Cleaning composition and cleaning method

Abstract

The present invention relates to a cleaning composition having reduced aerosolization properties, comprising at least one first surfactant, at least one second surfactant, and optionally a buffer system.

Description

[Technical Field]

[0001] Sequence listing reference This application includes a sequence listing in a computer-readable form, which is incorporated herein by reference.

[0002] The present invention relates to a cleaning composition having reduced aerosolization properties, comprising a first surfactant which is at least one low-spraying surfactant having 8 to 12 carbon units, and at least one second surfactant. The present invention also relates to a method for cleaning a surface, comprising foaming the composition onto the surface to be cleaned. [Background technology]

[0003] Most cleaning agents for industrial applications are high-pH (caustic) liquids. These cleaning agents pose risks to both the people performing the cleaning and the environment. One improvement is to apply the cleaning agent at room temperature rather than at higher temperatures such as 40-55°C. A second improvement is to reduce the amount of liquid cleaning agent applied, which can be achieved in two ways through the use of foam. Firstly, this will reduce the amount of liquid needed to cover the area to be cleaned. Secondly, this will provide a visual marker that will significantly reduce excessive spraying. However, since high pH improves the penetration and removal of dirt and organic matter such as fats, oils, and proteins, it will be difficult to achieve acceptable cleaning power if the pH of the applied liquid is lowered. Enzymes can be added to cleaning agents to lower the pH and still remove dirt and organic matter. However, historically, there have been safety concerns, such as the fact that aerosolizing enzymes can trigger allergic reactions. In addition, the stability of enzymes is another factor that limits the use of enzymes in industrial cleaning agents. [Overview of the project] [Problems that the invention aims to solve]

[0004] The remaining need is to provide a cleaning composition that can address at least one of the above problems. [Means for solving the problem]

[0005] The present invention relates to a cleaning composition having reduced aerosolization properties, (i) at least one first surfactant, the first surfactant having 8 to 12 carbon units, for example, 8 to 10 or 10 to 12 carbon units; (ii) at least one second surfactant, the second surfactant having 8 to 16 carbon units, for example, 8 to 10, 10 to 12 or 10 to 14; optionally, (iii) The present invention relates to a cleaning composition comprising a buffer system.

[0006] The cleaning composition of the present invention is less harmful to the environment and users compared to conventional high-pH detergents. The cleaning composition of the present invention is safe to apply as foam (reduced aerosolization level) and can be applied at room temperature and neutral pH while still providing a cleaning effect similar to or better than conventional detergents.

[0007] In addition, the foam resulting from applying the cleaning compositions of the present invention promotes more favorable cleaning performance by generating stable foam, providing effective stain removal and low surface tension. However, many of these compositions cannot provide long-term enzyme stability. Other compositions, such as concentrates, can be incorporated to provide long-term enzyme stability and thereby enhance safety and convenience for end-users.

[0008] The present invention relates to a method for cleaning a surface, such as a hard surface, and further comprises (a) foaming the cleaning composition described above onto the surface (e.g., by spraying or brushing), and optionally (b) rinsing the surface.

[0009] definition As used herein, the singular forms "a," "an," and "the" are intended to include the plural form unless otherwise specified in the context.

[0010] Unless otherwise specified, all references to percentages (%) in the disclosed compositions refer to weight percentages of each composition relative to its total weight.

[0011] Unless otherwise specifically defined or indicated by context, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention pertains.

[0012] Cleaning components: The term "cleaning components" is defined herein to mean the types of chemical substances that can be used in cleaning compositions. Examples of cleaning components include alkalis, surfactants, solvents, hydrotropes, builders, cobuilders, chelating agents or chelating agents, bleaching agents or bleaching components, polymers, foam enhancers, foam inhibitors, dispersants, stain transfer inhibitors, fragrances, bactericides, fungicides, corrosion inhibitors, stain floaters, soil release polymers, anti-redeposition agents, enzyme inhibitors or stabilizers, enzyme activators, antioxidants, preservatives, solvents and solubilizers.

[0013] Cleaning Composition: The term “cleaning composition” (which may also be referred to as “detergent composition”) refers to a composition used to remove undesirable compounds from an object to be cleaned, such as a hard surface. Cleaning compositions may be used, for example, for household and industrial cleaning. This term encompasses any materials / compounds selected to suit the specific type of cleaning composition and product form (e.g., liquid, gel, powder, granules, paste, or spray composition) of the desired cleaning composition. In addition to containing the surfactant of the present invention, the cleansing composition of the present invention may contain one or more enzymes selected from the group consisting of lipase, amylase, protease, cellulase, mannanase, pectinase, hemicellulase, peroxidase, xylanase, phospholipase, xyloglucanase, esterase, cutinase, laccase, keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, ligninase, pullulanase, maranase, β-glucanase, arabinosidase, hyaluronidase, deoxyribonuclease (DNase), RNase, hexosaminidase, peroxidase, and any mixture thereof, and / or at least one cleansing component described above.

[0014] Hard surfaces: The term “hard surfaces” includes surfaces in the home, such as showers, sinks, toilets, bathtubs, countertops, windows, mirrors, floors, chicken hatcheries, etc., as well as surfaces in industry, such as transport vehicles, storage tanks, bioreactors, fermenters, mixing vessels, pipelines, bottling process equipment, and biotechnology-based manufacturing, such as other equipment used in the manufacture of pharmaceuticals, food and beverages. The term may also include surfaces of medical or dental equipment that come into contact with patients, who may be human or animal. Exemplary hard surfaces may include steel, rubber, plastic, glass, ceramic, melamine, wood, coated surfaces, cement countertops, kitchen countertops, endoscopes, arthoscopes, scalpels, hemostatic forceps, Kocher forceps, tracheostomy knives, etc.

[0015] Aerosolization: Aerosolization is the process or act of converting a physical substance into particles small and light enough to be carried in the air, i.e., in the form of an aerosol. This term is often used, for example, in medicine to refer to the production of airborne particles (e.g., very small liquid droplets) containing infectious viruses or bacteria, or in enzyme-related applications where enzyme-containing droplets or particles may be generated when an enzyme solution (e.g., a cleaning composition) is sprayed, potentially causing allergic problems. In this invention, the aerosolization properties of the cleaning composition can be measured as particle aerosolization by the method described in Example 1 or as enzyme aerosolization by the method described in Example 2.

[0016] Mature polypeptide: The term "mature polypeptide" refers to the polypeptide in its final form after translation and any post-translational modifications such as N-terminal processing, C-terminal processing (e.g., removal of signal peptides), glycosylation, phosphorylation, etc.

[0017] Sequence identity: The relationship between two amino acid sequences or two nucleotide sequences is described by a parameter called "sequence identity."

[0018] For the purposes of this invention, sequence identity between two amino acid sequences is determined as output data called "longest identity" using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol. Biol. 48:443-453), preferably as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) version 6.6.0 or later. The parameters used are a gap-open penalty of 10, a gap-extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The nobrief option must be specified on the command line for the Needle program to report the longest identity. The Needle output data called "longest identity" is calculated as follows: (Number of identical residues × 100) / (Length of alignment - Total number of gaps in the alignment)

[0019] Mutant: The term "mutant" refers to a polypeptide that has the same activity as the parent enzyme but includes modifications (i.e., substitutions, insertions, and / or deletions) at one or more positions. Substitution means replacing an amino acid at a given position with a different amino acid, deletion means removing an amino acid at a given position, and insertion means adding an amino acid adjacent to and immediately following an amino acid at a given position.

[0020] A more complete understanding of the present invention can be obtained by referring to the detailed description and claims and considering them in conjunction with the drawings. [Brief explanation of the drawing]

[0021] [Figure 1] Figure 1 shows the volume of particles of various sizes generated during the application of foam using various detergent compositions. [Modes for carrying out the invention]

[0022] A first aspect of the present invention is a cleaning composition having reduced aerosolization properties, (i) at least one first surfactant, the first surfactant having 8 to 12 carbon units, for example, 8 to 10 or 10 to 12 carbon units; (ii) at least one second surfactant, the second surfactant having 8 to 16 carbon units, for example, 8 to 10, 10 to 12 or 10 to 14; optionally, (iii) The present invention relates to a cleaning composition comprising a buffer system.

[0023] composition ingredients The non-limiting list of compositional components illustrated below is suitable for use in the compositions and methods of the present invention and, preferably incorporated into specific embodiments of the invention, may, for example, assist or enhance cleaning performance for the treatment of hard surfaces to be cleaned, or, in the case of other components such as fragrances, colorants, dyes, etc., may modify the aesthetics of the composition. The exact properties of these components and the level of their incorporation will depend on the physical form of the composition and the nature of the cleaning operation in which it will be used. The components listed below are classified by general headings according to their specific functionality, but this should not be interpreted as limiting, as components may include additional functionality, as will be readily apparent to those skilled in the art.

[0024] Suitable component materials include, but are not limited to, surfactants, builders, chelating agents, dispersants, enzymes and enzyme stabilizers, catalysts, bleach activators, hydrogen peroxide, hydrogen peroxide sources, pre-formed peracids, polymer dispersants, soil removers, dyes, hydrotropes, processing aids, preservatives (2-phenoxyethanol and isothiazolionones (e.g., benzisothiazolinone (BIT), methylisothizolinone (MIT), chloromethylisothiazolinone (CMIT), etc.)) and solvents. In addition to the disclosures below, suitable examples of other such components and levels of use can be found in U.S. Patent No. 5,576,282, U.S. Patent No. 6,306,812 and U.S. Patent No. 6,326,348, which are incorporated herein by reference.

[0025] Examples of enzyme stabilizers include glycerol, sorbitol, sucrose, glucose, NaCl, KCl, sodium formate, citric acid, trisodium citrate dihydrate, hexanediol, heptanediol, PEG300 and PEG600, and mixtures thereof. The enzyme stabilizer may be present in the composition of the present invention at a level of 0.5 to 25% by weight, preferably 1 to 20% by weight, more preferably 3 to 15% by weight, for example, 5 to 15% by weight or 4 to 12% by weight.

[0026] surfactants The detergent or cleaning composition of the present invention comprises at least one first surfactant and at least one second surfactant to achieve foam stability. The first surfactant is for generating sufficient foam regardless of the means used to generate the foam. The second surfactant in the present invention is expected to lower the surface tension to a desired level, increase foam stability, and enhance foam generation.

[0027] The first surfactant may be a detergent surfactant having 8 to 12 carbon units. Any value exceeding 12 carbon units may result in insufficient foam formation. Any value less than 10 carbon units may also result in insufficient foam formation, and the resulting foam may have insufficient stability.

[0028] The first surfactant may be selected from the group consisting of sodium salts, potassium salts, or ammonium salts and mixtures thereof.

[0029] The first surfactant may include one or more anionic surfactants, such as linear alkylbenzene sulfonic acid (LAS), alcohol ether sulfate (AEOS) and / or alkyl sulfate (AS), particularly sodium lauryl sulfate (SLS), sodium laureth sulfate, sodium lauryl ether sulfate, sodium dodecyl sulfate, ammonium decyl ether sulfate, sodium decyl sulfate and ammonium alkyl ether sulfate.

[0030] The first surfactant may be an anionic surfactant such as sulfate and sulfonate cleaning surfactants.

[0031] Suitable sulfonate surfactants include alkylbenzene sulfonates, and in one embodiment, C 10~13 Alkylbenzene sulfonates are included. Suitable alkylbenzene sulfonates (LAS) can be obtained by sulfonating commercially available linear alkylbenzenes (LAB); suitable LABs include lower 2-phenyl LABs such as Isochem® or Petrelab®, and other suitable LABs include higher 2-phenyl LABs such as Hyblene®. Suitable anionic cleaning surfactants include alkylbenzene sulfonates obtained by the DETAL catalytic process, but other synthetic routes such as HF may also be suitable. In one embodiment, the magnesium salt of LAS is used.

[0032] Suitable sulfate surfactants include alkylalkoxylated sulfates, in one embodiment alkylethoxylated sulfates, and in one embodiment C 8~12 Alkylalkoxylated sulfates, in another embodiment, C 8~12 It is an alkylethoxylated sulfate.

[0033] Alkyl sulfates, alkylalkoxylated sulfates, and alkylbenzene sulfonates may be linear or branched, substituted or unsubstituted.

[0034] Non-limiting examples of anionic surfactants include sulfates and sulfonates, particularly linear alkylbenzene sulfonates (LAS), LAS isomers, branched alkylbenzene sulfonates (BABS), phenylalkane sulfonates, alpha-olefin sulfonates (AOS), olefin sulfonates, alkene sulfonates, alkane-2,3-diyrbis(sulfate), hydroxyalkane sulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS) or sodium lauryl sulfate (SLS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), and alcohol ether sulfates (AES or AE). This includes OS or FES (also known as alcohol ethoxysulfate or fatty alcohol ether sulfate), sodium laureth sulfate (SLES), secondary alkanesulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerol esters, alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES) including methyl ester sulfonates (MES), alkyl or alkenyl succinates, dodecenyl / tetradecenyl succinates (DTSA), alkyl ether sulfates, fatty acid derivatives of amino acids, diesters and monoesters of sulfosuccinates or soaps, and combinations thereof.

[0035] The first surfactant is present at levels of 0.2-60% by weight, 0.5-40% by weight, 0.8-30% by weight, 1-50% by weight, 1-40% by weight, 1-30% by weight, 1-20% by weight, 3-10% by weight, 3-5% by weight, 5-40% by weight, 5-30% by weight, 5-15% by weight, 2-20% by weight, 3-6% by weight, 8-12% by weight, 10-12% by weight, 20-25% by weight, or 25-60% by weight.

[0036] The second surfactant may be a detergent surfactant having 8 to 16 carbon units, for example, 8 to 10, 10 to 12, or 10 to 14 carbon units. Preferably, the second surfactant is selected from the group consisting of amine oxides, betaines, primary fatty alcohols, monoethanolamides, diethanolamides, and mixtures thereof.

[0037] The second surfactant preferably has almost the same number of carbon units as the first surfactant in order to achieve more suitable foaming properties (ease of foam formation, stability, adhesion, etc.).

[0038] Ideally, the second surfactant should be low volatility. In this invention, the terms "volatile" or "volatilizing" can be used interchangeably with "atomizing" or "atomizing."

[0039] The volatility of the surfactant is indirectly evaluated by particle aerosolization according to Example 2.

[0040] The second surfactant is selected from amine oxides, decylamine oxides, lauramine oxides, and mixtures thereof. In one embodiment, the second surfactant may further include primary fatty alcohols and / or linear alcohol (C12-C13) ethoxylates such as 1-dodecaol, 1-decanol, and / or linear alcohol (C12-C13) ethoxylate POE-3.

[0041] The second surfactant is present at levels of 0.01-10% by weight, 0.02-8% by weight, 0.05-6% by weight, 0.1-5% by weight, 0.5-4% by weight, 1-3% by weight, 1.5-8% by weight, or 2-6% by weight.

[0042] The amount of the second surfactant is less than the amount of the first surfactant in the composition. The weight ratio of the second surfactant to the first surfactant may be in the range of 0.001 to 1, for example, in the range of 0.005 to 0.9, 0.01 to 0.8, 0.1 to 0.6, 0.08 to 0.5, or 0.2 to 0.4.

[0043] The surface tension of the cleaning composition of the present invention is ideally 35 mN / m or less, for example, 30 mN / m or less, 28 mN / m or less, or more preferably 25 mN / m or less or 20 mN / m or less. Lower surface tension provides a more favorable chemical cleaning effect and more favorable enzyme penetration (as a result, the enzyme can provide a favorable cleaning function). Surface tension can be measured by methods known in the art. Surface tension can be adjusted, for example, by adjusting the amount / type of the first surfactant and the second surfactant of the present invention.

[0044] The washing composition may further include a buffer system to stabilize the enzyme and / or maximize its performance. A buffer system is a type of solution that can withstand changes in its pH when small amounts of acidic or basic substances are added. Buffer systems typically contain a conjugate base and an acid system.

[0045] In one embodiment, the buffer system includes TRIS-HCl / TRIS base, citrate / citrate, sodium carbonate / sodium bicarbonate, or potassium carbonate / potassium bicarbonate.

[0046] The buffering system is included in the cleaning composition at levels of 0.02 to 10% by weight, for example, 0.05 to 8% by weight, 0.1 to 6% by weight, 0.5 to 5% by weight, 1 to 4% by weight, 2% to 6% by weight, or 3% to 5% by weight.

[0047] The reduction in the aerosolization effect of the composition can be evaluated as particle aerosolization by the 20-minute foam test according to Example 1. Thus, in one embodiment, when the composition is evaluated by the 20-minute foam test according to Example 1, the volume of particles with a size of 0.3 um is 15×10 6 m 3 or less, for example, 10×10 6 m 3 、8×10 6 m 3 、6×10 6 m 3 、5×10 6 m 3 or 2×10 6 m 3 or less. In another embodiment, when the composition is evaluated by the 20-minute foam test according to Example 1, the volume of particles with a size of 0.5 um is 15×10 6 m 3 or less, for example, 10×10 6 m 3 、8×10 6 m 3 、6×10 6 m 3 、5×10 6 m 3 or 2×10 6 m​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​3 , 6×10 6 m 3 , 5×10 6 m 3 or 2 × 10 6 m 3 It is less than.

[0048] The decrease in the aerosolization effect of the composition can be evaluated as the enzyme aerosolization concentration by the method of Example 2. Preferably, the enzyme aerosolization concentration of the washing composition is 100 ppb or less, for example, 80 ppb or less, 60 ppb or less, 50 ppb or less, 30 ppb or less, 20 ppb or less, 15 ppb or less, or 10 ppb or less.

[0049] The composition is preferably formulated to be suitable for foam-type detergents.

[0050] The present invention can be formulated to have a pH lower than that of ordinary cleaning compositions, for example, less than 11, preferably in the range of 6 to 9.5, 6.5 to 9, 6.8 to 8.5, or 7 to 8, and more preferably a neutral pH of 6.5 to 7.5, such as pH 7.0, 7.1, or 7.

[0051] The present invention can be formulated as a ready-to-use detergent or as a concentrated detergent (e.g., 2x, 5x, 10x, or 15x concentrated) that can be diluted on-site before application. Enzymes may be added when formulating the concentrate or added to the on-site diluted product.

[0052] According to the present invention, the foam produced by the cleaning composition of the present invention has the following characteristics: Firstly, it is easily formed without requiring high pressure to generate foam; secondly, it adheres to all types of surfaces, especially vertical and horizontal surfaces; thirdly, it is stable and allows the enzyme to provide a cleaning effect for a sufficient amount of time; fourthly, it allows the enzyme to penetrate dirt and stains on the surface so that the enzyme can provide a cleaning effect; and fifthly, it significantly reduces the aerosolization of the enzyme and minimizes exposure associated with allergic sensitization.

[0053] In one embodiment, in addition to the enzyme(s) of the present invention, the detergent composition further comprises enzymes selected from the group consisting of proteases, cellulases, mannanases, amylases, lipases, hemicellulases, peroxidases, xylanases, phospholipases, esterases, cutinases, keratinases, reductases, oxidases, phenol oxidases, lipoxygenases, ligninases, pullulanases, β-glucanases, arabinosidases, hyaluronidases, laccases, deoxyribonucleases (DNases), hexosaminidases, and / or any mixtures thereof. Typical combinations include, for example, an enzyme cocktail that may include proteases and lipases together with alpha-amylase, phospholipase, cutinase, pectinase, mannanase, pectinate lyase, phosphodiesterase (PDE), deoxyribonuclease (DNase), xanthanase, dispersin, or mixtures thereof.

[0054] Generally, the properties of the selected enzyme(s) should be compatible with the selected detergent (i.e., optimal pH, compatibility with other enzymatic and non-enzymatic components, etc.), and the enzyme(s) should be present in an effective amount. Generally, when present in a cleaning composition, the enzyme(s) may be present at levels of at least 0.001 mg of enzyme protein, at least 0.006 mg of enzyme protein, at least 0.008 mg of enzyme protein, at least 0.01 mg of enzyme protein, at least 0.1 mg of enzyme protein, at least 0.5 mg of enzyme protein, at least 1 mg of enzyme protein, at least 1.5 mg of enzyme protein, at least 2 mg of enzyme protein, at least 5 mg of enzyme protein, at least 10 mg of enzyme protein, or at least 15 mg of enzyme protein.

[0055] Cellulase: In one embodiment, suitable cellulases include single components and mixtures of enzymes of bacterial or fungal origin. Chemically modified or protein-manipulated mutants are also intended. Cellulases may be single components or mixtures of single-component endo-1,4-beta-glucanase, also known as endoglucanase.

[0056] Suitable cellulases include those derived from the genera Bacillus, Pseudomonas, Humicola, Myceliophthora, Fusarium, Thielavia, Trichoderma, and Acremonium. Exemplary cellulases include those derived from Humicola insolens (U.S. Patent No. 4,435,307) or Trichoderma, such as T. reesei or T. viride. Other suitable cellulases include those derived from the genus Thielavia, for example, Thielavia terrestris as described in International Publication No. 96 / 29397, or fungal cellulases produced from Myceliophthora thermophila and Fusarium oxysporum as disclosed in U.S. Patents Nos. 5,648,263, 5,691,178, and 5,776,757, International Publication No. 89 / 09259, and International Publication No. 91 / 17244. Cellulases derived from the genus Bacillus as described in International Publication No. 02 / 099091 and Japanese Patent No. 2000210081 are also suitable. Suitable cellulases include alkaline or neutral cellulases with care effects. Examples of cellulases are described in European Patent Application Publication No. 0495257, European Patent Application Publication No. 0531372, International Publication Brochure No. 96 / 11262, International Publication Brochure No. 96 / 29397, and International Publication Brochure No. 98 / 08940.Other examples include cellulase variants described in International Publication No. 94 / 07998, European Patent Application Publication No. 0531315, U.S. Patent Nos. 5,457,046, 5,686,593, and 5,763,254, as well as International Publication No. 95 / 24471 and International Publication No. 98 / 12307.

[0057] In one embodiment, an enzyme suitable for replacing a whiteness-maintaining agent is an enzyme having cellulase activity. Suitable cellulases can be selected from glycoside hydrolase family 5 (GH5), glycoside hydrolase family 7 (GH7), glycoside hydrolase family 12 (GH12), glycoside hydrolase family 44 (GH44), and glycoside hydrolase family 45 (GH45), preferably family GH45 cellulases.

[0058] Other cellulases are end-beta-1,4-glucanase enzymes or family 44 xyloglucanases having a sequence with at least 97% identity to the amino acid sequence of positions 1-773 of Sequence ID No. 2 of International Publication No. 2002 / 099091, and xyloglucanase enzymes having a sequence with at least 60% identity to positions 40-559 of Sequence ID No. 2 of International Publication No. 2001 / 062903.

[0059] Commercially available cellulases include Celluzyme, Carezyme, Carezyme Elite, Carezyme Premium, Luminous, Celluclean, Celluclean Classic, Cellusoft, Whitezyme, Celluclean 4500T, and Celluclean 5000L (all registered trademarks of Novozymes A / S), Clazinase and Puradax HA (registered trademarks of Genencor International Inc.), KAC-500(B) (registered trademark of Kao Corporation), and Revitalez 2000 (registered trademark of Danisco / Dupont), as well as Biotouch FLX1, Biotouch FCL75, Biotouch DCL, and Biotouch FCC45 (registered trademarks of AB Enzymes), and Lavergy C from BASF.

[0060] Protease: In one embodiment, a protease suitable for the present invention may be of any origin, but is preferably of bacterial or fungal origin, and optionally takes the form of a protein-engineered mutant or a chemically modified mutant. The protease may be an alkaline protease such as a serine protease or a metalloprotease. The serine protease may be from the S1 family, such as trypsin, or the S8 family, such as subtilisin. The metalloprotease may be another metalloprotease, such as thermolysin, for example, from the M4 family, or from the M5, M7, or M8 family.

[0061] The term "subtyralase" refers to a subgroup of serine proteases as defined by Siezen et al., Protein Eng. 4 (1991) 719-737 and Siezen et al., Protein Sci. 6 (1997) 501-523. Serine proteases are a subgroup of proteases characterized by having serine in their active site, forming a covalent intermediate with a substrate. Subtyralases can be further divided into six subgroups: the subtilisin family, thetermitase family, proteinase K family, lanthobiotic peptidase family, kexin family, and pyrrolicin family.

[0062] Proteases suitable for detergent use can be obtained from various organisms, including fungi such as Aspergillus, but detergent proteases are generally obtained from bacteria, particularly from the genera Bacillus and related genera (see Patel and Gupta, op. cit.). Examples of Bacillus species from which subtilases are derived include Bacillus lentus, Bacillus alkalophilus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, and Bacillus gibsonii. Specific subtilisins include subtilisin lentus, subtilisin nov, subtilisin Carlsberg, subtilisin BPN', subtilisin 309, subtilisin 147, and subtilisin 168, as well as protease PD138 (described in International Publication No. 93 / 18140). Other useful proteases are described, for example, in International Publication No. 01 / 16285 and International Publication No. 02 / 16547.

[0063] Examples of trypsin-like proteases include Fusarium proteases described in International Publication No. 94 / 25583 and International Publication No. 2005 / 040372, and chymotrypsin proteases derived from the genus Cellumonas described in International Publication No. 2005 / 052161 and International Publication No. 2005 / 052146.

[0064] Examples of metalloproteinases include neutral metalloproteinases described in International Publication No. 2007 / 044993, such as those derived from Bacillus amyloliquefaciens, as well as metalloproteinases described in International Publication No. 2015 / 158723 and International Publication No. 2016 / 075078, for example.

[0065] Examples of useful proteases include International Publication No. 89 / 06279, International Publication No. 92 / 19729, International Publication No. 96 / 34946, International Publication No. 98 / 20115, International Publication No. 98 / 20116, International Publication No. 99 / 11768, International Publication No. 01 / 44452, International Publication No. 03 / 006602, and There are protease variants described in International Publication No. 2004 / 003186, International Publication No. 2004 / 041979, International Publication No. 2007 / 006305, International Publication No. 2011 / 036263, International Publication No. 2014 / 207227, International Publication No. 2016 / 087617, and International Publication No. 2016 / 174234. Preferred protease variants include, for example, S3T, V4I, S9R, S9E, A15T, S24G, S24R, K27R, N42R, S55P, G59E, G59D, N60D, N60E, V66A, N74D, S85R, A96S, S97G, S97D, S97A, S97SD, S99E, S99D, S99G, S99M, S99N, S99R, S99H, S101A, V102I, V102Y, V102N, S104A, G116V, G116R, H118D, H118N, A120S, S12 6L, P127Q, S128A, S154D, A156E, G157D, G157P, S158E, Y161A, R164S, Q176E, N179E, S182E, Q185N, A188P, G189E, V193M, N198D, V199I, Q 200L, Y203W, S206G, L211Q, L211D, N212D, N212S, M216S, A226V, K229L, Q230H, Q239R, N246K, S253D, N255W, N255D, N255E, L256E, L256D It may contain one or more mutations selected from the group consisting of T268A and R269H, and the position numbers correspond to the positions of the Bacillus lentus protease shown in Sequence ID No. 1 of International Publication No. 2016 / 001449.Protease variants having one or more of these mutations are preferably variants of the Bacillus lentus protease (Savinase®, also known as subtilisin 309) shown in Sequence ID No. 1 of International Publication No. 2016 / 001449, or variants of the Bacillus amyloliquefaciens protease (BPN') shown in Sequence ID No. 2 of International Publication No. 2016 / 001449. Such protease variants preferably have at least 80% sequence identity with Sequence ID No. 1 or Sequence ID No. 2 of International Publication No. 2016 / 001449.

[0066] Other proteases of interest include, for example, the alkaline protease derived from Bacillus lentus DSM5483 as described in International Publication No. 91 / 02792, and its variants as described in, for example, International Publication No. 92 / 21760, International Publication No. 95 / 23221, European Patent No. 1921147, European Patent No. 1921148, and International Publication No. 2016 / 096711.

[0067] Alternatively, the protease may be a variant of the TY145 protease having SEQ ID NO: 1 in International Publication No. 2004 / 067737, for example, a variant containing substitutions at one or more positions corresponding to positions 27, 109, 111, 171, 173, 174, 175, 180, 182, 184, 198, 199 and 297 of SEQ ID NO: 1 in International Publication No. 2004 / 067737, wherein the protease variant has at least 75% but less than 100% sequence identity with respect to SEQ ID NO: 1 in International Publication No. 2004 / 067737.

[0068] In some embodiments of the present invention, the protease is a polypeptide having protease activity and has sequence identity of at least 60%, at least 65%, at least 70%, 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%, at least 99%, or 100% with respect to the polypeptide of SEQ ID NO: 1 or 2.

[0069] In one embodiment, the protease of the present invention is selected from the group consisting of: S3T, V4I, S9E, S9R, A15T, V68A, N76D, S99D, S99G, S99A, S99SE, S101E, S101N, S101R, S103A, V104I, G160S, Y167A, R170S, A194P, V199M, V205I, Q206L, Y209W, L217D, L217Q, N218D, M222S, Q245R, N261W, and L262E. This variant of the polypeptide of SEQ ID NO: 2 contains one or more substitutions, the position number corresponds to the position of SEQ ID NO: 1, and this variant has protease activity and has sequence identity of 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%, and at least 99% but less than 100% relative to SEQ ID NO: 2.

[0070] In one embodiment, the proteases of the present invention are: S3T, V4I, S9E, S9R, A15T, T22A, N43R, V68A, N76D, S87N, S99D, S99G, S99A, S99SE, S101E, S101N, S101R, S103A, V104I, G118M, S128Q, G160S, Y167A, R170S, N184E, A194P, V199M, V205I, Q206L, Y209W, L217D, L217Q, N218D, M222S, Q245R, S259D, N261W A variant of the polypeptide of SEQ ID NO: 2 comprising one or more substitutions selected from the group consisting of and L262E, wherein the position number corresponds to the position of SEQ ID NO: 1, this variant has protease activity, and has sequence identity of 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%, and at least 99% but less than 100% of SEQ ID NO: 2.

[0071] In one embodiment, the protease of the present invention is a variant of the polypeptide of SEQ ID NO: 2 containing the substitution S87N, the variant having protease activity, the position corresponding to the position of SEQ ID NO: 1, the variant having protease activity, and having sequence identity of 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% but less than 100% relative to SEQ ID NO: 2.

[0072] In one embodiment, the protease of the present invention is a variant of the polypeptide of SEQ ID NO: 2 containing the substitution Y167A+R170S+A194P, the position number corresponds to the position of SEQ ID NO: 1, and this variant has protease activity and has sequence identity of 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% but less than 100% of SEQ ID NO: 2.

[0073] In one embodiment, the protease of the present invention is a variant of the polypeptide of SEQ ID NO: 2 containing the substitution S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E, the position numbers correspond to the positions of SEQ ID NO: 1, and this variant has protease activity and has sequence identity of 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% but less than 100% of SEQ ID NO: 2.

[0074] In one embodiment, the protease of the present invention is a variant of the polypeptide of SEQ ID NO: 2 containing the substitution S3T+N43R+N76D+S87N+G118M+S128Q+N184E+V205I+Q206L+Y209W+S259D+N261W+L262E, the position numbers correspond to the positions of SEQ ID NO: 1, and this variant has protease activity and has sequence identity of 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% but less than 100% of SEQ ID NO: 2.

[0075] In one embodiment, the protease of the present invention is a variant of the polypeptide of SEQ ID NO: 2 containing the substitution T22A+N43R+S87N+V205L+Q206L+Y209W+S259D+N261W+L262E, the position numbers correspond to the positions of SEQ ID NO: 1, and this variant has protease activity and has sequence identity of 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% but less than 100% of SEQ ID NO: 2.

[0076] In one embodiment, the protease of the present invention is a variant comprising substitutions at one or more positions corresponding to positions 171, 173, 175, 179, or 180 of Sequence ID No. 1 of International Publication No. 2004 / 067737, wherein the variant has protease activity and has sequence identity of 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% but less than 100% of Sequence ID No. 1 of International Publication No. 2004 / 067737.

[0077] In one embodiment, the protease of the present invention is X3V, X9[E,R], X22[R,A], X43R, X61[E,D], X62[E,D], X76[D], X87N, X101[E,G,D,N,M], X103A, X104I, X118[V,R], X120V, X128[A,L,S], X129Q, X 130A,X160D,X185[E,D],188[E,D],X191N,X194P,X205I,X206L,X209W,X216V,X217 [Q,D,E], X218[D,E,S], X232V, X245R, X248D, X256[E,D], X259[E,D], X261[E,D,W] and A mutant selected from the group consisting of B and X262[E,D], containing one or more substitutions compared to the parent protease, where the position number corresponds to the position of BPN' (SEQ ID NO: 1), and "X" represents any amino acid residue present at a specified position in the parent protease. This mutant has protease activity and sequence identity of 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% but less than 100% compared to SEQ ID NO: 1 or SEQ ID NO: 2.

[0078] In one embodiment, the protease of the present invention is a mutant comprising one of the following substitution sets compared to the parent protease, wherein the parent protease has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or has at least 80% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, the position number corresponds to the position of BPN' (SEQ ID NO: 1), "X" represents any amino acid residue present at a specified position in the parent protease, and the substitution set is: i.X9R+X15T+X68A+X218D+X245R, ii. X9R + X15T + X68A + X245R, iii.X61E+X194P+X205I+X261D, iv.X61D+X205I+X245R, X61E + X194P + X205I + X261D, vi.X87N+X118V+X128L+X129Q+X130A, vii.X87N+X101M+X118V+X128L+X129Q+X130A, viii.X76D+X87R+X118R+X128L+X129Q+X130A, ix.X22A+X62D+X101G+X188D+X232V+X245R, x.X103A+X104I, xi.X22R+X101G+X232V+X245R, xii.X103A+X104I+X156D, xiii.X103A+X104I+X261E, xiv.X62D+X245R, xv.X101N+X128A+X217Q, xvi.X101E+X217Q, xvii.X101E+X217D, xviii.X9E+X43R+X262E, xix.X76D+X43R+X209W, xx.X205I+X206L+X209W, xxi.X185E+X188E+X205I, xxii.X256D+X261W+X262E, xxiii.X191N+X209W, xxiv.X261E+X262E, xxv.X261E+X262D and xxvi.X167A+X170S+X194P Selected from the group consisting of the following, this protease variant has sequence identity of 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% but less than 100% with respect to SEQ ID NO: 1 or 2.

[0079] Suitable commercially available protease enzymes include Alcalase(registered trademark), Duralase(trademark), Durazym(trademark), Relase(registered trademark), Relase(registered trademark) Ultra, Savinase(registered trademark), Savinase(registered trademark) Ultra, Primase(trademark), Polarzyme(registered trademark), Kannase(registered trademark), Liquanase(registered trademark), Liquanase(registered trademark) Ultra, Ovozyme(registered trademark), Coronase(registered trademark), Coronase(registered trademark) Ultra, Blaze(registered trademark), Blaze(registered trademark) Pro, Blaze Evity(registered trademark) 100T, Blaze Evity(registered trademark) 125T, Blaze Evity(registered trademark) 150T, Blaze Evity(registered trademark) 200T, Neutrase(registered trademark), Everlase(registered trademark), Esperase(registered trademark), Progress(registered trademark) Uno, Progress(registered trademark) In, and Progress(registered trademark) Excel Products sold under the brand name (Novozymes A / S), Maxatase (trademark), Maxacal (trademark), Maxapem (registered trademark), Purafect (registered trademark) Ox, Purafect (registered trademark) OxP, Puramax (registered trademark), FN2 (trademark), FN3 (trademark), FN4 ex(Trademark), Excellase (Registered Trademark), Excellenz (Trademark) P1000, Excellenz (Trademark) P1250, Eraser (Trademark), Preferenz (Registered Trademark) P100, Preferenz (Registered Trademark) P300, Purafect Prime, Preferenz P110 (Trademark), Effectenz P1000 (Trademark), Purafect (Registered Trademark), Effectenz P1050 (Trademark), Purafect (Registered Trademark) Ox, Effectenz (Trademark) P2000, Purafast (Trademark), Properase (Registered Trademark), Opticlean (Trademark) and Optimase (Registered Trademark) (Danisco / DuPont), BLAP (sequence shown in Figure 29 of US Patent No. 5352604) and its variant (Henkel AG), Puzhi manufactured by Bestzyme This may include 1.0L bottles and products sold under the brand name KAP (Bacillus alkalophilus subtilisin) by Kao Corporation.

[0080] Amylase: In one embodiment, a suitable amylase may be an alpha-amylase or glucoamylase and may be of bacterial or fungal origin. This includes chemically modified or protein-manipulated mutants. Examples of amylases include alpha-amylase obtained from specific strains of the genus Bacillus, such as Bacillus licheniformis, which is described in more detail in British Patent No. 1,296,839.

[0081] Suitable amylases include amylases having SEQ ID NO: 2 in International Publication No. 95 / 10603 or variants having 90% sequence identity with SEQ ID NO: 3. Preferred variants are those described in SEQ ID NO: 4 in International Publication Nos. 94 / 02597, 94 / 18314, 97 / 43424 and 99 / 019467, for example, variants having 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.

[0082] Other suitable amylases include the amylase having SEQ ID NO: 6 as described in International Publication No. 02 / 010355, or its variants having 90% sequence identity with SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 have deletions at positions 181 and 182 and a substitution at position 193.

[0083] Other suitable amylases include hybrid alpha-amylases containing residues 1-33 of alpha-amylase derived from B. amyloliquefaciens, as shown in SEQ ID NO. 6 of International Publication No. 2006 / 066594, and residues 36-483 of alpha-amylase from B. licheniformis, as shown in SEQ ID NO. 4 of International Publication No. 2006 / 066594, or variants having 90% sequence identity. Preferred variants of this hybrid alpha-amylase have substitutions, deletions, or insertions at one or more of the following positions: G48, T49, G107, H156, A181, N190, M197, I201, A209, and Q264. The most preferred variant of the hybrid alpha-amylase containing residues 1-33 of the alpha-amylase derived from B. amyloliquefaciens as shown in Sequence ID 6 of International Publication No. 2006 / 066594 and residues 36-483 of Sequence ID 4 is the substitution: M197T; H156Y+A181T+N190F+A209V+Q264S; or It has G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.

[0084] Further suitable amylases are amylases having SEQ ID NO: 6 as described in International Publication No. 99 / 019467, or variants thereof having 90% sequence identity to SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 have substitutions, deletions, or insertions at one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216, and K269. Particularly preferred amylases have deletions at positions R181 and G182 or H183 and G184.

[0085] Additional amylases that can be used are those having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 2, or SEQ ID NO: 7 from International Publication No. 96 / 023873, or their variants having 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 7. Preferred variants of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 7 have substitutions, deletions, or insertions at one or more of the following positions: 140, 181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304, and 476, using SEQ ID NO: 2 from International Publication No. 96 / 023873 for numbering. More preferred variants have deletions at two positions selected from 181, 182, 183, and 184, such as positions 181 and 182, 182 and 183, or 183 and 184. The most preferred amylase variants of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7 have deletions at positions 183 and 184 and substitutions at one or more positions 140, 195, 206, 243, 260, 304, and 476.

[0086] Other amylases that can be used are amylases having SEQ ID NO: 2 in International Publication No. 08 / 153815, SEQ ID NO: 10 in International Publication No. 01 / 66712, or variants thereof having 90% sequence identity to SEQ ID NO: 2 in International Publication No. 08 / 153815 or 90% sequence identity to SEQ ID NO: 10 in International Publication No. 01 / 66712. Preferred variants of SEQ ID NO: 10 in International Publication No. 01 / 66712 have substitutions, deletions, or insertions at one or more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211, and 264.

[0087] Further suitable amylases are those having SEQ ID NO: 2 in International Publication No. 09 / 061380 or variants thereof having 90% sequence identity to SEQ ID NO: 2. Preferred variants of SEQ ID NO: 2 have a truncation at the C-terminus and / or substitution, deletion or insertion at one or more of the following positions: Q87, Q98, S125, N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475. More preferred variants of SEQ ID NO: 2 have substitutions at one or more of the following positions: Q87E,R, Q98R, S125A, N128C, T131I, T165I, K178L, T182G, M201L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R, R309A, Q320R, Q359E, K444E and G475K, and / or deletions at positions R180 and / or S181 or T182 and / or G183. The most preferred amylase variant of SEQ ID NO: 2 has substitutions: N128C+K178L+T182G+Y305R+G475K; N128C+K178L+T182G+F202Y+Y305R+D319T+G475K; S125A+N128C+K178L+T182G+Y305R+G475K; or The mutant has S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K, and the mutant is C-terminus truncated and optionally further includes substitution at position 243 and / or deletion at positions 180 and / or 181.

[0088] Further suitable amylases are those having SEQ ID NO: 1 in International Publication No. 13184577 or variants thereof having 90% sequence identity with SEQ ID NO: 1. Preferred variants of SEQ ID NO: 1 have substitutions, deletions, or insertions at one or more of the following positions: K176, R178, G179, T180, G181, E187, N192, M199, I203, S241, R458, T459, D460, G476, and G477. A more preferred variant of SEQ ID NO: 1 has substitutions at one or more of the following positions: K176L, E187P, N192FYH, M199L, I203YF, S241QADN, R458N, T459S, D460T, G476K, and G477K, and / or deletions at positions R178 and / or S179 or T180 and / or G181. The most preferred amylase variant of SEQ ID NO: 1 has substitutions: E187P+I203Y+G476K It has E187P+I203Y+R458N+T459S+D460T+G476K, The variants may optionally further include substitutions at position 241 and / or deletions at positions 178 and / or 179.

[0089] Further suitable amylases are those having SEQ ID NO: 1 in International Publication No. 10104675 or its variants having 90% sequence identity with SEQ ID NO: 1. Preferred variants of SEQ ID NO: 1 have substitutions, deletions, or insertions at one or more of the following positions: N21, D97, V128, K177, R179, S180, I181, G182, M200, L204, E242, G477, and G478. More preferred variants of SEQ ID NO: 1 have substitutions at one or more of the following positions: N21D, D97N, V128I, K177L, M200L, L204YF, E242QA, G477K, and G478K, and / or deletions at the R179 and / or S180 positions or the I181 and / or G182 positions. The most preferred amylase variant of sequence number 1 is the substitution: It has N21D+D97N+V128I. The mutants may optionally further include substitutions at position 200 and / or deletions at positions 180 and / or 181.

[0090] Other suitable amylases are alpha-amylases having SEQ ID NO: 12 in International Publication No. 01 / 66712 or variants having at least 90% sequence identity to SEQ ID NO: 12. Preferred amylase variants have substitutions, deletions, or insertions at one or more of the following positions in SEQ ID NO: 12 in International Publication No. 01 / 66712: R28, R118, N174; R181, G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484. Certain preferred amylases include mutants having deletions of D183 and G184 and substitutions R118K, N195F, R320K and R458K, as well as mutants having additional substitutions at one or more positions selected from the group M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345 and A339, with mutants having additional substitutions at all of these positions being the most preferred.

[0091] Other examples include amylase variants, such as those described in International Publication No. 2011 / 098531, International Publication No. 2013 / 001078, and International Publication No. 2013 / 001087.

[0092] Commercially available amylases include Duramyl™, Teramyl™, Fungamyl™, Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X, and BAN™ (manufactured by Novozymes A / S), as well as Rapidase™, Purastar™ / Effectenz™, Powerase, Preferred S1000, Preferred S100, and Preferred S110 (manufactured by Genencor International Inc. / DuPont).

[0093] Mannanase: In one embodiment, suitable mannanases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included. Mannanases may be alkaline mannanases of family 5 or 26. They may be wild types derived from the genera Bacillus or Humicola, in particular B. agaradhaerens, B. licheniformis, B. halodurans, B. clausii, or H. insolens. Suitable mannanases are described in International Publication No. 1999 / 064619. Commercially available mannanases are Mannaway (Novozymes A / S) and Purabrite® (Danisco / DuPont).

[0094] Pectin lyase, DNase and / or PDE: Other preferred enzymes that may be additionally included in the composition of the present invention include pectin lyase, for example, those sold under the trademark names Pectawash®, Pectaway®, or Xpect®. Finally, the composition may also include deoxyribonuclease (DNase) and / or phosphodiesterase (PDE).

[0095] In one embodiment, the enzyme that may be included in the cleaning composition is a protease.

[0096] In one embodiment, the enzymes that may be included in the washing composition are proteases, lipases, carbohydrases (e.g., amylases) and / or combinations thereof.

[0097] In one embodiment, the enzymes that may be included in the washing composition are a mixture of protease, DNase, and hexosaminidase (e.g., dispersin).

[0098] In one embodiment, the enzymes that may be included in the washing composition are proteases, lipases, DNases, hexosaminidases (e.g., dispersin), and / or combinations thereof.

[0099] Builders and co-builders The detergent composition may contain about 0% to about 65% by weight of detergent builder or co-builder, or mixtures thereof. In dishwashing detergents, the builder level is typically 40-65%, and particularly about 50-65%. The builder and / or co-builder is particularly Ca 2+ and Mg 2+ It may be a chelating agent that forms a water-soluble complex with it. Any builder and / or co-builder known in the art for use in laundry detergents may be used. Non-limiting examples of builders include zeolites, diphosphates (pyrophosphates), tripphosphates such as sodium triphosphate (STP or STPP), carbonates such as sodium carbonate, soluble silicates such as sodium metasilicate, layered silicates (e.g., SKS-6 from Hoechst), disilicates, ethanolamines such as 2-aminoethane-1-ol (MEA), iminodiethanol (DEA) and 2,2',2”-nitrilotriethanol (TEA), and carboxymethyl inulin (CMI) and combinations thereof.

[0100] The detergent composition may also contain about 0% to about 65% by weight of detergent cobuilders or mixtures thereof. The detergent composition may contain cobuilders alone or in combination with builders, such as zeolite builders. Non-limiting examples of cobuilders include homopolymers or copolymers thereof of polyacrylates, poly(acrylic acid) (PAA) or copoli(acrylic acid / maleic acid) (PAA / PMA), etc. Further non-limiting examples include chelating agents such as citrates, aminocarboxylates, aminopolycarboxylates and phosphonates, and alkyl or alkenyl succinates.Further specific examples include 2,2',2"-nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N'-disuccinic acid (EDDS), methylglycinediacetic acid (MGDA), glutamic acid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diyrbis(phosphonic acid) (HEDP), and ethylenediaminetetrakis(methylene)tetrakis(phosphonic acid) )(EDTMPA), diethylenetriaminepentakis(methylene)pentakis(phosphonic acid)(DTPMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA), N-(2-sulfomethyl)aspartic acid (SMAS), N-(2-sulfoethyl)aspartic acid (SEAS), N-(2- Sulfomethyl)glutamic acid (SMGL), N-(2-sulfoethyl)glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA), and sulfomethyl These include tyl-N,N-diacetic acid (SMDA), N-(hydroxyethyl)-ethylidenediaminetriacetic acid (HEDTA), diethanolglycine (DEG), diethylenetriamine penta(methylenephosphonic acid) (DTPMP), aminotris(methylenephosphonic acid) (ATMP), and combinations thereof and salts. Further exemplary builders and / or co-builders are described, for example, in International Publication No. 09 / 102854 and U.S. Patent No. 5977053.

[0101] Bleaching type Detergents may contain 0 to 40% by weight of a bleaching system, such as about 5% to about 25%. Any bleaching system known in the art for use in laundry detergents may be used. Suitable bleaching system components include bleaching catalysts, photobleaching agents, bleaching activators, hydrogen peroxide sources such as sodium percarbonate and sodium perborate, pre-formed peracids, and mixtures thereof.

[0102] polymer The detergent may further contain 0 to 10% by weight of another type of polymer, such as 0.5-5%, 2-5%, 0.5-2%, or 0.2-1%. The polymer may function as the above-mentioned cobuilder or provide soil-releasing and grease-cleaning properties. Some polymers may have several of the above properties and / or several of the following properties. Any polymer known in the art for use in detergents may be utilized.

[0103] method Another aspect of the present invention relates to a method for cleaning a surface, comprising (a) foaming one of the cleaning compositions of the present invention onto the surface (e.g., by spraying or brushing), and optionally (b) rinsing the surface.

[0104] A further aspect of the present invention relates to a method for formulating a composition with reduced aerosolization (e.g., enzymatic aerosolization), comprising mixing at least one first surfactant and at least one second surfactant as described in the section on surfactants above, and optionally adding at least one enzyme.

[0105] Application system for foam generation Typically, foam can be generated using a pressurized system and applied, for example, as a spray or through a brush. Applying foam as a spray may generate more aerosolizing enzymes than applying it with a brush. In the case of foaming brush application, the foam is generated before it leaves the brush, thus minimizing enzyme aerosolization. Therefore, in the case of spray application, both the first and second surfactants are ideally low-volatility surfactants to reduce the risk of safety (allergies). Those skilled in the art can prepare suitable cleaning compositions for various foam-generating systems according to the concept of the present invention.

[0106] Enzyme formulation in cogranules The enzymes of the present invention can be formulated as granules, for example, as cogranules combining one or more enzymes. In this case, each enzyme will be present in more granules, ensuring a more uniform distribution of enzymes in the detergent. This also reduces the physical segregation of different enzymes due to differences in particle size. A method for producing multi-enzyme cogranules for the detergent industry is disclosed in IP.com disclosure IPCOM000200739D.

[0107] Another example of enzyme formulation using cogranules is disclosed in International Publication No. 2013 / 188331, which relates to a cleaning composition comprising (a) a polyenzyme cogranule; (b) less than 10 by weight of zeolite (anhydrous); and (c) less than 10 by weight of phosphate (anhydrous), wherein the enzyme cogranule comprises 10 to 98% by weight of a moisture sink component, and the composition further comprises 20 to 80% by weight of a detergent moisture sink component. International Publication No. 2013 / 188331 also relates to a method for treating and / or cleaning a surface, comprising the steps of (i) contacting the surface with the cleaning composition described in the claims and specification in an aqueous cleaning solution, and (ii) rinsing and / or drying the surface.

[0108] The multi-enzyme cogranules may contain the enzymes of the present invention as well as one or more enzymes selected from the group consisting of lipase, amylase, hemicellulase, peroxidase, xylanase, phospholipase, esterase, cutinase, keratinase, reductase, oxidase, phenol oxidase, lipoxygenase, ligninase, pullulanase, maranase, β-glucanase, arabinosidase, hyaluronidase, laccase, deoxyribonuclease (DNase), hexosaminidase, and any mixture thereof.

[0109] The present invention is further summarized in the following sections: 1. A cleaning composition having reduced aerosolization properties, (i) at least one first surfactant, the first surfactant having 8 to 12 carbon units, for example, 8 to 10 or 10 to 12 carbon units; (ii) at least one second surfactant, the second surfactant having 8 to 16 carbon units, for example, 8 to 10, 10 to 12 or 10 to 14; optionally, (iii) A cleaning composition comprising a buffer system. 2. The composition according to item 1, wherein the first surfactant is selected from the group consisting of sodium salts, potassium salts, or ammonium salts and mixtures thereof. 3. The composition according to item 1 or 2, wherein the second surfactant is selected from the group consisting of those having 10 to 12 carbon units, preferably selected from the group consisting of amine oxides, betaines, primary fatty alcohols, monoethanolamides, diethanolamides, and mixtures thereof. 4. The composition according to any one of items 1 to 3, wherein the first surfactant is a low-volatility surfactant. 5. The composition according to item 4, wherein the second surfactant is a low-volatility surfactant. 6. The composition according to either item 4 or 5, wherein the volatility of the surfactant is evaluated by particle aerosolization according to Example 2. 7. The second surfactant is a composition according to any one of claims 1 to 6, having the same carbon units as the first surfactant. 8. The composition according to any one of claims 1 to 7, wherein the first surfactant comprises one or more anionic surfactants, for example, linear alkylbenzene sulfonic acid (LAS), alcohol ether sulfate (AEOS) and / or alkyl sulfate (AS), in particular sodium lauryl sulfate (SLS), sodium laureth sulfate, sodium lauryl ether sulfate, ammonium decyl ether sulfate, sodium decyl sulfate, ammonium alkyl ether sulfate and sodium dodecyl sulfate. 9. The composition according to any one of claims 1 to 8, wherein the first surfactant comprises one or more nonionic surfactants. 10. The composition according to any one of claims 1 to 9, wherein the second surfactant is selected from amine oxides, decylamine oxides, lauramine oxides and mixtures thereof, and optionally comprises primary fatty alcohols such as 1-dodecaol and 1-decanol. 11. The composition according to any one of claims 1 to 10, wherein the first surfactant is present at a level of 0.2-60% by weight, 0.5-40% by weight, 0.8-30% by weight, 1-50% by weight, 1-40% by weight, 1-30% by weight, 1-20% by weight, 3-10% by weight, 3-5% by weight, 5-40% by weight, 5-30% by weight, 5-15% by weight, 2-20% by weight, 3-6% by weight, 8-12% by weight, 10-12% by weight, 20-25% by weight, or 25-60% by weight. 12. The composition according to any one of claims 1 to 11, wherein the second surfactant is present at a level of 0.01 to 10% by weight, 0.02 to 8% by weight, 0.05 to 6% by weight, 0.1 to 5% by weight, 0.5 to 4% by weight, 1 to 3% by weight, 1.5 to 8% by weight, or 2 to 6% by weight. 13. The composition according to any one of claims 1 to 12, wherein the weight ratio of the second surfactant to the first surfactant is in the range of 0.001 to 1, for example, in the range of 0.005 to 0.9, 0.01 to 0.8, 0.1 to 0.6, 0.08 to 0.5, or 0.2 to 0.4. 14. The buffer system is a composition according to any one of items 1 to 13, comprising TRIS-HCl / TRIS base, citric acid / citrate, sodium carbonate / sodium bicarbonate, or potassium carbonate / potassium bicarbonate. 15. The composition according to any one of claims 1 to 14, wherein the buffer system is included at a level of 0.02 to 10% by weight, for example, 0.05 to 8% by weight, 0.1 to 6% by weight, 0.5 to 5% by weight, 1 to 4% by weight, 2% to 6% by weight, or 3% to 5% by weight. 16. The composition according to any one of claims 1 to 15, further comprising one or more enzymes selected from the group consisting of proteases, lipases, amylases, cellulases, mannanases, pectinases, hemicellulases, peroxidases, xylanases, phospholipases, xyloglucanases, esterases, cutinases, laccases, keratinases, reductases, oxidases, phenol oxidases, lipoxygenases, ligninases, pullulanases, maranases, β-glucanases, arabinosidases, hyaluronidases, deoxyribonucleases (DNases), RNases, hexosaminidases, peroxidases, and any mixtures thereof. 17. The composition according to any one of claims 1 to 16, wherein the enzyme is a protease, lipase, DNase, hexosaminidase (e.g., dispersin) and / or a combination thereof. 18. The composition according to any one of claims 1 to 17, wherein the enzyme is a mixture or blend of proteases, DNases, and hexosaminidases (e.g., dispersins). 19. The composition according to any one of claims 1 to 18, wherein the protease has at least 60%, at least 65%, at least 70%, 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%, at least 99%, or 100% sequence identity with respect to the polypeptide of SEQ ID NO: 1 or 2. 20. The composition according to any one of claims 1 to 19, wherein the protease is a variant of the polypeptide of SEQ ID NO: 2, comprising modifications at one or more positions corresponding to positions 3, 4, 9, 15, 43, 68, 76, 99, 101, 103, 104, 160, 167, 170, 194, 199, 205, 206, 209, 217, 218, 222, 245, 261 and 262, the position numbers corresponding to positions of SEQ ID NO: 1, each modification is independently a substitution, deletion or insertion, the variant has protease activity and has sequence identity of 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% but less than 100% of the polypeptide of SEQ ID NO: 2. 21. Proteases are, (a) Includes one or more substitutions selected from the group consisting of S3T, V4I, S9E, S9R, A15T, V68A, N76D, S99D, S99G, S99A, S99SE, S101E, S101N, S101R, S103A, V104I, G160S, Y167A, R170S, A194P, V199M, V205I, Q206L, Y209W, L217D, L217Q, N218D, M222S, Q245R, N261W, and L262E. A variant of the polypeptide of SEQ ID NO: 2, wherein the position number corresponds to the position of SEQ ID NO: 1, the variant has protease activity, and has sequence identity of 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%, and at least 99% but less than 100% of SEQ ID NO: 2; (b) A variant of the polypeptide of SEQ ID NO: 2 comprising the substitution S87N, wherein the variant has protease activity, its position corresponds to the position of SEQ ID NO: 1, and the variant has protease activity and sequence identity of 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% but less than 100% relative to SEQ ID NO: 2; (c) A variant of the polypeptide of SEQ ID NO: 2 comprising the substitution Y167A+R170S+A194P, wherein the position number corresponds to the position of SEQ ID NO: 1, the variant has protease activity, and has sequence identity of 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% but less than 100% relative to SEQ ID NO: 2; (d) A variant of the polypeptide of SEQ ID NO: 2 comprising the substitution S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E, wherein the positional numbers correspond to the positions of SEQ ID NO: 1, the variant has protease activity, and has sequence identity of 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% but less than 100% relative to SEQ ID NO: 2; (e) A variant of the polypeptide of SEQ ID NO: 4 comprising the substitution A68S+T77N+T78I+G127S+A128P+G165Q+N184Q+A202V+N217S+S258P, wherein the positional numbers correspond to the positions of SEQ ID NO: 4, the variant has protease activity, and has sequence identity of 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% but less than 100% relative to SEQ ID NO: 4; (f) A variant comprising substitutions at one or more positions corresponding to positions 171, 173, 175, 179 or 180 of Sequence ID No. 1 of International Publication No. 2004 / 067737, wherein the variant has protease activity and has sequence identity of 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% but less than 100% of Sequence ID No. 1 of International Publication No. 2004 / 067737; (g)X3V, X9[E,R], X22[R,A], X43R, X61[E,D], X62[E,D], X76[D], X87N, X101[E,G,D, N,M], X103A, X104I, X118[V,R], X120V, X128[A,L,S], X129Q, X130A, X160D, X185[E, Select from the group consisting of D], 188[E,D], X191N, X194P, X205I, X206L, X209W, X216V, X217[Q,D,E], X218[D,E,S], X232V, X245R, X248D, X256[E,D], X259[E,D], X261[E,D,W] and X262[E,D] A protease mutant is selected, comprising one or more substitutions compared to the parent protease, wherein the position number corresponds to the position of BPN' (SEQ ID NO: 1), and "X" represents any amino acid residue present at a specified position in the parent protease, and the mutant has protease activity and sequence identity of 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%, but less than 100%, with respect to SEQ ID NO: 1 or SEQ ID NO: 2; and (h) A protease mutant comprising one of the following substitution sets compared to the parent protease, wherein the parent protease has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or has at least 80% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, the position number corresponds to the position of BPN' (SEQ ID NO: 1), "X" represents any amino acid residue present at a specified position in the parent protease, and the substitution set is: i.X9R+X15T+X68A+X218D+X245R, ii. X9R + X15T + X68A + X245R, iii.X61E+X194P+X205I+X261D, iv.X61D+X205I+X245R, X61E + X194P + X205I + X261D, vi.X87N+X118V+X128L+X129Q+X130A, vii.X87N+X101M+X118V+X128L+X129Q+X130A, viii.X76D+X87R+X118R+X128L+X129Q+X130A, ix.X22A+X62D+X101G+X188D+X232V+X245R, x.X103A+X104I, xi.X22R+X101G+X232V+X245R, xii.X103A+X104I+X156D, xiii.X103A+X104I+X261E, xiv.X62D+X245R, xv.X101N+X128A+X217Q, xvi.X101E+X217Q, xvii.X101E+X217D, xviii.X9E+X43R+X262E, xix.X76D+X43R+X209W, xx.X205I+X206L+X209W, xxi.X185E+X188E+X205I, xxii.X256D+X261W+X262E, xxiii.X191N+X209W, xxiv.X261E+X262E, xxv.X261E+X262D and xxvi.X167A+X170S+X194P The composition according to any one of claims 1 to 20, wherein the protease mutant is selected from the group consisting of and has sequence identity of 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% but less than 100% with respect to SEQ ID NO: 1 or 2. 22. The composition according to any one of claims 1 to 21, wherein the protease mutant comprises the amino acid sequence of SEQ ID NO: 2 having the substitution Y167A+R170S+A194P, the mutant has protease activity, and the position number corresponds to the position of SEQ ID NO: 1. 23. The protease mutant comprises the amino acid sequence of SEQ ID NO: 2 having the substitution S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E, the mutant has protease activity, and the position number corresponds to the position of SEQ ID NO: 1, the composition according to any one of items 1 to 22. 24. When the composition is evaluated by the 20-minute foam test according to Example 1, the volume of particles with a size of 0.3 μm is 15 × 10 6 m 3 For example, 10 x 10 6 m 3 , 8×10 6 ms 3 , 6×10 6 m 3 , 5×10 6 m 3 or 2 × 10 6 m 3 A composition according to any one of items 1 to 23, which is less than [amount missing]. 25. When the composition is evaluated by the 20-minute foam test according to Example 1, the volume of particles of size 0.5 μm is 15 × 10 6 m 3 For example, 10 x 10 6 m 3 , 8×10 6 m 3 , 6×10 6 m 3 , 5×10 6 m 3 or 2 × 10 6 m 3 A composition according to any one of items 1 to 24, which is less than [amount missing]. 26. When the composition is evaluated by the 20-minute foam test according to Example 1, the volume of particles with a size of 1 μm is 15 × 10 6 m 3 For example, 10 x 10 6 m 3 , 8×10 6 m3 , 6 × 10 6 m 3 , 5 × 10 6 m 3 or 2 × 10 6 m 3 less than, the composition according to any one of items 1 to 25. 27. When the composition is evaluated by the 20 - minute foam test according to Example 1, the volume of particles with a size of 2um is 15 × 10 6 m 3 or less, for example, 10 × 10 6 m 3 , 8 × 10 6 m 3 , 6 × 10 6 m 3 , 5 × 10 6 m 3 or 2 × 10 6 m 3 less than, the composition according to any one of items 1 to 26. 28. When the composition is evaluated by the method according to Example 2, the enzyme aerosolization concentration is 100 ppb or less, for example, 80 ppb or less, 60 ppb or less, 50 ppb or less, 30 ppb or less, 20 ppb or less, 15 ppb or less, or 10 ppb or less, the composition according to any one of items 1 to 27. 29. The surface tension of the composition is 35 mN / m or less, 30 mN / m or less, 28 mN / m or less, 25 mN / m or less, or 20 mN / m or less, the composition according to any one of items 1 to 28. 30. The said composition is a formulation suitable for a foam - type detergent, preferably having a pH of less than 11, for example, in the range of 6 - 9.5, 6.5 - 9, 6.8 - 8.5, or in the range of 7 - 8, and more preferably having a neutral pH of 6.5 - 7.5 such as pH 7.0, 7.1, or 7, the composition according to any one of items 1 to 29. 31. The said composition further contains a preservative, for example, 2 - phenoxyethanol, isothiazolinone (benzisothiazolinone (BIT), methylisothiazolinone (MIT), chloromethylisothiazolinone (CMIT), etc.) or a mixture thereof, the composition according to any one of items 1 to 30. 32. The preservative is the composition described in item 31, comprising 2-phenoxyethanol. 33. The composition according to item 31 or 32, wherein the preservative is present in the composition at a level of 0.01 to 10% by weight, 0.1 to 5% by weight, 0.5 to 3% by weight, or 1 to 2% by weight. 34. The composition according to any one of claims 1 to 33, further comprising an enzyme stabilizer selected from the group consisting of glycerol, sorbitol, sucrose, glucose, NaCl, KCl, sodium formate, citric acid, trisodium citrate dihydrate, hexanediol, heptanediol, PEG 300 and PEG 600 and mixtures thereof. 35. The composition according to item 34, wherein the enzyme stabilizer comprises glycerol and / or PEG 600. 36. The composition according to item 34 or 35, wherein the enzyme stabilizer is present in the composition at a level of 0.5 to 25% by weight, 1 to 20% by weight, 2% to 18% by weight, 4% to 15% by weight, or 5% to 10% by weight. 37. The composition according to any one of claims 1 to 36, which is formulated as a ready-to-use detergent or as a concentrated detergent (for example, 2x, 5x, 10x, or 15x concentrated). 38. The composition according to item 37, wherein the concentrated detergent comprises alkyl ether ammonium sulfate, and the second surfactant is selected from lauramine oxide, linear alcohol (C12-13) ethoxylate (e.g., linear alcohol (C12-13) ethoxylate POE-3) and mixtures thereof. 39. A method for cleaning a surface, comprising (a) foaming one of the cleaning compositions described in any of sections 1 to 38 onto the surface (for example, by spraying or brushing), and optionally (b) rinsing the surface, preferably the surface being a hard surface. 40. A method for formulating a composition in which aerosolization (e.g., enzymatic aerosolization) has been reduced, comprising mixing at least one first surfactant and at least one second surfactant as described in any of items 1 to 39, and optionally adding at least one enzyme.

[0110] The present invention is further illustrated in the following non-limiting embodiments. [Examples]

[0111] Assay I Testing of protease activity The proteolytic activity can be determined by a method using the Suc-AAPF-pNA substrate. Suc-AAPF-pNA is an abbreviation for N-succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide, a block peptide that can be cleaved by endoproteases. After proteolytic cleavage, yellow free pNA molecules are released and can be measured at a wavelength of 405 nm by visible spectrophotometric spectroscopy. The Suc-AAPF-pNA substrate was prepared by Bachem (catalog number L1400, dissolved in DMSO).

[0112] The protease sample to be analyzed is diluted in residual activity buffer (100 mM Tris pH 8.6). 30 μl of the diluted enzyme sample is transferred to a 96-well microtiter plate, and the assay is performed by adding 70 μl of substrate working solution (0.72 mg / ml in 100 mM Tris pH 9). The solution is mixed at room temperature, and absorption is measured every 20 seconds over 5 minutes at OD 405 nm.

[0113] The slope of the time-dependent absorption curve (absorbance per minute) is directly proportional to the activity of the protease under given conditions. The protease sample should be diluted to a level where the slope is linear.

[0114] Assay II Testing of DNase activity DNase activity can be determined by DNase Test Agar with Methyl Green (BD, Franklin Lakes, NJ, USA), which is prepared according to the supplier's instructions. Briefly, 21 g of agar is dissolved in 500 ml of water and autoclaved at 121°C for 15 minutes. The autoclaved agar is then heated to 48°C in a water bath, 20 ml of agar is poured into a petri dish, and allowed to solidify overnight at room temperature by incubation. 5 μl of enzyme solution is added to the solidified agar plate, and DNase activity is observed as a colorless zone around the spotted enzyme solution.

[0115] Assay III Testing of hexosaminidase activity Hexosaminidase activity can be determined using 4-nitrophenyl N-acetyl-β-D-glucosaminide (Sigma-Aldrich) as the substrate. The enzymatic reaction is performed in triplicate in a 96-well flat-bottom polystyrene microtiter plate (Thermo Scientific) under the following conditions: 100 μl total reaction volume of 50 mM 2-(N-morpholino)ethanesulfonic acid pH 6 buffer, 1.5 mg / ml 4-nitrophenyl N-acetyl-β-D-glucosaminide, and 10, 20, or 50 μg / ml purified enzyme sample. A blank sample without polypeptides is run simultaneously. The reaction is carried out in a Thermomixer comfort (Eppendorf) at 37°C. After 10 minutes of incubation, 5 μl of 1 M NaOH is added to each reaction mixture to stop the enzymatic reaction. Absorbance was read at 405 nm using a POLAR star Omega plate reader (BMG LABTECH) to estimate the formation of 4-nitrophenolate ions released due to enzymatic hydrolysis of the 4-nitrophenyl N-acetyl-β-D-glucosaminide substrate. Absorbance measured by reactions using the hexosaminidase polypeptide, which was higher than the absorbance of the blank without polypeptide, indicates that the polypeptide tested exhibits hexosaminidase activity.

[0116] Assay IV Lipase activity determined by p-nitrophenyl (pNP) assay The hydrolytic activity of lipases can be determined by a kinetic assay using p-nitrophenyl acyl esters as substrates. The substrates p-nitrophenyl butyrate (C4), p-nitrophenyl caproate (C6), p-nitrophenyl caprate (C10), p-nitrophenyl laurate (C12), and p-nitrophenyl palmitate (C16) (all from Sigma-Aldrich Danmark A / S, Kirkebjerg Alle 84, 2605 Brondby; catalog numbers: C3:N-9876, C6:N-0502, C10:N-0252, C12:N-2002, C16:N-2752) are each diluted in DMSO to final concentrations of 1 mM and 25 mM in assay buffer (50 mM Tris; pH 7.7; 0.4% Triton X-100). Lipase in 50 mM Hepes; pH 8.0; 10 ppm Triton X-100; + / - 20 mM CaCl2 was analyzed to the following final protein concentration: 0.01 mg / ml; 5 × 10⁻⁶ -3 mg / ml; 2.5 × 10 -4 mg / ml; and 1.25 × 10 -4 The substrate solution is added at a concentration of mg / ml in a 96-well NUNC plate (catalog number 260836, Kamstrupvej 90, DK-4000, Roskilde). The release of p-nitrophenol by hydrolysis of p-nitrophenylacyl can be monitored at 405 nm for 5 minutes at 10-second intervals using a Spectra max 190 (Molecular Devices GmbH, Bismarckring 39, 88400 Biberach an der Riss, GERMANY).

[0117] detergent

[0118] [Table 1]

[0119] [Table 2]

[0120] Enzymes used in the following examples Protease: Polypeptide of Sequence ID No. 2 with the following mutation: Y167A+R170S+A194P, position number based on the numbering of Sequence ID No. 1. Nuclease: Polypeptide of SEQ ID NO: 3 with the following mutation: T1I+S13Y+T22P+S27L+L33K+S39P+S42G+D56I+S57W+S59V+T65V+V76L+Q109R+S116D+T127V+S144P+A147H+S167L+G175D. Hexosaminidase: Polypeptide of sequence number 4 with the following mutation: Q3I+H15Y+A49W+N59E+S163P+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q.

[0121] Example 1 - Cleaning composition with reduced particle aerosolization according to the present invention Experiments were conducted to determine the reduction in particle aerosolization of the detergent composition of the present invention.

[0122] Test Procedure The particle distribution of six detergents was evaluated. For this test, particle distribution was used as an indirect measure of enzymatic aerosolization. The detergents were sprayed onto a wall in a sealed area for 20 minutes using a 5-gallon Smart Foam unit (FoamIt). A high particle count indicated that the detergent would easily generate aerosolized particles and would be unsuitable for use in the enzymatic test. Conversely, a low particle count indicated that the detergent would not aerosolize and would be a suitable candidate for the detergent. The following data were collected: Particle count was measured using a Bench-Top Particle Counter (Met One Instruments, Grants Pass OR). Volume (m³) of particles of sizes 0.3, 0.5, 1, 2, 5, and 10 μm were collected. 3The results were recorded during a 20-minute test. The distribution of generated particles was plotted for each detergent, as shown in Figure 1.

[0123] Figure 1 shows that detergent 2, containing two different second surfactants at higher relative concentrations, had the largest total volume of particles produced. Conversely, detergents 1, 3, 4, 5, and 6 resulted in a significantly reduced total volume of particles produced. A possible reason for this difference may be related to the volatility (or atomization) of the chemicals used in the detergents. Surfactants such as aliphatic primary linear alcohols volatilize (or atomize) rapidly upon application of foam, allowing more particles to enter the air. These findings suggest that suitable detergents should contain small amounts of volatile surfactants or be completely free of them.

[0124] In addition, sodium lauryl sulfate (SLS) has a higher molecular weight and therefore is more cohesive than sodium dodecyl sulfate (SDS), and thus SLS is less volatile. Furthermore, SLS interacts more strongly with fatty alcohols than SDS. This may explain why detergent 1 has significantly lower particle dispersion compared to detergent 3, with detergent 1 being used to determine particle dispersion when using SLS and fatty alcohols, and detergent 3 being used to determine particle dispersion when using fatty alcohols and SDS. Therefore, SLS, etc., are preferred first surfactants when a volatile second surfactant is used and are considered suitable first surfactants for applications where on-site mixing (enzymes are added to the chemical immediately before use) is conceivable.

[0125] Example 2 - Reduction of enzyme aerosolization by the method to which the present invention is applied. The experiment was performed to determine the reduction in enzymatic aerosolization by the application of the present invention, which includes subtilisin protease (polypeptide of Sequence ID No. 2 with the following mutation: Y167A+R170S+A194P, position number based on the numbering of Sequence ID No. 1). The protease is present in detergent 7 at active enzyme protein (AEP) concentrations of 15 and 30 ppm.

[0126] Test Procedure We evaluated four different foam application methods: Application 1: Foam unit (FoamIt) with heavy-duty trigger gun. Application 2: Smart foam unit (FoamIt) with foam spray nozzle. Application 3: Smart foam unit (FoamIt) with brush head structure #1 (Lowes). Application 4: Smart foam unit (FoamIt) with brush head structure #2 (Lowes).

[0127] For each test, foam was applied to the wall within a sealed area for 20 minutes using one of the application methods. During this time, the following data was collected: 1) Particle count was measured using a Bench-Top Particle Counter (Met One Instruments, Grants Pass OR). Volume (m³) of particles of sizes 0.3, 0.5, 1, 2, 5, and 10 μm. 3 The results were recorded during a 20-minute test. The distribution of generated particles was plotted for each detergent. 2) Enzyme concentration in the air. 25L using a pump. -1 Air was passed through the filter at a certain rate. The aerosolized enzyme, obtained by foam application, was collected by the filter. The filter was analyzed using MICT along with the target enzyme protease for detection. The obtained enzyme concentrations were plotted against the particle distribution and grouped by application method.

[0128] [Table 3]

[0129] In general, the enzymatic aerosolization generated by the detergents of the present invention was lower than that of conventional detergents. Table 2 shows that detergent 7 produced the lowest amount of enzymatic aerosolization in all applications 1-4 tested, compared to the other two test detergents (detergents 4 and 6).

[0130] Example 3 - Enzyme-stabilized detergent composition of the present invention This embodiment determines the stability of the enzyme detergent composition of the present invention.

[0131] Detergent 7 is suitable for use as a ready-to-use formulation. Detergents 8 and 9 (see Table 1) are both 15x concentrated formulations. For concentrated products, the first surfactant (e.g., sodium lauryl sulfate) may denature and inactivate enzymes, so the first surfactant may be modified. For this reason, a first surfactant suitable for concentrated formulations may be ammonium alkyl ether sulfate, etc.

[0132] Experiments were conducted to determine the concentrations of stabilizers (such as citric acid, trisodium citrate dihydrate, glycerol, and PEG 600) required for protease, nuclease, and carbohydrase enzymes in order to produce a stable concentrated detergent. Stabilization was defined as maintaining >80% enzyme activity over 8 weeks.

[0133] Enzymes tested: (1) Enzyme blend (60% protease, 20% nuclease, 20% hexosaminidase); (2) Single enzyme: nuclease. The sequence information for these enzymes is as described in the enzyme section above.

[0134] Test procedure: As shown in Table 3 below, various combinations of enzyme stabilizers (glycerol and PEG 600) were evaluated for their ability to provide enzyme stability. Each combination was used to assess the stability of single nuclease enzymes and blends of proteases, nucleases, and hexosaminidases in a total of 14 tests. Each test was exposed to four different temperatures (4°C, 25°C, 30°C, and 37°C). Enzyme activity was measured at each temperature over 2, 4, and 8 weeks (activity measurement). Samples stored at -18°C were used as control samples at 2, 4, and 8-week intervals (control activity). The reported enzyme activity for each temperature and time point was calculated as a percentage (%) of the measured activity relative to the control activity. The results are shown in Tables 4 and 5 below.

[0135] Table 3 below outlines the combinations of glycerol and PEG 600 determined in stability experiments. The combinations are denoted with the reference letter "F". Tables 4 and 5 summarize the stability results for enzyme blends and individual enzymes. Based on Table 4, F13 provided sufficient stability for 2, 4, and 8 weeks at 4°C and 25°C. From Table 5, it could be concluded that the nuclease was stable in all test combinations (however, F10 and F14 were not as stable as the other combinations). F4 provided the best stability conditions for the nuclease enzyme at all temperatures and time points.

[0136] [Table 4]

[0137] [Table 5]

[0138] [Table 6]

Claims

1. A cleaning composition having reduced aerosolization properties, (i) at least one first surfactant, the first surfactant having 8 to 12, for example, 8 to 10 or 10 to 12 carbon units; (ii) at least one second surfactant, wherein the second surfactant has 8 to 16 carbon units, for example, 8 to 10, 10 to 12 or 10 to 14 carbon units; optionally, (iii) A cleaning composition comprising a buffer system.

2. The composition according to claim 1, wherein the first surfactant is selected from the group consisting of sodium salts, potassium salts, or ammonium salts and mixtures thereof.

3. The composition according to claim 1 or 2, wherein the second surfactant is selected from the group consisting of detergent surfactants having 10 to 12 carbon units, and preferably from the group consisting of amine oxides, betaines, primary fatty alcohols, monoethanolamides, diethanolamides, and mixtures thereof.

4. The composition according to any one of claims 1 to 3, wherein the volatility of the surfactant is evaluated by particle aerosolization according to Example 2.

5. The composition according to any one of claims 1 to 4, wherein the second surfactant has the same number of carbon units as the first surfactant.

6. The composition according to any one of claims 1 to 5, wherein the first surfactant comprises one or more anionic surfactants, for example, linear alkylbenzene sulfonic acid (LAS), alcohol ether sulfate (AEOS) and / or alkyl sulfate (AS), particularly sodium lauryl sulfate (SLS), sodium laureth sulfate, sodium lauryl ether sulfate and sodium dodecyl sulfate, ammonium decyl ether sulfate, sodium decyl sulfate and ammonium alkyl ether sulfate.

7. The composition according to any one of claims 1 to 6, wherein the second surfactant is selected from amine oxides, decylamine oxides, lauramine oxides, and mixtures thereof, and optionally comprises primary fatty alcohols such as 1-dodecaol and 1-decanol.

8. The composition according to any one of claims 1 to 7, wherein the first surfactant is present in a level of 0.2 to 60% by weight, 0.5 to 40% by weight, 0.8 to 30% by weight, 1 to 50% by weight, 1 to 40% by weight, 1 to 30% by weight, 1 to 20% by weight, 3 to 10% by weight, 3 to 5% by weight, 5 to 40% by weight, 5 to 30% by weight, 5 to 15% by weight, 2 to 20% by weight, 3 to 6% by weight, 8 to 12% by weight, 10 to 12% by weight, 20 to 25% by weight, or 25 to 60% by weight.

9. The composition according to any one of claims 1 to 8, wherein the second surfactant is present in a level of 0.01 to 10% by weight, 0.02 to 8% by weight, 0.05 to 6% by weight, 0.1 to 5% by weight, 0.5 to 4% by weight, 1 to 3% by weight, 1.5 to 8% by weight, or 2 to 6% by weight.

10. The composition according to any one of claims 1 to 9, wherein the weight ratio of the second surfactant to the first surfactant is in the range of 0.001 to 1, for example, in the range of 0.005 to 0.9, 0.01 to 0.8, 0.1 to 0.6, 0.08 to 0.5, or 0.2 to 0.

4.

11. The composition according to any one of claims 1 to 10, wherein the buffer system comprises TRIS-HCl / TRIS base, citric acid / citrate, sodium carbonate / sodium bicarbonate, potassium carbonate / potassium bicarbonate.

12. The composition according to any one of claims 1 to 11, wherein the buffering system is included in a level of 0.02 to 10% by weight, for example, 0.05 to 8% by weight, 0.1 to 6% by weight, 0.5 to 5% by weight, 1 to 4% by weight, 2 to 6% by weight, or 3 to 5% by weight.

13. The composition according to any one of claims 1 to 12, further comprising one or more enzymes selected from the group consisting of protease, lipase, amylase, cellulase, mannanase, pectinase, hemicellulase, peroxidase, xylanase, phospholipase, xyloglucanase, esterase, cutinase, laccase, keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, ligninase, pullulanase, maranase, β-glucanase, arabinosidase, hyaluronidase, deoxyribonuclease (DNase), RNase, hexosaminidase, peroxidase, and any mixture thereof.

14. When the composition is evaluated by the foam test for 20 minutes according to Example 1, the volume of particles with a size of 0.5 um is 15×10 ６ m ３ or less, for example, 10×10 ６ m ３ , 8×10 ６ m ３ , 6×10 ６ m ３ , 5×10 ６ m ３ or 2×10 ６ m ３ The composition according to any one of claims 1 to 13, which is less than.

15. The composition according to any one of claims 1 to 14, wherein, when the composition is evaluated by the method according to Example 2, the enzyme aerosolization concentration is 100 ppb or less, for example, 80 ppb or less, 60 ppb or less, 50 ppb or less, 30 ppb or less, 20 ppb or less, 15 ppb or less, or 10 ppb or less.

16. The composition according to any one of claims 1 to 15, wherein the surface tension of the composition is 35 mN / m or less, 30 mN / m or less, 28 mN / m or less, 25 mN / m or less, or 20 mN / m or less.

17. A method for cleaning a surface, comprising (a) foaming (e.g., spraying) one of the cleaning compositions described in any one of claims 1 to 16 onto the surface, and optionally (b) rinsing the surface, preferably the surface being a hard surface.

18. A method for formulating a composition in which aerosolization (e.g., enzymatic aerosolization) has been reduced, comprising mixing at least one first surfactant and at least one second surfactant and optionally a buffer system as described in any one of claims 1 to 17, and optionally adding at least one enzyme.

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