Liquid antifouling composition

Abstract

A liquid composition comprising (a) at least one chlorinated hydroxydiphenyl ether according to formula (I) wherein R1, R2 and R3 are selected from H and Cl, and wherein at least one of R1, R2 and R3 is Cl, (b) at least one compound providing DNase activity, and (c) at least one compound selected from anionic and nonionic surfactants.

Description

[0001] This invention relates to liquid compositions comprising at least one compound selected from chlorinated hydroxydiphenyl ethers, at least one compound providing DNase activity, and at least one compound selected from anionic and nonionic surfactants. The invention also relates to the use of liquid compositions for (a) reducing and / or inhibiting biofilm formation on the surface of devices that are periodically in contact with water (e.g., cleaning devices such as washing machines) and / or (b) reducing and / or inhibiting microbial growth on materials that are periodically in contact with water (such as objects to be cleaned like hard surfaces and / or textiles).

[0002] Microbial growth distinguishes between planktonic and biofilm growth patterns. Microbial cells growing in biofilms are physiologically different from planktonic cells of the same organism, which are single cells capable of floating or swimming in a liquid medium. A “biofilm” typically comprises a clump of microorganisms in which cells adhere to each other and often also to a surface. On the one hand, biofilms are said to begin forming as freely floating microorganisms attach to a surface. Once colonization begins, biofilms grow through cell division and recruitment. Cells within a biofilm are often embedded in an extracellular matrix, commonly referred to as extracellular polymeric substances (EPS). EPS typically contains extracellular polysaccharides, proteins, lipids, and DNA. These bacteria growing in biofilms are a source of hygiene problems, such as microbial contamination that can cause disease or at least unpleasant odors.

[0003] Biofilms can form on solid surfaces as well as soft surfaces such as textiles, and are generally resistant to conventional disinfection methods. Increased resistance to detergent application, ultraviolet radiation, and antibiotics has also been observed. Notably, biofilms are often sticky, and soil can adhere to surfaces containing such biofilms. Furthermore, biofilms can be an undesirable source of odor, such as on washed items or in washing machine ducts, possibly due to biofilm decomposition. Biofilms can adversely affect the long-term functionality of cleaning equipment.

[0004] One problem this invention aims to solve is to provide detergent formulations with anti-biofilm activity, which means inhibiting or reducing biofilm formation on the surfaces of devices and / or objects to be cleaned.

[0005] The present invention addresses the problem by providing a liquid composition comprising (a) at least one compound selected from chlorohydroxydiphenyl ethers, (b) at least one compound providing DNase activity, and (c) at least one compound selected from anionic and nonionic surfactants.

[0006] Throughout the specification (including the claims), unless otherwise stated, the terms “comprising one” or “comprising one” shall be understood to be synonymous with the term “comprising at least one”, and “between” shall be understood to be the boundary of inclusion.

[0007] The terms “one,” “a,” and “the” are used to refer to one or more (i.e., at least one) of the grammatical objects of the article.

[0008] The term “and / or” includes the meanings of both “and” and “or”.

[0009] The word “may” is used in this specification to refer to various embodiments, not just optional features.

[0010] It should be noted that when specifying any range of concentration, weight ratio or amount, any particular higher concentration, weight ratio or amount may be associated with any particular lower concentration, weight ratio or amount.

[0011] In one aspect, the present invention relates to a liquid composition comprising:

[0012] Component (a): at least one compound selected from chlorinated hydroxydiphenyl ethers according to formula (I),

[0013] Component (b): at least one compound that provides DNA enzyme activity and

[0014] Component (c): at least one compound selected from anionic and nonionic surfactants.

[0015] In one embodiment, the composition of the present invention is a liquid at 20°C and 101.3 kPa. The liquid comprises a castable liquid, preferably with a viscosity of less than 1500 mPa*s at 25°C. In another embodiment, the viscosity at 25°C is less than 1000 mPa*s, more preferably less than 800 mPa*s.

[0016] Component (a)

[0017] Component (a) comprises at least one compound selected from the group consisting of chlorinated hydroxydiphenyl ether compounds according to formula (I):

[0018]

[0019] R1, R2, and R3 are selected from H and Cl, and at least one of R1, R2, and R3 is Cl.

[0020] In one embodiment, the compound according to formula (I) is characterized in that at least two of R1, R2 and R3 are Cl.

[0021] In a preferred embodiment, R1 and R2 are Cl, while R3 is H (4,4'-dichloro-2-hydroxydiphenyl ether; such compounds are also referred to herein as DCPP or hydroxydichlorodiphenyl ether (Diclosan)).

[0022] In another embodiment, R1, R2 and R3 are all Cl (2,4,4'-trichloro-2'-hydroxydiphenyl ether; the compound is also referred to herein as trichlorohydroxydiphenyl ether (Triclosan)).

[0023] Component (b)

[0024] The compositions of the present invention comprise at least one compound that provides DNase activity. Compounds providing DNase activity include DNases that typically catalyze the hydrolysis and cleavage of phosphodiester bonds in DNA. DNases are classified, for example, in EC 3.1.11, EC 3.1.12, EC 3.1.15, EC 3.1.16, EC 3.1.21.X (where X = 1, 2, 3, 4, 5, 6, 7, 8, or 9), EC 3.1.22.Y (where Y = 1, 2, 4, or 5), EC 3.1.23, EC 3.1.24, EC 3.1.25, and EC 3.1.30.Z (where Z = 1 or 2), and EC 3.1.31.1. Preferably, the DNase is selected from any one of EC3.1.21.x (where x = 1, 2, 3, 4, 5, 6, 7, 8 or 9), EC3.1.22.y (where y = 1, 2, 4 or 5), EC3.1.30.z (where z = 1 or 2), EC3.1.31.1, and mixtures thereof.

[0025] • EC3.1.21.x type nucleases cleave at the 3' hydroxyl group to release 5' phosphate monoesters. Preferred, especially, nucleases with x=1.

[0026] • EC3.1.22.y class nucleases cleave at the 5' hydroxyl group to release 3' phosphomonoesters. EC3.1.30.z class enzymes may be preferred because they act on DNA and RNA and release 5'-phosphomonoesters. Suitable examples of EC3.1.31.2 class are described in US2012 / 0135498A, such as SEQ ID NO:3 therein.

[0027] • EC3.1.31.1 type nucleases produce 3' phosphate monoesters.

[0028] DNAse activity, or DNA degradation activity, refers to the catalytic activity exerted by an enzyme (preferably DNAse), typically expressed as units per milligram of enzyme (specific activity), which relates to the substrate molecules converted per minute per enzyme molecule (molecular activity). DNAse activity is determined using test protocols known to those skilled in the art. For example, DNAse activity can be determined on DNase test agar containing methyl green (BD, Franklin Lakes, NJ, USA), which should be prepared according to the supplier's manual. In short, 21 g of agar is dissolved in 500 ml of water and then autoclaved at 121°C for 15 min. The autoclaved agar is then heated in a water bath to 10–48°C, and 20 ml of agar is poured into a Piper plate and incubated at room temperature for curing. On the cured agar plate, 5 μl of enzyme solution is added, and DNAse activity is observed by the colorless area surrounding the spotted enzyme solution. Alternatively, DNAseAlert can be used according to the supplier's manual. TM Kit (11-02-01-04, IDT Integrated DNA Technologies) was used to determine DNase activity. In short, 95 μl of DNase sample was mixed with 5 μl of substrate in a microtiter plate and immediately titrated using BMG Labtech's [technology / method / equipment]. Fluorescence is measured using a microplate reader (536nm excitation, 556nm emission). Any suitable assay protocol can be used to determine DNAse activity.

[0029] DNAse is an enzyme. In the context of this invention, enzymes are identified by their "peptide sequence" (also referred to herein as "amino acid sequence"). The term "enzyme" in this invention is synonymous with the term "peptide" which has enzymatic activity.

[0030] The polypeptide sequence refers to the parent enzyme and / or its variant enzymes that have enzymatic activity. The enzymes in this article are polypeptides that have enzymatic activity or undergo enzymatic transformation, meaning that the enzyme acts on a substrate and converts it into a product.

[0031] The term "enzyme" as used herein preferably excludes inactive variants of the enzyme. Therefore, the variant enzymes according to the invention are preferably functional variants with enzymatic activity, wherein the enzymatic activity is of the same type as that of their respective parent enzymes.

[0032] A polypeptide sequence has a well-defined three-dimensional structure, including the enzyme's "active site," which determines the enzyme's catalytic activity. Polypeptide sequences can be identified by SEQ ID NO. In accordance with WIPO standard ST.25 (1998), amino acids in the text are represented by three-letter codes with the first letter capitalized, or by the corresponding single letter. Based on the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB), the enzyme name is known to those skilled in the art. The enzyme name includes: EC (Enzyme Committee) number, recommended name, alternative name (if any), catalytic activity, and other factors.

[0033] A “parent” sequence (parent protein or enzyme, also known as a “parent enzyme”) is a starting sequence into which changes are introduced (e.g., by introducing one or more amino acid substitutions, insertions, deletions, or combinations thereof), resulting in a “variant” of the parent sequence. The term parent sequence includes wild-type enzyme sequences and synthetically produced sequences used as starting sequences for introducing (further) changes. In this text, the terms “parent sequence” and “parent enzyme” are used interchangeably.

[0034] The terms "enzyme variant," "sequence variant," or "variant enzyme" refer to an enzyme whose amino acid sequence differs to some extent from that of its parent enzyme. Unless otherwise stated, a variant enzyme "possesses enzymatic activity."

[0035] When describing sequence variants, use the terminology described below:

[0036] Amino acid substitutions are described by providing the original amino acid of the parent enzyme, followed by its position number in the amino acid sequence, and then the substituted amino acid. Amino acid deletions are described by providing the original amino acid of the parent enzyme, followed by its position number in the amino acid sequence, and then an asterisk "*". Amino acid insertions are described by providing the original amino acid of the parent enzyme, followed by its position number in the amino acid sequence, and then the original amino acid and the additional amino acid. In the case of inserting an amino acid residue that is identical to an existing amino acid residue, degeneracy of nomenclature will obviously occur. If different changes can be introduced at a position, the different changes are separated by commas.

[0037] In one implementation, an enzyme variant is defined by its sequence identity when compared to the parent enzyme. Sequence identifiers are typically provided as “% sequence identity” or “% identity”. For the calculation of sequence identity, sequence alignment must be performed in the first step. According to the invention, pairwise global sequence alignment must be performed, meaning that the two sequences must be compared in their total length, which is typically accomplished using mathematical methods known as alignment algorithms.

[0038] According to the present invention, the pairing is performed using the Needleman and Wunsch (J. Mol. Biol. (1979) 48, 443-453) algorithm. Preferably, the program "NEEDLE" (The European Molecular Biology Open Software Suite (EMBOSS)) is used for the purposes of the present invention, using the program's default parameters (gap start = 10.0, gap extension = 0.5 and matrix = EBLOSUM62).

[0039] According to the present invention, the following %-identity is calculated as follows: %-identity = (length of identical residues / pairing regions, which show the total length of each sequence of the present invention) * 100.

[0040] In one embodiment, the enzyme variant is described as an amino acid sequence that is at least n% identical to the amino acid sequence of the corresponding parent enzyme, where "n" is an integer between 10 and 100. In one embodiment, the variant enzyme is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, 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% identical to the full-length amino acid sequence of the parent enzyme, wherein the enzyme variant has enzymatic activity.

[0041] The variant enzyme, as used herein, refers to an enzyme that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% of the enzyme activity of its parent enzyme. In one embodiment, the variant enzyme has increased enzyme activity compared to its parent enzyme. Increased means more than 100% of the enzyme activity compared to its parent enzyme. In a preferred embodiment, increased enzyme activity means that after storing the enzyme at a specific temperature for a certain period of time, its activity increases compared to its parent enzyme.

[0042] Previously described polypeptides with DNase activity, such as those describing fungal DNases WO2015 / 155350 (Novozymes A / S) and WO 2015 / 155351 (Novozymes A / S). DNase can be obtained from *Vibrisseaflavovirens*, *Penicillium reticulisporum*, *Acremonium dichromosporum*, *Preussia aemulans*, *Colletotrichum circinans*, Clavicipitaceae, *Trichurus spiralis*, *Pyrenochaetopsis sp.*, *Aspergillus sydowii*, *Cladosporium cladosporioides*, *Rhinocladiella sp.*, *Pyronema domesticum*, *Aspergillus niger*, *Phialophora geniculata*, *Paradendryphiella salina*, *Aspergillus insuetus*, and *Purpureocillium*. lilacinum), Warcupiella spinulosa, Stenocarpella maydis, Acrophialophora fusispora, Chaetomium luteum, or Arthrinium arundinis.

[0043] The DNase was obtained from Bacillus species, such as Bacillus cibi, Bacillus horikoshii, Bacillus horneckiae, Bacillus idriensis, Bacillus algicola, Bacillus vietnamensis, Bacillus hwajinpoensis, Paenibacillus mucilanginosus, Bacillus indicus, Bacillus luciferensis, Bacillus marisflavi, and their variants.

[0044] In one embodiment, the at least one DNase included in component (a) is selected from polypeptides having at least 80% identical amino acid sequences to SEQ ID NO:1-24 and SEQ ID NO:27-28 of WO2019 / 081724 or WO2019 / 081721. The variant DNase comprises one or two motifs: [D,M,L][S,T]GYSR[D,N] (SEQ ID NO:25 of WO 2019 / 081724 or WO 2019 / 081721) and ASXNRSKG (SEQ ID NO:26 of WO 2019 / 081724 or WO 2019 / 081721).

[0045] In one embodiment, at least one DNAseed is selected from SEQ ID NO:4 and its variants disclosed in US 9675736, SEQ ID NOs:1-3 and their variants disclosed in EP 3088502, and SEQ ID NOs:1-6 and their variants disclosed in WO 2017 / 001471.

[0046] In one embodiment, at least one DNAseed is selected from polypeptides containing one or more motifs, said motifs being selected from [G,Y,W,F,A,H]NI[R,Q,D,E,V](SEQ ID NO:73 of WO 2017 / 060493), SDH[D,H,L]P(SEQ ID NO:74 of WO 2017 / 060493), and GGNI[R,Q](SEQ ID NO:75 of WO 2017 / 060493). Preferably, the DNase has an amino acid sequence that is at least 80%, at least 82%, at least 85%, at least 90%, at least 92%, at least 95%, or at least 99% identical to the polypeptides SEQ ID NO: 6, 7, 10, 27, 30, 33, 36, 39, 42, 45, 48, 51, 57, 60, 63, 66, 69, and 72 disclosed in WO 2017 / 060493. In one embodiment, at least one DNase is selected from polypeptides having at least 85% sequence identity with the polypeptides SEQ ID NO: 10, 27, and 63 disclosed in WO 2017 / 060493. In one embodiment, at least one DNase is selected from polypeptides having at least 95% sequence identity with the polypeptides SEQ ID NO: 7, 39, 48, and 60 disclosed in WO 2017 / 060493. In one embodiment, at least one DNAseed is selected from a polypeptide having at least 99% sequence identity with the polypeptide SEQ ID NO:42 disclosed in WO2017 / 060493.

[0047] In one embodiment, the at least one DNase contained in component (b) has at least 75%, at least 80%, at least 85%, at least 90%, 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% identity when compared to the full-length polypeptide sequence SEQ ID NO:1 according to the sequence list of this application. Preferably, the DNase has the polypeptide sequence SEQ ID NO:1 according to the sequence list of this application.

[0048] SEQ ID NO:1 is characterized by having the following amino acid sequence:

[0049] MKKWMAGLFLAAAVLLCLMVPQQIQGASLYDKVLYFPLSRY-PETGDHIKDAIADGHS-DICTIDRDGADKRRQESLKGIPTKPGYDRDEWPMAVCEEGGAGADVRYVTPSDNRGAGSWVGNQMSGYPDGTRVLFIVQ

[0050] Component (c)

[0051] Component (c) contains at least one surfactant selected from anionic surfactants and nonionic surfactants.

[0052] Anionic surfactants (AIS)

[0053] In one embodiment, the liquid composition of the present invention comprises at least one anionic surfactant (AIS). Anionic surfactants include, but are not limited to, surfactant compounds containing a hydrophobic group and at least one water-soluble anionic group to form a water-soluble compound, wherein the water-soluble anionic group is typically selected from sulfates, sulfonates, and carboxylates.

[0054] In one embodiment, the liquid composition of the present invention comprises at least one anionic surfactant selected from compounds of general formula (AIS I):

[0055]

[0056] The variables in the general formula (AIS I) are defined as follows:

[0057] R 1 Selected from C1-C 23 -alkyl (e.g., 1-, 2-, 3-, 4-C1-C) 23 -alkyl) and C2-C 23 -Alkenyl, wherein the alkyl and / or alkenyl group is straight-chain or branched, and wherein it is 2-, 3-, or 4-alkyl; an example is n-C7H 15 n-C9H 19 nC 11 H 23 nC 13 H 27 nC 15 H 31 nC 17 H 35 i-C9H 19 iC 12 H 25 .

[0058] R 2 Selected from H, C1-C 20 -alkyl and C2-C 20 -Alkenyl, wherein the alkyl and / or alkenyl groups are straight-chain or branched.

[0059] R 3 and R 4 Each is independently selected from C1-C 16-alkyl, wherein the alkyl group is straight-chain or branched; examples are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, 1,2-dimethylpropyl, isopentyl, n-hexyl, isohexyl, sec-hexyl, n-heptyl, n-octyl, 2-ethylhexyl, n-nonyl, n-decyl, isodecyl.

[0060] A - Selected from -RCOO - -SO3 - and RSO3 - , wherein R is selected from straight-chain or branched C1-C8-alkyl and C1-C4 hydroxyalkyl, wherein alkyl. When A - SO3 - When this is the case, the compound can be called a (fatty) alcohol / alkyl (ethoxy / ether) sulfonate [(F)A(E)S], where A - For -RCOO - When this is the case, the compound can be called a (fatty) alcohol / alkyl (ethoxy / ether) carboxylic acid ester [(F)A(E)C].

[0061] M + Selected from H and salt-forming cations. Salt-forming cations can be monovalent or polyvalent; therefore, M + Equal to 1 / v M v+ Examples include, but are not limited to, ammonium salts of sodium, potassium, magnesium, calcium, ammonium, and monoethanolamine, diethanolamine, and triethanolamine.

[0062] m is in the range of 0 to 200, preferably 1 to 80, more preferably 3 to 20; n and o are each independently in the range of 0 to 100; n is preferably in the range of 1 to 10, more preferably in the range of 1 to 6; o is preferably in the range of 1 to 50, more preferably in the range of 4 to 25. The sum of m, n and o is at least 1, preferably in the range of 5 to 100, more preferably in the range of 9 to 50.

[0063] Anionic surfactants of general formula (AIS I) can be of any structure, block copolymers or random copolymers.

[0064] In one embodiment, the liquid composition of the present invention comprises at least one anionic surfactant according to formula (AIS I), wherein R 1 It is nC 11 H 23 R 2 It is H, A - It is SO3 - m, n, and o are 0. M + NH4 is preferred. + The compound may be referred to herein as ammonium dodecyl sulfate (ALS).

[0065] In one embodiment, the liquid composition of the present invention comprises at least one anionic surfactant according to formula (AIS I), wherein R 1 It is nC 11 H 23 R 2 Selected from H, A - It is SO3 - m is 2-5, preferably 3, and n and o are 0. M + Na is preferred. + The compound may be referred to herein as laurylethersulfate (LES), preferably sodium laurylethersulfate (SLES).

[0066] In one embodiment, the liquid composition of the present invention comprises at least two anionic surfactants selected from compounds of general formula (AIS I), wherein one of the anionic surfactants is characterized by R 1 It is C 11 R 2 H is 2, m is 2, n and o = 0, A - It is SO3 - M + Yes + And another surfactant is characterized by R 1 It is C 13 R 2 H is 2, m is 2, n and o = 0, A - It is SO3 - M + Yes + .

[0067] Other suitable anionic surfactants include C 12 -C 18 sulfonyl fatty acid alkyl esters (such as C 12 -C 18 sulfonated fatty acid methyl esters), C 10 -C 18 -alkyl aryl sulfonic acids (such as nC) 10 -C 18 -alkylbenzenesulfonic acid) and C 10 -C 18 Salts of alkylalkoxycarboxylic acids (M) + In all cases, M + All are selected from salt-forming cations. Salt-forming cations can be monovalent or polyvalent; therefore, M + Equal to 1 / v M v+Examples include, but are not limited to, ammonium salts of sodium, potassium, magnesium, calcium, ammonium, and monoethanolamine, diethanolamine, and triethanolamine.

[0068] Other suitable non-limiting examples of anionic surfactants include alkylbenzenesulfonate (BABS), benzene sulfonates, α-olefin sulfonates (AOS), olefin sulfonates, alkene sulfonates, alkane-2,3-diacylbis(sulfates), hydroxyalkyl sulfonates and disulfonates, secondary alkyl sulfonates (SAS), paraffin sulfonates (PS), sulfonated fatty acid glycerides, alkyl or alkenyl succinic acids, fatty acid derivatives of amino acids, diesters and monoesters of sulfosuccinic acids.

[0069] In one embodiment, the liquid composition comprises at least one anionic surfactant selected from compounds of general formula (AIS II):

[0070]

[0071] In equation (AIS II), R 1 It is C 10 -C 13 Alkyl group. In one embodiment, the liquid composition of the present invention comprises a salt of a compound according to formula (AIS II), preferably a sodium salt. In one embodiment, the liquid composition of the present invention comprises at least two anionic surfactants selected from compounds of general formula (AIS II), wherein one of the anionic surfactants is characterized by R 1 It is C 10 Another surfactant is characterized by R 1 It is C 13 In one embodiment, the liquid composition of the present invention comprises at least two anionic surfactants selected from sodium salts of compounds of general formula (AIS II), wherein one of the anionic surfactants is characterized by R 1 It is C 10 Another surfactant is characterized by R 1 It is C 13 The compound is also referred to herein as LAS (linear alkylbenzene sulfonate).

[0072] In one embodiment, the liquid composition of the present invention comprises at least one anionic surfactant selected from compounds of general formula (AIS III), which may also be referred to as an N-acyl amino acid surfactant:

[0073]

[0074] The variables in the general formula (AIS III) are defined below:

[0075] R 19 Selected from straight or branched C6-C 22 -Alkyl groups and straight-chain or branched C6-C 22 -Alkenyl, for example, oleylene.

[0076] R 20 Selected from H and C1-C4-alkyl.

[0077] R 21 Selected from H, methyl, -(CH2)3NHC(NH)NH2, -CH2C(O)NH2, -CH2C(O)OH, -(CH2)2C(O)NH2, -(CH2)2C(O)OH, (imidazol-4-yl)-methyl, -CH(CH3)C2H5, -CH2CH(CH3)2, -(CH2)4NH2, benzyl, hydroxymethyl, -CH(OH)CH3, (indole-3-yl)-methyl, (4-hydroxy-phenyl)-methyl, isopropyl, -(CH2)2SCH3 and -CH2SH.

[0078] R 22 Selected from -COOX and -CH2SO3X, where X is selected from Li + Na + and K + .

[0079] Non-limiting examples of suitable N-acyl amino acid surfactants are monocarboxylates and dicarboxylates of N-acylated glutamate (e.g., sodium, potassium, ammonium, and ammonium salts of monoethanolamine, diethanolamine, and triethanolamine), such as sodium cocoyl glutamate, sodium lauroyl glutamate, sodium myristoyl glutamate, sodium palmitoyl glutamate, sodium stearoyl glutamate, disodium cocoyl glutamate, disodium stearoyl glutamate, potassium cocoyl glutamate, potassium lauroyl glutamate, and potassium myristoyl glutamate; and carboxylates of N-acylated alanine (e.g., sodium, potassium, ammonium, and monoethanolamine). Ammonium salts of amines, diethanolamine, and triethanolamine, such as sodium cocoyl alanine and lauroyl alanine triethanolamine; carboxylates of N-acylated glycine (e.g., sodium, potassium, ammonium, and monoethanolamine, diethanolamine, and triethanolamine), such as sodium cocoyl glycine and potassium cocoyl glycine; carboxylates of N-acylated sarcosine (e.g., sodium, potassium, ammonium, and monoethanolamine, diethanolamine, and triethanolamine), such as sodium lauroyl sarcosine, sodium cocoyl sarcosine, sodium myristoyl sarcosine, sodium oleoyl sarcosine, and ammonium lauroyl sarcosine.

[0080] In one embodiment, the liquid composition of the present invention comprises at least one anionic surfactant selected from soaps. Suitable are saturated and unsaturated C... 12 -C 18Fatty acids such as lauric acid, myristic acid, palmitic acid, stearic acid, benzolic acid, oleic acid, and salts of (hydrated) erucic acid (M + M + Selected from salt-forming cations. Salt-forming cations can be monovalent or polyvalent; therefore, M + Equal to 1 / v M v+ Examples include, but are not limited to, ammonium salts of sodium, potassium, magnesium, calcium, ammonium, and monoethanolamine, diethanolamine, and triethanolamine.

[0081] Other non-limiting examples of suitable soaps include soap blends derived from natural fatty acids such as tallow, coconut oil, palm kernel oil, laurel oil, olive oil, or rapeseed oil. These soap blends contain varying amounts of lauric acid and / or myristic acid and / or palmitic acid and / or stearic acid and / or oleic acid and / or linoleic acid, depending on the natural fatty acids from which the soap is extracted.

[0082] Other non-limiting examples of suitable anionic surfactants include salts of sulfated, sulfonated, or carboxylic acid esters derived from natural fatty acids such as tallow, coconut oil, palm kernel oil, laurel oil, olive oil, or rapeseed oil (M... + The anionic surfactant contains varying amounts of sulfates, sulfonates, or carboxylates of lauric acid and / or myristic acid and / or palmitic acid and / or stearic acid and / or oleic acid and / or linoleic acid, depending on the natural fatty acids from which the soap is derived.

[0083] In one embodiment, the liquid composition of the present invention comprises a mixture of two or more different anionic surfactants. In one embodiment, the liquid composition comprises at least two anionic surfactants selected from compounds of general formula (AIS I), and one of said anionic surfactants is characterized by R 1 It is C 11 R 2 H is 2, m is 2, n and o = 0, A - It is SO3 - M + Yes + Another surfactant is characterized by R 1 It is C 13 R 2 H is 2, m is 2, n and o = 0, A - It is SO3 - M + Yes + In another embodiment, the liquid composition comprises at least two anionic surfactants selected from compounds of general formula (AIS II), and one of said anionic surfactants is characterized by R 1 It is C 10Another surfactant is characterized by R 1 It is C 13 .

[0084] In one embodiment, the liquid composition of the present invention comprises at least one anionic surfactant, wherein the total amount of the anionic surfactant may be 0% to 50% by weight, preferably 0.5% to 45% by weight, relative to the total weight of the liquid composition.

[0085] In one embodiment, the liquid composition of the present invention comprises at least two anionic surfactants in amounts of 0.5 wt% to 25 wt%, 1 wt% to 20 wt%, and 1.5 wt% to 15 wt%, respectively, relative to the total weight of the liquid composition. The at least two anionic surfactants are selected from compounds of general formula (AIS I), wherein one of the anionic surfactants is characterized by R... 1 It is C 11 R 2 H is 2, m is 2, n and o = 0, A - It is SO3 - M + Yes + Another surfactant is characterized by R 1 It is C 13 R 2 H is 2, m is 2, n and o = 0, A - It is SO3 - M + Yes + .

[0086] In one embodiment, the liquid composition of the present invention comprises at least two anionic surfactants in amounts of 0.5 wt% to 25 wt%, 1 wt% to 20 wt%, and 1.5 wt% to 15 wt%, respectively, relative to the total weight of the liquid composition. The at least two anionic surfactants are selected from compounds of general formula (AIS II), wherein one of the anionic surfactants is characterized by R... 1 It is C 10 Another surfactant is characterized by R 1 It is C 13 .

[0087] Nonionic surfactants (NIS)

[0088] In one embodiment, the liquid composition of the present invention comprises at least one nonionic surfactant.

[0089] In one embodiment, the liquid composition of the present invention comprises at least one nonionic surfactant (NIS) selected from compounds of general formulas (NIS Ia) and (NIS Ib):

[0090]

[0091] The variables in the general formulas (NIS Ia) and (NIS Ib) are defined as follows:

[0092] R 1 Selected from C1-C 23 Alkyl and C2-C 23 Alkenyl, wherein the alkyl and / or alkenyl groups are straight-chain or branched; an example is n-C7H. 15 n-C9H 19 nC 11 H 23 nC 13 H 27 nC 15 H 31 nC 17 H 35 i-C9H 19 iC 12 H 25 .

[0093] R 2 Selected from H, C1-C 20 Alkyl and C2-C 20 Alkenyl groups, wherein the alkyl and / or alkenyl groups are straight-chain or branched.

[0094] R 3 and R 4 Each is independently selected from C1-C 16 Alkyl groups, wherein the alkyl group is straight-chain or branched; examples are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, 1,2-dimethylpropyl, isopentyl, n-hexyl, isohexyl, sec-hexyl, n-heptyl, n-octyl, 2-ethylhexyl, n-nonyl, n-decyl, isodel.

[0095] R 5 Selected from H and C1-C 18 Alkyl groups, wherein the alkyl group is straight-chain or branched.

[0096] The integers of the general formulas (NIS Ia) and (NIS Ib) are defined below:

[0097] m is 0 to 200, preferably 1 to 80, more preferably 3 to 20; n and o are each independently 0 to 100; n is preferably 1 to 10, more preferably 1 to 6; o is preferably 1 to 50, more preferably 4 to 25. The sum of m, n and o is at least 1, preferably 5 to 100, more preferably 9 to 50.

[0098] In this text, compounds according to formula (NIS Ia) may also be referred to as alkyl polyethylene glycol ethers (AEO). Compounds according to formula (NIS Ib) may also be referred to as alkylphenol polyethylene glycol ethers (APEO).

[0099] In one embodiment, the liquid composition of the present invention comprises at least one nonionic surfactant selected from general formula (NIS Ia), wherein m is 3 to 11, preferably not exceeding 7; n and o are 0, R 1 It is C 12 -C 14 R 2 and R 5 It is H. Preferably, the liquid composition comprises at least two nonionic surfactants selected from compounds of general formula (NIS Ia), wherein one of the nonionic surfactants is characterized by R 1 It is C 12 R 2 and R 5 It is H, m is 7, n and o = 0, while another surfactant is characterized by R 1 It is C 14 R 2 and R 5 H is 7, m is 7, and n and o = 0.

[0100] In one embodiment, the liquid composition of the present invention comprises at least two nonionic surfactants selected from compounds of general formula (NIS Ia), wherein one of the nonionic surfactants is characterized by R 1 It is nC 11 H 23 R 5 It is H, m is 7, n and o = 0, while another surfactant is characterized by R 1 It is C 13 H 27 R 5 H is 7, m is 7, and n and o = 0.

[0101] Nonionic surfactants of general formulas (NIS Ia) and (NIS Ib) can have any structure, whether bulk or random, and are not limited to the sequences shown in formulas (NIS Ia) and (NIS Ib).

[0102] In one embodiment, the liquid composition of the present invention comprises at least one nonionic surfactant selected from compounds of general formula (NIS II), also referred to as an alkyl-polyglycoside (APG):

[0103]

[0104] The variables in the general formula (NIS II) are defined as follows:

[0105] R 1 Selected from C1-C 17 Alkyl and C2-C 17 Alkenyl, wherein the alkyl and / or alkenyl groups are straight-chain or branched, an example being n-C7H. 15 n-C9H 19 nC 11 H 23 nC 13 H 27 nC 15 H 31 nC 17 H 35 i-C9H 19 iC 12 H 25 .

[0106] R 2 Selected from H, C1-C 17 Alkyl and C2-C 17 Alkenyl groups, wherein the alkyl and / or alkenyl groups are straight-chain or branched.

[0107] G 1 Selected from monosaccharides containing 4 to 6 carbon atoms, such as glucose and xylose.

[0108] In the general formula (NIS II), the integer w ranges from 1.1 to 4, and w is an average.

[0109] In one embodiment, the liquid composition of the present invention comprises at least one nonionic surfactant selected from compounds of general formula (NIS III):

[0110]

[0111] The variables in general formula (III) are defined as follows:

[0112] AO is selected from ethylene oxide (EO), propylene oxide (PO), butane oxide (BO), and mixtures thereof.

[0113] R 6 Selected from C5-C 17 Alkyl and C5-C 17 Alkenyl groups, wherein the alkyl and / or alkenyl groups are straight-chain or branched.

[0114] R 7 Selected from H, C1-C 18 -alkyl, wherein the alkyl group is straight-chain or branched.

[0115] The integers in the general formula (NIS III) are numbers from 1 to 70, preferably from 7 to 15.

[0116] In one embodiment, the liquid composition of the present invention comprises at least one nonionic surfactant of general formula (NIS IV):

[0117]

[0118] In formula (NIS IV), the following applies:

[0119] AO is the same or different epoxide alkane, selected from CH2-CH2-O, (CH2)3-O, (CH2)4-O, CH2CH(CH3)-O, CH(CH3)-CH2-O- and CH2CH(n-C3H7)-O.

[0120] R 1 Selected from straight chains (straight chain; n-) or branched chains C4-C 30 -alkyl group, and selected from straight-chain or branched C4-C bonds containing at least one C-double bond. 30 -Alkenyl. R 1 Preferably selected from straight or branched C4-C 30 -alkyl, n-C4-C 30 -alkyl, n-C7-C 15 Alkyl or nC 10 -C 12 -alkyl.

[0121] R 2 Selected from straight chains (straight chain; n-) or branched chains C1-C 30 -alkyl group, and selected from straight-chain or branched C2-C groups containing at least one C-C double bond. 30 -Alkenyl. R 2 Preferably selected from straight or branched C6-C 20 -alkyl group, preferably straight-chain or branched C8-C 12 -alkyl, more preferably straight-chain or branched C 10 -C 12 -alkyl.

[0122] The integer x in the general formula (NIS IV) is preferably a number of 5 to 70, 10 to 60, 15 to 50 or 20 to 40.

[0123] In a preferred embodiment, the detergent formulation of the present invention comprises at least one nonionic surfactant according to formula (NIS IV), wherein R 1 It is n-C3-C 17 Alkyl, R 2 Is it a straight chain or a branched chain C8-C? 14 Alkyl group. Preferably, AO is selected from -(CH2CH2O). x2-(CH2CH(CH3)-O) x3 -(CH2CH2O) x2 -(CH(CH3)CH2-O) x3 and -(CH2CH2O) x4 x2 and x4 are numbers in the range of 15 to 20, while x3 is a number in the range of 1 to 15.

[0124] In a preferred embodiment, the detergent formulation of the present invention comprises at least one nonionic surfactant according to formula (NIS IV), wherein R 1 It is an n-C8 alkyl group, R 2 It is a branch C 11 Alkyl, AO is CH2-CH2-O, and x is 22.

[0125] In a preferred embodiment, the detergent formulation of the present invention comprises at least one nonionic surfactant according to formula (NIS IV), wherein R 1 It is an n-C8 alkyl group, R 2 It is n-C8-C 10 Alkyl, AO is CH2-CH2-O, and x is 40.

[0126] In a preferred embodiment, the detergent formulation of the present invention comprises at least one nonionic surfactant according to formula (NIS IV), wherein R 1 It is an n-C8 alkyl group, R 2 It is nC 10 Alkyl group, AO is selected from -(CH2CH2O). x2 -(CH2CH(CH3)-O) x3 -(CH2CH2O) x2 -(CH(CH3)CH2-O) x3 , where x2 is 22 and x3 is 1.

[0127] In one embodiment, at least one nonionic surfactant is selected from sorbitol esters and / or ethoxylated or propoxylated sorbitol esters. Non-limiting examples include products sold under the trademarks SPAN and TWEEN.

[0128] The nonionic surfactant may be further selected from alkoxylated monoalkylamines or dialkylamines, fatty acid monoethanolamides (FAMA), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fatty acid amides or N-acyl N-alkyl derivatives of glucosamine (glucosamide, GA or fatty acid glucosamide, FAGA) and combinations thereof.

[0129] In one embodiment, the liquid composition of the present invention comprises at least one nonionic surfactant, wherein the total amount of the nonionic surfactant may be in the range of 0% to 80% by weight, preferably in the range of 0.5% to 70% by weight, relative to the total weight of the liquid composition.

[0130] In one embodiment, the liquid composition of the present invention comprises at least one nonionic surfactant in amounts of 0.3% to 30%, 0.4% to 20%, or 0.5% to 10% by weight, all relative to the total weight of the liquid composition. In one embodiment, the at least one nonionic surfactant is selected from surfactants according to general formula (NIS Ia), wherein m is 7; n and o are 0, and R... 1 It is C 12 -C 14 R 2 and R 5 It is H. In one embodiment, the liquid composition of the present invention comprises two nonionic surfactants selected from compounds of general formula (NIS Ia), wherein one of the nonionic surfactants is characterized by R 1 It is C 12 R 2 and R 5 It is H, m is 7, n and o = 0, and another surfactant is characterized by R 1 It is C 14 R 2 and R 5 H is 7, m is 7, and n and o = 0.

[0131] In one embodiment, a detergent formulation comprising at least one nonionic surfactant according to formula (NIS IV) (preferably those described above) is an automatic dishwasher detergent. Preferably, the automatic dishwasher detergent comprises at least one compound according to formula (NIS IV) at approximately 0.3% to 10%, approximately 0.5% to 5%, or approximately 1% to 3% by weight, relative to the total weight of the detergent formulation. In one embodiment, the at least one nonionic surfactant is a compound according to formula (NIS IV), wherein R 1 It is an n-C8 alkyl group, R 2 It is a branch C 11 Alkyl group, AO is CH2-CH2-O, and x is 22.

[0132] In one embodiment, the liquid composition of the present invention comprises a mixture of two or more different nonionic surfactants.

[0133] In one aspect of the invention, component (c) comprises at least one anionic surfactant and at least one nonionic surfactant, wherein the weight ratio of the anionic to the nonionic surfactant is 5:1 to 1:5, preferably 2:1 to 1:4.

[0134] In another aspect of the invention, the liquid composition comprises anionic and nonionic surfactants in a weight ratio of 4:1 to 1:4. Preferably, the liquid composition is a detergent for laundry and / or manual dishwashing and / or cleaning of medical devices.

[0135] Component (d)

[0136] On one hand, the liquid composition of the present invention comprises component (d), which contains at least one hydrolase different from DNase. In one embodiment, the liquid composition of the present invention contains at least one protease and / or at least one amylase and / or at least one cellulase and / or at least one lipase and / or at least one mannanase. Preferably, the composition of the present invention contains at least one hydrolase different from DNase, which is capable of degrading extracellular matrix components, preferably selected from extracellular polymeric substances. Examples of EPS include, but are not limited to, polysaccharides, proteins, and lipids.

[0137] In one implementation, a hydrolytic enzyme, different from a DNA enzyme, can hydrolyze proteins.

[0138] In one embodiment, the hydrolase, which is different from a DNA enzyme, can hydrolyze β-1,4-glycosidic bonds, and preferably can degrade substrates selected from amylopectin, amylose and cellulose.

[0139] In one embodiment, a hydrolase different from a DNase can hydrolyze β-1,6-glycosidic bonds, preferably degrading poly(N-acetylglucosamine), and a protease.

[0140] In one aspect, the compositions of the present invention comprise at least one protease. Proteases belong to EC 3.4 class members and include aminopeptidases (EC 3.4.11), dipeptidases (EC 3.4.13), dipeptidyl-peptidases and tripeptidyl-peptidases (EC 3.4.14), peptidyl-dipeptidases (EC 3.4.15), serine carboxypeptidases (EC 3.4.16), metallocarboxypeptidases (EC 3.4.17), cysteine ​​carboxypeptidases (EC 3.4.18), ω-peptidases (EC 3.4.19), serine endopeptidases (EC 3.4.21), cysteine ​​endopeptidases (EC 3.4.22), aspartate endopeptidases (EC 3.4.23), metalloendopeptidases (EC 3.4.24), threonine endopeptidases (EC 3.4.25), or endopeptidases with unknown catalytic mechanisms (EC 3.4.99).

[0141] In a preferred embodiment, at least one protease is selected from serine proteases (EC 3.4.21). Serine proteases or serine peptidases are characterized by having a serine residue at the catalytically active site, which forms a covalent adduct with the substrate during the catalytic reaction. In the context of this invention, serine proteases are selected from chymotrypsin (e.g., EC 3.4.21.1), elastase (e.g., EC 3.4.21.36), elastase (e.g., EC 3.4.21.37 or EC 3.4.21.71), granzyme (e.g., EC 3.4.21.78 or EC 3.4.21.79), kallikrein (e.g., EC 3.4.21.34, EC 3.4.21.35, EC 3.4.21.118 or EC 3.4.21.119), plasmin (e.g., EC 3.4.21.7), trypsin (e.g., EC 3.4.21.4), thrombin (e.g., EC 3.4.21.5), and subtilisin. Subtilisin is also known as subtilis peptidase, for example, EC 3.4.21.62, which is also referred to hereinafter as "subtilisin".

[0142] A protease is an active protein that possesses "protease activity" or "protein hydrolytic activity." Protein hydrolytic activity is related to the rate at which a protease or proteolytic enzyme degrades a protein within a specified time.

[0143] Methods for analyzing proteolytic activity are well-known in the literature (see Gupta et al. (2002), Appl. Microbiol. Biotechnol. 60: 381-395). Proteolytic activity was determined using succinyl-Ala-Ala-Pro-Phe-p-nitroaniline (Suc AAPF pNA, abbreviated as AAPF; see, for example, DelMar et al. (1979), Analytical Biochem 99316-320) as a substrate. pNA is cleaved from the substrate molecule through proteolytic cleavage, releasing yellow free pNA, which can be measured by measuring OD. 405 Quantify it.

[0144] In a preferred embodiment of the invention, at least one protease is selected from the following: Bacillus subtilis protease from Bacillus amyloliquefaciens BPN (described in Vasantha et al. (1984) J. Bacteriol., Vol. 159, pp. 811-819 and JA Wells et al. (1983) Nucleic Acids Research, Vol. 11, pp. 7911-7925); Bacillus subtilis protease from Bacillus licheniformis (Bacillus subtilis protease Carlsberg; disclosed in EL Smith et al. (1968) J. Biol Chem, Vol. 243, pp. 2184-2191 and Jacobs et al. (1985) Nucleic Acids Res, Vol. 13, pp. 8913-8926); Bacillus subtilis protease PB92 (the original sequence of alkaline protease PB92 is described in EP... In WO 89 / 06279, the Bacillus subtilis proteases 147 and / or 309 (as disclosed in WO 283075A2) are respectively... The following are examples of Bacillus subtilis proteases disclosed in WO 91 / 02792, such as those from Bacillus subtilis DSM 5483 or variants of Bacillus subtilis DSM 5483 as described in WO 95 / 23221; the following are examples of Bacillus subtilis proteases from Bacillus alcalophilus (DSM 11233) disclosed in DE 10064983; the following are examples of Bacillus subtilis proteases from Bacillus gibsonii (DSM 14391) disclosed in WO 2003 / 054184; the following are examples of Bacillus subtilis proteases from Bacillus sp. (DSM 14390) disclosed in WO 2003 / 056017; and the following are examples of Bacillus subtilis proteases from Bacillus sp. (DSM 14390) disclosed in WO 2003 / 055974. The following are examples of Bacillus subtilis protease: 14392; Bacillus subtilis protease from Bacillus gibsonii (DSM 14393) disclosed in WO 2003 / 054184; Bacillus subtilis protease having SEQ ID NO:4 as described in WO 2005 / 063974; Bacillus subtilis protease having SEQ ID NO:4 as described in WO 2005 / 103244; Bacillus subtilis protease having SEQ ID NO:7 as described in WO 2005 / 103244; and Bacillus subtilis protease having SEQ ID NO:2 as described in application DE 102005028295.4.

[0145] In one embodiment, the composition of the present invention contains at least the subtilisin 309 (which may be referred to herein as Savinase) disclosed as sequence a) in Table I of WO 89 / 06279, or a variant thereof that is at least 80% identical to it and has proteolytic activity.

[0146] When compared with the full-length polypeptide sequence of the parent enzyme disclosed above, a suitable protease includes a protease variant having at least 80%, at least 85%, at least 90%, 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% of the same proteolytic activity.

[0147] In one embodiment, at least one protease has SEQ ID NO:22 as described in EP 1921147 (which is a mature alkaline protease sequence from Bacillus lentus DSM 5483) or a protease that is at least 80% identical to it and has proteolytic activity. As described in EP 1921147, a preferred variant of the subtilisin derived from SEQ ID NO:22 is characterized by one or more amino acid substitutions at the following positions: 3, 4, 9, 15, 24, 27, 33, 36, 57, 68, 76, 77, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 131, 154, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252 and 274 (according to BPN' number), which has proteolytic activity. Most preferably, the protease is not mutated at positions Asp32, His64, and Ser221 (according to BPN' numbering).

[0148] In one embodiment, the protease is characterized by having the amino acid glutamic acid (E), or aspartic acid (D), or asparagine (N), or glutamine (Q), or alanine (A), or glycine (G) or serine (S) at position 101 (according to BPN' number), and has proteolytic activity. In one embodiment, the protease comprises one or more further substitutions: (a) threonine at position 3 (3T), (b) isoleucine at position 4 (4I), (c) alanine, threonine, or arginine at position 63 (63A, 63T, or 63R), (d) aspartic acid or glutamic acid at position 156 (156D or 156E), (e) proline at position 194 (194P), (f) methionine at position 199 (199M), (g) isoleucine at position 205 (205I), (h) aspartic acid, glutamic acid, or glycine at position 217 (217D, 217E, or 217G), and (i) a combination of two or more amino acids according to (a) through (h). At least one protease may be at least 80% identical to SEQ ID NO:22 as described in EP 1921147, and is characterized by comprising (according to (a)-(h)) one amino acid or a combination thereof according to (i), and amino acids 101E, 101D, 101N, 101Q, 101A, 101G, or 101S (according to BPN' number), and having proteolytic activity. In one embodiment, the protease is characterized by comprising the mutant (according to BPN' number) R101E, or S3T+V4I+V205I, or R101E and S3T, V4I and V205I, or S3T+V4I+V199M+V205I+L217D.

[0149] In one embodiment, as described in EP 1921147, the protease according to SEQ ID NO:22 is characterized by containing the mutation (according to BPN' number) S3T+V4I+S9R+A15T+V68A+D99S+R101S+A103S+I104V+N218D.

[0150] According to the present invention, the liquid composition of the present invention comprises two or more of the above-described proteases.

[0151] Compositions containing at least one protease typically also contain at least one protease stabilizer selected from those disclosed herein.

[0152] amylase

[0153] In one embodiment, the liquid composition of the present invention comprises at least one amylase. The "amylase" (α and / or β) according to the present invention includes amylases of bacterial or fungal origin (EC 3.2.1.1 and 3.2.1.2, respectively). Chemically modified or protein-engineered mutants are also included.

[0154] The amylase according to the present invention possesses "starch-degrading activity" or "amylase activity" related to the (internal) hydrolysis of glycosidic bonds in polysaccharides. α-Amylase activity can be determined by analyses known to those skilled in the art for measuring α-amylase activity. Examples of analyses for measuring α-amylase activity are as follows:

[0155] α-Amylase activity can be determined using Phadebas tablets as a substrate (Phadebas amylase assay, provided by Magle Life Science). Starch is hydrolyzed by α-amylase to produce a soluble blue fragment. The absorbance of the resulting blue solution (measured spectrophotometrically at 620 nm) is a function of α-amylase activity. Under given conditions, the measured absorbance is directly proportional to the specific activity of the α-amylase in question (activity of pure α-amylase protein / mg).

[0156] In one embodiment, at least one amylase is selected from the following:

[0157] • An amylase from Bacillus licheniformis with SEQ ID NO:2, as described in WO 95 / 10603. Suitable variants are described in WO 95 / 10603, which contain one or more substitutions at the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408, and 444, which have starch-degrading activity. Variants are described in SEQ ID NO:4 of WO 94 / 02597, WO 94 / 018314, WO 97 / 043424, and WO 99 / 019467.

[0158] • The amylase of Bacillus stearothermophilus with SEQ ID NO:6 disclosed in WO 02 / 10355, or an amylase selectively having a C-terminal truncation on the wild-type sequence. Suitable variants of SEQ ID NO:6 include variants containing deletions at positions 181 and / or 182 and / or substitutions at position 193.

[0159] • An amylase of SEQ ID NO:6 from Bacillus genus 707, disclosed in WO 99 / 19467, and functional variants thereof. Preferred variants of SEQ ID NO:6 are variants having substitutions, deletions, or insertions at one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216, and K269.

[0160] The amylase from Bacillus halmapalus with SEQ ID NO:2 or SEQ ID NO:7 described in WO 96 / 23872 is also described herein as SP-722. Preferred variants are described in WO 97 / 3296, WO 99 / 194671 and WO 2013 / 001078.

[0161] Amylase of Bacillus genus DSM 12649 (with SEQ ID NO:4) and its functional variants disclosed in WO 00 / 22103.

[0162] Amylase of Bacillus TS-23 (with SEQ ID NO:2) and its functional variants disclosed in WO 2009 / 061380.

[0163] • An amylase of the genus Cytophaga disclosed in WO 2013 / 184577, having SEQ ID NO:1.

[0164] Amylase of Bacillus megaterium DSM90 (with SEQ ID NO:1, and functional variant thereof) disclosed in WO 2010 / 104675.

[0165] Amylases of the genus Bacillus with amino acids 1-485 of SEQ ID NO:2, as described in WO 00 / 60060, and their functional variants.

[0166] Amylases from Bacillus amyloliquefaciens and functional variants thereof disclosed in WO 2016 / 092009, wherein the amylases are preferably selected from the amylases according to SEQ ID NO:3.

[0167] The amylase having SEQ ID NO:12 as described in WO 2006 / 002643, and the amylase variant containing Y295F and M202LITV substitutions in SEQ ID NO:12.

[0168] The amylase having SEQ ID NO:6 as described in WO 2011 / 098531, or an amylase variant containing substitutions at one or more positions, wherein the substitutions are selected from SEQ ID NO:6 of 193[G,A,S,T or M], 195[F,W,Y,L,I or V], 197[F,W,Y,L,I or V], 198[Q or N], 200[F,W,Y,L,I or V], 203[F,W,Y,L,I or V], 206[F,W,Y,N,L,I,V,H,Q,D or E], 210[F,W,Y,L,I or V], 212[F,W,Y,L,I or V], 213[G,A,S,T or M] and 243[F,W,Y,L,I or V].

[0169] The amylase having SEQ ID NO:1 as described in WO 2013 / 001078, or the amylase variant containing two or more (several) positional changes corresponding to positions G304, W140, W189, D134, E260, F262, W284, W347, W439, W469, G476, and G477.

[0170] • An amylase or amylase variant having SEQ ID NO:2 as described in WO 2013 / 001087, comprising a deletion at position 181+182 or 182+183 or 183+184 within SEQ ID NO:2, optionally comprising one or more modifications at any position within SEQ ID NO:2 corresponding to W140, W159, W167, Q169, W189, E194, N260, F262, W284, F289, G304, G305, R320, W347, W439, W469, G476 and G477.

[0171] • Amylases derived from the above-mentioned hybrid α-amylases (such as those described in WO 2006 / 066594) and their functional variants;

[0172] • The hybrid amylase according to WO 2014 / 183920 has A and B domains that are at least 90% identical to SEQ ID NO:2 of WO 2014 / 183920, and C domain that is at least 90% identical to SEQ ID NO:6 of WO 2014 / 183920, wherein the hybrid amylase has starch-degrading activity; preferably, the hybrid α-amylase is at least 95% identical to SEQ ID NO:23 of WO 2014 / 183920 and has starch-degrading activity;

[0173] • The hybrid amylase according to WO 2014 / 183921, wherein its A and B domains are at least 75% identical to SEQ ID NO:2, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:26, SEQ ID NO:32 and SEQ ID NO:39 disclosed in WO 2014 / 183921, and its C domain is at least 90% identical to SEQ ID NO:6 of WO 2014 / 183921, wherein the hybrid amylase has starch-degrading activity; preferably, the hybrid α-amylase is at least 95% identical to SEQ ID NO:30 disclosed in WO 2014 / 183921 and has starch-degrading activity.

[0174] In one embodiment, the liquid composition of the present invention comprises two or more amylases disclosed above.

[0175] Cellulase

[0176] In one embodiment, the liquid composition of the present invention comprises at least one cellobiase selected from cellobiase (1,4-PD-glucan-cellobiase, EC 3.2.1.91), endo-ss-1,4-glucanase (endo-1,4-PD-glucan-4-glucanase, EC 3.2.1.4), and ss-glucosidase (EC 3.2.1.21). Preferably, the composition comprises at least one cellulase of glycosyl hydrolase family 7 (GH7, pfam00840), preferably selected from endo-glucanase (EC 3.2.1.4).

[0177] "Cellulases," "cellulases," or "cellulose hydrolases" are enzymes involved in the hydrolysis of cellulose. Analyses used to measure "cellulase activity" or "cellulose decomposition activity" are known to those skilled in the art. For example, according to Hoffman, WS, J. Biol. Chem. 120, 51 (1937), cellulose decomposition activity can be determined by the hydrolysis of carboxymethyl cellulose by cellulase to reduce carbohydrates, and its reducing power can be determined by a ferricyanide reaction colorimetric method.

[0178] The cellulases according to the invention include cellulases derived from bacteria or fungi. In one embodiment, at least one cellulase is selected from cellulases containing a cellulose-binding domain. In another embodiment, at least one cellulase is selected from cellulases containing only a catalytic domain, meaning that the cellulase lacks a cellulose-binding domain.

[0179] In one embodiment, the liquid composition comprises at least one cellulase whose polypeptide sequence is 80% identical to that of SEQ ID NO:2 according to WO 95 / 02675. In one embodiment, the composition comprises at least one cellulase whose polypeptide sequence is 80% identical to that of SEQ ID NO:4 according to WO2004 / 053039. In one embodiment, the liquid composition comprises at least one cellulase whose polypeptide sequence is 80% identical to that of SEQ ID NO:2 according to WO 2002 / 99091.

[0180] In one embodiment, the liquid composition of the present invention comprises two or more cellulases, preferably the endoglucanase disclosed above (EC 3.2.1.4).

[0181] Lipase

[0182] On one hand, the liquid composition of the present invention comprises at least one lipase. “Lipase,” “lipolytic enzyme,” and “lipoesterase” all refer to enzymes of EC category 3.1.1 (“carboxylate hydrolases”). Lipase refers to an active protein having lipase activity (or lipolytic activity; triacylglycerol lipase, EC 3.1.1.3), keratinase activity (EC 3.1.1.74; enzymes having keratinase activity are referred to herein as keratinases), sterol esterase activity (EC 3.1.1.13), and / or wax ester hydrolase activity (EC 3.1.1.50).

[0183] Methods for determining lipase activity are well known in the literature (see, for example, Gupta et al. (2003), Biotechnol. Appl. Biochem. 37, pp. 63-71). For instance, lipase activity can be measured by hydrolyzing the ester bond in the substrate p-nitrophenyl palmitate (pNP-palmitate, C:16) to release yellow pNP, which can be detected at 405 nm.

[0184] Lipases include lipases derived from bacteria or fungi, and useful lipases are known to those skilled in the art.

[0185] In one embodiment, at least one lipase is selected from fungal triglyceride lipases (EC category 3.1.1.3). The fungal triglyceride lipase may be selected from *Thermomyces lanuginosa* lipases. In one embodiment, at least one *Thermomyces lanuginosa* lipase is selected from amino acids 1-269 of SEQ ID NO:2 in US 5869438 and its lipolytic variants. Preferably, at least one *Thermomyces lanuginosa* lipase is at least 80% identical to SEQ ID NO:2 in US 5869438, characterized by having amino acids T231R and N233R. The *Thermomyces lanuginosa* lipase variant preferably further comprises one or more of the following amino acid exchanges: Q4V, V60S, A150G, L227G, P256K.

[0186] In one embodiment, the liquid composition of the present invention comprises at least two lipases disclosed above.

[0187] Mannan-degrading enzymes

[0188] In one embodiment, the liquid composition of the present invention comprises at least one mannan-degrading enzyme selected from β-mannosidase (EC 3.2.1.25), endo-1,4-β-mannosidase (EC 3.2.1.78), and 1,4-β-mannobiose glycosidase (EC 3.2.1.100). Preferably, at least one mannan-degrading enzyme is selected from the endo-1,4-β-mannosidase group (EC 3.2.1.78), which is referred to herein as endo-β-1,4-D-mannanase, β-mannanase, or mannanase.

[0189] Peptides exhibiting mannanase activity can be tested for mannanase activity according to standard test methods known in the art, for example, by applying the test solution to 4 mm diameter wells punched into an agar plate containing 0.2% AZCL galactomannan (caryomannan), which is derived from Megazyme (Megazyme's website address: http: / / www.megazyme.com / Purchase / index.html Substrate for endoglucanase assay sold under Cat No. I-AZGMA.

[0190] In one embodiment, the liquid composition of the present invention comprises at least one mannanase selected from families 5 or 26. The term "alkaline mannanase" means mannanase comprising at least 40% of which has its maximum activity within a given pH range of 7 to 12, preferably 7.5 to 10.5.

[0191] In one embodiment, at least one mannanase included in the composition of the present invention is selected from mannanases derived from Bacillus organisms, such as those described in: JP-0304706 [β-mannanase from Bacillus], JP-63056289 [alkaline, thermostable β-mannanase], JP-63036774 [β-mannanase and β-mannosidase produced by Bacillus microorganism FERM P-8856 at alkaline pH], JP-08051975 [alkaline β-mannanase from Bacillus alkalophilus AM-001], WO 97 / 11164 [mannanase from Bacillus amyloliquefaciens], WO 91 / 18974 [mannanase active at extreme pH and temperature], WO 2014 / 100018 [Endonuclease-(3-mannanase 1 (Bleman1; see US5476775) cloned from Bacillus circulans or Bacillus lentus strain CMG1240]. The appropriate mannanase is described in WO 99 / 064619.

[0192] In one embodiment, the at least one mannanase included in the liquid composition of the present invention is a polypeptide that is 80% identical to SEQ ID NO:2 in US6566114. The at least one mannanase included in the liquid composition of the present invention is selected from mannanases derived from Trichoderma organisms, such as those disclosed in WO 93 / 24622 or WO 2011 / 085747.

[0193] When compared with the full-length polypeptide sequence of the corresponding parent enzyme disclosed above, the mannanase variant according to the invention is at least 75%, at least 80%, at least 85%, at least 90%, 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% identical.

[0194] In one embodiment, the liquid composition of the present invention comprises two or more mannan-degrading enzymes, preferably endo-1,4-β-mannosidase (EC 3.2.1.78) as disclosed above.

[0195] Other components

[0196] solvent

[0197] In one embodiment, the innovative composition of the present invention comprises water in the range of 5% to 95% by weight, 5% to 70% by weight, 5% to 50% by weight, and 50% to 95% by weight, all relative to the total weight of the liquid composition.

[0198] In one embodiment, the liquid composition of the present invention comprises at least one organic solvent, preferably selected from water-miscible organic solvents. Examples of such organic solvents include, but are not limited to, acetaldehyde, acetic acid, acetone, acetonitrile, butane-2-ol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2-butoxyethanol, 2-(2-butoxyethoxy)ethanol, butyric acid, 2-ethoxyethanol, diethanolamine, diethylene glycol, diethylenetriamine, dipropylene glycol, dimethylformamide, dimethoxyethane, dimethyl sulfoxide, 1,4-dioxane, ethanol, ethylamine, ethylene glycol, formic acid, etc. Furfuryl alcohol, hexanediol, methanol, 2-methoxyethanol, methyl diethanolamine, methyl isocyanate, N-methyl-2-pyrrolidone, 1-propanol, 1,2-propanediol, 1,3-propanediol, 1,2,3-propanetriol, 1,2-pentanediol, 1,5-pentanediol, 2-propanol, propionic acid, propylene glycol, 2-propoxyethanol, 2-(2-propoxyethoxy)ethanol, pyridine, tetrahydrofuran, triethylenglycol.

[0199] Preferably, the liquid composition of the present invention comprises at least one organic solvent selected from ethanol, n-propanol, isopropanol, sec-butanol, ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,2,3-propanetriol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, diethylene glycol, 2-(2-butoxyethoxy)ethanol, 2-(2-propoxyethoxy)ethanol, hexanediol, 2-methoxyethanol, 2-ethoxyethanol, and 2-propoxyethanol, with ethanol, isopropanol, or 1,2-propanediol being more preferred.

[0200] The liquid compositions of the present invention typically contain an organic solvent in amounts ranging from 0% to 30%, 2% to 25%, and 5% to 20% by weight relative to the total weight of the liquid composition. In one embodiment, the liquid composition of the present invention contains 5% to 10% of an organic solvent, preferably 1,2-propanediol.

[0201] The liquid composition to be sealed in a water-soluble container (e.g., a bag made of a water-soluble polymer film) preferably contains as little water as possible to ensure the storage stability of the bag. Storage stability of the bag, as used herein, means that the polymer film does not become leaky during storage before the bag is used in a washing process.

[0202] Compounds that stabilize the liquid composition itself

[0203] "A compound that stabilizes the liquid composition itself" means any compound that can establish the storage stability of the liquid composition in an effective amount to ensure storage stability.

[0204] For those skilled in the art, storage stability in the context of liquid compositions typically includes aspects of product appearance and dosage uniformity.

[0205] The appearance of a product is affected by its pH value, as well as the presence of compounds such as antioxidants, viscosity modifiers, and emulsifiers.

[0206] Dosage uniformity is usually related to product uniformity.

[0207] The compositions according to the invention can be alkaline or have a neutral or slightly acidic pH value, for example 6-14, 6.5-13, 8-10.5 or 8.5-9.0.

[0208] In one embodiment, the liquid composition of the present invention comprises at least one preservative selected from 2-phenoxyethanol, glutaraldehyde, 2-bromo-2-nitropropane-1,3-diol, and acidic formic acid or a salt thereof.

[0209] In one embodiment, the liquid composition contains the preservative, preferably 2-phenoxyethanol, at a concentration of 0.01% to 5% or 0.1% to 2% by weight relative to the total weight of the detergent formulation.

[0210] In another embodiment, the liquid composition contains a preservative, preferably 2-bromo-2-nitropentane-1,3-diol, at a concentration of 5 ppm to 5000 ppm or 20 ppm to 1000 ppm of the total weight of the detergent formulation.

[0211] In another embodiment, the liquid composition contains glutaraldehyde at a concentration of 2 ppm to 5000 ppm or 10 ppm to 2000 ppm of the total weight of the detergent formulation.

[0212] In yet another embodiment, the liquid composition contains formic acid (as an acid or a salt thereof) at a concentration of 0.01% to 3% by weight or 0.05% to 0.5% by weight relative to the total weight of the detergent formulation.

[0213] On one hand, the liquid composition of the present invention comprises at least one enzyme stabilizer selected from boron-containing compounds, polyols, peptide aldehydes, other stabilizers and mixtures thereof.

[0214] Boron-containing compounds are typically selected from boric acids and their derivatives, borous acids and their derivatives such as arylboronic acids and their derivatives, borates and mixtures thereof. Boric acid in this article may also be referred to as orthoboric acid.

[0215] In one embodiment, at least one boron-containing compound is selected from arylboronic acids and their derivatives. In one embodiment, the boron-containing compound is selected from phenylboronic acid (BBA), also known as phenylboronic acid (PBA), its derivatives, and mixtures thereof. In one embodiment, the phenylboronic acid derivative is selected from derivatives of formulas (1a) and (1b):

[0216]

[0217] in

[0218] R1 is selected from hydrogen, hydroxyl, unsubstituted or substituted C1-C6 alkyl, and unsubstituted or substituted C1-C6 alkenyl; in a preferred embodiment, R is selected from hydroxyl and unsubstituted C1 alkyl.

[0219] R2 is selected from hydrogen, hydroxyl, unsubstituted or substituted C1-C6 alkyl, and unsubstituted or substituted C1-C6 alkenyl; in a preferred embodiment, R is selected from H, hydroxyl and unsubstituted C1 alkyl.

[0220] In one embodiment, the phenylboronic acid derivative is selected from 4-formylphenylboronic acid (4-FPBA), 4-carboxyphenylboronic acid (4-CPBA), 4-(hydroxymethyl)phenylboronic acid (4-HMPBA), and p-tolylboronic acid (p-TBA).

[0221] In one embodiment, the liquid composition of the present invention comprises at least one polyol selected from polyols containing 2 to 6 hydroxyl groups. Preferably, the at least one polyol is selected from ethylene glycol, propylene glycol, 1,2-propanediol, 1,2-butanediol, 1,2-pentanediol, ethylene glycol, hexanediol, glycerol, sorbitol, mannitol, erythritol, glucose, fructose, lactose, and mixtures thereof.

[0222] In one embodiment, the liquid composition comprises at least one peptide aldehyde (in one of the B1-BO-R forms, selected from dipeptides, tripeptides, or tetrapeptide aldehydes and aldehyde analogs, where R is H, CH3, CX3, CHX2, or CH2X (X = halogen), BO is a single amino acid residue (in one embodiment, having an optionally substituted aliphatic or aromatic side chain); and B1 consists of one or more amino acid residues (in one embodiment, one, two, or three), which optionally contains an N-terminal protecting group, or as described in WO 09 / 118375 and WO 98 / 13459, or a protein-type protease inhibitor, such as RASI, BASI, WASI (a bifunctional α-amylase / subtilis protease inhibitor for rice, barley, and wheat), or CI2 or SSI.

[0223] In some embodiments, at least one peptide stabilizer is selected from compounds of formula (2) and their salts:

[0224]

[0225] R in equation (2) 1 R 2 R 3 R 4 R 5 Z is defined as follows:

[0226] R 1 It is a group that makes NH-CHR 1 -CO is an L or D-amino acid residue of Gly, Ala, Val, Leu, Ile, Met, Pro, Phe, Trp, Ser, Thr, Asp, Gln, Tyr, Cys, Lys, Arg, His, Asn, Glu, meta-tyrosine, 3,4-dihydroxyphenylalanine, Nva, or Nle. Preferably, R 1 It is this group that makes NH-CHR 1 -CO is an L or D-amino acid residue of Ala, Val, Gly, Arg, Leu, Phe, Ile, His, or Thr. More preferably, R 1 It is this group that makes NH-CHR 1 -CO is an L or D-amino acid residue of Ala, Val, Gly, Arg, Leu, Ile, or His.

[0227] R 2 It is this group that makes NH-CHR 2 -CO is an L or D-amino acid residue of Gly, Ala, Val, Leu, Ile, Met, Pro, Phe, Trp, Ser, Thr, Asp, Gln, Tyr, Cys, Lys, Arg, His, Asn, Glu, meta-tyrosine, 3,4-dihydroxyphenylalanine, Nva, or Nle. Preferably, R 2 It is this group that makes NH-CHR 2 -CO is an L or D-amino acid residue of Ala, Cys, Gly, Pro, Ser, Thr, Val, Nva, or Nle. More preferably, R 2 It is this group that makes NH-CHR 2 -CO is an L or D-amino acid residue of Ala, Gly, Pro, or Val.

[0228] R 3 It is this group that makes NH-CHR 3 -CO is an L or D-amino acid residue of Tyr, meta-tyrosine, 3,4-dihydroxyphenylalanine, Phe, Val, Ala, Met, Nva, Leu, Ile, or Nle, or other non-natural amino acids containing an alkyl group. Preferably, R3 is a group that makes NH-CHR 3 -CO is an L or D-amino acid residue of Tyr, Phe, Val, Ala, or Leu.

[0229] In one implementation, R1 It is this group that makes NH-CHR 1 -CO is an L or D-amino acid residue of Gly or Val; R 2 It is this group that makes NH-CHR 2 -CO is an L or D-amino acid residue of Ala; and R 3 It is this group that makes NH-CHR 3 -CO is an L or D-amino acid residue of Tyr, Ala, or Leu.

[0230] In one implementation, R 1 It is this group that makes NH-CHR 1 -CO is an L or D-amino acid residue of Val; R 2 It is this group that makes NH-CHR 2 -CO is an L or D-amino acid residue of Ala; and R 3 It is this group that makes NH-CHR 3 -CO is an L or D-amino acid residue of Leu.

[0231] In one implementation, R 4 and R 5 Each is independently selected from methyl, ethyl, isopropyl, 2-butyl, or 3-pentyl. More preferably, R 4 and R 5 They are all methyl, ethyl, isopropyl, 2-butyl, or 3-pentyl.

[0232] Z is selected from hydrogen, an N-terminal protecting group, and optionally one or more amino acid residues containing an N-terminal protecting group. Preferably, Z is an N-terminal protecting group.

[0233] If Z is one or more amino acid residues containing an N-terminal protecting group, the N-terminal protecting group is preferably a small aliphatic group, such as formyl, acetyl, fluorenylmethoxycarbonyl (Fmoc), tert-butoxycarbonyl (Boc), methoxycarbonyl (Moc); methoxyacetyl (Mac); methyl carbamate or methylaminocarbonyl / methylurea. For tripeptides, the N-terminal protecting group is preferably a bulky aromatic group, such as benzoyl (Bz), benzyloxycarbonyl (Cbz), p-methoxybenzylcarbonyl (MOZ), benzyl (Bn), p-methoxybenzyl (PMB), or p-methoxyphenyl (PMP).

[0234] Other suitable N-terminal protecting groups are described in Peter GMWuts’ Greene’s Protective Groups in Organic Synthesis, 5th edition, John Wiley & Sons, Inc., 2014, and in Isidro-Llobet et al., Amino Acid-Protecting Groups, Chem. Rev. 2009 109(6), 2455-2504.

[0235] In a preferred embodiment, the peptide stabilizer is selected from compounds according to formula (2), wherein

[0236] ·R 1 and R 2 It is this group that makes NH-CHR 1 -CO and NH-CHR 2 -CO are each an L or D-amino acid residue selected from Ala, Cys, Gly, Pro, Ser, Thr, Val, Nva, or Nle, and R 3 It is this group that makes NH-CHR 3 -CO is an L or D amino acid residue selected from Tyr, meta-tyrosine, 3,4-dihydroxyphenylalanine, Phe, Val, Ala, Met, Nva, Leu, Ile, or Nle;

[0237] and

[0238] The N-terminal protecting group Z is selected from benzyloxycarbonyl (Cbz), p-methoxybenzylcarbonyl (MOZ), benzyl (Bn), benzoyl (Bz), p-methoxybenzyl (PMB), p-methoxyphenyl (PMP), formyl, acetyl (Ac), methoxy, alkoxycarbonyl, methoxycarbonyl, fluorenylmethoxycarbonyl (Fmoc) or tert-butoxycarbonyl (Boc).

[0239] In one embodiment, the liquid composition comprises about 0.1% to 2% by weight of at least one peptide stabilizer relative to the total weight of the enzyme preparation. Preferably, the liquid composition comprises about 0.15% to 1%, 0.2% to 0.5%, or about 0.3% by weight of at least one peptide stabilizer relative to the total weight of the enzyme preparation. More preferably, the liquid composition comprises about 0.3% by weight of a peptide stabilizer according to formula (2) relative to the total weight of the liquid composition, characterized in that:

[0240] ·R 1 It is this group that makes NH-CHR 1 -CO is an L or D-amino acid residue of Val; R 2It is this group that makes NH-CHR 2 -CO is an L or D-amino acid residue of Ala; and R 3 It is this group that makes NH-CHR 3 -CO is an L or D-amino acid residue of Leu; and

[0241] The N-terminal protecting group Z is benzyloxycarbonyl (Cbz).

[0242] In one embodiment, the liquid composition of the present invention comprises at least one polyol described above and at least one peptide stabilizer described above.

[0243] In one embodiment, the liquid composition of the present invention comprises a salt preferably selected from NaCl, KCl, lactic acid base salts, formic acid, and mixtures thereof.

[0244] In one embodiment, the liquid composition of the present invention contains water-soluble zinc (II), calcium (II) and / or magnesium (II) ions (E) (which provide said ions for the enzyme) and other metal ions (e.g., barium (II), scandium (II), iron (II), manganese (II), aluminum (III), tin (II), cobalt (II), copper (II), nickel (II) and vanadium oxide (IV)) in the finished composition.

[0245] As described herein, a compound that stabilizes a liquid composition refers to any compound other than enzyme stabilizers and antimicrobial compounds required to establish the storage stability of the liquid composition in an effective amount to ensure storage stability.

[0246] For those skilled in the art, storage stability in the context of liquid compositions typically includes aspects of product appearance and dosage uniformity.

[0247] The appearance of a product is affected by its pH value and the presence of compounds such as antioxidants, viscosity modifiers, and emulsifiers.

[0248] Dosage uniformity is usually related to product uniformity.

[0249] The enzyme preparations of the present invention may be alkaline or have a neutral or slightly acidic pH value, such as 6-14, 6.5-13, 8-10.5 or 8.5-9.0.

[0250] Component (a) is used to provide a homogeneous liquid composition comprising component (b).

[0251] In one aspect, the present invention provides a composition containing a homogeneous liquid enzyme having anti-biofilm activity. Homogeneity, as used herein, means that the liquid is a homogeneous liquid without phase separation.

[0252] The present invention provides the use of component (a) disclosed herein for providing a homogeneous liquid composition comprising component (b), wherein the liquid composition preferably has anti-biofilm activity.

[0253] This invention relates to a method for providing a liquid antibacterial composition by adding at least one chlorinated hydroxydiphenyl ether according to formula (I) to a liquid composition having DNase activity. Preferably, the composition has anti-biofilm activity.

[0254] In one embodiment, the liquid antimicrobial composition is stably stored at 4°C for at least 7 days. In another embodiment, the liquid antimicrobial composition is stably stored at 30°C or 37°C for at least 7 days.

[0255] In one embodiment, storage stability refers to the stability of the homogeneity of the liquid antimicrobial composition. Preferably, the stability of the homogeneity of the liquid antimicrobial composition means that phase separation does not occur during storage.

[0256] In another embodiment, storage stability refers to the storage stability of DNase activity. Storage stability of DNase activity preferably means that the remaining DNase activity after storage is at least 60% compared to the DNase activity before storage. The remaining DNase activity after storage is preferably at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the DNase activity before storage.

[0257] Uses of liquid compositions

[0258] The liquid compositions of this invention may be referred to herein as “detergent formulations” or “cleaning formulations,” meaning formulations specified for cleaning soiled materials. Cleaning may mean rinsing or hard surface cleaning. Soiled materials according to this invention include textiles and / or hard surfaces.

[0259] The term "cleaning" encompasses both household and industrial cleaning, and refers to the process of treating textiles with a solution containing the detergent formulation of this invention. The cleaning process can be carried out using technical equipment such as household or industrial cleaning machines. Alternatively, the cleaning process can be performed manually.

[0260] The term “textiles” means any textile material, including yarn (threads made of natural or synthetic fibers for knitting or weaving), yarn intermediates, fibers, nonwoven materials, natural materials, synthetic materials, and fabrics made of said materials (textiles made of woven, knitted or felted fibers), such as clothing (any garments made of textiles), clothing, and other articles.

[0261] The term "fiber" includes natural fibers, synthetic fibers, and mixtures thereof. Examples of natural fibers are plant fibers (such as flax, jute, and cotton) or animal fibers that contain proteins such as collagen, keratin, and fibroin (e.g., silk, wool, angora, mohair, cashmere). Examples of synthetic fibers are polyurethane fibers such as spandex. Or Leica Polyester fibers, polyolefins such as elastofin, or polyamide fibers such as nylon. The fibers can be monofilaments or part of textiles such as knitted fabrics, woven fabrics, or nonwoven fabrics.

[0262] The term "hard surface cleaning" is defined herein as the cleaning of hard surfaces, which can include any hard surface in the home, such as floors, furniture, walls, sanitary ceramics, glass, and metal surfaces including cutlery or plates. Therefore, the term "hard surface cleaning" also refers to "dishwashing," which encompasses all forms of dishwashing, such as manual or automatic dishwashing (ADW). Dishwashing includes, but is not limited to, cleaning all forms of cutlery such as plates, cups, glasses, bowls, spoons, knives, forks, and utensils, as well as ceramics, plastics such as melamine resin, metals, porcelain, glass, and acrylic resins.

[0263] In one aspect, the present invention relates to a method for inhibiting and / or reducing biofilm formation on hard and / or flexible and / or textile surfaces in washing and cleaning equipment, which involves:

[0264] (1) Using the liquid composition of the present invention

[0265] (2) Contact the surface with the liquid composition, and

[0266] (3) thereby preventing one or more microorganisms from attaching to the surface and / or growing on the surface and / or forming a biofilm on the surface.

[0267] Preferably, the liquid composition of the present invention is diluted with water in a washing device or container before or during contact with the surface. Preferably, the liquid composition is diluted in water at a ratio of 1:100 to 1:3000.

[0268] Reduction in microbial growth means, in any case, a reduction of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% in the formation of biofilm on hard and soft surfaces or textile surfaces in contact with the detergent formulation of the present invention, compared to the formation of biofilm on the surfaces in contact with a detergent formulation lacking component (a) and / or component (b). In one embodiment, complete or near-complete inhibition of biofilm formation means “substantially no biofilm” formation. “Substantially no biofilm” means less than about 20%, less than about 18%, less than about 15%, or less than about 12% of biofilm formation on equipment or fabric surfaces in contact with a detergent formulation lacking component (a) and / or component (b).

[0269] In one embodiment, the biofilm according to the above embodiment comprises at least one microorganism selected from bacteria and fungi.

[0270] Based on the Gram staining retention characteristics, the bacteria contained in the biofilm of this invention can be divided into two major categories: Gram-positive and Gram-negative. This is due to the significant differences in the ultrastructure and chemical composition of the bacterial cell wall.

[0271] In one aspect of the invention, at least one microorganism grown in the biofilm is selected from Gram-positive bacteria, preferably cocci and bacilli. In one embodiment, at least one microorganism is selected from catalase-positive cocci, such as Staphylococcus and Micrococcus. In another embodiment, at least one microorganism is selected from Gram-negative cocci, such as Streptococcus. Staphylococci can be further subdivided into coagulase-positive (e.g., Staphylococcus aureus) and coagulase-negative (e.g., Staphylococcus epidermidis, Staphylococcus saprophyticus). In a preferred embodiment, at least one microorganism is selected from coagulase-positive staphylococci. More preferably, at least one microorganism is Staphylococcus aureus.

[0272] Gram-positive bacteria can also be selected from the genera *Aerobicum*, *Microbacterium*, and *Micrococcus*.

[0273] In one aspect of the invention, at least one microorganism growing in the biofilm according to the invention is selected from Gram-negative bacteria, preferably from Gram-negative Proteus bacteria. Proteus bacteria are generally subdivided into the following classes: α-Proteus, β-Proteus, γ-Proteus, δ-Proteus, ε-Proteus, ζ-Proteus, Thiobacillus acidophilus, hydrogenophilic bacteria, and Oligoflexia.

[0274] Alpha Proteus includes, but is not limited to, the following genera: Brucella, Rhizobium, Agrobacterium, Stembacterium, Rickettsia, and Wolbachia. Preferably, at least one microorganism growing in the biofilm according to the invention originates from the order Stembacterales, more preferably from the genus Brevundimonas.

[0275] Beta-Proteus includes, but is not limited to, the following genera: Bordetella, Ralstonia, Neisseria, and Nitrosomonas.

[0276] Gamma Proteus includes, but is not limited to, the following genera: *Escherichia*, *Shigella*, *Salmonella*, *Stenotrophomonas*, *Yersinia*, *Buchnera*, *Haemophilus*, *Vibrio*, and *Pseudomonas*. Preferably, at least one microorganism is selected from the orders Enterobacteriaceae, Xanthomonaes, and Pseudomonas. Preferably, at least one microorganism is selected from *Escherichia coli*, *Pseudomonas aeruginosa*, and *Pseudomonas putida*.

[0277] The fungi can be selected from the class Microbotryomycetes, preferably from the genus Rhodotorula, such as species selected from Rhodotorula mucilaginosa, R. glutinis, and R. minuta.

[0278] Fungi may also be selected from yeasts, preferably from the order Saccharales, and more preferably from the genus Candida, such as Candida albicans.

[0279] Individual detergent components and their uses in detergent formulations are known to those skilled in the art. Suitable detergent components include, in particular, surfactants, builders, polymers, alkalis, bleaching systems, optical brighteners, foam inhibitors and stabilizers, solubilizers, and corrosion inhibitors. Further examples are described, for instance, in the “Complete Technology Book on Detergents with Formulations (Detergent Cake, Dishwashing Detergents, Liquid & Paste Detergents, Enzyme Detergents, Cleaning Powder & Spray-Dried Washing Powder)”, Institute of Indian Engineers (EIRI), 6th edition (2015). Another reference for those skilled in the art is the “Detergent Formulations Encyclopedia”, Solverchem Publishing, 2016.

[0280] It should be understood that if the components are mixed into a liquid detergent formulation and provided as a composition, the detergent formulation containing the composition of the present invention needs to be adjusted by concentration, provided that the components are effective for the desired purpose. This purpose includes the product's stability against degradation and its washing performance.

[0281] Detergent components can have multiple functions in the final application of a detergent formulation; therefore, any detergent component mentioned in the specific function section of this article may also have another function in the final application of a detergent formulation. In the final application of a detergent formulation, the function of a particular detergent component typically depends on its amount in the detergent formulation, i.e., the effective amount of the detergent component.

[0282] The term "effective amount" includes the amount of a single component that provides effective detergency and / or effective cleaning conditions (e.g., pH value, foaming amount), the amount of a specific component that effectively provides optical benefits (e.g., optical brightening, anti-dye migration), and / or the amount of a specific component that effectively assists in treatment (e.g., maintaining physical properties during processing, storage and use; e.g., viscosity modifiers, water-soluble growth promoters, desiccants).

[0283] In one embodiment, the detergent formulation is a formulation of two or more detergent ingredients, wherein at least one ingredient effectively removes stains, at least one ingredient effectively provides optimal cleaning conditions, and at least one ingredient effectively maintains the physical properties of the detergent.

[0284] The liquid detergent formulation of the present invention comprises, with respect to the total weight of the liquid detergent formulation, a component (a) and a component (b) having a weight percentage of ...% ...%.

[0285] In one embodiment, the detergent formulation of the present invention is a liquid at 20°C and 101.3 kPa.

[0286] In one embodiment of the invention, the liquid detergent formulation comprises one or more viscosity modifiers. Non-limiting examples of suitable viscosity modifiers include agar-agar, carrageenan, tragacanth gum, gum arabic, xanthan gum, sodium alginate, pectin, hydroxyethyl cellulose, hydroxypropyl cellulose, starch, gelatin, locust bean gum, cross-linked poly(meth)acrylates such as polyacrylic acid cross-linked with bis(meth)acrylamide, and further comprising silicic acid, clays such as, but not limited to, montmorillonite, zeolite, dextrin, and casein. The viscosity modifier may be included in an amount that effectively provides the desired viscosity.

[0287] In one embodiment of the invention, the liquid detergent formulation comprises one or more hydrotropes, which may be organic solvents such as ethanol, isopropanol, ethylene glycol, 1,2-propanediol, and other organic solvents miscible with water under normal conditions, but are not limited thereto. Other examples of suitable hydrotropes are sodium salts of toluenesulfonic acid, xylenesulfonic acid, and cumenesulfonic acid. The amount of hydrotropes generally helps or enables the dissolution of compounds with limited solubility in water.

[0288] In one embodiment, the liquid detergent formulation of the present invention is free of bleach, such as inorganic peroxide compounds or chlorine bleach such as sodium hypochlorite, meaning that in each case, the liquid detergent formulation according to the present invention contains a total of 0.01% or less of inorganic peroxide compounds and chlorine bleach by weight relative to the total weight of the liquid detergent formulation.

[0289] laundry

[0290] In one embodiment, the liquid composition of the present invention is a liquid detergent. Typically, the detergent contains a relatively high amount of surfactant, preferably selected from at least one nonionic surfactant according to formula (NIS-I) and / or at least one anionic surfactant according to formula (AS-I) and / or at least one anionic surfactant according to formula (AS-II).

[0291] In one embodiment, the liquid detergent contains an anionic surfactant in an amount of about 5% to 50% by weight, preferably about 10% to 40% by weight, relative to the total weight of the liquid detergent composition.

[0292] In one embodiment, the liquid detergent contains a nonionic surfactant in an amount of about 5% to 50% by weight, preferably about 5% to 40% by weight, relative to the total weight of the liquid detergent composition.

[0293] In one embodiment, the liquid detergent of the present invention comprises at least one hydrolytic enzyme disclosed herein. Preferably, at least one protease disclosed herein is combined with one or more detergent components by incorporation into the liquid composition of the present invention. Preferably, at least one protease is contained in an amount of about 0.005% to 0.15% by weight, more preferably about 0.01% to 0.1% by weight, relative to the total weight of the detergent formulation.

[0294] In one embodiment, the liquid detergent preferably contains at least one lipase disclosed herein in an amount of about 0.001% to 0.005% by weight, more preferably 0.001% to 0.002% by weight, relative to the total weight of the detergent formulation. The at least one lipase is selected from fungal triglyceride lipases, specifically from the Thermomyces lanuginosa lipase and its variants disclosed herein.

[0295] In one embodiment, the liquid detergent preferably contains at least one α-amylase disclosed herein in an amount of about 0.001% to 0.015% by weight, more preferably 0.002% to 0.015% by weight, relative to the total weight of the detergent formulation.

[0296] In one embodiment, the liquid detergent preferably contains at least one cellulase disclosed herein in an amount of about 0.001% to 0.01% by weight, more preferably 0.002% to 0.009% by weight, relative to the total weight of the detergent formulation. The at least one cellulase is selected from the endoglucanases disclosed herein (EC 3.2.1.4), preferably endoglucanases and variants thereof having the amino acid sequences disclosed in Figures 14A-E of WO 91 / 17244.

[0297] In one embodiment, the liquid detergent preferably contains at least one mannanase disclosed herein in an amount of about 0.0005% to 0.005% by weight, more preferably 0.0005% to 0.002% by weight, relative to the total weight of the detergent formulation. The at least one mannanase is selected from the endo-1,4-β-mannosidase (EC3.2.1.78) disclosed herein.

[0298] In one embodiment, the liquid detergent contains at least one aminocarboxylic acid ester disclosed herein, selected from ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), methylglycine diacetic acid (MGDA), and glutamic acid diacetic acid (GLDA), in an amount ranging from 1% to 10% by weight, 1.5% to 7% by weight, or 2% to 5% by weight, relative to the total weight of the liquid detergent.

[0299] In one embodiment, the liquid detergent contains citric acid in an amount ranging from 0.1% to 10% by weight, 0.5% to 8% by weight, 1% to 5% by weight, or 2% to 4% by weight relative to the total weight of the detergent formulation; in one embodiment, a mixture of citric acid and formate is provided, such as sodium citrate:sodium formate = 9:1.

[0300] In one embodiment, the liquid detergent contains at least one phosphonate disclosed herein, preferably selected from HEDP and DTPMP, in an amount of 0.5% to 3.0% or 1.0% to 2.5% by weight relative to the total weight of the detergent formulation.

[0301] In one embodiment, the liquid detergent comprises at least one ethoxylated polyethyleneimine polymer based on a polyethylene core and a polyethylene oxide shell. Preferably, the polyethyleneimine core molecule has an average molecular weight M0. W The molecular weight is 500–5000 g / mol of polyethyleneimine. More preferably, it is 500–1000 g / mol, and even more preferably, it is M... W The concentration is 600–800 g / mol. The ethoxylated polymer has an average of 5–50, preferably 10–30, and even more preferably 15–25 EO (ethoxylated) groups / -NH groups, resulting in an average molecular weight M... W The concentration is 5,000 to 200,000 g / mol, preferably 8,000 to 100,000 g / mol, more preferably 8,000 to 50,000 g / mol, even more preferably 10,000 to 30,000 g / mol, and most preferably 10,000 to 20,000 g / mol. Preferably, it contains at least one ethoxylated polyethyleneimine polymer in an amount of about 0.5% to 5% by weight, about 1% to 4% by weight, or about 1.5% to 3% by weight relative to the total weight of the liquid detergent.

[0302] In one embodiment, the liquid detergent comprises at least one ethoxylated hexamethylene diamine polymer, preferably quaternized and—optionally but preferably—sulfated, with an average molecular weight Mw preferably of 2,000 to 10,000 g / mol, more preferably 3,000 to 8,000 g / mol, and most preferably 4,000 to 6,000 g / mol. Preferably, the quaternized and—optionally but preferably—sulfated ethoxylated hexamethylene diamine polymer contains an average of 10 to 50, preferably 15 to 40, and even more preferably 20 to 30 EO (ethoxylated) groups / -NH groups, resulting in an average molecular weight Mw of 2,000 to 10,000 g / mol. W The concentration is 2,000–10,000 g / mol, more preferably 3,000–8,000 g / mol, and most preferably 4,000–6,000 g / mol. In a preferred embodiment, the ethoxylated hexamethylenediamine is quaternized and also sulfated, preferably containing two cationic ammonium groups and two anionic sulfate groups. Preferably, at least one ethoxylated hexamethylenediamine polymer is contained in an amount of about 0.5% to 5% by weight, about 1% to 4% by weight, or about 1.5% to 3% by weight relative to the total weight of the liquid detergent.

[0303] Automated dishwashing

[0304] In one embodiment, the liquid composition of the present invention is a liquid automatic dishwasher detergent.

[0305] Typically, automatic dishwasher detergents do not contain large amounts of anionic surfactants. In one aspect of the invention, the liquid automatic dishwasher detergent contains anionic surfactants in an amount of about 0% to 3% by weight, preferably less than 1% by weight, and more preferably without anionic surfactants, relative to the total weight of the liquid automatic dishwasher detergent composition.

[0306] In one aspect of the invention, the liquid automatic dishwasher detergent comprises a nonionic surfactant in an amount of about 0% to 10% by weight, preferably less than 5% by weight, and more preferably less than 3% by weight, relative to the total weight of the liquid automatic dishwasher detergent composition.

[0307] Preferably, the liquid automatic dishwasher detergent of the present invention comprises at least one nonionic surfactant according to formula (NIS-IV), more preferably, wherein R 1 It is an n-C8 alkyl group, R 2 It is a branch C 11Alkyl group, AO is CH2-CH2-O, and x is 22. All are relative to the total weight of the liquid automatic dishwasher detergent, which preferably contains the compound in an amount of about 0.3% to 10% by weight, about 0.5% to 5% by weight, or about 1% to 3% by weight.

[0308] In one embodiment, the liquid automatic dishwasher detergent contains at least one aminocarboxylic acid ester disclosed herein in an amount of 5% to 15% by weight relative to the total weight of the detergent formulation. Preferably, the automatic dishwasher detergent comprises a detergent aid system containing:

[0309] The ethylenediaminetetraacetic acid (EDTA) and / or diethylenetriaminepentaacetic acid (DTPA) and / or methylglycine diacetate (MGDA) and / or glutamic acid diacetate (GLDA) disclosed herein are present in amounts of 0.1% to 15% by weight, 1% to 10% by weight, 3% to 8% by weight, or 2.5% to 5% by weight, relative to the total weight of the detergent formulation.

[0310] • Optionally, citric acid is provided in an amount of 0.1% to 10% by weight, 0.5% to 8% by weight, 1% to 5% by weight, or 2% to 4% by weight relative to the total weight of the detergent formulation; in one embodiment, citric acid is provided as a mixture with a formate, such as sodium citrate:sodium formate = 9:1;

[0311] Optionally, at least one phosphonate, preferably selected from polyphosphonic acid derivatives such as diphosphonic acid derivatives (such as sodium salts of HEDP), and aminopolyphosphonic acid derivatives such as aminoalkylphosphonic acid derivatives (such as DTPMP), are present in an amount of 0.1% to 5% by weight, 0.5% to 3% by weight, or 1% to 2% by weight relative to the total weight of the detergent formulation.

[0312] Optionally, at least one polycarboxylate, selected from homopolymers of repeating monomers of the same unsaturated carboxylic acid (e.g., polyacrylic acid (PAA)) and copolymers of repeating monomers of at least two different unsaturated carboxylic acids (e.g., copolymers of acrylic acid and methacrylic acid, copolymers of acrylic acid or methacrylic acid and maleic acid and / or fumaric acid), are present in an amount of 0% to 10% by weight, 0.5% to 7% by weight, 1% to 5% by weight, or 2.5% to 5% by weight relative to the total weight of the detergent formulation; the polyacrylic acid homopolymer may be partially neutralized or sulfonated.

[0313] In one embodiment, the liquid automatic dishwasher detergent of the present invention comprises at least one hydrolytic enzyme selected from the proteases, amylases, and lipases disclosed herein. The at least one protease disclosed herein is combined with one or more detergent components by incorporating the liquid composition of the present invention. Preferably, at least one protease is contained in an amount of about 0.10% to 0.25% by weight, preferably about 0.12% to 0.21% by weight, relative to the total weight of the liquid automatic dishwasher detergent.

[0314] In one embodiment, the liquid automatic dishwasher detergent preferably contains at least one α-amylase disclosed herein in an amount of about 0.002% to 0.015% by weight, more preferably 0.004% to 0.01% by weight, relative to the total weight of the detergent formulation.

[0315] In one embodiment, the liquid automatic dishwasher detergent comprises at least one zinc salt. The zinc salt is preferably selected from water-soluble and water-insoluble zinc salts. In this context, water-insoluble refers to those zinc salts having a solubility of 0.1 g / L or less in distilled water at 25°C. Therefore, in the context of this invention, zinc salts with higher solubility in water will be referred to as water-soluble zinc salts.

[0316] The zinc salt can be selected from zinc benzoate, zinc gluconate, zinc lactate, zinc formate, ZnCl2, ZnSO4, zinc acetate, zinc citrate, Zn(NO3)2, Zn(CH3SO3)2 and zinc gallate, with ZnCl2, ZnSO4, zinc acetate, zinc citrate, Zn(NO3)2, Zn(CH3SO3)2 and zinc gallate being preferred.

[0317] In another embodiment of the invention, the zinc salt is selected from ZnO, ZnO·aq, Zn(OH)2, and ZnCO3. ZnO·aq is preferred.

[0318] In one embodiment of the invention, the zinc salt is selected from zinc oxide with an average particle size (weight average) in the range of 10 nm to 100 μm.

[0319] The cations in zinc salts can exist in complexed forms, such as complexed with amino or aqueous ligands, and particularly in hydrated forms. For the sake of simplicity, in the context of this invention, the ligand is usually omitted if it is an aqueous ligand.

[0320] According to the method of adjusting the pH of the mixture according to the invention, the zinc salt can be changed. Therefore, it is possible to use zinc acetate or ZnCl2, for example, to prepare the formulation according to the invention, but when the pH of the aqueous environment is 8 or 9, the zinc salt is converted to ZnO, Zn(OH)2 or ZnO·aq, which can exist in non-complexed or complexed forms.

[0321] Zinc salts are preferably present in liquid detergent formulations in dissolved or colloidal form.

[0322] In one embodiment of the invention, in each case, based on the solids content of the formulation, the innovative automatic dishwasher formulation contains a total of 0.05% to 0.4% zinc salt by weight. Hereinafter, the zinc salt portion is given in the form of zinc or zinc ions. Therefore, it is possible to calculate the counterion portion. Example

[0323] Example 1

[0324] In summary, biofilms were cultured in nutrient medium in a microtiter plate in the presence of components (a) and / or (b) disclosed above. After culturing, the biofilms were stained with a dye (saffron), and then the dye was redissolved in a solvent. The absorbance of the dye solution at 540 nm is a measure of the total amount of biofilm grown in the wells.

[0325] Component (a) is 4,4'-dichloro-2-hydroxydiphenyl ether (CAS-No. 3380-30-1), added in the form of a 30% solution in 1,2-propylene glycol, which is commercially available as Tinosan HP 100.

[0326] Component (b) is a lyophilized powder containing a DNase at a concentration of approximately 33%, the DNase having the protein sequence MKKWMAGLFLAAAVLLCLMVPQQIQGASLYDKVLYFPLSRY-PETGDHIKDAIADGHS-DICTIDRDGADKRRQESLKGIPTKPGYDRDEWPMAVCEEGGAGADVRYVTPSDNRGAGSWVGNQMSGYPDGTRVLFIVQ.

[0327] The test bacteria, Pseudomonas aeruginosa DSM 1117 and Staphylococcus aureus DSM 20231, were cultured on trypsin-soy agar at 35°C for 24 hours. The first subculture was stored at +4°C for 9 days.

[0328] Inoculum preparation, by suspension:

[0329] (i) For Pseudomonas aeruginosa, 5 single colonies and

[0330] (ii) For Staphylococcus aureus, 2 single colonies

[0331] For the first subculture, the culture was placed in a 200 ml shake flask containing 200 ml of 30% TSB* diluted with water and 2.5 g / L glucose, and shaken at approximately 160 rpm for 24 h at 35 °C. The optical density (OD) of the overnight culture was measured photometrically at 595 nm (OD595 nm), and adjusted to OD595 nm = 0.4 in 60% TSB + 5 g / L glucose.

[0332] *TSB 100% trypsin-based soy broth: 1L of liquid culture medium contains 17g casein peptone, 3g soy peptone, 5g NaCl, and 2.5g K2HPO4.

[0333] Component (a), component (b), and component (a) + (b) solutions were prepared in deionized water at twice the final test concentration, aseptically filtered, and 75 μl was added to the wells of a clear 96-well microtiter plate. 75 μl of a cell suspension (inoculum) with LOD595nm = 0.4 was transferred to the wells of the microtiter plate to produce a total volume of 150 μL. The biofilm was then cultured in 30% TSB + 2.5 g / L glucose. The culture plate was incubated in a humidifier at 33°C and 40 rpm for 24 hours.

[0334] After culturing, remove the supernatant containing planktonic cells using a pipette, and rinse the wells three times with 195 μL of 0.85% NaCl solution. After removing the NaCl solution, gently tap the plate on a paper towel to minimize residual NaCl in the wells, and then dry the empty plates in laminar flow.

[0335] Biofilms were stained with Gram's Safranin (Sigma-Aldrich): 175 μL was used per well and the mixture was incubated at room temperature (RT) for 30 min.

[0336] Remove the supernatant with a pipette, wash the wells four times with 195 μL of 0.85% NaCl solution, and finally remove the liquid with a pipette. Tap the plate gently on a paper towel to minimize residual NaCl in the wells. Dry the empty plate in laminar flow. Fill the wells with 175 μL of 30% acetic acid (dye solvent) dissolved in deionized water. Further dissolve the dye by pipetting up and down, and transfer the liquid to a new microtiter plate.

[0337] The absorbance of the saffron solution was measured at 540 nm using a plate reader.

[0338] Perform parallel tests on each composition in at least three wells. Take the average of the three results. Subtract the blank background absorbance value (the average of at least three wells containing growth medium but without bacteria) from each average absorbance value. This net average absorbance is a measure of the total amount of biofilm formed in the well.

[0339] The highest amount of biofilm was found in the blank experiment, and the biofilm inhibition effect was 0% (by definition). The active ingredient that does not form a biofilm (net absorbance = 0) showed 100% biofilm inhibition.

[0340] Five systems were tested:

[0341] (1) No anti-biofilm substances were added to the biofilm growth medium; instead, a salt solution was added. (“Reagent-free blank experiment”)

[0342] (2) Add 10 ppm of component (b) to the growth medium, where ppm refers to the weight of the lyophilized powder in mg relative to the liquid volume in L per well of the microtiter plate.

[0343] (3) Add 20 ppm of component (b) to the growth medium, where ppm refers to the weight of the lyophilized powder in mg relative to the liquid volume in L per well of the microtiter plate.

[0344] (4) Add 0.6 ppm of component (a) (i.e., 4,4'-dichloro-2-hydroxydiphenyl ether) to the growth medium, where ppm refers to the weight of component (a) in mg relative to the liquid volume in L per well of the microtiter plate.

[0345] (5) Add a combination of 2 ppm component (a) and 10 ppm component (b)

[0346]

[0347]

[0348] Example 2

[0349] The following liquid detergent formulations can be prepared:

[0350]

[0351]

[0352] AEO: Lutensol AO7 (BASF); two nonionic surfactants selected from compounds of general formula (Ia), one of which is characterized by R 1 It is C 12 R 2 and R 5 It is H, m is 7, n and o = 0, while another surfactant is characterized by R 1 It is C 14 R 5H is given, m is 7, and n and o = 0.

[0353] AES: Texapon 70 (BASF); Two anionic surfactants selected from compounds of general formula (AIS Ia), one of which is characterized by R 1 It is C 11 R 2 H is 2, m is 2, n and o = 0, A - It is SO3 - M + Yes + Another surfactant is characterized by R 1 It is C 13 R 2 H is 2, m is 2, n and o = 0, A - It is SO3 - M + Yes + .

[0354] LAS: Maranil DBS / LC (BASF); two anionic surfactants selected from compounds of general formula (AIS II), one of which is characterized by R 1 It is C 10 Another surfactant is characterized by R 1 It is C 13 .

[0355] Coconut fatty acids: Edenoor K12-18 (Emery Oleochemicals)

[0356] Optional protease: a protease having the polypeptide sequence of SEQ ID NO:22 as described in EP 1921147 and having an R101E substitution.

[0357] The above formulation was prepared by first preparing a premix containing surfactant, solvent, fatty acid, citric acid, and NaOH, as well as up to 90% water. This premix was prepared by adding all components to an appropriate amount of water and stirring at room temperature. Subsequently, the pH was adjusted to pH 8.5 using NaOH. Then, the final formulation was prepared by stirring at room temperature: 90% premix, an appropriate concentration of a composition containing / without protease and water, reaching 100%. sequence list <110> BASF Europe <120> Liquid anti-biofilm composition <130> 200048EP01 <150> EP20214664.3 <151> 16 / 12 / 2020 <160> 1 <170> According to Wipo Std 25 <210> 1 <211> 136 <212> PRT <213> Bacillus intestinalis <220> <223> <220> <221> <222> <223> <400> 1 Met Lys Lys Trp Met Ala Gly Leu Phe Leu Ala Ala Ala Val Leu Leu 1 5 10 15 Cys Leu Met Val Pro Gln Gln Ile Gln Gly Ala Ser Leu Tyr Asp Lys 20 25 30 Val Leu Tyr Phe Pro Leu Ser Arg Tyr Pro Glu Thr Gly Asp His Ile 35 40 45 Lys Asp Ala Ile Ala Asp Gly His Ser Asp Ile Cys Thr Ile Asp Arg 50 55 60 Asp Gly Ala Asp Lys Arg Arg Gln Glu Ser Leu Lys Gly Ile Pro Thr 65 70 75 80 Lys Pro Gly Tyr Asp Arg Asp Glu Trp Pro Met Ala Val Cys Glu Glu 85 90 95 Gly Gly Ala Gly Ala Asp Val Arg Tyr Val Thr Pro Ser Asp Asn Arg 100 105 110 Gly Ala Gly Ser Trp Val Gly Asn Gln Met Ser Gly Tyr Pro Asp Gly 115 120 125 Thr Arg Val Leu Phe Ile Val Gln 130 135 <210> 2 <211> 7 <212> PRT <213> Artificial <220> <223> Motif of WO 2019 / 081724 SEQ ID NO: 25 <220> <221> MISC_FEATURE <222> (1)..(1) <223> Xaa = Asp (D) or Met (M) or Leu (L) <220> <221> MISC_FEATURE <222> (2)..(2) <223> Xaa = Ser (S) or Thr (T) <220> <221> MISC_FEATURE <222> (7)..(7) <223> Xaa = Asp (D) or Asn (N) <400> 25 Xaa Xaa Gly Tyr Ser Arg Xaa 1 5 <210> 3 <211> 8 <212> PRT <213> Artificial <220> <223> Motif of WO 2019 / 081724 SEQ ID NO: 26 <220> <221> MISC_FEATURE <222> (3)..(3) <223> Xaa = any amino acid <400> 26 Ala Ser Xaa Asn Arg Ser Lys Gly 1 5 <210> 4 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Motif of WO 2017 / 060493 SEQ ID NO: 73 <220> <221> MISC_FEATURE <222> (1)..(1) <223> Xaa = Gly (G) or Tyr (Y) or Trp (W) or Phe (F) or Ala (A) or His (H) <220> <221> MISC_FEATURE <222> (4)..(4) <223> Xaa = Arg (R) or Gln (Q) or Asp (D) or Glu (E) or Val (V) <400> 73 Xaa Asn Ile Xaa 1 <210> 5 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Motif of WO 2017 / 060493 SEQ ID NO: 74 <220> <221> MISC_FEATURE <222> (4)..(4) <223> Xaa = Asp (D) or His(H) or Leu (L) <400> 74 Ser Asp His Xaa Pro 1 5 <210> 6 <211> 5 <212> PRT <213> artificial hierarchy <220> <223> The sequence SEQ ID NO: 75 for WO 2017 / 060493 <220> <221> MISC_FEATURE <222> (5)..(5) <223> Xaa = Arg (R) or Gln (Q) <400> 75 Gly Gly Asn Ile Xaa 1 5

Claims

1. A liquid composition containing (a) At least one chlorinated hydroxydiphenyl ether according to formula (I): (I) R1, R2, and R3 are selected from H and Cl, and at least one of R1, R2, and R3 is Cl. (b) at least one compound that provides DNA enzyme activity, and (c) At least one compound selected from anionic and nonionic surfactants, in, When compared with the polypeptide sequence SEQ ID NO:1, the at least one compound of component (b) that provides DNA enzyme activity has at least 80% sequence identity.

2. The composition according to claim 1, wherein component (c) comprises at least one anionic surfactant and at least one nonionic surfactant.

3. The composition according to claim 1 or 2, further comprising at least one hydrolytic enzyme selected from proteases, lipases, amylases, cellulases, mannan-degrading enzymes, and mixtures thereof.

4. A method of providing a liquid anti-biofilm composition, comprising adding at least one chlorinated hydroxydiphenyl ether according to formula (I) to a liquid composition comprising DNase activity, wherein the liquid composition comprising DNase activity contains at least one compound providing DNase activity, wherein, When compared with the polypeptide sequence SEQ ID NO:1, the at least one compound providing DNA enzyme activity has at least 80% sequence identity. The formula (I) is as follows: (I) R1, R2, and R3 are selected from H and Cl, and at least one of R1, R2, and R3 is Cl.

5. A method for reducing and / or inhibiting biofilm formation, said method being used for washing and cleaning by using a liquid composition comprising components (a) and (b) according to any one of claims 1 to 3, wherein a reduction and / or inhibition of microbial growth is determined compared to microbial growth in the absence of components (a) and (b).

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