C(=S)-C(=0)-;
xxiii. y is 0, 1 or 2 for (A)y, and A is independently a single amino acid residue connected to L via the /V-terminal of the (A)y amino acid, with the proviso that if L is absent then A is absent;
xxiv. P is selected from the group consisting of hydrogen and an /V-terminal protection group, with the proviso that if L is absent then P is an /V-terminal protection group;
xxv. R is independently selected from the group consisting of C1-6 alkyl, C6-io aryl or C7-10 arylalkyl optionally substituted with one or more, identical or different, sub- stituent's R';
xxvi. R' is independently selected from the group consisting of halogen, -OH, -OR", - SH, -SR", -NH2, -NHR", -NR"2, -C02H, -CONH2, -CONHR", -CONR"2, -
NHC(=N)NH2; and
xxvii. R" is a C1-6 alkyl group.
3. The laundry soap of any of the preceding claims, wherein the hydrosulfite adduct of a peptide aldehyde is of the formula P-(A)y-L-(B)x-N(H)-CHR-CH(OH)-S03M, wherein xvii. M is hydrogen or an alkali metal; xviii. x is 1 , 2 or 3 for (B)x, and B is independently a single amino acid connected to B° via the C-terminal of the (B)x amino acid
xix. L is absent or L is independently a linker group of the formula -C(=0)-,
-C(=0)-C(=0)-, -C(=S)-, -C(=S)-C(=S)- or -C(=S)-C(=0)-;
xx. y is 0, 1 or 2 for (A)y, and A is independently a single amino acid residue connected to L via the /V-terminal of the (A)y amino acid, with the proviso that if L is absent then A is absent;
xxi. P is selected from the group consisting of hydrogen and an /V-terminal protection group, with the proviso that if L is absent then P is an /V-terminal protection group;
xxii. R is independently selected from the group consisting of C1-6 alkyl, C6-io aryl or C7-10 arylalkyi optionally substituted with one or more, identical or different, sub- stituent's R';
xxiii. R' is independently selected from the group consisting of halogen, -OH, -OR", - SH, -SR", -NH2, -NHR", -NR"2, -C02H, -CONH2, -CONHR", -CONR"2, - NHC(=N)NH2; and
xxiv. R" is a C1-6 alkyl group.
4. The laundry soap bar of claim 3, wherein M is Na or K and R is a C7 arylalkyi substituted with -OH.
5. The laundry soap bar of any of claims 2 to 4, wherein B° is selected from the group consisting of D- or L-form of arginine (Arg), 3,4-dihydroxyphenylalanine, isoleucine (lie), leucine (Leu), methionine (Met), norleucine (NIe), norvaline (Nva), phenylalanine (Phe), m- tyrosine, p-tyrosine (Tyr) and valine (Val).
6. The laundry soap bar of any of claims 2 to 5, wherein
a. B1 can be selected from the group consisting of alanine (Ala), cysteine (Cys), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (NIe), norvaline (Nva), proline (Pro), serine (Ser), threonine (Thr) and valine (Val),
b. B2 can be selected from the group consisting of alanine (Ala), arginine (Arg), cap- reomycidine (Cpd), cysteine (Cys), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (NIe), norvaline (Nva), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), and valine (Val), or c. B3 can be selected from the group consisting of isoleucine (lie), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), phenylglycine, tyrosine (Tyr), tryptophan (Trp) and valine (Val).
7. The laundry soap bar of any of claims 2 to 6, wherein x is 1 , 2 or 3.
8. The laundry soap bar of any of claims 2 to 7, wherein
a. A1 can be selected from the group consisting of alanine (Ala), arginine (Arg), capreomycidine (Cpd), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), threonine (Thr), tyrosine (Tyr), tryptophan (Trp) and valine (Val) or,
b. A2 can be selected from the group consisting of arginine (Arg), isoleucine (lie), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), phenylglycine, Tyrosine (Tyr), tryptophan (Trp) and valine (Val).
9. The laundry soap bar of any of claims 2 to 7, wherein L is absent and A is absent.
10. The laundry soap bar of any of claims 2 to 7 and 9, wherein P is selected from the group consisting of formyl, acetyl (Ac), benzoyl (Bz), trifluoroacetyl, methoxysuccinyl, fluo- renylmethyloxycarbonyl (Fmoc), methoxycarbonyl (MEO-CO), (fluoromethoxy)carbonyl, benzyloxycarbonyl (Cbz), t-butyloxycarbonyl (Boc), adamantyloxycarbonyl, p- methoxybenzyl carbonyl (Moz), benzyl (Bn), p-methoxybenzyl (PMB), p-methoxyphenyl (PMP), methoxyacetyl, methylamino carbonyl (MeNCO), methylsulfonyl (MeS02), ethyl- sulfonyl (EtS02), benzylsulfonyl (PhCH2S02), methylphosphoramidyl (MeOP(OH)(=0)) and benzylphosphoramidyl (PhCH20-P(OH)(0)).
1 1 . The laundry soap bar of any of claims 1 to 7 and 9 to 10, wherein the peptide aldehyde adduct is L-Alaninamide, /V-[(phenylmethoxy)carbonyl]glycyl-/V-[2-hydroxy-1-[(4- hydroxyphenyl)methyl]-2-sulfoethyl]-, sodium salt (1 :1 ).
12. The laundry soap bar of any of claims 2 to 10, wherein the protease inhibitor is one of the following peptide aldehydes or a adduct thereof: Cbz-Arg-Ala-Tyr-H, Ac-Gly-Ala-Tyr-
H, Cbz-Gly-Ala-Tyr-H, Cbz-Gly-Ala-Leu-H, Cbz-Val-Ala-Leu-H, Cbz-Gly-Ala-Phe-H, Cbz-
Gly-Ala-Val-H, Cbz-Gly-Gly-Tyr-H, Cbz-Gly-Gly-Phe-H, Cbz-Arg-Val-Tyr-H, Cbz-Leu-
Val-Tyr-H, Ac-Leu-Gly-Ala-Tyr-H, Ac-Phe-Gly-Ala-Tyr-H, Ac-Tyr-Gly-Ala-Tyr-H, Ac-Phe-
Gly-Ala-Leu-H, Ac-Phe-Gly-Ala-Phe-H, Ac-Phe-Gly-Val-Tyr-H, Ac-Phe-Gly-Ala-Met-H,
Ac-Trp-Leu-Val-Tyr-H, MeO-CO-Val-Ala-Leu-H, MeNCO-Val-Ala-Leu-H, MeO-CO-Phe- Gly-Ala-Leu-H, MeO-CO-Phe-Gly-Ala-Phe-H, MeS02-Phe-Gly-Ala-Leu-H, MeS02-Val- Ala-Leu-H, PhCH20-P(OH)(0)-Val-Ala-Leu-H, EtS02-Phe-Gly-Ala-Leu-H, PhCH2S02- Val-Ala-Leu-H, PhCH20-P(OH)(0)-Leu-Ala-Leu-H, PhCH20-P(OH)(0)-Phe-Ala-Leu-H, or MeO-P(OH)(0)-Leu-Gly-Ala-Leu-H, α-ΜΑΡΙ, β-ΜΑΡΙ, Phe-C(=0)-Arg-Val-Tyr-H, Phe- C(=0)-Gly-Gly-Tyr-H, Phe-C(=0)-Gly-Ala-Phe-H, Phe-C(=0)-Gly-Ala-Tyr-H, Phe-C(=0)- Gly-Ala-L-H, Phe-C(=0)-Gly-Ala-Nva-H, Phe-C(=0)-Gly-Ala-Nle-H, Tyr-C(=0)-Arg-Val- Tyr-H, Tyr-C(=0)-Gly-Ala-Tyr-H, Phe-C(=S)-Arg-Val-Phe-H, Phe-C(=S)-Arg-Val-Tyr-H, Phe-C(=S)-Gly-Ala-Tyr-H, Antipain, GE20372A, GE20372B, Chymostatin A, Chymo- statin B, Chymostatin C,
Cbz-Gly-Ala-N(H)-CH(CH2-p-C6H40H)-CH(OH)-S03Na,
Cbz-Gly-Ala-N(H)-CH(CH2Ph)-CH(OH)-S03Na,
MeO-CO_Val-Ala-N(H)-CH(CH2CH(CH3)2)-CH(OH)-S03Na,
Cbz-Arg-Ala-NHCH(CH2C6H4OH)C(OH)(S03M)-H where M=Na,
Ac-Gly-Ala-NHCH(CH2C6H4OH)C(OH)(S03M)-H, where M=Na,
Cbz-Gly-Ala-NHCH(CH2C6H4OH)C(OH)(S03M)-H, where M=Na,
Cbz-Gly-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H, where M=Na,
Cbz-Val-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H, where M=Na,
Cbz-Gly-Ala-NHCH(CH2Ph)C(OH)(S03M)-H, where M=Na,
Cbz-Gly-Ala-NHCH(CH(CH3)2)C(OH)(S03M)-H, where M=Na,
Cbz-Gly-Gly-NHCH(CH2C6H4OH)C(OH)(S03M)-H, where M=Na,
Cbz-Gly-Gly-NHCH(CH2Ph)C(OH)(S03M)-H, where M=Na,
Cbz-Arg-Val-NHCH(CH2C6H4OH)C(OH)(S03M)-H, where M=Na,
Cbz-Leu-Val-NHCH(CH2C6H4OH)C(OH)(S03M)-H, where M=Na,
Ac-Leu-Gly-Ala-NHCH(CH2C6H4OH)C(OH)(S03M)-H, where M=Na,
Ac-Phe-Gly-Ala-NHCH(CH2C6H4OH)C(OH)(S03M)-H, where M=Na,
Ac-Tyr-Gly-Ala-NHCH(CH2C6H4OH)C(OH)(S03M)-H, where M=Na,
Ac-Phe-Gly-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H, where M=Na,
Ac-Phe-Gly-Ala-NHCH(CH2Ph)C(OH)(S03M)-H, where M=Na,
Ac-Phe-Gly-Val-NHCH(CH2C6H4OH)C(OH)(S03M)-H, where M=Na ,
Ac-Phe-Gly-Ala-NHCH(CH2CH2SCH3)(S03M)-H, where M=Na,
Ac-Trp-Leu-Val-NHCH(CH2C6H4OH)C(OH)(S03M)-H, where M=Na,
MeO-CO-Val-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H, where M=Na,
MeNCO-Val-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H, where M=Na,
MeO-CO-Phe-Gly-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H, where M=Na,
MeO-CO-Phe-Gly-Ala-NHCH(CH2Ph)C(OH)(S03M)-H, where M=Na,
MeS02-Phe-Gly-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H, where M=Na, MeS02-Val-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H! where M=Na, PhCH20(OH)(0)P-Val-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H! where M=Na , EtS02-Phe-Gly-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H! where M=Na,
PhCH2S02-Val-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H! where M=Na,
PhCH20(OH)(0)P-Leu-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H! where M=Na, PhCH20(OH)(0)P-Phe-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H! where M=Na, MeO(OH)(0)P-Leu-Gly-Ala-NHCH(CH2CH(CH3)2))C(OH)(S03M)-H, where M=Na, and Phe-urea-Arg-Val-NHCH(CH2C6H40H)C(OH)(S03M)-H where M=Na.
13. A process of preparing the laundry soap bars according to any of claims 1 to 12 comprising the steps of:
a) mixing a soap; a protease; a protease inhibitor which is a peptide aldehyde or a hy- drosulfite adduct or hemiacetal adduct thereof; and a salt of a monovalent cation and an organic anion; and
b) plodding the mixture from step (a).
14. A premix to be used in the process of claim 13, comprising a protease; a protease inhibitor which is a peptide aldehyde or a hydrosulfite adduct or hemiacetal adduct thereof; and a salt of a monovalent cation and/or a monovalent organic anion.
15. Use of a laundry soap bar of any of claims 1 to 12 for washing a textile, preferably, wherein the washing is done by hand.
PCT/US2013/045091 2012-06-13 2013-06-11 Laundry soap bars WO2013188344A2 (en)

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BR112014030846A BR112014030846A2 (en) 2012-06-13 2013-06-11 bar of soap, process for preparing bar of soap, premixing, and use of a bar of soap.
AP2014008122A AP3789A (en) 2012-06-13 2013-06-11 Laundry soap bars.
MX2014015186A MX2014015186A (en) 2012-06-13 2013-06-11 Laundry soap bars.
CN201380031099.5A CN104583382B (en) 2012-06-13 2013-06-11 Laundry soap bar

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US10538720B2 (en) 2016-03-08 2020-01-21 The Procter & Gamble Company Particles including enzyme
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AP2014008122A0 (en) 2014-12-31
MX2014015186A (en) 2015-09-25
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AR091423A1 (en) 2015-02-04
CN104583382B (en) 2017-10-20
CO7240383A2 (en) 2015-04-17
CN104583382A (en) 2015-04-29
AP3789A (en) 2016-08-31

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