WO2018161899A1 - Use of one or more enzymes in preventing, inhibiting or reducing microbe growth on a surface - Google Patents

Use of one or more enzymes in preventing, inhibiting or reducing microbe growth on a surface Download PDF

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Publication number
WO2018161899A1
WO2018161899A1 PCT/CN2018/078160 CN2018078160W WO2018161899A1 WO 2018161899 A1 WO2018161899 A1 WO 2018161899A1 CN 2018078160 W CN2018078160 W CN 2018078160W WO 2018161899 A1 WO2018161899 A1 WO 2018161899A1
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WIPO (PCT)
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seq
protease
enzyme
microbe
swatches
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PCT/CN2018/078160
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French (fr)
Inventor
Wenwen TAO
Yanfei Wang
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Novozymes A/S
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Priority to CN201880006185.3A priority Critical patent/CN110381973A/en
Publication of WO2018161899A1 publication Critical patent/WO2018161899A1/en
Priority to PH12019501420A priority patent/PH12019501420A1/en

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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21062—Subtilisin (3.4.21.62)

Definitions

  • the present invention relates to a novel use of one or more enzymes in preventing, inhibiting or reducing microbe growth on a surface, wherein said microbe have an increase in the TTC detectable time of at least 20%on said surface after use of said enzymes.
  • the present invention also relates to a method of preventing, inhibiting or reducing microbe growth on such surface, by treating the surface with enzymes to an extent that the microbe has a TTC detectable time on said surface after the treatment which increases at least 20%.
  • the present invention also relates to a method of demonstrating the microbe growth inhibition of an enzyme on a surface.
  • Microorganism growth can be a problem, even after the surfaces have been treated via conventional cleaning processes such as laundry. In these regions, the problem of microbe growth on a surface can be especially pronounced after being washed, due to the humidity of the washed surface itself and the prolonged exposure in the humid air during the air-drying process.
  • the growth of the microbes on the surfaces may be visualized as dark spots on the surface, and the microbial growth may further give rise to malodors, which are undesirable and unpleasant.
  • the present invention relates to a novel use of one or more enzymes in preventing, inhibiting or reducing microbe growth on a surface, wherein said microbe have an increase in the TTC detectable time of at least 20%on said surface after use of said enzymes.
  • Another aspect of the invention relates to a method of of preventing, inhibiting or reducing microbe growth on surface, by treating the surface with enzymes for a pre-determined period to the extent that the TTC detectable time of microbes on said surface after the treatment of enzyme increases at least 20%as compared to those on a surface not treated with said enzymes.
  • Another aspect of the invention relates to a method of demonstrating the microbe growth inhibition or deep cleansing benefit of an enzyme or enzyme combination on a surface, comprising the steps of:
  • washing A with a detergent composition comprising no enzyme, and washing A’with said detergent composition comprising one or more enzymes,
  • Figure 1 relates to PrestoBlue fluorescene unit detection of Candida Parapsilosis on two differently treated swatches.
  • SEQ ID NO: 1 is the amino acid sequence of a protease
  • SEQ ID NO: 2 is the amino acid sequence of a protease
  • SEQ ID NO: 3 is the amino acid sequence of a protease
  • SEQ ID NO: 4 is the amino acid sequence of a protease
  • SEQ ID NO: 5 is the amino acid sequence of a protease
  • SEQ ID NO: 6 is the amino acid sequence of a protease
  • SEQ ID NO: 7 is the amino acid sequence of a protease
  • SEQ ID NO: 8 is the amino acid sequence of a protease
  • SEQ ID NO: 9 is the amino acid sequence of an amylase
  • SEQ ID NO: 10 is the amino acid sequence of an amylase
  • SEQ ID NO: 11 is the amino acid sequence of an amylase (SEQ ID NO 2 of WO2000/060060)
  • SEQ ID NO: 12 is the amino acid sequence of an amylase (SEQ ID NO 2 of WO96/023873)
  • SEQ ID NO: 13 is the amino acid sequence of an amylase (SEQ ID NO 3 of WO2008/112459
  • SEQ ID NO: 14 is the amino acid sequence of an subtilase (SEQ ID NO: 1 of WO2004/067737)
  • SEQ ID NO: 15 is the amino acid sequence of a cellulase
  • SEQ ID NO: 16 is the amino acid sequence of a cellulase
  • SEQ ID NO: 17 is the amino acid sequence of a cellulase
  • SEQ ID NO: 18 is the amino acid sequence of a cellulase
  • TTC Detectable Time This term determines the time when the microbes on the surfaces become identifieable in situ, as shown by the color change of triphenyltetrazolium chloride (TTC) .
  • TTC is a reduction–oxidation reaction indicator, which can be used to differentiate metabolically active and inactive tissues/organisms.
  • TTC is white in color, and when it is enzymatically reduced to water-insoluble TPF (1, 3, 5-triphenylformazan) by various dehydrogenases in living organism, the color will turn to red.
  • TTC Assay In situ detection of bacterial
  • This term determines the time when the microbes, particularly the fungi on the surface become identifiable in situ, through microscope observation. This is an additional tool for monitoring the growth of microbes on the surface beyond the above mentioned TTC detectable time assay.
  • the spore forming time assay method can be preferred in the situation where the spore has a color which may mask the TTC color change. Later paragraphs of this text, specifcally the “Fungi Spore Formation Time Assay” section in the Assays part, discloses the details of the procedure.
  • Spore Density Score This term determines the growth of fungi as represented by visible marks on household surfaces caused by the spores and/or mycelium of the fungi which grow on said surface. At a scale of 0 to 7, 0 represents no fungi growth on the surface, and 7 being the surface are overgrown with fungi. For different fungi, the color of the spore can be different, but the same 0-7 scale can apply for different fungi growth and spore density measurement. A group of trained panellists can be trained to understand the scale, and score the spore dentisty of each surfaces according to a preset procedure.
  • Detergent Composition refers to compositions that find use in the removal of undesired compounds from textiles to be cleaned, such as textiles.
  • the detergent composition may be used to e.g. clean textiles for both household cleaning and industrial cleaning.
  • the terms encompass any materials/compounds selected for the particular type of detergent composition desired and the form of the product (e.g., liquid, gel, powder, granulate, paste, or spray compositions) and includes, but is not limited to, detergent compositions (e.g., liquid and/or solid laundry detergents and fine fabric detergents; fabric fresheners; fabric softeners; and textile and laundry pre-spotters/pre-treatment) .
  • the detergent formulation may contain one or more additional enzymes (such as proteases, amylases, lipases, cutinases, cellulases, endoglucanases, xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidases, haloperoxygenases, catalases and mannanases, or any mixture thereof) , ingredients such as surfactants, builders, chelators or chelating agents, bleach system or bleach components, polymers, fabric conditioners, foam boosters, suds suppressors, dyes, perfume, tannish inhibitors, optical brighteners, bactericides, fungicides, soil suspending agents, anti-corrosion agents, enzyme inhibitors or stabilizers, enzyme activators, transferase (s) , hydrolytic enzymes, oxido reductases, bluing agents and fluorescent dyes, antioxidants, and solubilizers.
  • additional enzymes such as proteases, amy
  • Delta remission value ( ⁇ Rem) :
  • the terms “Delta remission” or “Delta remission value” are defined herein as the result of a reflectance or remission measurement at a certain wavelength which typically is 460 nm.
  • the swatch is measured with one swatch of similar colour as background, preferably a swatch from a repetition wash. A swatch representing each swatch type is measured before the wash.
  • the Delta remission is the remission value of the washed swatch minus the remission value of the unwashed swatch.
  • Delta enzyme performance value ( ⁇ Rem enzyme value) :
  • the term “Delta enzyme remission value” is defined herein as the result of a reflectance or remission measurement at 460 nm.
  • the swatch is measured with one swatch of similar colour as background, preferably a swatch from a repetition wash. A swatch representing each swatch type is measured before wash.
  • the Delta enzyme remission is the remission value of the swatch washed in detergent with an enzyme present minus the remission value of a similar swatch washed in a detergent without enzyme present.
  • Dish wash refers to all forms of washing dishes, e.g., by hand or automatic dish wash.
  • Washing dishes includes, but is not limited to, the cleaning of all forms of crockery such as plates, cups, glasses, bowls, all forms of cutlery such as spoons, knives, forks and serving utensils as well as ceramics, plastics such as melamine, metals, china, glass and acrylics.
  • Dish washing composition refers to compostions intended for cleaning dishes, table ware, pots, pans, cutlery and all forms of compositions for cleaning hard surfaces areas in kitchens.
  • the present invention is not restricted to any particular type of dish wash composition or any particular detergent.
  • Enzyme Detergency benefit is defined herein as the advantageous effect an enzyme may add to a detergent compared to the same detergent without the enzyme.
  • Important detergency benefits which can be provided by enzymes are stain removal with no or very little visible soils after washing and/or cleaning, prevention or reduction of redeposition of soils released in the washing process (an effect that also is termed anti-redeposition) , restoring fully or partly the whiteness of textiles which originally were white but after repeated use and wash have obtained a greyish or yellowish appearance (an effect that also is termed whitening) .
  • Textile care benefits which are not directly related to catalytic stain removal or prevention of redeposition of soils, are also important for enzyme detergency benefits.
  • textile care benefits are prevention or reduction of dye transfer from one fabric to another fabric or another part of the same fabric (an effect that is also termed dye transfer inhibition or anti-backstaining) , removal of protruding or broken fibers from a fabric surface to decrease pilling tendencies or remove already existing pills or fuzz (an effect that also is termed anti-pilling) , improvement of the fabric-softness, colour clarification of the fabric and removal of particulate soils which are trapped in the fibers of the fabric or garment.
  • Enzymatic bleaching is a further enzyme detergency benefit where the catalytic activity generally is used to catalyze the formation of bleaching components such as hydrogen peroxide or other peroxides.
  • Hard surface cleaning is defined herein as cleaning of hard surfaces wherein hard surfaces may include floors, tables, walls, roofs etc. as well as surfaces of hard objects such as cars (car wash) and dishes (dish wash) . Dish washing includes but are not limited to cleaning of plates, cups, glasses, bowls, cutlery such as spoons, knives, forks, serving utensils, ceramics, plastics, metals, china, glass and acrylics.
  • Laundering relates to both household laundering and industrial laundering and means a process of treating textiles and/or fabrics with a solution containing a detergent composition .
  • the laundering process can for example be carried out using, e.g., a household or an industrial washing machine or can be carried out by hand.
  • a peroxidase according to the invention is a peroxidase enzyme comprised by the enzyme classification EC 1.11.1.7, as set out by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB) , or any fragment derived therefrom, exhibiting peroxidase activity.
  • IUBMB International Union of Biochemistry and Molecular Biology
  • Protease is defined herein as an enzyme that hydrolyzes peptide bonds. It includes any enzyme belonging to the EC 3.4 enzyme group (including each of the thirteen subclasses thereof) .
  • the EC number refers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego, California, including supplements 1-5 published in Eur. J. Biochem. 1223: 1-5 (1994) ; Eur. J. Biochem. 232: 1-6 (1995) ; Eur. J. Biochem. 237: 1-5 (1996) ; Eur. J. Biochem. 250: 1-6 (1997) ; and Eur. J. Biochem. 264: 610-650 (1999) ; respectively.
  • proteases in the detergent industry such as laundry and dish wash are the serine proteases or serine peptidases which is a subgroup of proteases characterised by having a serine in the active site, which forms a covalent adduct with the substrate.
  • subtilases and the serine proteases are characterized by having two active site amino acid residues apart from the serine, namely a histidine residue and an aspartic acid residue.
  • Subtilase refer to a sub-group of serine protease according to Siezen et al., 1991, Protein Engng. 4: 719-737 and Siezen et al., 1997, Protein Science 6: 501-523.
  • the subtilases may be divided into 6 sub-divisions, i.e., the Subtilisin family, the Thermitase family, the Proteinase K family, the Lantibiotic peptidase family, the Kexin family and the Pyrolysin family.
  • the term “protease activity” means a proteolytic activity (EC 3.4) .
  • Proteases usable in detergents are mainly endopeptidases (EC 3.4.21) .
  • protease activity types There are several protease activity types: The three main activity types are: trypsin-like where there is cleavage of amide substrates following Arg or Lys at P1, chymotrypsin-like where cleavage occurs following one of the hydrophobic amino acids at P1, and elastase-like with cleavage following an Ala at P1.
  • Sequence identity The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity” .
  • sequence identity is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) , preferably version 5.0.0 or later.
  • the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
  • Needle labeled “longest identity” (obtained using the –nobrief option) is used as the percent identity and is calculated as follows: (Identical Residues x 100) / (Length of Alignment –Total Number of Gaps in Alignment) .
  • sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EM-BOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra) , prefer-ably version 5.0.0 or later.
  • the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.
  • the output of Needle labeled “longest identity” (obtained using the –nobrief option) is used as the percent identity and is calculated as follows: (Identical Deoxyribonucleotides x 100) / (Length of Alignment –Total Number of Gaps in Alignment) .
  • Stains in the context of the present invention can be from various sources, including but not limited to starch stains, proteineous stains, amylose stains, lipid-based stains; food stains, beverage stains, environmental stains, work place stains, et al.
  • Textile means any textile material including yarns, yarn intermediates, fibers, non-woven materials, natural materials, synthetic materials, and any other textile material, fabrics made of these materials and products made from fabrics (e.g., garments and other articles) .
  • the textile or fabric may be in the form of knits, wovens, denims, non-wovens, felts, yarns, and towelling.
  • the textile may be cellulose based such as natural cellulosics, including cotton, flax/linen, jute, ramie, sisal or coir or manmade cellulosics (e.g.
  • the textile or fabric may also be non-cellulose based such as natural polyamides including wool, camel, cashmere, mohair, rabbit and silk or synthetic polymers such as nylon, aramid, polyester, acrylic, polypropylene and spandex/elastane, or blends thereof as well as blends of cellulose based and non-cellulose based fibers.
  • non-cellulose based such as natural polyamides including wool, camel, cashmere, mohair, rabbit and silk or synthetic polymers such as nylon, aramid, polyester, acrylic, polypropylene and spandex/elastane, or blends thereof as well as blends of cellulose based and non-cellulose based fibers.
  • blends are blends of cotton and/or rayon/viscose with one or more companion material such as wool, synthetic fiber (e.g.
  • Fabric may be conventional washable laundry, for example stained household laundry.
  • fabric or garment it is intended to include the broader term textiles as well.
  • textile is used interchangeably with fabric and cloth.
  • Variant means a polypeptide having enzyme activity but which comprises an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions compared to the partent or reference enzyme.
  • a substitution means replacement of the amino acid occupying a position with a different amino acid;
  • a deletion means removal of the amino acid occupying a position;
  • an insertion means adding one or more (e.g., several) amino acids, e.g., 1, 2, 3, 4 or 5 amino acids adjacent to and immediately following the amino acid occupying a position.
  • Wash liquor is defined herein as the solution or mixture of water and detergent components optionally including the enzyme.
  • malodour By the term ” malodour it means an odor which is not desired.
  • malodour include compounds with an unpleasant smell, which may be produced by microorganisms. These microorganisms can be from the body of humans or animals, the inner space of the washing machine, or from the rest of the environment.
  • Some examples of such unpleasant smelling compounds are hexanal (grassy smell) , 3-Octanone (musty and moldy) , 2, 3-Butanedione (skink smell) , Benzonitrile (almond-like odor) , Benzene (gasoline-like odor) or Toluene (pungent, benzene-like smell) .
  • the present invention relates to the use of enzymes for preventing, inhibiting or reducing growth of microorganism on surfaces e.g. textiles. Textiles which are air-dried are exposed to various microorgansms which may thrive and colonize on the textiles. The microbial growth and surfaces may be visualized as dark stains and/or a malodors such moldy, damp or unpleasant smells.
  • the present invention relates to use of one or more enzymes in preventing, inhibiting or reducing microbe growth on a surface, wherein after use of said enzymes on said surface said microbe have an increase in its TTC detectable time of at least 20%.
  • the prolonged TTC detectable time shows that the use of enzyme on such surface prevented, inhibited or reduced the growth of the microbes.
  • the TTC detectable time increases at least at least 30%.
  • the TTC detectable time increases at least at least 40%.
  • the TTC detectable time increases at least at least 50%.
  • the TTC detectable time increases at least at least 70%.
  • the TTC detectable time increases at least at least 80%.
  • the TTC detectable time increases at least at least 100%.
  • the effect of the use of enzymes on a surface for prevention of microbial growth could be measured as an increase in spore forming time.
  • the microbe is a fungi, and after use of said enzymes on said surface the fungi spore forming time has an increase of at least 20%compared to the spore forming time on a surface bit treated with enzymes.
  • the prolonged spore forming time after use of the enzyme on such surfaces also shows that the microbe’s growth is prevented, inhibited or reduced.
  • the TTC detectable time increases at least at least 30%.
  • the spore forming time increases at least at least 40%.
  • the spore forming time increases at least at least 50%.
  • the spore forming time increases at least at least 70%.
  • the spore forming time increases at least at least 80%.
  • the spore forming time increases at least at least 100%.
  • microorganism When the microorganism is a fungi the effect of the use of enzymes on a surface for prevention of microbial growth could be measured as a decrease of spore density.
  • One aspect relates to the use of one or more enzymes for prevention of microbial growth on a surface, wherein the microbe is a fungi and wherein decrease of spore density is at least 1, on a density scale from 0 to 7, when compared to the spore density of a surface not treated with enzymes.
  • the decrease in spore density score is at least 2.
  • the decrease in spore density score is at least 3.
  • the decrease in spore density score is at least 4.
  • the decrease in spore density score is at least 5.
  • the decrease in spore density score is at least 6.
  • Residual stains often provide nutrient for microbes to survive and thrive on the surfaces. Without being bound by theory, it is believed that enzymes work in part by removing or reducing the residual stains on the surfaces.
  • One aspect of the present invention relates to the use of one or more enzymes for prevention of microbial growth on a surface, wherein said microbe growth is on the residual stain area of said surface.
  • the present inventions further relates to use of one or more enzymes in inhibiting or reducing malodor caused by microbe on a textile surface.
  • the present inventions further relates to use of one or more enzymes in preventing or reducing microbe attachment onto a textile surface. It is known that certain cellulase provides color care and whiteness improvement benefit to the textile being laundered. But it is not known that cellulase can be used in preventing or reducing microbe. Without being bound by theory, it is believed that the pills and damaged fibers are more prone to be transformed into amorphous cellulase removes the pills formed the fabric surface and damaged fiber, and some cellulase are very efficient at hydrolyzing amorphous cellulose and thereby these regions of the fiber are enzymatically removed. Removal of these pills and damaged fibers may contribute to the prevention or reduction of microbe that can be attached onto it, among other unknown factors.
  • the microbe targeted by the use of the enzymes in the present invention can be those that tend to multiply relatively fast on household surfaces in a hot and humid environment. It can be a fungi or a bacterial.
  • the microbe is a fungi or bacterial selecting from a group consisting of Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Pseudomonas putida, Micrococcus luteus, Staphylococcus epidermidis, Enhydrobacter aerosaccus, Corynebacterium jeikeium, Propionibacterium acnes, Brevundimonas vesicularis, Streptomyces griseus, Streptomyces odorifer, Exophiala phaeomuriformis, Fusarium oxysporum, Alternaria alternate, Aspergillus flavus, Aspergillus fumigatus, Asper
  • the microbe is a fungi, and preferably Aspergillus niger.
  • the microbe is a bacterial, and preferably Escherichia coli or Pseudomonas putida.
  • the enzyme useful in the present invention can be selected from a group consisting of amylase, protease, lipase, mannase, cellulase, pectinase, and combinations thereof.
  • the enzyme is an amylase or protease.
  • the enzyme is not oxidase or peroxidase.
  • the enzyme is an amylase, protese, cellulase or combinations thereof.
  • the present inventions further relates to use of one or more enzymes in preventing or reducing microbe attachment onto a textile surface.
  • the enzyme useful in the present invention may be added to a detergent composition in an amount corresponding to 0.001-200 mg of protein, such as 0.005-100 mg of protein, preferably 0.01-50 mg of protein, more preferably 0.05-20 mg of protein, even more preferably 0.1-10 mg of protein per liter of wash liquor.
  • the enzyme (s) useful in the present invention may be stabilized using conventional stabilizing agents, e.g. a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g. an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in, for example, WO 92/19709 and WO 92/19708.
  • a polyol such as propylene glycol or glycerol
  • a sugar or sugar alcohol lactic acid, boric acid, or a boric acid derivative, e.g. an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid
  • Suitable proteases include those of bacterial, fungal, plant, viral or animal origin e.g. vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included. It may be an alkaline protease, such as a serine protease or a metalloprotease. A serine protease may for example be of the S1 family, such as trypsin, or the S8 family such as subtilisin. A metalloproteases protease may for example be a thermolysin from e.g. family M4 or other metalloprotease such as those from M5, M7 or M8 families.
  • subtilases refers to a sub-group of serine protease according to Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523.
  • Serine proteases are a subgroup of proteases characterized by having a serine in the active site, which forms a covalent adduct with the substrate.
  • the subtilases may be divided into 6 sub-divisions, i.e. the Subtilisin family, the Thermitase family, the Proteinase K family, the Lantibiotic peptidase family, the Kexin family and the Pyrolysin family.
  • subtilases are those derived from Bacillus such as Bacillus lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii described in; US7262042 and WO09/021867, and subtilisin lentus, subtilisin Novo, subtilisin Carlsberg, Bacillus licheniformis, subtilisin BPN’, subtilisin 309, subtilisin 147 and subtilisin 168 described in WO89/06279 and protease PD138 described in (WO93/18140) .
  • proteases may be those described in WO92/175177, WO01/016285, WO02/026024 and WO02/016547.
  • trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO89/06270, WO94/25583 and WO05/040372, and the chymotrypsin proteases derived from Cellumonas described in WO05/052161 and WO05/052146.
  • a further preferred protease is the alkaline protease from Bacillus lentus DSM 5483, as described for example in WO95/23221, and variants thereof which are described in WO92/21760, WO95/23221, EP1921147 and EP1921148.
  • metalloproteases are the neutral metalloprotease as described in WO07/044993 (Genencor Int. ) such as those derived from Bacillus amyloliquefaciens.
  • Suitable commercially available protease enzymes include those sold under the trade names Duralase Tm , Durazym Tm , Ultra, Ultra, Ultra, Ultra, Blaze 100T, Blaze 125T, Blaze 150T, Progress and (Novozymes A/S) , those sold under the tradename Purafect Purafect Excellenz P1000 TM , Excellenz P1250 TM , Preferenz P100 TM , Purafect Preferenz P110 TM , Effectenz P1000 TM , Effectenz P1050 TM , Purafect Effectenz P2000 TM , and (Danisco/DuPont) , Axapem TM (Gist-Brocases N.V. ) , BLAP (sequence shown in Figure 29
  • the protease useful in the present invention is selected from a group consisting of:
  • polypeptides having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at leaset 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%sequence identity to any of SEQ ID NO: 1-8; and combinations thereof.
  • the protease is a peptide having at least 60%sequence identity to SEQ ID NO: 1 or 2, 3, 4, 5, 6, 7, 8 or 14.
  • the protease is a peptide having at least 65%sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.
  • the protease is a peptide having at least 70%sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.
  • the protease is a peptide having at least 75%sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.
  • the protease is a peptide having at least 80%sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.
  • the protease is a peptide having at least 85%sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.
  • the protease is a peptide having at least 90%sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.
  • the protease is a peptide having at least 95%sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.
  • the protease is a peptide having at least 96%sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.
  • the protease is a peptide having at least 97%sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.
  • the protease is a peptide having at least 98%sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.
  • the protease is a peptide having at least 99%sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.
  • the protease is a peptide selected from a group consisting of:
  • a protease comprising a substitution compared to the protease shown in SEQ ID NO 1 or SEQ ID NO 2 in one or more of the following positions: 3, 4, 9, 15, 24, 27, 42, 55, 59, 60, 66, 74, 85, 96, 97, 98, 99, 100, 101, 102, 104, 116, 118, 121, 126, 127, 128, 154, 156, 157, 158, 161, 164, 176, 179, 182, 185, 188, 189, 193, 198, 199, 200, 203, 206, 211, 212, 216, 218, 226, 229, 230, 239, 246, 255, 256, 268 and 269, wherein the positions correspond to the positions of the protease shown in SEQ ID NO 1, or
  • protease variant of a protease parent, wherein the protease variant comprises one or more mutation selected from the group consisting of S3T, V4I, S9R, S9E, A15T, S24G, S24R, K27R, N42R, S55P, G59E, G59D, N60D, N60E, V66A, N74D, N85S, N85R, G96S, G96A, S97G, S97D, S97A, S97SD, S99E, S99D, S99G, S99M, S99N, S99R, S99H, S101A, V102I, V102Y, V102N, S104A, G116V, G116R, H118D, H118N, N120S, S126L, P127Q, S128A, S154D, A156E, G157D, G157P, S158E, Y161A, R164S, Q176E, N179E
  • protease comprising a substitution at one or more positions corresponding to positions 171, 173, 175, 179, or 180 of SEQ ID NO: 14, compared to the protease shown in SEQ ID NO 48, wherein the protease variant has a sequence identity of at least 75%but less than 100%to SEQ ID NO 14,
  • a protease comprising the amino acid sequence shown in SEQ ID NO 1 or 2 or a protease having at least 80%sequence identity to; the polypeptide comprising amino acids 1-269 of SEQ ID NO 1 or the polypeptide comprising amino acids 1-275 of SEQ ID NO 2,
  • protease variants selected from the group:
  • Suitable amylases which can be used together with the enzyme useful in the present invention may be an alpha-amylase or a glucoamylase and may be of bacterial or fungal origin. Chemically modified or protein engineered mutants are included.
  • Amylases can include, for example, alpha-amylases obtained from Bacillus, e.g., a special strain of Bacillus licheniformis, described in more detail in GB 1, 296, 839.
  • Suitable amylases include amylases having SEQ ID NO: 2 in WO 95/10603 or variants having 90%sequence identity to SEQ ID NO: 3 thereof.
  • Preferred variants are described in WO 94/02597, WO 94/18314, WO 97/43424 and SEQ ID NO: 4 of WO 99/019467, such as variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408, and 444.
  • amylases having SEQ ID NO: 6 in WO 02/010355 or variants thereof having 90%sequence identity to SEQ ID NO: 6.
  • Preferred variants of SEQ ID NO: 6 are those having a deletion in positions 181 and 182 and a substitution in position 193.
  • amylases which are suitable are hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006/066594 and residues 36-483 of the B. licheniformis alpha-amylase shown in SEQ ID NO: 4 of WO 2006/066594 or variants having 90%sequence identity thereof.
  • Preferred variants of this hybrid alpha-amylase are those having a substitution, a deletion or an insertion in one of more of the following positions: G48, T49, G107, H156, A181, N190, M197, I201, A209 and Q264.
  • hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006/066594 and residues 36-483 of SEQ ID NO: 4 are those having the substitutions:
  • amylases which are suitable are amylases having SEQ ID NO: 6 in WO 99/019467 or variants thereof having 90%sequence identity to SEQ ID NO: 6.
  • Preferred variants of SEQ ID NO: 6 are those having a substitution, a deletion or an insertion in one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216 and K269.
  • Particularly preferred amylases are those having deletion in positions R181 and G182, or positions H183 and G184.
  • Additional amylases which can be used are those having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 7 of WO 96/023873 or variants thereof having 90%sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 7.
  • Preferred variants of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 7 are those having a substitution, a deletion or an insertion in one or more of the following positions: 140, 181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304 and 476, using SEQ ID 2 of WO 96/023873 for numbering.
  • More preferred variants are those having a deletion in two positions selected from 181, 182, 183 and 184, such as 181 and 182, 182 and 183, or positions 183 and 184.
  • Most preferred amylase variants of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 7 are those having a deletion in positions 183 and 184 and a substitution in one or more of positions 140, 195, 206, 243, 260, 304 and 476.
  • amylases which can be used are amylases having SEQ ID NO: 2 of WO 08/153815, SEQ ID NO: 10 in WO 01/66712 or variants thereof having 90%sequence identity to SEQ ID NO: 2 of WO 08/153815 or 90%sequence identity to SEQ ID NO: 10 in WO 01/66712.
  • Preferred variants of SEQ ID NO: 10 in WO 01/66712 are those having a substitution, a deletion or an insertion in one of more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211 and 264.
  • amylases having SEQ ID NO: 2 of WO 09/061380 or variants having 90%sequence identity to SEQ ID NO: 2 thereof.
  • Preferred variants of SEQ ID NO: 2 are those having a truncation of the C-terminus and/or a substitution, a deletion or an insertion in one of more of the following positions: Q87, Q98, S125, N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475.
  • More preferred variants of SEQ ID NO: 2 are those having the substitution in one of 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 deletion in position R180 and/or S181 or of T182 and/or G183.
  • Most preferred amylase variants of SEQ ID NO: 2 are those having the substitutions:
  • variants are C-terminally truncated and optionally further comprises a substitution at position 243 and/or a deletion at position 180 and/or position 181.
  • amylases having SEQ ID NO: 1 of WO13184577 or variants having 90%sequence identity to SEQ ID NO: 1 thereof.
  • Preferred variants of SEQ ID NO: 1 are those having a substitution, a deletion or an insertion in one of more of the following positions: K176, R178, G179, T180, G181, E187, N192, M199, I203, S241, R458, T459, D460, G476 and G477.
  • More preferred variants of SEQ ID NO: 1 are those having the substitution in one of more of the following positions: K176L, E187P, N192FYH, M199L, I203YF, S241QADN, R458N, T459S, D460T, G476K and G477K and/or deletion in position R178 and/or S179 or of T180 and/or G181.
  • Most preferred amylase variants of SEQ ID NO: 1 are those having the substitutions:
  • variants optionally further comprises a substitution at position 241 and/or a deletion at position 178 and/or position 179.
  • amylases having SEQ ID NO: 1 of WO10104675 or variants having 90%sequence identity to SEQ ID NO: 1 thereof.
  • Preferred variants of SEQ ID NO: 1 are those having a substitution, a deletion or an insertion in one of 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 are those having the substitution in one of more of the following positions: N21D, D97N, V128I K177L, M200L, L204YF, E242QA, G477K and G478K and/or deletion in position R179 and/or S180 or of I181 and/or G182.
  • Most preferred amylase variants of SEQ ID NO: 1 are those having the substitutions:
  • variants optionally further comprises a substitution at position 200 and/or a deletion at position 180 and/or position 181.
  • amylases are the alpha-amylase having SEQ ID NO: 12 in WO01/66712 or a variant having at least 90%sequence identity to SEQ ID NO: 12.
  • Preferred amylase variants are those having a substitution, a deletion or an insertion in one of more of the following positions of SEQ ID NO: 12 in WO01/66712: 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.
  • Particular preferred amylases include variants having a deletion of D183 and G184 and having the substitutions R118K, N195F, R320K and R458K, and a variant additionally having substitutions in one or more position selected from the group: M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345 and A339, most preferred a variant that additionally has substitutions in all these positions.
  • amylase variants such as those described in WO2011/098531, WO2013/001078 and WO2013/001087.
  • amylases are Duramyl TM , Termamyl TM , Fungamyl TM , Stainzyme TM , Stainzyme Plus TM , Natalase TM , Liquozyme X, Amplify Achieve and BAN TM (from Novozymes A/S) , and Rapidase TM , Purastar TM /Effectenz TM , Powerase, Preferenz S1000, Preferenz S100 and Preferenz S110 (from Genencor International Inc. /DuPont) .
  • amylase is selected from the group consisting of:
  • variants comprising one or more substitutions in the following positions: 9, 26, 30, 33, 82, 37, 106, 118, 128, 133, 149, 150, 160, 178, 182, 186, 193, 195, 202, 203, 214, 231, 256, 257, 258, 269, 270, 272, 283, 295, 296, 298, 299, 303, 304, 305, 311, 314, 315, 318, 319, 320, 323, 339, 345, 361, 378, 383, 419, 421, 437, 441, 444, 445, 446, 447, 450, 458, 461, 471, 482, 484, wherein the positions corresponds to positions of SEQ ID NO 11;
  • variants exhibiting at least 95 percent identity with SEQ ID NO 13, comprising mutations in one or more of the following positions M202, M208, S255, R172 and/or M261,
  • v a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at leaset 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%sequence identity to SEQ ID NO: 9 or 10,
  • the protease is a peptide having at least 60%sequence identity to SEQ ID NO: 9 or 10.
  • the protease is a peptide having at least 65%sequence identity to SEQ ID NO: 9 or 10.
  • the protease is a peptide having at least 70%sequence identity to SEQ ID NO: 9 or 10.
  • the protease is a peptide having at least 75%sequence identity to SEQ ID NO: 9 or 10.
  • the protease is a peptide having at least 80%sequence identity to SEQ ID NO: 9 or 10.
  • the protease is a peptide having at least 85%sequence identity to SEQ ID NO: 9 or 10.
  • the protease is a peptide having at least 90%sequence identity to SEQ ID NO: 9 or 10.
  • the protease is a peptide having at least 95%sequence identity to SEQ ID NO: 9 or 10.
  • the protease is a peptide having at least 96%sequence identity to SEQ ID NO: 9 or 10.
  • the protease is a peptide having at least 97%sequence identity to SEQ ID NO: 9 or 10.
  • the protease is a peptide having at least 98%sequence identity to SEQ ID NO: 9 or 10.
  • the protease is a peptide having at least 99%sequence identity to SEQ ID NO: 9 or 10.
  • cellulase denotes an enzyme that hydrolyses cellulose.
  • the cellulase is an endoglucanase.
  • cellulase activity is defined herein as an enzyme catalyzed hydrolysis of 1, 4-beta-D-glucosidic linkages in beta-1, 4-glucan (cellulose) .
  • cellulase activity is determined using AZCL-HE-cellulose (from Megazyme) as the reaction substrate, as shown in Assay IV.
  • Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included.
  • Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757 and WO 89/09259.
  • cellulases are the alkaline or neutral cellulases having color care benefits.
  • Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940.
  • Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471, WO 98/12307 and WO99/001544.
  • cellulases are endo-beta-1, 4-glucanase enzyme having a sequence of at least 97%identity to the amino acid sequence of position 1 to position 773 of SEQ ID NO: 2 of WO 2002/099091 or a family 44 xyloglucanase, which a xyloglucanase enzyme having a sequence of at least 60%identity to positions 40-559 of SEQ ID NO: 2 of WO 2001/062903.
  • Celluzyme TM Commercially available cellulases include Celluzyme TM , and Carezyme TM (Novozymes A/S) , Carezyme Premium TM (Novozymes A/S) , Celluclean TM (Novozymes A/S) , Celluclean Classic TM (Novozymes A/S) , Cellusoft TM (Novozymes A/S) , Whitezyme TM (Novozymes A/S) , Clazinase TM , and Puradax HA TM (Genencor International Inc. ) , and KAC-500 (B) TM (Kao Corporation) , Revitalenz TM 1000, Revitalenz TM 2000, Revitalenz TM 3000 (Dupont) .
  • Carezyme TM Novozymes A/S
  • Carezyme Premium TM Novozymes A/S
  • Celluclean TM Novozymes A/S
  • Celluclean Classic TM Novozymes A
  • the present invention relates to the use of one or more enzymes in preventing or reducing microbe attachment onto a textile surface, wherein the enzyme is a cellulase.
  • the present invention relates to the use of one or more enzymes in 2 in inhibiting or reducing malodor caused by microbe on a textile, wherein the enzyme is a cellulase.
  • the cellulase useful in the present invention comprise a polypeptide having cellulase activity, which comprise the amino acid sequence of SEQ ID NO: 15.
  • the cleaning composition comprise a polypeptide having cellulase activity, which comprises an amino acid sequence at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, even more preferably at least 97%, most preferably at least 98%, or even most preferably at least 99%, identity SEQ ID NO: 15.
  • the amylase useful in the present invention comprise a polypeptide having cellulase activity, which comprise the amino acid sequence of SEQ ID NO: 16.
  • the cleaning composition can comprise a polypeptide having cellulase activity, which comprises an amino acid sequence at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, even more preferably at least 97%, most preferably at least 98%, or even most preferably at least 99%, identity SEQ ID NO: 16.
  • the amylase useful in the present invention comprise a polypeptide having cellulase activity, which comprise the amino acid sequence of SEQ ID NO: 17.
  • the cleaning composition comprise a polypeptide having cellulase activity, which comprises an amino acid sequence at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, even more preferably at least 97%, most preferably at least 98%, or even most preferably at least 99%, identity SEQ ID NO: 17.
  • the amylase useful in the present invention comprise a polypeptide having cellulase activity, which comprise the amino acid sequence of SEQ ID NO: 18.
  • the cleaning composition comprise a polypeptide having cellulase activity, which comprises an amino acid sequence at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, even more preferably at least 97%, most preferably at least 98%, or even most preferably at least 99%, identity SEQ ID NO: 18.
  • the invention is directed to use of one or more enzyme which is comprised in a detergent composition for preventing, inhibiting or reducing microbe growth on a surface, wherein after use of said enzymes said microbe on said surface have an increase in its TTC detectable time of at least 20%, at least 30%, at least 50%, at least 70%, at least 80%, or at least 100%.
  • the detergent compositions comprising an enzyme useful in the present invention can comprise one or more additional cleaning composition components.
  • additional components is within the skill of the artisan and includes conventional ingredients, including the exemplary non-limiting components set forth below.
  • the choice of components may include, for textile care, the consideration of the type of textile to be cleaned, the type and/or degree of soiling, the temperature at which cleaning is to take place, and the formulation of the detergent product.
  • components mentioned below are categorized by general header according to a particular functionality, this is not to be construed as a limitation, as a component may comprise additional functionalities as will be appreciated by the skilled artisan.
  • the invention is directed to use of one or more enzyme which is comprised in an ADW (Automatic Dish Wash) compositions in combination with one or more additional ADW composition components.
  • ADW Automatic Dish Wash
  • additional components is within the skill of the artisan and includes conventional ingredients, including the exemplary non-limiting components set forth below.
  • the invention is directed to detergent composition comprising essentially no biocide. It is believed that by replacing biocides at least partially with enzymes thereby reducing the need of biocide, less harm brought by the biocide to the environment can be at least partially prevented.
  • the invention relates to use of one or more enzyme in preventing, inhibiting or reducing microbe growth on surfaces, wherein said microbe growth is on the residual stain area of said surface.
  • the residual stains can serve as the nutrient, such as the carbon source and the nitrogen source, on which the microbe’s growth is relying.
  • the present invention relates to use of one or more enzyme in preventing, inhibiting or reducing microbe growth on surfaces, where the surface can be hard surface or soft surface, such as fabric/textile surface.
  • the surface is a textile made of nature fiber, synthetic fiber, or a mixture thereof.
  • the surface is made of cotton, polyester or blends thereof.
  • the surface is a porous surface, such as a sponge.
  • the surface is a hard surface, such as kitchen counter, chopping board and so on.
  • the detergent composition may comprise one or more surfactants, which may be anionic and/or cationic and/or non-ionic and/or semi-polar and/or zwitterionic, or a mixture thereof.
  • the detergent composition includes a mixture of one or more nonionic surfactants and one or more anionic surfactants.
  • the surfactant (s) is typically present at a level of from about 0.1%to 60%by weight, such as about 1%to about 40%, or about 3%to about 20%, or about 3%to about 10%.
  • the surfactant (s) is chosen based on the desired cleaning application, and may include any conventional surfactant (s) known in the art.
  • the detergent When included therein the detergent will usually contain from about 1%to about 40%by weight of an anionic surfactant, such as from about 5%to about 30%, including from about 5%to about 15%, or from about 15%to about 20%, or from about 20%to about 25%of an anionic surfactant.
  • an anionic surfactant such as from about 5%to about 30%, including from about 5%to about 15%, or from about 15%to about 20%, or from about 20%to about 25%of an anionic surfactant.
  • Non-limiting examples of anionic surfactants include sulfates and sulfonates, in particular, linear alkylbenzenesulfonates (LAS) , isomers of LAS, branched alkylbenzenesulfonates (BABS) , phenylalkanesulfonates, alpha-olefinsulfonates (AOS) , olefin sulfonates, alkene sulfonates, alkane-2, 3-diylbis (sulfates) , hydroxyalkanesulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS) , fatty alcohol sulfates (FAS) , primary alcohol sulfates (PAS) , alcohol ethersulfates (AES or AEOS or FES, also known as alcohol ethoxysulfates or fatty alcohol ether sul
  • the detergent When included therein the detergent will usually contain from about from about 1%to about 40%by weigh of a cationic surfactant, for example from about 0.5%to about 30%, in particular from about 1%to about 20%, from about 3%to about 10%, such as from about 3%to about 5%, from about 8%to about 12%or from about 10%to about 12%.
  • a cationic surfactant for example from about 0.5%to about 30%, in particular from about 1%to about 20%, from about 3%to about 10%, such as from about 3%to about 5%, from about 8%to about 12%or from about 10%to about 12%.
  • Non-limiting examples of cationic surfactants include alkyldimethylethanolamine quat (ADMEAQ) , cetyltrimethylammonium bromide (CTAB) , dimethyldistearylammonium chloride (DSDMAC) , and alkylbenzyldimethylammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, ester quats, and combinations thereof.
  • ADMEAQ alkyldimethylethanolamine quat
  • CTAB cetyltrimethylammonium bromide
  • DMDMAC dimethyldistearylammonium chloride
  • AQA alkoxylated quaternary ammonium
  • the detergent When included therein the detergent will usually contain from about 0.2%to about 40%by weight of a nonionic surfactant, for example from about 0.5%to about 30%, in particular from about 1%to about 20%, from about 3%to about 10%, such as from about 3%to about 5%, from about 8%to about 12%, or from about 10%to about 12%.
  • a nonionic surfactant for example from about 0.5%to about 30%, in particular from about 1%to about 20%, from about 3%to about 10%, such as from about 3%to about 5%, from about 8%to about 12%, or from about 10%to about 12%.
  • Non-limiting examples of nonionic surfactants include alcohol ethoxylates (AE or AEO) , alcohol propoxylates, propoxylated fatty alcohols (PFA) , alkoxylated fatty acid alkyl esters, such as ethoxylated and/or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates (APE) , nonylphenol ethoxylates (NPE) , alkylpolyglycosides (APG) , alkoxylated amines, fatty acid monoethanolamides (FAM) , fatty acid diethanolamides (FADA) , ethoxylated fatty acid monoethanolamides (EFAM) , propoxylated fatty acid monoethanolamides (PFAM) , polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamides, GA, or fatty acid glucamides, FAGA)
  • the detergent When included therein the detergent will usually contain from about 0.5%to about 50%, preferfably from about 1%to about 25%, by weight of a semipolar surfactant.
  • semipolar surfactants include amine oxides (AO) such as alkyldimethylamineoxide, N- (coco alkyl) -N, N-dimethylamine oxide and N- (tallow-alkyl) -N, N-bis (2-hydroxyethyl) amine oxide, , and combinations thereof.
  • AO amine oxides
  • the detergent When included therein the detergent will usually contain from about 0.5%to about 50%, preferfably from about 1%to about 25%, by weight of a zwitterionic surfactant.
  • zwitterionic surfactants include betaines such as alkyldimethylbetaines, sulfobetaines, and combinations thereof.
  • the detergent may contain 0-10%by weight, for example 0-5%by weight, such as about 0.5 to about 5%, or about 3%to about 5%, of a hydrotrope.
  • Any hydrotrope known in the art for use in detergents may be utilized.
  • Non-limiting examples of hydrotropes include sodium benzenesulfonate, sodium p-toluene sulfonate (STS) , sodium xylene sulfonate (SXS) , sodium cumene sulfonate (SCS) , sodium cymene sulfonate, amine oxides, alcohols and polyglycolethers, sodium hydroxynaphthoate, sodium hydroxynaphthalene sulfonate, sodium ethylhexyl sulfate, and combinations thereof.
  • the detergent composition may contain about 0-65%by weight, such as about 5%to about 50% of a detergent builder or co-builder, or a mixture thereof.
  • the level of builder is typically 40-65%, particularly 50-65%.
  • the builder and/or co-builder may particularly be a chelating agent that forms water-soluble complexes with Ca and Mg. Any builder and/or co-builder known in the art for use in laundry/ADW/hard surface cleaning detergents may be utilized.
  • Non-limiting examples of builders include zeolites, diphosphates (pyrophosphates) , triphosphates 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) , ethanolamines such as 2-aminoethan-1-ol (MEA) , diethanolamine (DEA, also known as 2, 2’-iminodiethan-1-ol) , triethanolamine (TEA, also known as 2, 2’, 2”-nitrilotriethan-1-ol) , and (carboxymethyl) inulin (CMI) , and combinations thereof.
  • zeolites such as 2-aminoethan-1-ol (MEA) , diethanolamine (DEA, also known as 2, 2’-iminodiethan-1-ol) , triethanolamine (TEA, also known as 2, 2’, 2”-nitrilot
  • the detergent composition may also contain 0-50%by weight, such as about 5%to about 30%, of a detergent co-builder.
  • the detergent composition may include include a co-builder alone, or in combination with a builder, for example a zeolite builder.
  • co-builders include homopolymers of polyacrylates or copolymers thereof, such as poly (acrylic acid) (PAA) or copoly (acrylic acid/maleic acid) (PAA/PMA) .
  • Further non-limiting examples include citrate, chelators such as aminocarboxylates, aminopolycarboxylates and phosphonates, and alkyl-or alkenylsuccinic acid.
  • NTA 2, 2’, 2”-nitrilotriacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • DTPA diethylenetriaminepentaacetic acid
  • IDS iminodisuccinic acid
  • EDDS ethylenediamine-N, N’-disuccinic acid
  • MGDA methylglycinediacetic acid
  • GLDA glutamic acid-N, N-diacetic acid
  • HEDP ethylenediaminetetra (methylenephosphonic acid)
  • DTMPA or DTPMPA diethylenetriaminepentakis (methylenephosphonic acid)
  • EDG 2, 2’, 2”-nitrilotriacetic acid
  • ASMA aspartic acid-N-monoacetic acid
  • ASMA aspartic acid-N-monoacetic acid
  • ASMA aspartic acid-N, N-diacetic acid
  • the detergent composition may contain 0-30%by weight, such as about 1%to about 20%, of a bleaching system.
  • a bleaching system comprising components known in the art for use in cleaning detergents may be utilized. Suitable bleaching system components include sources of hydrogen peroxide; sources of peracids; and bleach catalysts or boosters.
  • Suitable sources of hydrogen peroxide are inorganic persalts, including alkali metal salts such as sodium percarbonate and sodium perborates (usually mono-or tetrahydrate) , and hydrogen peroxide-urea (1/1) .
  • Peracids may be (a) incorporated directly as preformed peracids or (b) formed in situ in the wash liquor from hydrogen peroxide and a bleach activator (perhydrolysis) or (c) formed in situ in the wash liquor from hydrogen peroxide and a perhydrolase and a suitable substrate for the latter, e.g., an ester.
  • Suitable preformed peracids include, but are not limited to, peroxycarboxylic acids such as peroxybenzoic acid and its ring-substituted derivatives, peroxy- ⁇ -naphthoic acid, peroxyphthalic acid, peroxylauric acid, peroxystearic acid, ⁇ -phthalimidoperoxycaproic acid [phthalimidoperoxyhexanoic acid (PAP) ] , and o-carboxybenzamidoperoxycaproic acid; aliphatic and aromatic diperoxydicarboxylic acids such as diperoxydodecanedioic acid, diperoxyazelaic acid, diperoxysebacic acid, diperoxybrassylic acid, 2-decyldiperoxybutanedioic acid, and diperoxyphthalic, -isophthalic and -terephthalic acids; perimidic acids; peroxymonosulfuric acid; peroxydisulfuric acid;
  • Suitable bleach activators include those belonging to the class of esters, amides, imides, nitriles or anhydrides and, where applicable, salts thereof. Suitable examples are tetraacetylethylenediamine (TAED) , sodium 4- [ (3, 5, 5-trimethylhexanoyl) oxy] benzene-1-sulfonate (ISONOBS) , sodium 4- (dodecanoyloxy) benzene-1-sulfonate (LOBS) , sodium 4- (decanoyloxy) benzene-1-sulfonate, 4- (decanoyloxy) benzoic acid (DOBA) , sodium 4- (nonanoyloxy) benzene-1-sulfonate (NOBS) , and/or those disclosed in WO98/17767.
  • TAED tetraacetylethylenediamine
  • ISONOBS sodium 4- [ (3, 5, 5-trimethylhexanoyl) oxy]
  • ATC acetyl triethyl citrate
  • ATC or a short chain triglyceride like triacetin has the advantage that they are environmentally friendly.
  • acetyl triethyl citrate and triacetin have good hydrolytically stability in the product upon storage and are efficient bleach activators.
  • ATC is multifunctional, as the citrate released in the perhydrolysis reaction may function as a builder.
  • the bleaching system may also include a bleach catalyst or booster.
  • bleach catalysts that may be used in the compositions of the present invention include manganese oxalate, manganese acetate, manganese-collagen, cobalt-amine catalysts and manganese triazacyclononane (MnTACN) catalysts; particularly preferred are complexes of manganese with 1, 4, 7-trimethyl-1, 4, 7-triazacyclononane (Me3-TACN) or 1, 2, 4, 7-tetramethyl-1, 4, 7-triazacyclononane (Me4-TACN) , in particular Me3-TACN, such as the dinuclear manganese complex [ (Me3-TACN) Mn (O) 3Mn (Me3-TACN) ] (PF6) 2, and [2, 2', 2”-nitrilotris (ethane-1, 2-diylazanylylidene- ⁇ N-methanylylidene) triphenolato- ⁇
  • an organic bleach catalyst or bleach booster may be used having one of the following formulae:
  • each R1 is independently a branched alkyl group containing from 9 to 24 carbons or linear alkyl group containing from 11 to 24 carbons, preferably each R1 is independently a branched alkyl group containing from 9 to 18 carbons or linear alkyl group containing from 11 to 18 carbons, more preferably each R1 is independently selected from the group consisting of 2-propylheptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, dodecyl, tetradecyl, hexadecyl, octadecyl, isononyl, isodecyl, isotridecyl and isopentadecyl.
  • Suitable bleaching systems are described, e.g. in WO2007/087258, WO2007/087244, WO2007/087259, EP1867708 (Vitamin K) and WO2007/087242.
  • Suitable photobleaches may for example be sulfonated zinc or aluminium phthalocyanines.
  • the detergent may contain 0-10%by weight, such as 0.5-5%, 2-5%, 0.5-2%or 0.2-1%of a polymer. Any polymer known in the art for use in detergents may be utilized.
  • the polymer may function as a co-builder as mentioned above, or may provide antiredeposition, fiber protection, soil release, dye transfer inhibition, grease cleaning and/or anti-foaming properties. Some polymers may have more than one of the above-mentioned properties and/or more than one of the below-mentioned motifs.
  • Exemplary polymers include (carboxymethyl) cellulose (CMC) , poly (vinyl alcohol) (PVA) , poly (vinylpyrrolidone) (PVP) , poly (ethyleneglycol) or poly (ethylene oxide) (PEG) , ethoxylated poly (ethyleneimine) , carboxymethyl inulin (CMI) , and polycarboxylates such as PAA, PAA/PMA, poly-aspartic acid, and lauryl methacrylate/acrylic acid copolymers , hydrophobically modified CMC (HM-CMC) and silicones, copolymers of terephthalic acid and oligomeric glycols, copolymers of poly (ethylene terephthalate) and poly (oxyethene terephthalate) (PET-POET) , PVP, poly (vinylimidazole) (PVI) , poly (vinylpyridine-N-oxide) (PVPO or PVPNO) and
  • exemplary polymers include sulfonated polycarboxylates, polyethylene oxide and polypropylene oxide (PEO-PPO) and diquaternium ethoxy sulfate.
  • PEO-PPO polypropylene oxide
  • diquaternium ethoxy sulfate diquaternium ethoxy sulfate.
  • Other exemplary polymers are disclosed in, e.g., WO 2006/130575. Salts of the above-mentioned polymers are also contemplated.
  • the detergent compositions may also include fabric hueing agents such as dyes or pigments, which when formulated in detergent compositions can deposit onto a fabric when said fabric is contacted with a wash liquor comprising said detergent compositions and thus altering the tint of said fabric through absorption/reflection of visible light.
  • fabric hueing agents alter the tint of a surface as they absorb at least a portion of the visible light spectrum.
  • Suitable fabric hueing agents include dyes and dye-clay conjugates, and may also include pigments.
  • Suitable dyes include small molecule dyes and polymeric dyes. Suitable small molecule dyes include small molecule dyes selected from the group consisting of dyes falling into the Colour Index (C. I.
  • the detergent composition preferably comprises from about 0.00003 wt%to about 0.2 wt%, from about 0.00008 wt%to about 0.05 wt%, or even from about 0.0001 wt%to about 0.04 wt%fabric hueing agent.
  • the composition may comprise from 0.0001 wt%to 0.2 wt%fabric hueing agent, this may be especially preferred when the composition is in the form of a unit dose pouch. Suitable hueing agents are also disclosed in, e.g. WO 2007/087257 and WO2007/087243.
  • the detergent additive as well as the detergent composition may comprise one or more additional enzymes such as a protease, lipase, cutinase, an amylase, carbohydrase, cellulase, pectinase, mannanase, arabinase, galactanase, xylanase, oxidase, e.g., a laccase, and/or peroxidase.
  • additional enzymes such as a protease, lipase, cutinase, an amylase, carbohydrase, cellulase, pectinase, mannanase, arabinase, galactanase, xylanase, oxidase, e.g., a laccase, and/or peroxidase.
  • the properties of the selected enzyme (s) should be compatible with the selected detergent, (i.e., pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc. ) , and the enzyme (s) should be present in effective amounts.
  • Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757 and WO 89/09259.
  • cellulases are the alkaline or neutral cellulases having colour care benefits.
  • Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940.
  • Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471, WO 98/12307 and WO99/001544.
  • cellulases are endo-beta-1, 4-glucanase enzyme having a sequence of at least 97%identity to the amino acid sequence of position 1 to position 773 of SEQ ID NO: 2 of WO 2002/099091 or a family 44 xyloglucanase, which a xyloglucanase enzyme having a sequence of at least 60%identity to positions 40-559 of SEQ ID NO: 2 of WO 2001/062903.
  • Celluzyme TM Commercially available cellulases include Celluzyme TM , and Carezyme TM (Novozymes A/S) Carezyme Premium TM (Novozymes A/S) , Celluclean TM (Novozymes A/S) , Celluclean Classic TM (Novozymes A/S) , Cellusoft TM (Novozymes A/S) , Whitezyme TM (Novozymes A/S) , Clazinase TM , and Puradax HA TM (Genencor International Inc. ) , and KAC-500 (B) TM (Kao Corporation) .
  • Carezyme TM Novozymes A/S
  • Carezyme Premium TM Novozymes A/S
  • Celluclean TM Novozymes A/S
  • Celluclean Classic TM Novozymes A/S
  • Cellusoft TM Novozymes A/S
  • Whitezyme TM Novozymes A/S
  • Suitable cellulases include complete cellulases or mono-component endoglucanases of bacterial or fungal origin. Chemically or genetically modified mutants are included.
  • the cellulase may for example be a mono-component or a mixture of mono-component endo-1, 4-beta-glucanase often just termed endoglucanases.
  • Suitable cellulases include a fungal cellulase from Humicola insolens (US 4,435,307) or from Trichoderma, e.g. T. reesei or T. viride. Examples of cellulases are described in EP 0 495 257. Other suitable cellulases are from Thielavia e.g.
  • Thielavia terrestris as described in WO 96/29397 or Fusarium oxysporum as described in WO 91/17244 or from Bacillus as described in, WO 02/099091 and JP 2000210081.
  • Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471, WO 98/12307
  • Commercially available cellulases include and (Novozymes A/S) Puradax HA, and Puradax EG (available from Genencor) .
  • Suitable mannanases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included.
  • the mannanase may be an alkaline mannanase of Family 5 or 26. It may be a wild-type from Bacillus or Humicola, particularly B. agaradhaerens, B. licheniformis, B. halodurans, B. clausii, or H. insolens.
  • Suitable mannanases are described in WO 1999/064619. A commercially available mannanase is Mannaway (Novozymes A/S) .
  • Suitable lipases and cutinases include those of bacterial or fungal origin. Chemically modified or protein engineered mutant enzymes are included. Examples include lipase from Thermomyces, e.g. from T. lanuginosus (previously named Humicola lanuginosa) as described in EP258068 and EP305216, cutinase from Humicola, e.g. H. insolens (WO96/13580) , lipase from strains of Pseudomonas (some of these now renamed to Burkholderia) , e.g. P. alcaligenes or P. pseudoalcaligenes (EP218272) , P.
  • Thermomyces e.g. from T. lanuginosus (previously named Humicola lanuginosa) as described in EP258068 and EP305216
  • cutinase from Humicola e.g. H. insolens (WO96/135
  • lipase variants such as those described in EP407225, WO92/05249, WO94/01541, WO94/25578, WO95/14783, WO95/30744, WO95/35381, WO95/22615, WO96/00292, WO97/04079, WO97/07202, WO00/34450, WO00/60063, WO01/92502, WO07/87508 and WO09/109500.
  • Preferred commercial lipase products include include Lipolase TM , Lipex TM ; Lipolex TM and Lipoclean TM (Novozymes A/S) , Lumafast (originally from Genencor) and Lipomax (originally from Gist-Brocades) .
  • lipases sometimes referred to as acyltransferases or perhydrolases, e.g. acyltransferases with homology to Candida antarctica lipase A (WO10/111143) , acyltransferase from Mycobacterium smegmatis (WO05/56782) , perhydrolases from the CE 7 family (WO09/67279) , and variants of the M. smegmatis perhydrolase in particular the S54V variant used in the commercial product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd (WO10/100028) .
  • Suitable peroxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinopsis, e.g., from C. cinerea (EP 179, 486) , and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257.
  • a peroxidase according to the invention also include a haloperoxidase enzyme, such as chloroperoxidase, bromoperoxidase and compounds exhibiting chloroperoxidase or bromoperoxidase activity.
  • haloperoxidases are classified according to their specificity for halide ions. Chloroperoxidases (E.C. 1.11.1.10) catalyze formation of hypochlorite from chloride ions.
  • the haloperoxidase of the invention is a chloroperoxidase.
  • the haloperoxidase is a vanadium haloperoxidase, i.e., a vanadate-containing haloperoxidase.
  • the vanadate-containing haloperoxidase is combined with a source of chloride ion.
  • Haloperoxidases have been isolated from many different fungi, in particular from the fungus group dematiaceous hyphomycetes, such as Caldariomyces, e.g., C. fumago, Alternaria, Curvularia, e.g., C. verruculosa and C. inaequalis, Drechslera, Ulocladium and Botrytis.
  • Caldariomyces e.g., C. fumago
  • Alternaria Curvularia
  • Curvularia e.g., C. verruculosa and C. inaequalis
  • Drechslera Ulocladium and Botrytis.
  • Haloperoxidases have also been isolated from bacteria such as Pseudomonas, e.g., P. pyrrocinia and Streptomyces, e.g., S. aureofaciens.
  • the haloperoxidase is derivable from Curvularia sp., in particular Curvularia verruculosa or Curvularia inaequalis, such as C. inaequalis CBS 102.42 as described in WO 95/27046; or C. verruculosa CBS 147.63 or C. verruculosa CBS 444.70 as described in WO 97/04102; or from Drechslera hartlebii as described in WO 01/79459, Dendryphiella salina as described in WO 01/79458, Phaeotrichoconis crotalarie as described in WO 01/79461, or Geniculosporium sp. as described in WO 01/79460.
  • Curvularia verruculosa or Curvularia inaequalis such as C. inaequalis CBS 102.42 as described in WO 95/27046; or C. verruculosa CBS 147.63 or C. verruculosa
  • An oxidase according to the invention include, in particular, any laccase enzyme comprised by the enzyme classification EC 1.10.3.2, or any fragment derived therefrom exhibiting laccase activity, or a compound exhibiting a similar activity, such as a catechol oxidase (EC 1.10.3.1) , an o-aminophenol oxidase (EC 1.10.3.4) , or a bilirubin oxidase (EC 1.3.3.5) .
  • Preferred laccase enzymes are enzymes of microbial origin.
  • the enzymes may be derived from plants, bacteria or fungi (including filamentous fungi and yeasts) .
  • Suitable examples from fungi include a laccase derivable from a strain of Aspergillus, Neurospora, e.g., N. crassa, Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus, Trametes, e.g., T. villosa and T. versicolor, Rhizoctonia, e.g., R. solani, Coprinopsis, e.g., C. cinerea, C. comatus, C. friesii, and C. plicatilis, Psathyrella, e.g., P. condelleana, Panaeolus, e.g., P.
  • papilionaceus Myceliophthora, e.g., M. thermophila, Schytalidium, e.g., S. thermophilum, Polyporus, e.g., P. pinsitus, Phlebia, e.g., P. radiata (WO 92/01046) , or Coriolus, e.g., C. hirsutus (JP 2238885) .
  • Suitable examples from bacteria include a laccase derivable from a strain of Bacillus.
  • a laccase derived from Coprinopsis or Myceliophthora is preferred; in particular a laccase derived from Coprinopsis cinerea, as disclosed in WO 97/08325; or from Myceliophthora thermophila, as disclosed in WO 95/33836.
  • the detergent enzyme (s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes.
  • a detergent additive of the invention i.e., a separate additive or a combined additive, can be formulated, for example, as a granulate, liquid, slurry, etc.
  • Preferred detergent additive formulations are granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries.
  • Non-dusting granulates may be produced, e.g. as disclosed in US 4,106,991 and 4,661,452 and may optionally be coated by methods known in the art.
  • waxy coating materials are polyethyleneglycol (PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono-and di-and triglycerides of fatty acids.
  • PEG polyethyleneglycol
  • film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591.
  • Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods.
  • Protected enzymes may be prepared according to the method disclosed in EP 238, 216.
  • the detergent additive as well as the detergent composition may also comprise one or more microorganisms, such as one or more fungi, yeast, or bacteria.
  • the one or more microorganisms are dehydrated (for example by lyophilization) bacteria or yeast, such as a strain of Lactobacillus.
  • the microrganisms are one or more microbial spores (as opposed to vegetative cells) , such as bacterial spores; or fungal spores, conidia, hypha.
  • the one or more spores are Bacillus endospores; even more preferably the one or more spores are endospores of Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, or Bacillus megaterium.
  • microrganisms may be included in the detergent composition or additive in the same way as enzymes (see above) .
  • any detergent components known in the art for use in laundry/ADW/hard surface cleaning detergents may also be utilized.
  • Other optional detergent components include anti-corrosion agents, anti-shrink agents, anti-soil redeposition agents, anti-wrinkling agents, bactericides, binders, corrosion inhibitors, disintegrants/disintegration agents, dyes, enzyme stabilizers (including boric acid, borates, CMC, and/or polyols such as propylene glycol) , fabric conditioners including clays, fillers/processing aids, fluorescent whitening agents/optical brighteners, foam boosters, foam (suds) regulators, perfumes, soil-suspending agents, softeners, suds suppressors, tarnish inhibitors, and wicking agents, either alone or in combination.
  • Any ingredient known in the art for use in laundry/ADW/hard surface cleaning detergents may be utilized. The choice of such ingredients is well within the skill of the artisan.
  • the detergent compositions useful in the present invention can also contain dispersants.
  • powdered detergents may comprise dispersants.
  • Suitable water-soluble organic materials include the homo-or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms.
  • Suitable dispersants are for example described in Powdered Detergents, Surfactant science series volume 71, Marcel Dekker, Inc.
  • the detergent compositions useful in the present invention may also include one or more dye transfer inhibiting agents.
  • Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof.
  • the dye transfer inhibiting agents may be present at levels from about 0.0001 %to about 10%, from about 0.01%to about 5%or even from about 0.1%to about 3%by weight of the composition.
  • the detergent compositions useful in the present invention will preferably also contain additional components that may tint articles being cleaned, such as fluorescent whitening agent or optical brighteners. Where present the brightener is preferably at a level of about 0.01%to about 0.5%.
  • Any fluorescent whitening agent suitable for use in a laundry detergent composition may be used in the composition of the present invention.
  • the most commonly used fluorescent whitening agents are those belonging to the classes of diaminostilbene-sulfonic acid derivatives, diarylpyrazoline derivatives and bisphenyl-distyryl derivatives.
  • diaminostilbene-sulfonic acid derivative type of fluorescent whitening agents include the sodium salts of: 4, 4'-bis- (2-diethanolamino-4-anilino-s-triazin-6-ylamino) stilbene-2, 2'-disulfonate, 4, 4'-bis- (2, 4-dianilino-s-triazin-6-ylamino) stilbene-2.2'-disulfonate, 4, 4'-bis- (2-anilino-4- (N-methyl-N-2-hydroxy-ethylamino) -s-triazin-6-ylamino) stilbene-2, 2'-disulfonate, 4, 4'-bis- (4-phenyl-1, 2, 3-triazol-2-yl) stilbene-2, 2'-disulfonate and sodium 5- (2H-naphtho [1, 2-d] [1, 2, 3] triazol-2-yl) -2- [ (E) -2-phenylvinyl)
  • Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBS available from Ciba-Geigy AG, Basel, Switzerland.
  • Tinopal DMS is the disodium salt of 4, 4'-bis- (2-morpholino-4-anilino-s-triazin-6-ylamino) stilbene-2, 2'-disulfonate.
  • Tinopal CBS is the disodium salt of 2, 2'-bis- (phenyl-styryl) -disulfonate.
  • fluorescent whitening agents is the commercially available Parawhite KX, supplied by Paramount Minerals and Chemicals, Mumbai, India.
  • Other fluorescers suitable for use in the invention include the 1-3-diaryl pyrazolines and the 7-alkylaminocoumarins.
  • Suitable fluorescent brightener levels include lower levels of from about 0.01, from 0.05, from about 0.1 or even from about 0.2 wt %to upper levels of 0.5 or even 0.75 wt%.
  • the detergent compositions useful in the present invention may also include one or more soil release polymers which aid the removal of soils from fabrics such as cotton and polyester based fabrics, in particular the removal of hydrophobic soils from polyester based fabrics.
  • the soil release polymers may for example be nonionic or anionic terephthalte based polymers, polyvinyl caprolactam and related copolymers, vinyl graft copolymers, polyester polyamides see for example Chapter 7 in Powdered Detergents, Surfactant science series volume 71, Marcel Dekker, Inc.
  • Another type of soil release polymers are amphiphilic alkoxylated grease cleaning polymers comprising a core structure and a plurality of alkoxylate groups attached to that core structure.
  • the core structure may comprise a polyalkylenimine structure or a polyalkanolamine structure as described in detail in WO 2009/087523 (hereby incorporated by reference) .
  • random graft co-polymers are suitable soil release polymers. Suitable graft co-polymers are described in more detail in WO 2007/138054, WO 2006/108856 and WO 2006/113314 (hereby incorporated by reference) .
  • Other soil release polymers are substituted polysaccharide structures especially substituted cellulosic structures such as modified cellulose deriviatives such as those described in EP 1867808 or WO 2003/040279 (both are hereby incorporated by reference) .
  • Suitable cellulosic polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides and mixtures thereof. Suitable cellulosic polymers include anionically modified cellulose, nonionically modified cellulose, cationically modified cellulose, zwitterionically modified cellulose, and mixtures thereof. Suitable cellulosic polymers include methyl cellulose, carboxy methyl cellulose, ethyl cellulose, hydroxyl ethyl cellulose, hydroxyl propyl methyl cellulose, ester carboxy methyl cellulose, and mixtures thereof.
  • the detergent compositions useful in the present invention may also include one or more anti-redeposition agents such as carboxymethylcellulose (CMC) , polyvinyl alcohol (PVA) , polyvinylpyrrolidone (PVP) , polyoxyethylene and/or polyethyleneglycol (PEG) , homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimines.
  • CMC carboxymethylcellulose
  • PVA polyvinyl alcohol
  • PVP polyvinylpyrrolidone
  • PEG polyethyleneglycol
  • homopolymers of acrylic acid copolymers of acrylic acid and maleic acid
  • ethoxylated polyethyleneimines ethoxylated polyethyleneimines.
  • the cellulose based polymers described under soil release polymers above may also function as anti-redeposition agents.
  • the detergent compositions useful in the present invention may also include one or more rheology modifiers, structurants or thickeners, as distinct from viscosity reducing agents.
  • the rheology modifiers are selected from the group consisting of non-polymeric crystalline, hydroxy-functional materials, polymeric rheology modifiers which impart shear thinning characteristics to the aqueous liquid matrix of a liquid detergent composition.
  • the rheology and viscosity of the detergent can be modified and adjusted by methods known in the art, for example as shown in EP 2169040.
  • adjunct materials include, but are not limited to, anti-shrink agents, anti-wrinkling agents, bactericides, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, hydrotropes, perfumes, pigments, sod suppressors, solvents, and structurants for liquid detergents and/or structure elasticizing agents.
  • the detergent compositions useful in the present invention may be in any convenient form, e.g., a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid.
  • Pouches can be configured as single or multicompartments. It can be of any form, shape and material which is suitable for hold the composition, e.g. without allowing the release of the composition to release of the composition from the pouch prior to water contact.
  • the pouch is made from water soluble film which encloses an inner volume. Said inner volume can be divided into compartments of the pouch.
  • Preferred films are polymeric materials preferably polymers which are formed into a film or sheet.
  • Preferred polymers, copolymers or derivates thereof are selected polyacrylates, and water soluble acrylate copolymers, methyl cellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, malto dextrin, poly methacrylates, most preferably polyvinyl alcohol copolymers and, hydroxypropyl methyl cellulose (HPMC) .
  • the level of polymer in the film for example PVA is at least about 60%.
  • Preferred average molecular weight will typically be about 20,000 to about 150,000.
  • Films can also be of blended compositions comprising hydrolytically degradable and water soluble polymer blends such as polylactide and polyvinyl alcohol (known under the Trade reference M8630 as sold by MonoSol LLC, Indiana, USA) plus plasticisers like glycerol, ethylene glycerol, propylene glycol, sorbitol and mixtures thereof.
  • the pouches can comprise a solid laundry cleaning composition or part components and/or a liquid cleaning composition or part components separated by the water soluble film.
  • the compartment for liquid components can be different in composition than compartments containing solids: US2009/0011970 A1.
  • Detergent ingredients can be separated physically from each other by compartments in water dissolvable pouches or in different layers of tablets. Thereby negative storage interaction between components can be avoided. Different dissolution profiles of each of the compartments can also give rise to delayed dissolution of selected components in the wash solution.
  • a liquid or gel detergent which is not unit dosed, may be aqueous, typically containing at least 20%by weight and up to 95%water, such as up to about 70%water, up to about 65%water, up to about 55%water, up to about 45%water, up to about 35%water.
  • Other types of liquids including without limitation, alkanols, amines, diols, ethers and polyols may be included in an aqueous liquid or gel.
  • An aqueous liquid or gel detergent may contain from 0-30%organic solvent.
  • a liquid or gel detergent may be non-aqueous.
  • the enzyme useful in the present invention for preventing microbe growth on household surfaces may be formulated as a granule for example as a co-granule that combines one or more enzymes. Each enzyme will then be present in more granules securing a more uniform distribution of enzymes in the detergent. This also reduces the physical segregation of different enzymes due to different particle sizes.
  • Methods for producing multi-enzyme co-granulates for the detergent industry are disclosed in the IP. com disclosure IPCOM000200739D.
  • WO 2013/188331 Another example of formulation of enzymes by the use of co-granulates are disclosed in WO 2013/188331, which relates to a detergent composition comprising (a) a multi-enzyme co- granule; (b) less than 10 wt zeolite (anhydrous basis) ; and (c) less than 10 wt phosphate salt (anhydrous basis) , wherein said enzyme co-granule comprises from 10 to 98 wt%moisture sink component and the composition additionally comprises from 20 to 80 wt%detergent moisture sink component.
  • WO 2013/188331 also relates to a method of treating and/or cleaning a surface, preferably a fabric surface comprising the steps of (i) contacting said surface with the detergent composition as claimed and described herein in an aqueous wash liquor, (ii) rinsing and/or drying the surface.
  • the multi-enzyme co-granule may comprise an enzyme of the invention and (a) one or more enzymes selected from the group consisting of first-wash lipases, cleaning cellulases, xyloglucanases, perhydrolases, peroxidases, lipoxygenases, laccases and mixtures thereof; and (b) one or more enzymes selected from the group consisting of hemicellulases, proteases, care cellulases, cellobiose dehydrogenases, xylanases, phospho lipases, esterases, cutinases, pectinases, mannanases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, ligninases, pullulanases, tannases, pentosanases, lichenases glucanases, arabinosidases, hyaluronidase, chondroitinase, amy
  • the present invention in another aspect of the present invention, relates to a method of preventing, inhibiting or reducing microbe growth on surface, by treating the surface with enzymes defined in previous paragraphs and aspects under Enzyme of the Present Invention section to the extent that the microbe TTC detectable time on said surface after the treatment increases at least 20%.
  • the microbe is a fungi
  • the fungi on said surface has an increase of spore forming time of at least 20%after the surface is treated by the enzyme useful in the present invention.
  • the microbe is a fungi
  • the fungi has a decrease of spore density score of at least 1 in a scale of from 0 to 7 on said surface after the surface is treated by the enzyme useful in the present invention.
  • washing A with a detergent composition comprising no enzyme, and washing A’with said detergent composition comprising one or more enzymes,
  • the surface mentioned here can be any household surface. can be hard surface or soft surface, such as fabric/textile surface.
  • the surface is a textile made of nature fiber, synthetic fiber, or a mixture thereof.
  • the surface is made of cotton, polyester or blends thereof.
  • the microbe growth indicator is selected from a group consisting of triphenyltetrazolium chloride (TTC) , INT (2- (4-iodophenyl) -3- (4-nitrophenyl) -5-phenyl-2H-tetrazolium) , MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) , XTT (2, 3-bis- (2-methoxy-4-nitro-5-sulfophenyl) -2H-tetrazolium-5-carboxanilide) , MTS (3- (4, 5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium) , WSTs (Water-soluble Tetrazolium salts) and CCK-8 (Cell Counting Kit-
  • TTC triphenyltetrazolium chloride
  • detergent compositions can incorporate the enzyme suitable for the use and method of the present invention.
  • Powder Detergent Model X
  • composition of Ariel Sensitive White &Color liquid detergent composition
  • Ingredients 5-15%Anionic surfactants, Oxygen-based bleaching agents, ⁇ 5%Non-ionic surfactants, Phosphonates, Polycarboxylates, Zeolites, Optical brighteners, Enzymes, Perfumes, Butylphenyl Methylpropional, Coumarin, Hexyl Cinnamal.
  • microbe is a fungi
  • the fungi has an increase in the spore forming time of at least 20%, and/or an decrease in spore density score of at least 1 in a spore density scale of from 0 to 7, compared to those on a surface not treated with enzymes.
  • paragraph 1 or 2 in inhibiting or reducing malodor caused by microbe on a textile surface.
  • paragraph 1 or 2 in preventing or reducing microbe attachment onto a textile surface.
  • microbe is a fungi or bacteria selected from a group consisting of Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Pseudomonas putida, Micrococcus luteus, Staphylococcus epidermidis, Enhydrobacter aerosaccus, Corynebacterium jeikeium, Propionibacterium acnes, Brevundimonas vesicularis, Streptomyces griseus, Streptomyces odorifer, Exophiala phaeomuriformis, Fusarium oxysporum, Alternaria alternate, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aureobasidium pullulans, Chaetomium globosum, Cladosporium sphaerospermum
  • said one or more enzymes are selected from a group consisting of amylase, protease, lipase, mannase, cellulase, pectinase, and combinations thereof.
  • protease is selected from a group consisting of:
  • a protease comprising a substitution compared to the protease shown in SEQ ID NO 1 or SEQ ID NO 2 in one or more of the following positions: 3, 4, 9, 15, 24, 27, 42, 55, 59, 60, 66, 74, 85, 96, 97, 98, 99, 100, 101, 102, 104, 116, 118, 121, 126, 127, 128, 154, 156, 157, 158, 161, 164, 176, 179, 182, 185, 188, 189, 193, 198, 199, 200, 203, 206, 211, 212, 216, 218, 226, 229, 230, 239, 246, 255, 256, 268 and 269, wherein the positions correspond to the positions of the protease shown in SEQ ID NO 1, or
  • protease variant of a protease parent, wherein the protease variant comprises one or more mutation selected from the group consisting of S3T, V4I, S9R, S9E, A15T, S24G, S24R, K27R, N42R, S55P, G59E, G59D, N60D, N60E, V66A, N74D, N85S, N85R, G96S, G96A, S97G, S97D, S97A, S97SD, S99E, S99D, S99G, S99M, S99N, S99R, S99H, S101A, V102I, V102Y, V102N, S104A, G116V, G116R, H118D, H118N, N120S, S126L, P127Q, S128A, S154D, A156E, G157D, G157P, S158E, Y161A, R164S, Q176E, N179E
  • protease comprising a substitution at one or more positions corresponding to positions 171, 173, 175, 179, or 180 of SEQ ID NO: 14, compared to the protease shown in SEQ ID NO 14, wherein the protease variant has a sequence identity of at least 75%but less than 100%to SEQ ID NO 14,
  • a protease comprising the amino acid sequence shown in SEQ ID NO 1 or 2 or a protease having at least 80%sequence identity to; the polypeptide comprising amino acids 1-269 of SEQ ID NO 1 or the polypeptide comprising amino acids 1-275 of SEQ ID NO 2,
  • protease variants selected from the group:
  • amylase is selected from the group consisting of:
  • variants comprising one or more substitutions in the following positions: 9, 26, 30, 33, 82, 37, 106, 118, 128, 133, 149, 150, 160, 178, 182, 186, 193, 195, 202, 203, 214, 231, 256, 257, 258, 269, 270, 272, 283, 295, 296, 298, 299, 303, 304, 305, 311, 314, 315, 318, 319, 320, 323, 339, 345, 361, 378, 383, 419, 421, 437, 441, 444, 445, 446, 447, 450, 458, 461, 471, 482, 484, wherein the positions corresponds to positions of SEQ ID NO 11;
  • variants exhibiting at least 95 percent identity with SEQ ID NO 13, comprising mutations in one or more of the following positions M202, M208, S255, R172 and/or M261,
  • v a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at leaset 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%sequence identity to SEQ ID NO: 9 or 10, and combinations thereof.
  • said surface is a textile made of nature fiber such as cotton, synthetic fiber such as polyester, or a mixture thereof.
  • a method of preventing, inhibiting or reducing microbe growth on surface comprising the steps of :
  • a method of demonstrating the microbe growth inhibition or deep cleansing benefit of an enzyme or enzyme combination as defined in paragraphs 10-12 on a surface comprising the steps of:
  • washing A with a detergent composition comprising no enzyme, and washing A’with said detergent composition comprising one or more enzymes,
  • microbe growth indicator is triphenyltetrazolium chloride (TTC) .
  • the Tergo-To-Meter is a medium scale model wash system that can be applied to test 16 different wash conditions simultaneously.
  • a TOM is basically a large temperature controlled water bath with up to 16 open metal beakers submerged into it. Each beaker constitutes one small top loader style washing machine and during an experiment, each of them will contain a solution of a specific detergent/enzyme system and the soiled and unsoiled fabrics its performance is tested on. Mechanical stress is achieved by a rotating stirring arm, which stirs the liquid within each beaker. Because the TOM beakers have no lid, it is possible to withdraw samples during a TOM experiment and assay for information on-line during wash.
  • the TOM model wash system is mainly used in medium scale testing of detergents and enzymes at US or LA/AP wash conditions.
  • factors such as the ballast to soil ratio and the fabric to wash liquor ratio can be varied. Therefore, the TOM provides the link between small scale experiments, such as AMSA and mini-wash, and the more time consuming full scale experiments in top loader washing machines.
  • the water bath with 16 steel beakers and 1 rotating arm per beaker with capacity of 500 to 1200 mL of detergent solution. Temperature ranges from 3.5 to 60°C.
  • the water bath has to be filled up with deionised water. Rotational speed can be set up to 40 to 200 rpm/min.
  • wash solution with desired amount of detergent and water hardness are prepared in a bucket.
  • the detergent is allowed to dissolve during magnet stirring for 10 min, and measure pH of detergent solution after 10 min stirring. Wash solution shall be used within 30 to 60 min after preparation.
  • 1000 ml wash solution is added into a TOM beaker.
  • the wash solution is agitated at 120 rpm and let rotate until the temperature is correct.
  • the swatches are sprinkled into the beaker and the ballast load and then optionally one or more enzymes are added to the beaker.
  • Time measurement starts when the swatches and ballast are added to the beaker.
  • the swatches are washed for 20 minutes after which agitation is terminated.
  • the wash load is subsequently transferred from the TOM beaker to a container and rinse with cold tap water.
  • the soiled swatches are separated from the ballast load.
  • the soil swatches are transferred to a 5 L beaker with cold tap water under running water for 5 minutes.
  • the ballast load is kept separately for the coming inactivation.
  • the water is gently pressed out of the swatches by hand and placed on a tray covered with a paper. Another paper is placed on top of the swatches.
  • the swatches are allowed to dry overnight before subjecting the swatches to analysis, such as measuring the color intensity using a Color Eye as described herein.
  • FSW i.e. to test product performance in washing machines under scientifically designed conditions
  • the FSW system can be used at US or LA, AP and EU wash conditions according to different regional machines. Test swatches and ballast are added to each wash together with detergent and enzyme.
  • Enzyme The enzymes can be added on basis of e.g. Molar Concentration, Protein amount, or on Activity basis.
  • Ballast Clean white cloth (without optical whitener) made of cotton, polyester or cotton/polyester.
  • the composition of the ballast is a mix of different items at a cotton/polyester ratio of 65/35 based on weight.
  • the ballast weight, dryness and item composition must be the same in each wash. After each wash the ballast is inactivated in an industrial washer at 85°C /15 min or in a 90°C wash (EU machine) without detergent.
  • the ballast weight is adjusted with shirts, short sleeves to the right ballast.
  • Test swatches The test swatches, i.e. technical and natural stains, are either commercial or NZ-produced. The same batch must be used in all washes in a trial. Different swatch sizes can be used. The swatches are attached to tea towels by a stapler; the same swatch Type (stain) is placed on different towels or in different positions on the towel. Each swatch is marked individually for identification and to indicate the front side. It is important to keep the swatches in the dark and limit the exposure to light at all times as many stains are sensitive to light. The total weight of textiles include the weight of ballast and the tesed swatches.
  • Natural water contains different levels of metal ions (mainly Ca 2+ and Mg 2+ ) depending on geographical area.
  • the metal ions considered to make up the water hardness are the ones that precipitate fatty acid soaps (mainly Ca 2+ and Mg 2+ , but not for example Na + ) .
  • Natural water also contains hydrogencarbonate, HCO 3 - , at varying levels, on average 1.5 times more than the sum of Ca 2+ and Mg 2+ on a molar basis, and may contain a number of other anions, including Cl - and SO 4 2-. Presence of HCO 3 - is important for the buffer capacity of the water and affects the pH of wash solutions prepared by adding a detergent.
  • Artificial hard water is prepared by adding CaCl 2 , MgCl 2 and NaHCO 3 to Milli-Q water or deionized water.
  • wash conditions in various regions for normal heavy duty wash are exemplified in below table.
  • the enzyme solution or granulate is added –short mix (10 sec) .
  • Ballast and swatches are added to the washing machine.
  • the washing machine by default gives 2 rinses. Rinsing with water having the same hardness . The machine will automatically add 1 more rinse if over foaming is detected. Water consumption is registered automatically during this time.
  • test swatches are removed from the tea towels and placed on trays for drying -make sure the swatches are dried in completely darkness, as many stains are sensitive to light. The swatches are dried overnight and must be completely dry before measurement.
  • MiniLOM Minimum Launder-O-Meter
  • MiniLOM is a modified mini wash system of the Launder-O-Meter (LOM) , which is a medium scale model wash system that can be applied to test up to 32 different wash conditions simultaneously.
  • MiniLOM model wash system is mainly used to wash hygiene experiment.
  • a MiniLOM is a rotator with free 50 ml x 16 (usually 6-32) tube rotisserie accessory included, with capacity of 10 to 20 mL of detergent solution in each tube.
  • the water bath has to be filled up with sterilized deionised water.
  • Each tube will contain a solution of a specific detergent/enzyme system and the soiled and unsoiled fabrics its performance is tested on. Mechanical stress is achieved by a rotating axis mix them horizontally or vertically. It can be used in ambient temperatures from 4C to 55C, and the rotation range is from 10 to 70 rpm.
  • factors such as the microorganisms loading and the fabric pieces can be varied. The tubes shall be discarded after wash.
  • wash solution with desired amount of detergent and water hardness are prepared in a 1 L Beaker.
  • the detergent is allowed to dissolve during magnet stirring for 10 min, and measure pH of detergent solution after 10 min stirring. Wash solution shall be used within 30 to 60 min after preparation.
  • Wash performance is expressed as a delta remission value ( ⁇ Rem) .
  • ⁇ Rem delta remission value
  • Calculating the enzyme effect is done by taking the measurements from washed swatches with enzymes and subtract with the measurements from washed without enzyme for each stain. The total enzyme performance is calculated as the sum of individual ⁇ Rem enzyme .
  • Triphenyltetrazolium Chloride is a reduction–oxidation reaction indicator, and it can be used to differentiate metabolically active and inactive tissues/organisms. It is white in color, and the color will turn to red when TTC is enzymatically reduced to water-insoluble TPF (1, 3, 5-triphenylformazan) in living cells due to the activity of various dehydrogenases (enzymes important in oxidation of organic compounds and thus cellular metabolism) .
  • Scheme I below show the reaction from TTC to TPF. For this reason, TTC can be used for detecting microbial growth.
  • microbe growth indicators having TTC-like microbial growth indication function can be used in the present study. They can include the redox indicator, living cell dye, the colored metabolites and metabolites detecting assay.
  • the redox indicators include all redox assay, such as INT (2- (4-iodophenyl) -3- (4-nitrophenyl) -5-phenyl-2H-tetrazolium) , MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) , XTT (2, 3-bis- (2-methoxy-4-nitro-5-sulfophenyl) -2H-tetrazolium-5-carboxanilide) , MTS (3- (4, 5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium) , WSTs (Water-
  • the living cell dye can include acridine orange, Calcein-AM and Hoechst. All the colored metabolites assays/microorganisms should be included, such as all the carotenoid, luciferin, fluorescent protein/fluorescent producing microorganisms. Metabolites detecting assays include all the metabolites and enzyme detecting assay, such as ATP detect assay, using X-Gal or MUG to detect ⁇ -Galactosidase.
  • TTC Triphenyl tetrazolium chloride
  • Nutrient agar medium 5 g Peptone, 30 g Beef extract, 5 g NaCl, 15 g Agar, in 1 liter distilled water, adjust pH to 7.0-7.2. This medium is used for cultivating P. putida
  • Potato Dextrose Agar medium Boil 200 g sliced, unpeeled potatoes in 1 liter distilled water for 30 min. Filter through cheesecloth, saving effluent, which is potato infusion (or use commercial dehydrated form) . Mix with 20 g dextrose and add water to dilute it to 1 L. Distribute it to flasks, and add 15 g/L agar to each flask, then autoclave 15 min at 121°C. Then dispense 20-25 ml portions into sterile 15 ⁇ 100 mm petri dishes. This medium is used for cultivating fungi.
  • PDA Potato Dextrose Agar medium
  • the swatches are taken out from incubator at a 6 hour interval, and the evaluation can be conducted on scanner.
  • a scanner e.g., Epson Expression 10000XL
  • a predetermined setting e.g., the software of Silverfast can be used and is set to scan in 200dpi and 48 ⁇ 24 bit color
  • the software of Color vector program for RGB reading can be used, and a circled area with a diameter of 1.2 fold of the diameter of the stain are choosen for the color analysis purpose.
  • the intensity of the color on swatches is calculated according to the following equation. Then compare the intensity of the tested swatches with the intensity of the similarly stained and washed (but without enzyme useful in the present invention) swatch, if the change of the intensity is a decrease of 4, then record the time as TTC detectiable time.
  • the swatches are taken out for microscope detection at an interval of 12 hrs to check the spore formation.
  • Olympus SZX16 microscope can be used, and a 10 fold magnification can be used by randomly selecting 3 visual field under the microscope for any visible conidia.
  • their spore/mycelium may show a color of black, green, or red and so on. The color can be predetermined through studying the specification of the fungi.
  • the spore (conidia, sporangia or other type of spores) or mycelium is dected under 10 fold stereo microscopre screening, then proceed to higher magnification detection of 50 fold to confirm the size of the conidial head, if the conidial head has a diameter of no less than 20 ⁇ m (for Aspegillus niger) or the mycelium has a diameter of no less than 2 ⁇ m, then record the time as fungi spore formation time.
  • Spore Density Score is used to determines the growth of fungi as represented by visible marks on household surfaces caused by the spores/conidia of the fungi which grow on said surface.
  • the swatches in the TTC Assay section described above are taken out for measuring the spore density score after being cultivated for 7 days.
  • a scale of 0 to 7 is set for measuring the spore density value, where 0 represents no fungi growth on the surface, and 7 being the surface are overgrown with fungi. For different fungi, the color of the spore can be different, but the same 0-7 scale can apply for different fungi growth and spore density measurement.
  • a group of panellists is first trained to understand the scale and corresponding spore density value, and then score the spore dentisty by comparing each surfaces (swatches) as tested with the scale.
  • a filamentous fungi Aspergillus niger (Novozymes internal strain number 57825) is used. This strain is isolated from the moldy corn in Liaoning province of China in the year of 2014. It was isolated and cultured in Potato Dextrose Agar medium (PDA) medium at 25°C for 3 days. As an alternative, other commercially available Aspergillus niger can be used interchangeably with this strain in the Examples.
  • PDA Potato Dextrose Agar medium
  • Example 1 Prevention of fungi growth on soils on chopping board treated with enzymatic Hand Dish Wash (HDW) detergent
  • a filamentous fungi Aspergillus niger (Novozymes internal strain number 57825) , is used. This strain is isolated from the moldy corn in Liaoning province of China in the year of 2014. It was isolated and cultured in Potato Dextrose Agar medium (PDA) medium at 25°C for 3 days. It is believed that other commercially available Aspergillus niger can be used interchangeably with this strain in the Examples.
  • PDA Potato Dextrose Agar medium
  • Enzymes Protease (SEQ ID NO: 3) , Amylase (SEQ ID NO: 9)
  • 5%potato starches are gelatinized in tap water at 85°C for 5 min, which forms a hot paste, and then add 2%semi-skimmed milk and 2%egg yolk, and further add 1.3%soy sauce to make a homogeneous solution.
  • each group was added with its corresponding wash solution as listed in the Grouping table shown above.
  • the boards were then left standing still for 10 mins. Afterwards, the boards were sponge-scrubbed under flowing tap water with 5 stokes of scrubbing.
  • the growth of the fungi was observed every 12 hours. Record the timing when the visible black color sporange/spore is identified by naked eye by following the procedure described in the Fungi Spore formation Assay section in the Assays part.
  • the chopping boards from all the Test groups 1-3 which were enzymatically treated has much longer spore forming time than the blank group and the negative control group which are stained and respectively washed with the water only or washed liquid detergent itself comprising no such protease and amylase.
  • the test group has a spore forming time which is at least 1.5 fold, i.e., an increase of at least 50%compared with that of the negative control group.
  • Each of the chopping board was observed by a panel for spore density score evaluation after being cultivated for 7 days, by following the procedure described in the Spore Density Score Assay section of the Assay part in previous paragraphs.
  • the chopping boards from all the test groups 1-3 which were enzymatically treated has much lower Spore Density Score than the blank group (score 7) and at least one score lower than the negative control group (score 3) which are stained and washed with the water only or liquid detergent itself comprising no such protease and amylase.
  • Example 2 Inhibition of bacterial growth by washing with enzymatic liquid detergent comprising enzymes at different dosage
  • Enzyme Protease (SEQ ID NO: 3) , Amylase (SEQ ID NO: 9)
  • plastic chopping board (Jie neng brand purchased from supermarket, polypropylene) which has been sliced slightly with a saw to make a few lines on its surface to mimic the board in real life use.
  • the Baby food #2 soil was prepared according to the description in the Stain Recipe section in the Assays part. 1 mL of each soil solution was applied onto each respective chopping board. Then the boards were left for air dry for 3hrs at room temperature.
  • the board from each group was added with 1 mL its corresponding wash solution as listed in the Grouping table shown above.
  • the boards were then left standing still for 10 mins. Afterwards, the boards were sponge-scrubbed under flowing tap water with 5 stokes of scrubbing.
  • TTC detectable time was measured by following the procedure in the TTC Assay section in the Assay part.
  • Results in Table 3 show that for boards from all the test groups 1-3 which were respectively washed with water or detergent comprising blend of protease and amylase, the TTC detectable time that was were significantly prolonged (ratio>2) as compared to the negative control group. This suggests the bacterial growth was inhibited by using these enzymes in water or liquid detergent for plastic chopping board surface.
  • Example 3 Inhibition of fungi growth by washing with enzymatic liquid detergent comprising combination of amylase and protease
  • Microorganism-Aspergillus niger (Same as in Example 2)
  • Enzyme Protease (SEQ ID NO: 3) , Amylase (SEQ ID NO: 9)
  • Stained swatches are prepared according to the procedure described in the Wash Assay part above.
  • test group swatches which was washed with detergent comprising protease and amylase, at least the PCN01 and W30A showed the same result as the unstained and unwashed swatches.
  • the results from the test group and the blank control group are siginificantly longer than the two negative control groups, which are either stained but unwashed, or stained and washed with the liquid detergent itself comprising no such protease and amylase. This clearly suggests protease and amylase can be useful in inhibiting the growth of the fungi, Aspergillus niger.
  • Each of the swatches are observed by a panel, and given a score of the spore conidia size and density.
  • the measurement refers to the “Spore Density Score” section under the Assay part.
  • test group swatches which was washed with detergent comprising protease and amylase, at least the PCN01 and W30A swatches showed the same result as the unstained and unwashed swatches.
  • results from both the test group and the blank control group are siginificantly smaller than the two negative control groups, which are either stained but unwashed, or stained and washed with the detergent itself comprising no such protease and amylase.
  • protease and amylase can be useful in preventing, inhibiting, or reducing the growth of the fungi, Aspergillus niger.
  • Example 4 Inhibition of fungi growth by washing with enzymatic powder detergent comprising combination of amylase and protease
  • Microorganism-Aspergillus niger (same as in Example 2)
  • Enzyme blend of protease (SEQ ID NO: 1) and amylase (SEQ ID NO: 10) .
  • Stained swatches are prepared according to the procedure described in the Wash Assay part above.
  • Aspergillus niger is a black-spored filamentous fungus that forms asexual spores called conidospores (conidia) during growing.
  • the Aspergillus niger conidial head can be observed by naked eye. Record when can see the visible black conidia.
  • test group swatches which was washed with detergent comprising protease and amylase, at least the W30A swatch showed the same result as the unstained and unwashed swatches.
  • the above results show that the test group and the blank control group have siginificantly longer spore formation time than the two negative control groups, which are either stained but unwashed, or stained and washed with the powder detergent itself comprising no such protease and amylase. Both aspects of the results clearly suggests protease and amylase can be useful in inhibiting the growth of the fungi, Aspergillus niger.
  • Each of the swatches are observed by a panel, and given a score of the spore conidia size and density.
  • the measurement refers to the “Spore Density Score” section under the Assay part.
  • Example 5 Inhibition of fungi growth by washing with enzymatic liquid detergent comprising enzymes at different dosage
  • A single protease (SEQ ID NO: 3) ,
  • Stained swatches are prepared according to the procedure described in the Wash Assay part above.
  • the Aspergillus niger conidia were observed following the same procedure in the previous Examples. can be observed by naked eye. Record when can see the visible black conidia.
  • Each of the swatches are observed by a panel, and given a score of the spore conidia size and density.
  • the measurement refers to the “Spore Density Score” section under the Assay part.
  • the rusults in Table 9 shows the swatches from all three test groups A-C, and for both the PCN01 and W30A swatches, it showed at lower enzyme dosage the fungi density score were much smaller as compared to the negative control group, and at higher dosagethe fungi spore forming time was even the same as the blank control, which were unstained and unwashed.
  • protease and amylase can be useful in preventing, inhibiting, or reducing the growth of the fungi, Aspergillus niger.
  • CFU counting is conducted to quantify the fungi on swatches.
  • the PCN01 swatches from Test Group C was used for this purpose.
  • the spore amount is calculated from the CFU by this equation: ( (Average CFU with the dosage of 10 -n ) ⁇ 10 n /0.2 mL) ⁇ 10 Ml.
  • Table 10 clearly shows that the test group has significantly lower amount of fungi left on the swatches which are treated with enzymatic detergent comprising protease and amylase.
  • Example 6 Inhibition of bacterial growth by washing with with enzymatic liquid detergent comprising enzymes at different dosage
  • Enzyme Protease (SEQ ID NO: 3) , Amylase (SEQ ID NO: 9)
  • Stained swatches are prepared according to the procedure described in the Wash Assay part above.
  • TTC detectable time was measured by following the procedure in the TTC Assay section in the Assay part.
  • Example 7 Use of cellulase in reducing attachment of bacterial on textile
  • Microorganism Pseudomonas putida (China General Microbiological Culture Collection Center (CGMCC) , the strain number 1.3096)
  • Pigment soil WFK09V from Center For Testmaterials BV, CFT. It is a standard pigment mixture in all soiled/stained test fabrics containing 'Pigment' ) . Addition of pigment soil is to mimic the real wash situation where there are relatively dirty laundry releasing soil into the wash liquor.
  • Prewash of the swatch The swatches were first aged in Wascator machine, program 151, age time: 10 h. Then the test swatches were washed with model detergents either liquid dertergent Model A comprising 0.075 ppm Cellulase, SEQ ID NO: 15; or powder detergent X comprising 0.05 ppm Cellulase, SEQ ID NO: 15; and the control swatches were washed with respective Model Detergent only, in FSW. After wash for 20 cycles, the swatches were cut to round shape with a diameter of 1.9 cm and then sterilize in autoclave under 121°C, 15 min and dry at oven with 60°C.
  • Example 8 Use of cellulase in reducing attachment of fungi on textile
  • Example 8 was conducted essentially the same as the previous example with the exception in that the fungi Candida parapsilosis (China General Microbiological Culture Collection Center (strain number: 2.1846) ) replaced the bacterial Pseudomonas putida, the amount of Candida parapsilosis and the cultivation condition was accordingly different as well from previous Example.
  • the cultivation medium for Candida parapsilosis is Yeast Extract–Peptone–Dextrose (YPD) Medium: 10 g/L Yeast extract, 20 g/L Peptone, 20 g/L Dectrose (sterilize Dectrose separately with others, mix together after sterilization) , 20 g/L agar for solid medium.
  • YPD Yeast Extract–Peptone–Dextrose
  • the swatches were subject to the fluorescene detection from remaining microbes on the swatches using the PrestoBlue TM Cell Viability Reagent to compare the viability of microbes remained on it after the second wash.
  • PrestoBlue TM Cell Viability Reagent (Thermo Fisher Scientific, Catalog number: A13261) was used.
  • PrestoBlue is a cell permeable resazurin-based solution that functions as a cell viability indicator by using the reducing power of living cells to quantitatively measure the proliferation of cells.
  • the PrestoBlue reagent When added to cells, the PrestoBlue reagent is modified by the reducing environment of the viable cell and turns red in color, becoming highly fluorescent. This color change can be detected using fluorescence or absorbance measurements.
  • the swatches enzymatically prewashed showed a much slower flatter rise of the fluorescene unit valuem and this is significantwhen the swatches had been incubated for a period of time of more than 900 mins. This result shows cellulase can be effectively used in reducing the attachment of fungi onto textiles.
  • Example 9 Use of enzyme in inhibiting bacteria generated with enzymatic liquid detergent
  • Enzyme mixture 1 0.41 ppm protease (SEQ ID NO: 3) , 0.052 ppm amylase (SEQ ID NO: 9)
  • Enzyme mixture 2 0.62 ppm protease (SEQ ID NO: 3) , 0.052 ppm amylase (SEQ ID NO: 9) and 0.16 ppm celluclase (SEQ ID No: 17)
  • the baby food stained swatches or the braised beef stained swatches were prepared according to the procedure described in the Assays section: Preparation of stained swatches.
  • the swatches taken out from the incubator were allowed to balance at room temperature and room humidity for 30 min before moving into the panel room for evaluation.
  • the panelists are selected from experienced and qualified pool of candidates who. The panelists performed the evaluation without being able to see the swatches.
  • Two samples in two petri dishes which need to be evaluated were provided, the panellist smelled each sample for 2 second and then took some breath of fresh air for more than 10 s before smell another sample. After breathing fresh air for more than 10s, they can evaluate the next pair of samples following the same procedure.
  • Gerstel MPS SPME Incubator Agitator. Incubation Temperature: 60°C.
  • MS Information Acquisition Mode: Scan. Solvent Delay (minutes) : 1. Scan Parameters: Start Time: 1. Low Mass: 35. High Mass: 350. Threshold: 100. A/D Samples: 4. MS Zones: MS Source: 230 °C. MS Quad: 150 °C
  • the four VOC molecules are representative malodor molecules.
  • the seven VOC molecules can be representative malodor molecules, particularly 3-octanone has musty and moldy smell, 2, 3-Butanedione has skink smell.
  • Example 10 Inhibition of fungal malodor generation by washing with multi enzymatic liquid detergent comprising enzymes
  • Example 9 was conducted essentially the same as the previous example with the exception in that the fungi Aspergillus niger (stain information is same as in Example 2) replaced the bacterial Pseudomonas putida.
  • the swatches were only evaluated with GS-MS measurement for malodor.

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Abstract

Use of one or more enzymes in preventing, inhibiting or reducing microbe growth on a surface, wherein after use of said enzymes said microbe on said surface have an increase in its TTC detectable time of at least 20%, at least 30%, at least 50%, at least 70%, at least 80%, or at least 100%.

Description

USE OF ONE OR MORE ENZYMES IN PREVENTING, INHIBITING OR REDUCING MICROBE GROWTH ON A SURFACE
Reference to a Sequence Listing
This application comprises a Sequence Listing in computer readable form, which is incorporated herein by reference.
Field of the Invention
The present invention relates to a novel use of one or more enzymes in preventing, inhibiting or reducing microbe growth on a surface, wherein said microbe have an increase in the TTC detectable time of at least 20%on said surface after use of said enzymes. The present invention also relates to a method of preventing, inhibiting or reducing microbe growth on such surface, by treating the surface with enzymes to an extent that the microbe has a TTC detectable time on said surface after the treatment which increases at least 20%. The present invention also relates to a method of demonstrating the microbe growth inhibition of an enzyme on a surface.
Background of the Invention
In domestic household cleaning field, drying machines are often used to dry clothes and kitchen surfaces such as plates. However, machine dryers are not very environmental friendly and are less available in some regions such as Asia. When machine-dry is not accessible, air-dry for such surfaces are commonly adopted. But humidity can cause problems during the air drying process. Specifically, it not only takes longer to dry, but also causes sanity concerns.
In humid environments, such as places where precipitation is frequent, e.g., places which have a regular Monsoon season or regions where it is warm and humid all year round, microorganisms like fungi and bacteria grow fast on surfaces such as textiles and kitchen ware. Microorganism growth can be a problem, even after the surfaces have been treated via conventional cleaning processes such as laundry. In these regions, the problem of microbe growth on a surface can be especially pronounced after being washed, due to the humidity of the washed surface itself and the prolonged exposure in the humid air during the air-drying process. The growth of the microbes on the surfaces may be visualized as dark spots on the surface, and the microbial growth may further give rise to malodors, which are undesirable and unpleasant.
Numerous enzymes have conventionally been incorporated into the detergent compositions for cleaning benefits. In addition, some enzyms including but not limited to amylase, have been reported as useful in preventing, removing or reducing biofilms on surfaces, see WO2008112459A2 (Novozymes, A/S) , WO2006/031554 (Novozymes, A/S) . Besides, enzymes such as oxidase or peroxidase has been reported to be useful in inhibiting microorganisms in laundary together with the action of polycationic polymer in the wash liquor. US 6,287,585B1, Novozymes A/S.
However, no disclosure has been made about the use of enzymes in preventing, microbe growth which causes the aforementioned spot and malodor problems on household surfaces in particular in humid environments. It is therefore an object of the present invention to provide such use.
Summary of the Invention
The present invention relates to a novel use of one or more enzymes in preventing, inhibiting or reducing microbe growth on a surface, wherein said microbe have an increase in the TTC detectable time of at least 20%on said surface after use of said enzymes.
Another aspect of the invention relates to a method of of preventing, inhibiting or reducing microbe growth on surface, by treating the surface with enzymes for a pre-determined period to the extent that the TTC detectable time of microbes on said surface after the treatment of enzyme increases at least 20%as compared to those on a surface not treated with said enzymes.
Another aspect of the invention relates to a method of demonstrating the microbe growth inhibition or deep cleansing benefit of an enzyme or enzyme combination on a surface, comprising the steps of:
a. Providing two surfaces A and A’,
b. Applying a soil comprising a microbe growth indicator onto each of the surfaces A and A’,
c. Washing A with a detergent composition comprising no enzyme, and washing A’with said detergent composition comprising one or more enzymes,
d. Preparing an inoculation of microbe and apply it onto the surfaces A and A’,
e. Incubating A and A’under suitable conditions to allow microbe growth for a predetermined period of time,
f. Comparing the growth of microbe on A and A’.
Brief Description of the Figure
Figure 1 relates to PrestoBlue fluorescene unit detection of Candida Parapsilosis on two differently treated swatches.
Overview of sequences listing
SEQ ID NO: 1 is the amino acid sequence of a protease
SEQ ID NO: 2 is the amino acid sequence of a protease
SEQ ID NO: 3 is the amino acid sequence of a protease
SEQ ID NO: 4 is the amino acid sequence of a protease
SEQ ID NO: 5 is the amino acid sequence of a protease
SEQ ID NO: 6 is the amino acid sequence of a protease
SEQ ID NO: 7 is the amino acid sequence of a protease
SEQ ID NO: 8 is the amino acid sequence of a protease
SEQ ID NO: 9 is the amino acid sequence of an amylase
SEQ ID NO: 10 is the amino acid sequence of an amylase
SEQ ID NO: 11 is the amino acid sequence of an amylase (SEQ ID NO 2 of WO2000/060060)
SEQ ID NO: 12 is the amino acid sequence of an amylase (SEQ ID NO 2 of WO96/023873)
SEQ ID NO: 13 is the amino acid sequence of an amylase (SEQ ID NO 3 of WO2008/112459
SEQ ID NO: 14 is the amino acid sequence of an subtilase (SEQ ID NO: 1 of WO2004/067737)
SEQ ID NO: 15 is the amino acid sequence of a cellulase
SEQ ID NO: 16 is the amino acid sequence of a cellulase
SEQ ID NO: 17 is the amino acid sequence of a cellulase
SEQ ID NO: 18 is the amino acid sequence of a cellulase
Definitions
TTC Detectable Time: This term determines the time when the microbes on the surfaces become identifieable in situ, as shown by the color change of triphenyltetrazolium chloride (TTC) . TTC is a reduction–oxidation reaction indicator, which can be used to differentiate metabolically active and inactive tissues/organisms. TTC is white in color, and when it is enzymatically reduced to water-insoluble TPF (1, 3, 5-triphenylformazan) by various  dehydrogenases in living organism, the color will turn to red. Later paragraphs in this text, specifcally the “TTC Assay (In situ detection of bacterial) ” section in the Assays part, discloses the details of the in situ detection of microbe growth.
Spore Forming Time: This term determines the time when the microbes, particularly the fungi on the surface become identifiable in situ, through microscope observation. This is an additional tool for monitoring the growth of microbes on the surface beyond the above mentioned TTC detectable time assay. The spore forming time assay method can be preferred in the situation where the spore has a color which may mask the TTC color change. Later paragraphs of this text, specifcally the “Fungi Spore Formation Time Assay” section in the Assays part, discloses the details of the procedure.
Spore Density Score: This term determines the growth of fungi as represented by visible marks on household surfaces caused by the spores and/or mycelium of the fungi which grow on said surface. At a scale of 0 to 7, 0 represents no fungi growth on the surface, and 7 being the surface are overgrown with fungi. For different fungi, the color of the spore can be different, but the same 0-7 scale can apply for different fungi growth and spore density measurement. A group of trained panellists can be trained to understand the scale, and score the spore dentisty of each surfaces according to a preset procedure.
Detergent Composition: The term “detergent composition” refers to compositions that find use in the removal of undesired compounds from textiles to be cleaned, such as textiles. The detergent composition may be used to e.g. clean textiles for both household cleaning and industrial cleaning. The terms encompass any materials/compounds selected for the particular type of detergent composition desired and the form of the product (e.g., liquid, gel, powder, granulate, paste, or spray compositions) and includes, but is not limited to, detergent compositions (e.g., liquid and/or solid laundry detergents and fine fabric detergents; fabric fresheners; fabric softeners; and textile and laundry pre-spotters/pre-treatment) . In addition to containing a DNase of the invention, the detergent formulation may contain one or more additional enzymes (such as proteases, amylases, lipases, cutinases, cellulases, endoglucanases, xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidases, haloperoxygenases, catalases and mannanases, or any mixture thereof) , ingredients such as surfactants, builders, chelators or chelating agents, bleach system or bleach components, polymers, fabric conditioners, foam boosters, suds suppressors, dyes, perfume, tannish inhibitors, optical brighteners, bactericides, fungicides, soil suspending agents, anti-corrosion agents, enzyme inhibitors or stabilizers, enzyme activators, transferase (s) , hydrolytic enzymes,  oxido reductases, bluing agents and fluorescent dyes, antioxidants, and solubilizers.
Delta remission value (ΔRem) : The terms “Delta remission” or “Delta remission value” are defined herein as the result of a reflectance or remission measurement at a certain wavelength which typically is 460 nm. The swatch is measured with one swatch of similar colour as background, preferably a swatch from a repetition wash. A swatch representing each swatch type is measured before the wash. The Delta remission is the remission value of the washed swatch minus the remission value of the unwashed swatch.
One way of measuring the wash performance is the Delta enzyme performance value (ΔRem enzyme value) : The term “Delta enzyme remission value” is defined herein as the result of a reflectance or remission measurement at 460 nm. The swatch is measured with one swatch of similar colour as background, preferably a swatch from a repetition wash. A swatch representing each swatch type is measured before wash. The Delta enzyme remission is the remission value of the swatch washed in detergent with an enzyme present minus the remission value of a similar swatch washed in a detergent without enzyme present.
Dish wash refers to all forms of washing dishes, e.g., by hand or automatic dish wash. Washing dishes includes, but is not limited to, the cleaning of all forms of crockery such as plates, cups, glasses, bowls, all forms of cutlery such as spoons, knives, forks and serving utensils as well as ceramics, plastics such as melamine, metals, china, glass and acrylics.
Dish washing composition: The term “dish washing composition" refers to compostions intended for cleaning dishes, table ware, pots, pans, cutlery and all forms of compositions for cleaning hard surfaces areas in kitchens. The present invention is not restricted to any particular type of dish wash composition or any particular detergent.
Enzyme Detergency benefit: The term “enzyme detergency benefit” is defined herein as the advantageous effect an enzyme may add to a detergent compared to the same detergent without the enzyme. Important detergency benefits which can be provided by enzymes are stain removal with no or very little visible soils after washing and/or cleaning, prevention or reduction of redeposition of soils released in the washing process (an effect that also is termed anti-redeposition) , restoring fully or partly the whiteness of textiles which originally were white but after repeated use and wash have obtained a greyish or yellowish appearance (an effect that also is termed whitening) . Textile care benefits, which are not directly related to catalytic stain removal or prevention of redeposition of soils, are also important for enzyme detergency benefits. Examples of such textile care benefits are prevention or reduction of dye transfer from one fabric to another fabric or another part of the same fabric (an effect that is also termed dye transfer inhibition or anti-backstaining) , removal of protruding or broken fibers from a fabric  surface to decrease pilling tendencies or remove already existing pills or fuzz (an effect that also is termed anti-pilling) , improvement of the fabric-softness, colour clarification of the fabric and removal of particulate soils which are trapped in the fibers of the fabric or garment. Enzymatic bleaching is a further enzyme detergency benefit where the catalytic activity generally is used to catalyze the formation of bleaching components such as hydrogen peroxide or other peroxides.
Hard surface cleaning: The term “Hard surface cleaning” is defined herein as cleaning of hard surfaces wherein hard surfaces may include floors, tables, walls, roofs etc. as well as surfaces of hard objects such as cars (car wash) and dishes (dish wash) . Dish washing includes but are not limited to cleaning of plates, cups, glasses, bowls, cutlery such as spoons, knives, forks, serving utensils, ceramics, plastics, metals, china, glass and acrylics.
Laundering relates to both household laundering and industrial laundering and means a process of treating textiles and/or fabrics with a solution containing a detergent composition . The laundering process can for example be carried out using, e.g., a household or an industrial washing machine or can be carried out by hand.
Peroxidases/Oxidases: A peroxidase according to the invention is a peroxidase enzyme comprised by the enzyme classification EC 1.11.1.7, as set out by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB) , or any fragment derived therefrom, exhibiting peroxidase activity.
Protease is defined herein as an enzyme that hydrolyzes peptide bonds. It includes any enzyme belonging to the EC 3.4 enzyme group (including each of the thirteen subclasses thereof) . The EC number refers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego, California, including supplements 1-5 published in Eur. J. Biochem. 1223: 1-5 (1994) ; Eur. J. Biochem. 232: 1-6 (1995) ; Eur. J. Biochem. 237: 1-5 (1996) ; Eur. J. Biochem. 250: 1-6 (1997) ; and Eur. J. Biochem. 264: 610-650 (1999) ; respectively. The most widely used proteases in the detergent industry such as laundry and dish wash are the serine proteases or serine peptidases which is a subgroup of proteases characterised by having a serine in the active site, which forms a covalent adduct with the substrate. Further the subtilases (and the serine proteases) are characterized by having two active site amino acid residues apart from the serine, namely a histidine residue and an aspartic acid residue. Subtilase refer to a sub-group of serine protease according to Siezen et al., 1991, Protein Engng. 4: 719-737 and Siezen et al., 1997, Protein Science 6: 501-523. The subtilases may be divided into 6 sub-divisions, i.e., the Subtilisin family, the Thermitase family, the Proteinase K family, the Lantibiotic peptidase family, the Kexin family and the Pyrolysin family. The term “protease activity” means a proteolytic activity (EC 3.4) . Proteases usable in detergents are mainly endopeptidases (EC 3.4.21) . There  are several protease activity types: The three main activity types are: trypsin-like where there is cleavage of amide substrates following Arg or Lys at P1, chymotrypsin-like where cleavage occurs following one of the hydrophobic amino acids at P1, and elastase-like with cleavage following an Ala at P1.
Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity” . For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) , preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the –nobrief option) is used as the percent identity and is calculated as follows: (Identical Residues x 100) / (Length of Alignment –Total Number of Gaps in Alignment) . For purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EM-BOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra) , prefer-ably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the –nobrief option) is used as the percent identity and is calculated as follows: (Identical Deoxyribonucleotides x 100) / (Length of Alignment –Total Number of Gaps in Alignment) .
Residual Stains: This term means the stains that are not completely removed from the household surfaces after cleaning. There may be different level of residual stains, and they can be ranked from best to worse in term of cleaness of the surface with 1 being the best. Rating: 1=completely removed, 2=very good (acceptable) , 3=pretty good (borderline) , 4=poor (unacceptable) , 5=nothing removed (same as original) . In some instances, a visual notation can be made to designate a ranking for the residual stains. Here in the text of the present invention, residual stains include those ranked from 2 to 5. Stains in the context of the present invention can be from various sources, including but not limited to starch stains, proteineous stains,  amylose stains, lipid-based stains; food stains, beverage stains, environmental stains, work place stains, et al.
Textile: The term “textile” means any textile material including yarns, yarn intermediates, fibers, non-woven materials, natural materials, synthetic materials, and any other textile material, fabrics made of these materials and products made from fabrics (e.g., garments and other articles) . The textile or fabric may be in the form of knits, wovens, denims, non-wovens, felts, yarns, and towelling. The textile may be cellulose based such as natural cellulosics, including cotton, flax/linen, jute, ramie, sisal or coir or manmade cellulosics (e.g. originating from wood pulp) including viscose/rayon, cellulose acetate fibers (tricell) , lyocell or blends thereof. The textile or fabric may also be non-cellulose based such as natural polyamides including wool, camel, cashmere, mohair, rabbit and silk or synthetic polymers such as nylon, aramid, polyester, acrylic, polypropylene and spandex/elastane, or blends thereof as well as blends of cellulose based and non-cellulose based fibers. Examples of blends are blends of cotton and/or rayon/viscose with one or more companion material such as wool, synthetic fiber (e.g. polyamide fiber, acrylic fiber, polyester fiber, polyvinyl chloride fiber, polyurethane fiber, polyurea fiber, aramid fiber) , and/or cellulose-containing fiber (e.g. rayon/viscose, ramie, flax/linen, jute, cellulose acetate fiber, lyocell) . Fabric may be conventional washable laundry, for example stained household laundry. When the term fabric or garment is used it is intended to include the broader term textiles as well. In the context of the present invention, the term “textile” is used interchangeably with fabric and cloth.
Variant means a polypeptide having enzyme activity but which comprises an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions compared to the partent or reference enzyme. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding one or more (e.g., several) amino acids, e.g., 1, 2, 3, 4 or 5 amino acids adjacent to and immediately following the amino acid occupying a position.
Wash liquor: The term “wash liquor” is defined herein as the solution or mixture of water and detergent components optionally including the enzyme.
Malodor: By the term ” malodour” it means an odor which is not desired. Examples of malodour include compounds with an unpleasant smell, which may be produced by microorganisms. These microorganisms can be from the body of humans or animals, the inner space of the washing machine, or from the rest of the environment. Some examples of such  unpleasant smelling compounds are hexanal (grassy smell) , 3-Octanone (musty and moldy) , 2, 3-Butanedione (skink smell) , Benzonitrile (almond-like odor) , Benzene (gasoline-like odor) or Toluene (pungent, benzene-like smell) .
Detailed Description of the Invention
The present invention relates to the use of enzymes for preventing, inhibiting or reducing growth of microorganism on surfaces e.g. textiles. Textiles which are air-dried are exposed to various microorgansms which may thrive and colonize on the textiles. The microbial growth and surfaces may be visualized as dark stains and/or a malodors such moldy, damp or unpleasant smells.
In one aspect, the present invention relates to use of one or more enzymes in preventing, inhibiting or reducing microbe growth on a surface, wherein after use of said enzymes on said surface said microbe have an increase in its TTC detectable time of at least 20%. The prolonged TTC detectable time shows that the use of enzyme on such surface prevented, inhibited or reduced the growth of the microbes.
In one aspect, the TTC detectable time increases at least at least 30%.
In one aspect, the TTC detectable time increases at least at least 40%.
In one aspect, the TTC detectable time increases at least at least 50%.
In one aspect, the TTC detectable time increases at least at least 70%.
In one aspect, the TTC detectable time increases at least at least 80%.
In one aspect, the TTC detectable time increases at least at least 100%.
When the microorganism is a fungi the effect of the use of enzymes on a surface for prevention of microbial growth could be measured as an increase in spore forming time. In one aspect, the microbe is a fungi, and after use of said enzymes on said surface the fungi spore forming time has an increase of at least 20%compared to the spore forming time on a surface bit treated with enzymes. The prolonged spore forming time after use of the enzyme on such surfaces also shows that the microbe’s growth is prevented, inhibited or reduced.
In one aspect, the TTC detectable time increases at least at least 30%.
In one aspect, the spore forming time increases at least at least 40%.
In one aspect, the spore forming time increases at least at least 50%.
In one aspect, the spore forming time increases at least at least 70%.
In one aspect, the spore forming time increases at least at least 80%.
In one aspect, the spore forming time increases at least at least 100%.
When the microorganism is a fungi the effect of the use of enzymes on a surface for prevention of microbial growth could be measured as a decrease of spore density. One aspect relates to the use of one or more enzymes for prevention of microbial growth on a surface, wherein the microbe is a fungi and wherein decrease of spore density is at least 1, on a density scale from 0 to 7, when compared to the spore density of a surface not treated with enzymes.
In another aspect, the decrease in spore density score is at least 2.
In another aspect, the decrease in spore density score is at least 3.
In another aspect, the decrease in spore density score is at least 4.
In another aspect, the decrease in spore density score is at least 5.
In another aspect, the decrease in spore density score is at least 6.
Residual stains often provide nutrient for microbes to survive and thrive on the surfaces. Without being bound by theory, it is believed that enzymes work in part by removing or reducing the residual stains on the surfaces.
One aspect of the present invention relates to the use of one or more enzymes for prevention of microbial growth on a surface, wherein said microbe growth is on the residual stain area of said surface.
In one aspect, the present inventions further relates to use of one or more enzymes in inhibiting or reducing malodor caused by microbe on a textile surface.
In one aspect, the present inventions further relates to use of one or more enzymes in preventing or reducing microbe attachment onto a textile surface. It is known that certain cellulase provides color care and whiteness improvement benefit to the textile being laundered. But it is not known that cellulase can be used in preventing or reducing microbe. Without being bound by theory, it is believed that the pills and damaged fibers are more prone to be transformed into amorphous cellulase removes the pills formed the fabric surface and damaged fiber, and some cellulase are very efficient at hydrolyzing amorphous cellulose and thereby these regions of the fiber are enzymatically removed. Removal of these pills and damaged fibers may contribute to the prevention or reduction of microbe that can be attached onto it, among other unknown factors.
The microbe targeted by the use of the enzymes in the present invention can be those that tend to multiply relatively fast on household surfaces in a hot and humid environment. It can be a fungi or a bacterial. In one aspect, the microbe is a fungi or bacterial selecting from a group consisting of Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Pseudomonas putida, Micrococcus luteus, Staphylococcus epidermidis, Enhydrobacter aerosaccus, Corynebacterium jeikeium, Propionibacterium acnes, Brevundimonas vesicularis, Streptomyces griseus, Streptomyces odorifer, Exophiala phaeomuriformis, Fusarium  oxysporum, Alternaria alternate, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aureobasidium pullulans, Chaetomium globosum, Cladosporium sphaerospermum, Gliocladium virens, Mucor plumbeus, Penicillium chrysogenum, Penicillium commune, Penicillium crustosum, Penicillium rubens, Penicillium varians, Stachybotrys chartarum, Trichoderma viride, Trichophyton rubrum, Candida parapsilosis, Rhodotorula mucilaginosa, Debaryomyces hansenii, Meyerozyma guilliermondii, Candida albicans, and combinations thereof.
In one aspect, the microbe is a fungi, and preferably Aspergillus niger.
In one aspect, the microbe is a bacterial, and preferably Escherichia coli or Pseudomonas putida.
The enzyme useful in the present invention can be selected from a group consisting of amylase, protease, lipase, mannase, cellulase, pectinase, and combinations thereof.
In one aspect, the enzyme is an amylase or protease.
In one aspect, the enzyme is not oxidase or peroxidase.
In one aspect, the enzyme is an amylase, protese, cellulase or combinations thereof.
In one aspect, the present inventions further relates to use of one or more enzymes in preventing or reducing microbe attachment onto a textile surface.
Enzyme of the present invention
The enzyme useful in the present invention may be added to a detergent composition in an amount corresponding to 0.001-200 mg of protein, such as 0.005-100 mg of protein, preferably 0.01-50 mg of protein, more preferably 0.05-20 mg of protein, even more preferably 0.1-10 mg of protein per liter of wash liquor.
The enzyme (s) useful in the present inventionmay be stabilized using conventional stabilizing agents, e.g. a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g. an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in, for example, WO 92/19709 and WO 92/19708.
Proteases
Suitable proteases include those of bacterial, fungal, plant, viral or animal origin e.g. vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included. It may be an alkaline protease, such as a serine protease or a metalloprotease. A serine protease may for example be of the S1 family, such as trypsin, or  the S8 family such as subtilisin. A metalloproteases protease may for example be a thermolysin from e.g. family M4 or other metalloprotease such as those from M5, M7 or M8 families.
The term "subtilases" refers to a sub-group of serine protease according to Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523. Serine proteases are a subgroup of proteases characterized by having a serine in the active site, which forms a covalent adduct with the substrate. The subtilases may be divided into 6 sub-divisions, i.e. the Subtilisin family, the Thermitase family, the Proteinase K family, the Lantibiotic peptidase family, the Kexin family and the Pyrolysin family.
Examples of subtilases are those derived from Bacillus such as Bacillus lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii described in; US7262042 and WO09/021867, and subtilisin lentus, subtilisin Novo, subtilisin Carlsberg, Bacillus licheniformis, subtilisin BPN’, subtilisin 309, subtilisin 147 and subtilisin 168 described in WO89/06279 and protease PD138 described in (WO93/18140) . Other useful proteases may be those described in WO92/175177, WO01/016285, WO02/026024 and WO02/016547. Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO89/06270, WO94/25583 and WO05/040372, and the chymotrypsin proteases derived from Cellumonas described in WO05/052161 and WO05/052146.
A further preferred protease is the alkaline protease from Bacillus lentus DSM 5483, as described for example in WO95/23221, and variants thereof which are described in WO92/21760, WO95/23221, EP1921147 and EP1921148.
Examples of metalloproteases are the neutral metalloprotease as described in WO07/044993 (Genencor Int. ) such as those derived from Bacillus amyloliquefaciens. Suitable commercially available protease enzymes include those sold under the trade names
Figure PCTCN2018078160-appb-000001
Duralase Tm, Durazym Tm, 
Figure PCTCN2018078160-appb-000002
Ultra, 
Figure PCTCN2018078160-appb-000003
Ultra, 
Figure PCTCN2018078160-appb-000004
Figure PCTCN2018078160-appb-000005
Ultra, 
Figure PCTCN2018078160-appb-000006
Ultra, 
Figure PCTCN2018078160-appb-000007
Blaze
Figure PCTCN2018078160-appb-000008
100T, Blaze
Figure PCTCN2018078160-appb-000009
125T, Blaze
Figure PCTCN2018078160-appb-000010
150T, 
Figure PCTCN2018078160-appb-000011
Figure PCTCN2018078160-appb-000012
Progress
Figure PCTCN2018078160-appb-000013
and
Figure PCTCN2018078160-appb-000014
 (Novozymes A/S) , those sold under the tradename 
Figure PCTCN2018078160-appb-000015
Purafect
Figure PCTCN2018078160-appb-000016
Purafect
Figure PCTCN2018078160-appb-000017
Figure PCTCN2018078160-appb-000018
Excellenz P1000 TM, Excellenz P1250 TM, 
Figure PCTCN2018078160-appb-000019
Preferenz P100 TM, Purafect
Figure PCTCN2018078160-appb-000020
Preferenz P110 TM, Effectenz P1000 TM, 
Figure PCTCN2018078160-appb-000021
Effectenz P1050 TM, Purafect
Figure PCTCN2018078160-appb-000022
Effectenz P2000 TM, 
Figure PCTCN2018078160-appb-000023
and
Figure PCTCN2018078160-appb-000024
 (Danisco/DuPont) , Axapem TM (Gist-Brocases N.V. ) , BLAP (sequence shown in Figure 29 of  US5352604) and variants hereof (Henkel AG) and KAP (Bacillus alkalophilus subtilisin) from Kao.
In one aspect, the protease useful in the present invention is selected from a group consisting of:
a) a polypeptides having the amino acid sequence of any of SEQ ID NO: 1-8, 14;
b) a polypeptides having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at leaset 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%sequence identity to any of SEQ ID NO: 1-8; and combinations thereof.
In one aspect, the protease is a peptide having at least 60%sequence identity to SEQ ID NO: 1 or 2, 3, 4, 5, 6, 7, 8 or 14.
In one aspect, the protease is a peptide having at least 65%sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.
In one aspect, the protease is a peptide having at least 70%sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.
In one aspect, the protease is a peptide having at least 75%sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.
In one aspect, the protease is a peptide having at least 80%sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.
In one aspect, the protease is a peptide having at least 85%sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.
In one aspect, the protease is a peptide having at least 90%sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.
In one aspect, the protease is a peptide having at least 95%sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.
In one aspect, the protease is a peptide having at least 96%sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.
In one aspect, the protease is a peptide having at least 97%sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.
In one aspect, the protease is a peptide having at least 98%sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.
In one aspect, the protease is a peptide having at least 99%sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.
In one aspect, the protease is a peptide selected from a group consisting of:
i) a protease comprising a substitution compared to the protease shown in SEQ ID NO 1 or SEQ ID NO 2 in one or more of the following positions: 3, 4, 9, 15, 24, 27, 42, 55, 59, 60, 66, 74, 85, 96, 97, 98, 99, 100, 101, 102, 104, 116, 118, 121, 126, 127, 128, 154, 156, 157, 158, 161, 164, 176, 179, 182, 185, 188, 189, 193, 198, 199, 200, 203, 206, 211, 212, 216, 218, 226, 229, 230, 239, 246, 255, 256, 268 and 269, wherein the positions correspond to the positions of the protease shown in SEQ ID NO 1, or
ii) one or more protease variant of a protease parent, wherein the protease variant comprises one or more mutation selected from the group consisting of S3T, V4I, S9R, S9E, A15T, S24G, S24R, K27R, N42R, S55P, G59E, G59D, N60D, N60E, V66A, N74D, N85S, N85R, G96S, G96A, S97G, S97D, S97A, S97SD, S99E, S99D, S99G, S99M, S99N, S99R, S99H, S101A, V102I, V102Y, V102N, S104A, G116V, G116R, H118D, H118N, N120S, S126L, P127Q, S128A, S154D, A156E, G157D, G157P, S158E, Y161A, R164S, Q176E, N179E, S182E, Q185N, A188P, G189E, V193M, N198D, V199I, Y203W, S206G, L211Q, L211D, N212D, N212S, M216S, A226V, K229L, Q230H, Q239R, N246K, N255W, N255D, N255E, L256E, L256D T268A and R269H, wherein the positions correspond to the positions of the protease shown in SEQ ID NO 1, wherein the protease parent is selected from the protease shown in SEQ ID NO 1 and the Bacillus amylolichenifaciens protease (BPN’) shown in SEQ ID NO 2 and wherein the protease variant has at least 80%sequence identity to SEQ ID NO 1 or 80%sequence identity to SEQ ID NO 2,
iii) a protease comprising a substitution at one or more positions corresponding to positions 171, 173, 175, 179, or 180 of SEQ ID NO: 14, compared to the protease shown in SEQ ID NO 48, wherein the protease variant has a sequence identity of at least 75%but less than 100%to SEQ ID NO 14,
iv) a protease comprising the amino acid sequence shown in SEQ ID NO 1 or 2 or a protease having at least 80%sequence identity to; the polypeptide comprising  amino acids 1-269 of SEQ ID NO 1 or the polypeptide comprising amino acids 1-275 of SEQ ID NO 2,
v) One or more of the following protease variants selected from the group:
SEQ ID NO 1 with the alterations T22R+S99G+S101A+V102I+A226V+Q239R,
SEQ ID NO 2 with the alterationsS24G+S53G+S78N+S101N+G128A+Y217Q,
SEQ ID NO 2 with the alterations S24G+S53G+S78N+S101N+G128S+Y217Q,
SEQ ID NO 1 with the alterations S9E + N42R + N74D + V199I + Q200L + Y203W + S253D + N255W + L256E,
SEQ ID NO 1 with S9E+N42R+N74D+H118V+Q176E+A188P+V199I+Q200L Y203W+S250D+S253D+N255W+L256E,
SEQ ID NO 1 with S9E+N42R+N74D+Q176E+A188P+V199I+Q200L+Y203W S250D+S253D+N255W+L256E,
SEQ ID NO 1 with S3V+N74D+H118V+Q176E+N179E+S182E+V199I+Q200L Y203W+S210V+S250D+S253D+N255W+L256E
SEQ ID NO 1 with the alterations T22A+N60D+S99G+S101A+V102I+N114L+G157D +S182D+T207A+A226V+Q239R+N242D+E265F,
SEQ ID NO 1 with the alterations S9E + N42R + N74D + H118V + Q176E + A188P + V199I + Q200L + Y203W + S250D + S253D + N255W + L256E,
SEQ ID NO 1 with the alterations S9E + N42R + N74D + Q176E + A188P + V199I + Q200L + Y203W + S250D + S253D + N255W + L256E,
SEQ ID NO 1 with the alterations S9E + N42R + N74D + H118V + Q176E + A188P + V199I + Q200L + Y203W + S250D + N255W + L256E + *269aH + *269bH,
SEQ ID NO 1 with the alterations S3V + N74D + H118V + Q176E + N179E + S182E + V199I + Q200L + Y203W + S210V + S250D + N255W + L256E,
SEQ ID NO 1 with the alterations S9E + N74D + G113W + G157P + Q176E + V199I + Q200L + Y203W + S250D + T254E + N255W + L256E,
SEQ ID NO 1 with the alterations S3V + S9R + N74D + H118V + Q176E + N179E + S182E + V199I + Q200L + Y203W + S212V + S250D + N255W + L256E,
SEQ ID NO1 with the alteration S99E, and
SEQ ID NO 2 with the alteration L217D,
and combinations thereof.
Amylases:
Suitable amylases which can be used together with the enzyme useful in the present invention may be an alpha-amylase or a glucoamylase and may be of bacterial or fungal origin. Chemically modified or protein engineered mutants are included.
Amylases can include, for example, alpha-amylases obtained from Bacillus, e.g., a special strain of Bacillus licheniformis, described in more detail in GB 1, 296, 839.
Suitable amylases include amylases having SEQ ID NO: 2 in WO 95/10603 or variants having 90%sequence identity to SEQ ID NO: 3 thereof. Preferred variants are described in WO 94/02597, WO 94/18314, WO 97/43424 and SEQ ID NO: 4 of WO 99/019467, such as variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408, and 444.
Different suitable amylases include amylases having SEQ ID NO: 6 in WO 02/010355 or variants thereof having 90%sequence identity to SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are those having a deletion in positions 181 and 182 and a substitution in position 193.
Other amylases which are suitable are hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006/066594 and residues 36-483 of the B. licheniformis alpha-amylase shown in SEQ ID NO: 4 of WO 2006/066594 or variants having 90%sequence identity thereof. Preferred variants of this hybrid alpha-amylase are those having a substitution, a deletion or an insertion in one of more of the following positions: G48, T49, G107, H156, A181, N190, M197, I201, A209 and Q264. Most preferred variants of the hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006/066594 and residues 36-483 of SEQ ID NO: 4 are those having the substitutions:
M197T;
H156Y+A181T+N190F+A209V+Q264S; or
G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.
Further amylases which are suitable are amylases having SEQ ID NO: 6 in WO 99/019467 or variants thereof having 90%sequence identity to SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are those having a substitution, a deletion or an insertion in one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216 and K269.  Particularly preferred amylases are those having deletion in positions R181 and G182, or positions H183 and G184.
Additional amylases which can be used are those having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 7 of WO 96/023873 or variants thereof having 90%sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 7. Preferred variants of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 7 are those having a substitution, a deletion or an insertion in one or more of the following positions: 140, 181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304 and 476, using SEQ ID 2 of WO 96/023873 for numbering. More preferred variants are those having a deletion in two positions selected from 181, 182, 183 and 184, such as 181 and 182, 182 and 183, or positions 183 and 184. Most preferred amylase variants of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 7 are those having a deletion in positions 183 and 184 and a substitution in one or more of positions 140, 195, 206, 243, 260, 304 and 476.
Other amylases which can be used are amylases having SEQ ID NO: 2 of WO 08/153815, SEQ ID NO: 10 in WO 01/66712 or variants thereof having 90%sequence identity to SEQ ID NO: 2 of WO 08/153815 or 90%sequence identity to SEQ ID NO: 10 in WO 01/66712. Preferred variants of SEQ ID NO: 10 in WO 01/66712 are those having a substitution, a deletion or an insertion in one of more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211 and 264.
Further suitable amylases are amylases having SEQ ID NO: 2 of WO 09/061380 or variants having 90%sequence identity to SEQ ID NO: 2 thereof. Preferred variants of SEQ ID NO: 2 are those having a truncation of the C-terminus and/or a substitution, a deletion or an insertion in one of more of the following positions: Q87, Q98, S125, N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475. More preferred variants of SEQ ID NO: 2 are those having the substitution in one of 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 deletion in position R180 and/or S181 or of T182 and/or G183. Most preferred amylase variants of SEQ ID NO: 2 are those having the substitutions:
N128C+K178L+T182G+Y305R+G475K;
N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;
S125A+N128C+K178L+T182G+Y305R+G475K; or
S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K wherein the variants are C-terminally truncated and optionally further comprises a substitution at position 243 and/or a deletion at position 180 and/or position 181.
Further suitable amylases are amylases having SEQ ID NO: 1 of WO13184577 or variants having 90%sequence identity to SEQ ID NO: 1 thereof. Preferred variants of SEQ ID NO: 1 are those having a substitution, a deletion or an insertion in one of more of the following positions: K176, R178, G179, T180, G181, E187, N192, M199, I203, S241, R458, T459, D460, G476 and G477. More preferred variants of SEQ ID NO: 1 are those having the substitution in one of more of the following positions: K176L, E187P, N192FYH, M199L, I203YF, S241QADN, R458N, T459S, D460T, G476K and G477K and/or deletion in position R178 and/or S179 or of T180 and/or G181. Most preferred amylase variants of SEQ ID NO: 1 are those having the substitutions:
E187P+I203Y+G476K
E187P+I203Y+R458N+T459S+D460T+G476K
wherein the variants optionally further comprises a substitution at position 241 and/or a deletion at position 178 and/or position 179.
Further suitable amylases are amylases having SEQ ID NO: 1 of WO10104675 or variants having 90%sequence identity to SEQ ID NO: 1 thereof. Preferred variants of SEQ ID NO: 1 are those having a substitution, a deletion or an insertion in one of 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 are those having the substitution in one of more of the following positions: N21D, D97N, V128I K177L, M200L, L204YF, E242QA, G477K and G478K and/or deletion in position R179 and/or S180 or of I181 and/or G182. Most preferred amylase variants of SEQ ID NO: 1 are those having the substitutions:
N21D+D97N+V128I
wherein the variants optionally further comprises a substitution at position 200 and/or a deletion at position 180 and/or position 181.
Other suitable amylases are the alpha-amylase having SEQ ID NO: 12 in WO01/66712 or a variant having at least 90%sequence identity to SEQ ID NO: 12. Preferred amylase variants are those having a substitution, a deletion or an insertion in one of more of the following positions of SEQ ID NO: 12 in WO01/66712: 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. Particular preferred amylases include variants having a deletion of D183 and G184 and having the substitutions  R118K, N195F, R320K and R458K, and a variant additionally having substitutions in one or more position selected from the group: M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345 and A339, most preferred a variant that additionally has substitutions in all these positions.
Other examples are amylase variants such as those described in WO2011/098531, WO2013/001078 and WO2013/001087.
Commercially available amylases are Duramyl TM, Termamyl TM, Fungamyl TM, Stainzyme  TM, Stainzyme Plus TM, Natalase TM, Liquozyme X, Amplify
Figure PCTCN2018078160-appb-000025
Achieve
Figure PCTCN2018078160-appb-000026
and BAN TM (from Novozymes A/S) , and Rapidase TM , Purastar TM/Effectenz TM, Powerase, Preferenz S1000, Preferenz S100 and Preferenz S110 (from Genencor International Inc. /DuPont) .
In one aspect, the amylase is selected from the group consisting of:
i) variants comprising one or more substitutions in the following positions: 9, 26, 30, 33, 82, 37, 106, 118, 128, 133, 149, 150, 160, 178, 182, 186, 193, 195, 202, 203, 214, 231, 256, 257, 258, 269, 270, 272, 283, 295, 296, 298, 299, 303, 304, 305, 311, 314, 315, 318, 319, 320, 323, 339, 345, 361, 378, 383, 419, 421, 437, 441, 444, 445, 446, 447, 450, 458, 461, 471, 482, 484, wherein the positions corresponds to positions of SEQ ID NO 11;
ii) variants exhibiting at least 90 percent identit`y with SEQ ID NO 12, with deletions in the 183 and 184 positions,
iii) variants exhibiting at least 95 percent identity with SEQ ID NO 13, comprising mutations in one or more of the following positions M202, M208, S255, R172 and/or M261,
iv) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 9 or 10,
v) a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at leaset 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%sequence identity to SEQ ID NO: 9 or 10,
and combinations thereof.
In one aspect, the protease is a peptide having at least 60%sequence identity to SEQ ID NO: 9 or 10.
In one aspect, the protease is a peptide having at least 65%sequence identity to SEQ ID NO: 9 or 10.
In one aspect, the protease is a peptide having at least 70%sequence identity to SEQ ID NO: 9 or 10.
In one aspect, the protease is a peptide having at least 75%sequence identity to SEQ ID NO: 9 or 10.
In one aspect, the protease is a peptide having at least 80%sequence identity to SEQ ID NO: 9 or 10.
In one aspect, the protease is a peptide having at least 85%sequence identity to SEQ ID NO: 9 or 10.
In one aspect, the protease is a peptide having at least 90%sequence identity to SEQ ID NO: 9 or 10.
In one aspect, the protease is a peptide having at least 95%sequence identity to SEQ ID NO: 9 or 10.
In one aspect, the protease is a peptide having at least 96%sequence identity to SEQ ID NO: 9 or 10.
In one aspect, the protease is a peptide having at least 97%sequence identity to SEQ ID NO: 9 or 10.
In one aspect, the protease is a peptide having at least 98%sequence identity to SEQ ID NO: 9 or 10.
In one aspect, the protease is a peptide having at least 99%sequence identity to SEQ ID NO: 9 or 10.
Cellulase-Polypeptides having Cellulase activity
The term "cellulase" denotes an enzyme that hydrolyses cellulose. In a preferred embodiment of the invention, the cellulase is an endoglucanase. The term “cellulase activity” is defined herein as an enzyme catalyzed hydrolysis of 1, 4-beta-D-glucosidic linkages in beta-1, 4-glucan (cellulose) . For purposes of the present invention, cellulase activity is determined using AZCL-HE-cellulose (from Megazyme) as the reaction substrate, as shown in Assay IV. Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757 and WO 89/09259.
Especially suitable cellulases are the alkaline or neutral cellulases having color care benefits. Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants  such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471, WO 98/12307 and WO99/001544.
Other cellulases are endo-beta-1, 4-glucanase enzyme having a sequence of at least 97%identity to the amino acid sequence of position 1 to position 773 of SEQ ID NO: 2 of WO 2002/099091 or a family 44 xyloglucanase, which a xyloglucanase enzyme having a sequence of at least 60%identity to positions 40-559 of SEQ ID NO: 2 of WO 2001/062903.
Commercially available cellulases include Celluzyme TM, and Carezyme TM (Novozymes A/S) , Carezyme Premium TM (Novozymes A/S) , Celluclean  TM (Novozymes A/S) , Celluclean Classic TM (Novozymes A/S) , Cellusoft TM (Novozymes A/S) , Whitezyme TM (Novozymes A/S) , Clazinase TM, and Puradax HA TM (Genencor International Inc. ) , and KAC-500 (B)  TM (Kao Corporation) , Revitalenz TM 1000, Revitalenz TM 2000, Revitalenz TM 3000 (Dupont) .
In one aspect of the present invention, it relates to the use of one or more enzymes in preventing or reducing microbe attachment onto a textile surface, wherein the enzyme is a cellulase.
In one aspect of the present invention, it relates to the use of one or more enzymes in 2 in inhibiting or reducing malodor caused by microbe on a textile, wherein the enzyme is a cellulase.
In one aspect of the present invention, the cellulase useful in the present invention comprise a polypeptide having cellulase activity, which comprise the amino acid sequence of SEQ ID NO: 15. In one aspect of the present invention, the cleaning composition comprise a polypeptide having cellulase activity, which comprises an amino acid sequence at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, even more preferably at least 97%, most preferably at least 98%, or even most preferably at least 99%, identity SEQ ID NO: 15.
In one aspect of the present invention, the amylase useful in the present invention comprise a polypeptide having cellulase activity, which comprise the amino acid sequence of SEQ ID NO: 16. In one aspect of the present invention, the cleaning composition can comprise a polypeptide having cellulase activity, which comprises an amino acid sequence at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, even more preferably at least 97%, most preferably at least 98%, or even most preferably at least 99%, identity SEQ ID NO: 16.
In one aspect of the present invention, the amylase useful in the present invention comprise a polypeptide having cellulase activity, which comprise the amino acid sequence of SEQ ID NO: 17. In one aspect of the present invention, the cleaning composition comprise a polypeptide having cellulase activity, which comprises an amino acid sequence at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, even more preferably at least 97%, most preferably at least 98%, or even most preferably at least 99%, identity SEQ ID NO: 17.
In one aspect of the present invention, the amylase useful in the present invention comprise a polypeptide having cellulase activity, which comprise the amino acid sequence of SEQ ID NO: 18. In one aspect of the present invention, the cleaning composition comprise a polypeptide having cellulase activity, which comprises an amino acid sequence at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, even more preferably at least 97%, most preferably at least 98%, or even most preferably at least 99%, identity SEQ ID NO: 18.
Detergent compositions
In one aspect, the invention is directed to use of one or more enzyme which is comprised in a detergent composition for preventing, inhibiting or reducing microbe growth on a surface, wherein after use of said enzymes said microbe on said surface have an increase in its TTC detectable time of at least 20%, at least 30%, at least 50%, at least 70%, at least 80%, or at least 100%.
The detergent compositions comprising an enzyme useful in the present invention can comprise one or more additional cleaning composition components. The choice of additional components is within the skill of the artisan and includes conventional ingredients, including the exemplary non-limiting components set forth below.
The choice of components may include, for textile care, the consideration of the type of textile to be cleaned, the type and/or degree of soiling, the temperature at which cleaning is to take place, and the formulation of the detergent product. Although components mentioned below are categorized by general header according to a particular functionality, this is not to be construed as a limitation, as a component may comprise additional functionalities as will be appreciated by the skilled artisan.
In one aspect, the invention is directed to use of one or more enzyme which is comprised in an ADW (Automatic Dish Wash) compositions in combination with one or more additional ADW composition components. The choice of additional components is within the skill of the artisan and includes conventional ingredients, including the exemplary non-limiting components set forth below.
In one aspect, the invention is directed to detergent composition comprising essentially no biocide. It is believed that by replacing biocides at least partially with enzymes thereby reducing the need of biocide, less harm brought by the biocide to the environment can be at least partially prevented.
In one aspect, the invention relates to use of one or more enzyme in preventing, inhibiting or reducing microbe growth on surfaces, wherein said microbe growth is on the residual stain area of said surface. Without being bound by theory, it is believed that the residual stains can serve as the nutrient, such as the carbon source and the nitrogen source, on which the microbe’s growth is relying.
In one aspect, the present invention relates to use of one or more enzyme in preventing, inhibiting or reducing microbe growth on surfaces, where the surface can be hard surface or soft surface, such as fabric/textile surface.
In one aspect, the surface is a textile made of nature fiber, synthetic fiber, or a mixture thereof.
In one aspect, the surface is made of cotton, polyester or blends thereof.
In one aspect, the surface is a porous surface, such as a sponge.
In one aspect, the surface is a hard surface, such as kitchen counter, chopping board and so on.
Surfactants
The detergent composition may comprise one or more surfactants, which may be anionic and/or cationic and/or non-ionic and/or semi-polar and/or zwitterionic, or a mixture thereof. In a particular aspect, the detergent composition includes a mixture of one or more nonionic surfactants and one or more anionic surfactants. The surfactant (s) is typically present at a level of from about 0.1%to 60%by weight, such as about 1%to about 40%, or about 3%to about 20%, or about 3%to about 10%. The surfactant (s) is chosen based on the desired cleaning application, and may include any conventional surfactant (s) known in the art.
When included therein the detergent will usually contain from about 1%to about 40%by weight of an anionic surfactant, such as from about 5%to about 30%, including from about 5%to  about 15%, or from about 15%to about 20%, or from about 20%to about 25%of an anionic surfactant. Non-limiting examples of anionic surfactants include sulfates and sulfonates, in particular, linear alkylbenzenesulfonates (LAS) , isomers of LAS, branched alkylbenzenesulfonates (BABS) , phenylalkanesulfonates, alpha-olefinsulfonates (AOS) , olefin sulfonates, alkene sulfonates, alkane-2, 3-diylbis (sulfates) , hydroxyalkanesulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS) , fatty alcohol sulfates (FAS) , primary alcohol sulfates (PAS) , alcohol ethersulfates (AES or AEOS or FES, also known as alcohol ethoxysulfates or fatty alcohol ether sulfates) , 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 sulfonate (MES) , alkyl-or alkenylsuccinic acid, dodecenyl/tetradecenyl succinic acid (DTSA) , fatty acid derivatives of amino acids, diesters and monoesters of sulfo-succinic acid or salt of fatty acids (soap) , and combinations thereof.
When included therein the detergent will usually contain from about from about 1%to about 40%by weigh of a cationic surfactant, for example from about 0.5%to about 30%, in particular from about 1%to about 20%, from about 3%to about 10%, such as from about 3%to about 5%, from about 8%to about 12%or from about 10%to about 12%. Non-limiting examples of cationic surfactants include alkyldimethylethanolamine quat (ADMEAQ) , cetyltrimethylammonium bromide (CTAB) , dimethyldistearylammonium chloride (DSDMAC) , and alkylbenzyldimethylammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, ester quats, and combinations thereof.
When included therein the detergent will usually contain from about 0.2%to about 40%by weight of a nonionic surfactant, for example from about 0.5%to about 30%, in particular from about 1%to about 20%, from about 3%to about 10%, such as from about 3%to about 5%, from about 8%to about 12%, or from about 10%to about 12%. Non-limiting examples of nonionic surfactants include alcohol ethoxylates (AE or AEO) , alcohol propoxylates, propoxylated fatty alcohols (PFA) , alkoxylated fatty acid alkyl esters, such as ethoxylated and/or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates (APE) , nonylphenol ethoxylates (NPE) , alkylpolyglycosides (APG) , alkoxylated amines, fatty acid monoethanolamides (FAM) , fatty acid diethanolamides (FADA) , ethoxylated fatty acid monoethanolamides (EFAM) , propoxylated fatty acid monoethanolamides (PFAM) , polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamides, GA, or fatty acid glucamides, FAGA) , as well as products available under the trade names SPAN and TWEEN, and combinations thereof.
When included therein the detergent will usually contain from about 0.5%to about 50%, preferfably from about 1%to about 25%, by weight of a semipolar surfactant. Non-limiting  examples of semipolar surfactants include amine oxides (AO) such as alkyldimethylamineoxide, N- (coco alkyl) -N, N-dimethylamine oxide and N- (tallow-alkyl) -N, N-bis (2-hydroxyethyl) amine oxide, , and combinations thereof.
When included therein the detergent will usually contain from about 0.5%to about 50%, preferfably from about 1%to about 25%, by weight of a zwitterionic surfactant. Non-limiting examples of zwitterionic surfactants include betaines such as alkyldimethylbetaines, sulfobetaines, and combinations thereof.
Hydrotropes
The detergent may contain 0-10%by weight, for example 0-5%by weight, such as about 0.5 to about 5%, or about 3%to about 5%, of a hydrotrope. Any hydrotrope known in the art for use in detergents may be utilized. Non-limiting examples of hydrotropes include sodium benzenesulfonate, sodium p-toluene sulfonate (STS) , sodium xylene sulfonate (SXS) , sodium cumene sulfonate (SCS) , sodium cymene sulfonate, amine oxides, alcohols and polyglycolethers, sodium hydroxynaphthoate, sodium hydroxynaphthalene sulfonate, sodium ethylhexyl sulfate, and combinations thereof.
Builders and Co-Builders
The detergent composition may contain about 0-65%by weight, such as about 5%to about 50% of a detergent builder or co-builder, or a mixture thereof. In a dish wash detergent, the level of builder is typically 40-65%, particularly 50-65%. The builder and/or co-builder may particularly be a chelating agent that forms water-soluble complexes with Ca and Mg. Any builder and/or co-builder known in the art for use in laundry/ADW/hard surface cleaning detergents may be utilized. Non-limiting examples of builders include zeolites, diphosphates (pyrophosphates) , triphosphates 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) , ethanolamines such as 2-aminoethan-1-ol (MEA) , diethanolamine (DEA, also known as 2, 2’-iminodiethan-1-ol) , triethanolamine (TEA, also known as 2, 2’, 2”-nitrilotriethan-1-ol) , and (carboxymethyl) inulin (CMI) , and combinations thereof.
The detergent composition may also contain 0-50%by weight, such as about 5%to about 30%, of a detergent co-builder. The detergent composition may include include a co-builder alone, or in combination with a builder, for example a zeolite builder. Non-limiting examples of co-builders include homopolymers of polyacrylates or copolymers thereof, such as poly (acrylic acid) (PAA) or copoly (acrylic acid/maleic acid) (PAA/PMA) . Further non-limiting examples include citrate,  chelators such as aminocarboxylates, aminopolycarboxylates and phosphonates, and alkyl-or alkenylsuccinic acid. Additional 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-diphosphonic acid (HEDP) , ethylenediaminetetra (methylenephosphonic acid) (EDTMPA) , diethylenetriaminepentakis (methylenephosphonic acid) (DTMPA or 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-N, N-diacetic acid (SMDA) , N- (2-hydroxyethyl) ethylenediamine-N, N’, N”-triacetic acid (HEDTA) , diethanolglycine (DEG) , diethylenetriamine penta (methylenephosphonic acid) (DTPMP) , aminotris (methylenephosphonic acid) (ATMP) , and combinations and salts thereof. Further exemplary builders and/or co-builders are described in, e.g., WO 09/102854, US 5977053.
Bleaching Systems
The detergent composition may contain 0-30%by weight, such as about 1%to about 20%, of a bleaching system. Any bleaching system comprising components known in the art for use in cleaning detergents may be utilized. Suitable bleaching system components include sources of hydrogen peroxide; sources of peracids; and bleach catalysts or boosters.
·Sources of hydrogen peroxide
Suitable sources of hydrogen peroxide are inorganic persalts, including alkali metal salts such as sodium percarbonate and sodium perborates (usually mono-or tetrahydrate) , and hydrogen peroxide-urea (1/1) .
·Sources of peracids
Peracids may be (a) incorporated directly as preformed peracids or (b) formed in situ in the wash liquor from hydrogen peroxide and a bleach activator (perhydrolysis) or (c) formed in situ in the wash liquor from hydrogen peroxide and a perhydrolase and a suitable substrate for the latter, e.g., an ester.
a) Suitable preformed peracids include, but are not limited to, peroxycarboxylic acids such as peroxybenzoic acid and its ring-substituted derivatives, peroxy-α-naphthoic acid, peroxyphthalic acid, peroxylauric acid, peroxystearic acid, ε-phthalimidoperoxycaproic acid [phthalimidoperoxyhexanoic acid (PAP) ] , and o-carboxybenzamidoperoxycaproic acid; aliphatic and aromatic diperoxydicarboxylic acids such as diperoxydodecanedioic acid, diperoxyazelaic acid, diperoxysebacic acid, diperoxybrassylic acid, 2-decyldiperoxybutanedioic acid, and diperoxyphthalic, -isophthalic and -terephthalic acids; perimidic acids; peroxymonosulfuric acid; peroxydisulfuric acid; peroxyphosphoric acid; peroxysilicic acid; and mixtures of said compounds. It is understood that the peracids mentioned may in some cases be best added as suitable salts, such as alkali metal salts (e.g., 
Figure PCTCN2018078160-appb-000027
) or alkaline earth-metal salts.
b) Suitable bleach activators include those belonging to the class of esters, amides, imides, nitriles or anhydrides and, where applicable, salts thereof. Suitable examples are tetraacetylethylenediamine (TAED) , sodium 4- [ (3, 5, 5-trimethylhexanoyl) oxy] benzene-1-sulfonate (ISONOBS) , sodium 4- (dodecanoyloxy) benzene-1-sulfonate (LOBS) , sodium 4- (decanoyloxy) benzene-1-sulfonate, 4- (decanoyloxy) benzoic acid (DOBA) , sodium 4- (nonanoyloxy) benzene-1-sulfonate (NOBS) , and/or those disclosed in WO98/17767. A particular family of bleach activators of interest was disclosed in EP624154 and particularly preferred in that family is acetyl triethyl citrate (ATC) . ATC or a short chain triglyceride like triacetin has the advantage that they are environmentally friendly. Furthermore acetyl triethyl citrate and triacetin have good hydrolytically stability in the product upon storage and are efficient bleach activators. Finally ATC is multifunctional, as the citrate released in the perhydrolysis reaction may function as a builder.
·Bleach catalysts and boosters
The bleaching system may also include a bleach catalyst or booster. Some non-limiting examples of bleach catalysts that may be used in the compositions of the present invention include manganese oxalate, manganese acetate, manganese-collagen, cobalt-amine catalysts and manganese triazacyclononane (MnTACN) catalysts; particularly preferred are complexes of manganese with 1, 4, 7-trimethyl-1, 4, 7-triazacyclononane (Me3-TACN) or 1, 2, 4, 7-tetramethyl-1, 4, 7-triazacyclononane (Me4-TACN) , in particular Me3-TACN, such as the dinuclear manganese complex [ (Me3-TACN) Mn (O) 3Mn (Me3-TACN) ] (PF6) 2, and [2, 2', 2”-nitrilotris (ethane-1, 2-diylazanylylidene-κN-methanylylidene) triphenolato-κ3O] manganese (III) . The bleach catalysts may also be other metal compounds, such as iron or cobalt complexes.
In some embodiments, where a source of a peracid is included, an organic bleach catalyst or bleach booster may be used having one of the following formulae:
Figure PCTCN2018078160-appb-000028
(iii) and mixtures thereof; wherein each R1 is independently a branched alkyl group containing from 9 to 24 carbons or linear alkyl group containing from 11 to 24 carbons, preferably each R1 is independently a branched alkyl group containing from 9 to 18 carbons or linear alkyl group containing from 11 to 18 carbons, more preferably each R1 is independently selected from the group consisting of 2-propylheptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, dodecyl, tetradecyl, hexadecyl, octadecyl, isononyl, isodecyl, isotridecyl and isopentadecyl.
Other exemplary bleaching systems are described, e.g. in WO2007/087258, WO2007/087244, WO2007/087259, EP1867708 (Vitamin K) and WO2007/087242. Suitable photobleaches may for example be sulfonated zinc or aluminium phthalocyanines.
Polymers
The detergent may contain 0-10%by weight, such as 0.5-5%, 2-5%, 0.5-2%or 0.2-1%of a polymer. Any polymer known in the art for use in detergents may be utilized. The polymer may function as a co-builder as mentioned above, or may provide antiredeposition, fiber protection, soil release, dye transfer inhibition, grease cleaning and/or anti-foaming properties. Some polymers may have more than one of the above-mentioned properties and/or more than one of the below-mentioned motifs. Exemplary polymers include (carboxymethyl) cellulose (CMC) , poly (vinyl alcohol) (PVA) , poly (vinylpyrrolidone) (PVP) , poly (ethyleneglycol) or poly (ethylene oxide) (PEG) , ethoxylated poly (ethyleneimine) , carboxymethyl inulin (CMI) , and polycarboxylates such as PAA, PAA/PMA, poly-aspartic acid, and lauryl methacrylate/acrylic acid copolymers , hydrophobically modified CMC (HM-CMC) and silicones, copolymers of terephthalic acid and oligomeric glycols, copolymers of poly (ethylene terephthalate) and poly (oxyethene terephthalate) (PET-POET) , PVP, poly (vinylimidazole) (PVI) , poly (vinylpyridine-N-oxide) (PVPO or PVPNO) and polyvinylpyrrolidone-vinylimidazole (PVPVI) . Further exemplary polymers include sulfonated polycarboxylates, polyethylene oxide and polypropylene oxide (PEO-PPO) and diquaternium ethoxy sulfate. Other exemplary polymers are disclosed in, e.g., WO 2006/130575. Salts of the above-mentioned polymers are also contemplated.
Fabric hueing agents
The detergent compositions may also include fabric hueing agents such as dyes or pigments, which when formulated in detergent compositions can deposit onto a fabric when said fabric is contacted with a wash liquor comprising said detergent compositions and thus altering the tint of said fabric through absorption/reflection of visible light. Fluorescent whitening agents emit at least some visible light. In contrast, fabric hueing agents alter the tint of a surface as they absorb at least a portion of the visible light spectrum. Suitable fabric hueing agents include dyes and dye-clay conjugates, and may also include pigments. Suitable dyes include small molecule dyes and polymeric dyes. Suitable small molecule dyes include small molecule dyes selected from the group consisting of dyes falling into the Colour Index (C. I. ) classifications of Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, or mixtures thereof, for example as described in WO2005/03274, WO2005/03275, WO2005/03276 and EP1876226 (hereby incorporated by reference) . The detergent composition preferably comprises from about 0.00003 wt%to about 0.2 wt%, from about 0.00008 wt%to about 0.05 wt%, or even from about 0.0001 wt%to about 0.04 wt%fabric hueing agent. The composition may comprise from 0.0001 wt%to 0.2 wt%fabric hueing agent, this may be especially preferred when the composition is in the form of a unit dose pouch. Suitable hueing agents are also disclosed in, e.g. WO 2007/087257 and WO2007/087243.
Enzymes
The detergent additive as well as the detergent composition may comprise one or more additional enzymes such as a protease, lipase, cutinase, an amylase, carbohydrase, cellulase, pectinase, mannanase, arabinase, galactanase, xylanase, oxidase, e.g., a laccase, and/or peroxidase.
In general the properties of the selected enzyme (s) should be compatible with the selected detergent, (i.e., pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc. ) , and the enzyme (s) should be present in effective amounts.
Cellulases
Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757 and WO 89/09259.
Especially suitable cellulases are the alkaline or neutral cellulases having colour care benefits. Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471, WO 98/12307 and WO99/001544.
Other cellulases are endo-beta-1, 4-glucanase enzyme having a sequence of at least 97%identity to the amino acid sequence of position 1 to position 773 of SEQ ID NO: 2 of WO 2002/099091 or a family 44 xyloglucanase, which a xyloglucanase enzyme having a sequence of at least 60%identity to positions 40-559 of SEQ ID NO: 2 of WO 2001/062903.
Commercially available cellulases include Celluzyme TM, and Carezyme TM (Novozymes A/S) Carezyme Premium TM (Novozymes A/S) , Celluclean  TM (Novozymes A/S) , Celluclean Classic TM (Novozymes A/S) , Cellusoft TM (Novozymes A/S) , Whitezyme TM (Novozymes A/S) , Clazinase TM, and Puradax HA TM (Genencor International Inc. ) , and KAC-500 (B)  TM (Kao Corporation) .
Suitable cellulases include complete cellulases or mono-component endoglucanases of bacterial or fungal origin. Chemically or genetically modified mutants are included. The cellulase may for example be a mono-component or a mixture of mono-component endo-1, 4-beta-glucanase often just termed endoglucanases. Suitable cellulases include a fungal cellulase from Humicola insolens (US 4,435,307) or from Trichoderma, e.g. T. reesei or T. viride. Examples of cellulases are described in EP 0 495 257. Other suitable cellulases are from Thielavia e.g. Thielavia terrestris as described in WO 96/29397 or Fusarium oxysporum as described in WO 91/17244 or from Bacillus as described in, WO 02/099091 and JP 2000210081. Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471, WO 98/12307 Commercially available cellulases include
Figure PCTCN2018078160-appb-000029
and
Figure PCTCN2018078160-appb-000030
Figure PCTCN2018078160-appb-000031
 (Novozymes A/S) 
Figure PCTCN2018078160-appb-000032
Puradax HA, and Puradax EG (available from Genencor) .
Mannanases
Suitable mannanases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included. The mannanase may be an alkaline mannanase of Family 5 or 26. It may be a wild-type from Bacillus or Humicola, particularly B. agaradhaerens, B. licheniformis, B. halodurans, B. clausii, or H. insolens. Suitable mannanases are described in WO 1999/064619. A commercially available mannanase is Mannaway (Novozymes A/S) .
Lipases and Cutinases:
Suitable lipases and cutinases include those of bacterial or fungal origin. Chemically modified or protein engineered mutant enzymes are included. Examples include lipase from Thermomyces, e.g. from T. lanuginosus (previously named Humicola lanuginosa) as described in EP258068 and EP305216, cutinase from Humicola, e.g. H. insolens (WO96/13580) , lipase from strains of Pseudomonas (some of these now renamed to Burkholderia) , e.g. P. alcaligenes or P. pseudoalcaligenes (EP218272) , P. cepacia (EP331376) , P. sp. strain SD705 (WO95/06720 &WO96/27002) , P. wisconsinensis (WO96/12012) , GDSL-type Streptomyces lipases (WO10/065455) , cutinase from Magnaporthe grisea (WO10/107560) , cutinase from Pseudomonas mendocina (US5,389,536) , lipase from Thermobifida fusca (WO11/084412) , Geobacillus stearothermophilus lipase (WO11/084417) , lipase from Bacillus subtilis (WO11/084599) , and lipase from Streptomyces griseus (WO11/150157) and S. pristinaespiralis (WO12/137147) .
Other examples are lipase variants such as those described in EP407225, WO92/05249, WO94/01541, WO94/25578, WO95/14783, WO95/30744, WO95/35381, WO95/22615, WO96/00292, WO97/04079, WO97/07202, WO00/34450, WO00/60063, WO01/92502, WO07/87508 and WO09/109500.
Preferred commercial lipase products include include Lipolase TM, Lipex TM; Lipolex TM and Lipoclean TM (Novozymes A/S) , Lumafast (originally from Genencor) and Lipomax (originally from Gist-Brocades) .
Still other examples are lipases sometimes referred to as acyltransferases or perhydrolases, e.g. acyltransferases with homology to Candida antarctica lipase A (WO10/111143) , acyltransferase from Mycobacterium smegmatis (WO05/56782) , perhydrolases from the CE 7 family (WO09/67279) , and variants of the M. smegmatis perhydrolase in particular the S54V variant used in the commercial product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd (WO10/100028) .
Suitable peroxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinopsis, e.g., from C. cinerea (EP 179, 486) , and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257.
A peroxidase according to the invention also include a haloperoxidase enzyme, such as chloroperoxidase, bromoperoxidase and compounds exhibiting chloroperoxidase or bromoperoxidase activity. Haloperoxidases are classified according to their specificity for halide ions. Chloroperoxidases (E.C. 1.11.1.10) catalyze formation of hypochlorite from chloride ions.
In an aspect, the haloperoxidase of the invention is a chloroperoxidase. Preferably, the haloperoxidase is a vanadium haloperoxidase, i.e., a vanadate-containing haloperoxidase. In a preferred method of the present invention the vanadate-containing haloperoxidase is combined with a source of chloride ion.
Haloperoxidases have been isolated from many different fungi, in particular from the fungus group dematiaceous hyphomycetes, such as Caldariomyces, e.g., C. fumago, Alternaria, Curvularia, e.g., C. verruculosa and C. inaequalis, Drechslera, Ulocladium and Botrytis.
Haloperoxidases have also been isolated from bacteria such as Pseudomonas, e.g., P. pyrrocinia and Streptomyces, e.g., S. aureofaciens.
In an preferred aspect, the haloperoxidase is derivable from Curvularia sp., in particular Curvularia verruculosa or Curvularia inaequalis, such as C. inaequalis CBS 102.42 as described in WO 95/27046; or C. verruculosa CBS 147.63 or C. verruculosa CBS 444.70 as described in WO 97/04102; or from Drechslera hartlebii as described in WO 01/79459, Dendryphiella salina as described in WO 01/79458, Phaeotrichoconis crotalarie as described in WO 01/79461, or Geniculosporium sp. as described in WO 01/79460.
An oxidase according to the invention include, in particular, any laccase enzyme comprised by the enzyme classification EC 1.10.3.2, or any fragment derived therefrom exhibiting laccase activity, or a compound exhibiting a similar activity, such as a catechol oxidase (EC 1.10.3.1) , an o-aminophenol oxidase (EC 1.10.3.4) , or a bilirubin oxidase (EC 1.3.3.5) .
Preferred laccase enzymes are enzymes of microbial origin. The enzymes may be derived from plants, bacteria or fungi (including filamentous fungi and yeasts) .
Suitable examples from fungi include a laccase derivable from a strain of Aspergillus, Neurospora, e.g., N. crassa, Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus, Trametes, e.g., T. villosa and T. versicolor, Rhizoctonia, e.g., R. solani, Coprinopsis, e.g., C. cinerea, C. comatus, C. friesii, and C. plicatilis, Psathyrella, e.g., P. condelleana, Panaeolus, e.g., P. papilionaceus, Myceliophthora, e.g., M. thermophila, Schytalidium, e.g., S. thermophilum, Polyporus, e.g., P. pinsitus, Phlebia, e.g., P. radiata (WO 92/01046) , or Coriolus, e.g., C. hirsutus (JP 2238885) .
Suitable examples from bacteria include a laccase derivable from a strain of Bacillus.
A laccase derived from Coprinopsis or Myceliophthora is preferred; in particular a laccase derived from Coprinopsis cinerea, as disclosed in WO 97/08325; or from Myceliophthora thermophila, as disclosed in WO 95/33836.
The detergent enzyme (s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes. A detergent additive of the invention, i.e., a separate additive or a combined additive, can be formulated, for example, as a granulate, liquid, slurry, etc. Preferred detergent additive formulations are granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries.
Non-dusting granulates may be produced, e.g. as disclosed in US 4,106,991 and 4,661,452 and may optionally be coated by methods known in the art. Examples of waxy coating materials are polyethyleneglycol (PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono-and di-and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591. Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Protected enzymes may be prepared according to the method disclosed in EP 238, 216.
Microorgansims
The detergent additive as well as the detergent composition may also comprise one or more microorganisms, such as one or more fungi, yeast, or bacteria.
In an aspect, the one or more microorganisms are dehydrated (for example by lyophilization) bacteria or yeast, such as a strain of Lactobacillus.
In another aspect, the microrganisms are one or more microbial spores (as opposed to vegetative cells) , such as bacterial spores; or fungal spores, conidia, hypha. Preferably, the one or more spores are Bacillus endospores; even more preferably the one or more spores are endospores of Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, or Bacillus megaterium.
The microrganisms may be included in the detergent composition or additive in the same way as enzymes (see above) .
Adjunct materials
Any detergent components known in the art for use in laundry/ADW/hard surface cleaning detergents may also be utilized. Other optional detergent components include anti-corrosion agents, anti-shrink agents, anti-soil redeposition agents, anti-wrinkling agents, bactericides,  binders, corrosion inhibitors, disintegrants/disintegration agents, dyes, enzyme stabilizers (including boric acid, borates, CMC, and/or polyols such as propylene glycol) , fabric conditioners including clays, fillers/processing aids, fluorescent whitening agents/optical brighteners, foam boosters, foam (suds) regulators, perfumes, soil-suspending agents, softeners, suds suppressors, tarnish inhibitors, and wicking agents, either alone or in combination. Any ingredient known in the art for use in laundry/ADW/hard surface cleaning detergents may be utilized. The choice of such ingredients is well within the skill of the artisan.
Dispersants
The detergent compositions useful in the present invention can also contain dispersants. In particular powdered detergents may comprise dispersants. Suitable water-soluble organic materials include the homo-or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms. Suitable dispersants are for example described in Powdered Detergents, Surfactant science series volume 71, Marcel Dekker, Inc.
Dye Transfer Inhibiting Agents
The detergent compositions useful in the present invention may also include one or more dye transfer inhibiting agents. Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof. When present in a subject composition, the dye transfer inhibiting agents may be present at levels from about 0.0001 %to about 10%, from about 0.01%to about 5%or even from about 0.1%to about 3%by weight of the composition.
Fluorescent whitening agent
The detergent compositions useful in the present invention will preferably also contain additional components that may tint articles being cleaned, such as fluorescent whitening agent or optical brighteners. Where present the brightener is preferably at a level of about 0.01%to about 0.5%. Any fluorescent whitening agent suitable for use in a laundry detergent composition may be used in the composition of the present invention. The most commonly used fluorescent whitening agents are those belonging to the classes of diaminostilbene-sulfonic acid derivatives, diarylpyrazoline derivatives and bisphenyl-distyryl derivatives. Examples of the diaminostilbene-sulfonic acid derivative type of fluorescent whitening agents include the sodium salts of: 4, 4'-bis-  (2-diethanolamino-4-anilino-s-triazin-6-ylamino) stilbene-2, 2'-disulfonate, 4, 4'-bis- (2, 4-dianilino-s-triazin-6-ylamino) stilbene-2.2'-disulfonate, 4, 4'-bis- (2-anilino-4- (N-methyl-N-2-hydroxy-ethylamino) -s-triazin-6-ylamino) stilbene-2, 2'-disulfonate, 4, 4'-bis- (4-phenyl-1, 2, 3-triazol-2-yl) stilbene-2, 2'-disulfonate and sodium 5- (2H-naphtho [1, 2-d] [1, 2, 3] triazol-2-yl) -2- [ (E) -2-phenylvinyl] benzenesulfonate. Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBS available from Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is the disodium salt of 4, 4'-bis- (2-morpholino-4-anilino-s-triazin-6-ylamino) stilbene-2, 2'-disulfonate. Tinopal CBS is the disodium salt of 2, 2'-bis- (phenyl-styryl) -disulfonate. Also preferred are fluorescent whitening agents is the commercially available Parawhite KX, supplied by Paramount Minerals and Chemicals, Mumbai, India. Other fluorescers suitable for use in the invention include the 1-3-diaryl pyrazolines and the 7-alkylaminocoumarins.
Suitable fluorescent brightener levels include lower levels of from about 0.01, from 0.05, from about 0.1 or even from about 0.2 wt %to upper levels of 0.5 or even 0.75 wt%.
Soil release polymers
The detergent compositions useful in the present invention may also include one or more soil release polymers which aid the removal of soils from fabrics such as cotton and polyester based fabrics, in particular the removal of hydrophobic soils from polyester based fabrics. The soil release polymers may for example be nonionic or anionic terephthalte based polymers, polyvinyl caprolactam and related copolymers, vinyl graft copolymers, polyester polyamides see for example Chapter 7 in Powdered Detergents, Surfactant science series volume 71, Marcel Dekker, Inc. Another type of soil release polymers are amphiphilic alkoxylated grease cleaning polymers comprising a core structure and a plurality of alkoxylate groups attached to that core structure. The core structure may comprise a polyalkylenimine structure or a polyalkanolamine structure as described in detail in WO 2009/087523 (hereby incorporated by reference) . Furthermore random graft co-polymers are suitable soil release polymers. Suitable graft co-polymers are described in more detail in WO 2007/138054, WO 2006/108856 and WO 2006/113314 (hereby incorporated by reference) . Other soil release polymers are substituted polysaccharide structures especially substituted cellulosic structures such as modified cellulose deriviatives such as those described in EP 1867808 or WO 2003/040279 (both are hereby incorporated by reference) . Suitable cellulosic polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides and mixtures thereof. Suitable cellulosic polymers include anionically modified cellulose, nonionically modified cellulose, cationically modified cellulose, zwitterionically modified cellulose, and mixtures thereof. Suitable  cellulosic polymers include methyl cellulose, carboxy methyl cellulose, ethyl cellulose, hydroxyl ethyl cellulose, hydroxyl propyl methyl cellulose, ester carboxy methyl cellulose, and mixtures thereof.
Anti-redeposition agents
The detergent compositions useful in the present invention may also include one or more anti-redeposition agents such as carboxymethylcellulose (CMC) , polyvinyl alcohol (PVA) , polyvinylpyrrolidone (PVP) , polyoxyethylene and/or polyethyleneglycol (PEG) , homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimines. The cellulose based polymers described under soil release polymers above may also function as anti-redeposition agents.
Rheology Modifiers
The detergent compositions useful in the present invention may also include one or more rheology modifiers, structurants or thickeners, as distinct from viscosity reducing agents. The rheology modifiers are selected from the group consisting of non-polymeric crystalline, hydroxy-functional materials, polymeric rheology modifiers which impart shear thinning characteristics to the aqueous liquid matrix of a liquid detergent composition. The rheology and viscosity of the detergent can be modified and adjusted by methods known in the art, for example as shown in EP 2169040.
Other suitable adjunct materials include, but are not limited to, anti-shrink agents, anti-wrinkling agents, bactericides, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, hydrotropes, perfumes, pigments, sod suppressors, solvents, and structurants for liquid detergents and/or structure elasticizing agents.
Formulation of detergent products
The detergent compositions useful in the present inventionmay be in any convenient form, e.g., a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid.
Pouches can be configured as single or multicompartments. It can be of any form, shape and material which is suitable for hold the composition, e.g. without allowing the release of the composition to release of the composition from the pouch prior to water contact. The pouch is made from water soluble film which encloses an inner volume. Said inner volume can be divided  into compartments of the pouch. Preferred films are polymeric materials preferably polymers which are formed into a film or sheet. Preferred polymers, copolymers or derivates thereof are selected polyacrylates, and water soluble acrylate copolymers, methyl cellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, malto dextrin, poly methacrylates, most preferably polyvinyl alcohol copolymers and, hydroxypropyl methyl cellulose (HPMC) . Preferably the level of polymer in the film for example PVA is at least about 60%. Preferred average molecular weight will typically be about 20,000 to about 150,000. Films can also be of blended compositions comprising hydrolytically degradable and water soluble polymer blends such as polylactide and polyvinyl alcohol (known under the Trade reference M8630 as sold by MonoSol LLC, Indiana, USA) plus plasticisers like glycerol, ethylene glycerol, propylene glycol, sorbitol and mixtures thereof. The pouches can comprise a solid laundry cleaning composition or part components and/or a liquid cleaning composition or part components separated by the water soluble film. The compartment for liquid components can be different in composition than compartments containing solids: US2009/0011970 A1.
Detergent ingredients can be separated physically from each other by compartments in water dissolvable pouches or in different layers of tablets. Thereby negative storage interaction between components can be avoided. Different dissolution profiles of each of the compartments can also give rise to delayed dissolution of selected components in the wash solution.
A liquid or gel detergent , which is not unit dosed, may be aqueous, typically containing at least 20%by weight and up to 95%water, such as up to about 70%water, up to about 65%water, up to about 55%water, up to about 45%water, up to about 35%water. Other types of liquids, including without limitation, alkanols, amines, diols, ethers and polyols may be included in an aqueous liquid or gel. An aqueous liquid or gel detergent may contain from 0-30%organic solvent.
A liquid or gel detergent may be non-aqueous.
Formulation of enzyme in co-granule
The enzyme useful in the present invention for preventing microbe growth on household surfaces may be formulated as a granule for example as a co-granule that combines one or more enzymes. Each enzyme will then be present in more granules securing a more uniform distribution of enzymes in the detergent. This also reduces the physical segregation of different enzymes due to different particle sizes. Methods for producing multi-enzyme co-granulates for the detergent industry are disclosed in the IP. com disclosure IPCOM000200739D.
Another example of formulation of enzymes by the use of co-granulates are disclosed in WO 2013/188331, which relates to a detergent composition comprising (a) a multi-enzyme co- granule; (b) less than 10 wt zeolite (anhydrous basis) ; and (c) less than 10 wt phosphate salt (anhydrous basis) , wherein said enzyme co-granule comprises from 10 to 98 wt%moisture sink component and the composition additionally comprises from 20 to 80 wt%detergent moisture sink component. WO 2013/188331 also relates to a method of treating and/or cleaning a surface, preferably a fabric surface comprising the steps of (i) contacting said surface with the detergent composition as claimed and described herein in an aqueous wash liquor, (ii) rinsing and/or drying the surface.
The multi-enzyme co-granule may comprise an enzyme of the invention and (a) one or more enzymes selected from the group consisting of first-wash lipases, cleaning cellulases, xyloglucanases, perhydrolases, peroxidases, lipoxygenases, laccases and mixtures thereof; and (b) one or more enzymes selected from the group consisting of hemicellulases, proteases, care cellulases, cellobiose dehydrogenases, xylanases, phospho lipases, esterases, cutinases, pectinases, mannanases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, ligninases, pullulanases, tannases, pentosanases, lichenases glucanases, arabinosidases, hyaluronidase, chondroitinase, amylases, and mixtures thereof.
Methods and Uses
In another aspect of the present invention, it relates to a method of preventing, inhibiting or reducing microbe growth on surface, by treating the surface with enzymes defined in previous paragraphs and aspects under Enzyme of the Present Invention section to the extent that the microbe TTC detectable time on said surface after the treatment increases at least 20%.
In one aspect, it relates to the method mentioned above, wherein the microbe is a fungi, and the fungi on said surface has an increase of spore forming time of at least 20%after the surface is treated by the enzyme useful in the present invention.
In one aspect, it relates to the method mentioned above, wherein the microbe is a fungi, and the fungi has a decrease of spore density score of at least 1 in a scale of from 0 to 7 on said surface after the surface is treated by the enzyme useful in the present invention.
In another aspect of the present invention, it relates to a method of demonstrating the microbe growth inhibition or deep cleansing benefit of an enzyme as defined in previous paragraphs under the section name of Enzyme of the Present Invention on a surface, comprising the steps of:
a. Providing two surfaces A and A’,
b. Applying a soil comprising a microbe growth indicator onto each of the surfaces A and A’,
c. Washing A with a detergent composition comprising no enzyme, and washing A’with said detergent composition comprising one or more enzymes,
d. Preparing an inoculation of microbe and apply it onto the surfaces A and A’,
e. Incubating A and A’under suitable conditions to allow microbe growth for a predetermined period of time,
f. Comparing the growth of microbe on A and A’.
The surface mentioned here can be any household surface. can be hard surface or soft surface, such as fabric/textile surface. In one aspect, the surface is a textile made of nature fiber, synthetic fiber, or a mixture thereof. In one aspect, the surface is made of cotton, polyester or blends thereof.
In one aspect, it relates to the method mentioned in previous aspects, wherein the microbe growth indicator is selected from a group consisting of triphenyltetrazolium chloride (TTC) , INT (2- (4-iodophenyl) -3- (4-nitrophenyl) -5-phenyl-2H-tetrazolium) , MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) , XTT (2, 3-bis- (2-methoxy-4-nitro-5-sulfophenyl) -2H-tetrazolium-5-carboxanilide) , MTS (3- (4, 5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium) , WSTs (Water-soluble Tetrazolium salts) and CCK-8 (Cell Counting Kit-8) , carotenoid, luciferin, fluorescent protein/fluorescent producing microorganisms, ATP, X-Gal, MUG and combinations thereof. In one aspect, the microbe growth indicator is triphenyltetrazolium chloride (TTC) .
In one aspect, it relates to the method mentioned in previous aspects, wherein the surfaces A and A’provided in step a. described in previous aspects are made of essentially the same material.
Commerical detergent compositions and textile pretreatment compositions
The below mentioned detergent compositions can incorporate the enzyme suitable for the use and method of the present invention.
Liquid Detergent: Model A
Figure PCTCN2018078160-appb-000033
Liquid Detergent: Model N
Figure PCTCN2018078160-appb-000034
Liquid Detergent: Model O
Figure PCTCN2018078160-appb-000035
Powder Detergent: Model X
Figure PCTCN2018078160-appb-000036
Liquid Detergent: Model SEA
Figure PCTCN2018078160-appb-000037
Biotex black (liquid)
5-15%Anionic surfactants, <5%Nonionic surfactants, perfume, enzymes, DMDM and hydantoin.
Composition of Ariel Sensitive White &Color, liquid detergent composition
Aqua, Alcohol Ethoxy Sulfate, Alcohol Ethoxylate, Amino Oxide, Citric Acid, C12-18 topped palm kernel fatty acid, Protease, Glycosidase, Amylase, Ethanol, 1, 2 Propanediol, Sodium Formate, Calcium Chloride, Sodium hydroxide, Silicone Emulsion, Trans-sulphated EHDQ (the ingredients are listed in descending order) .
Composition of model detergent A (liquid)
Ingredients: 12%LAS, 11%AEO Biosoft N25-7 (NI) , 7%AEOS (SLES) , 6%MPG (monopropylene glycol) , 3%ethanol, 3%TEA, 2.75%cocoa soap, 2.75%soya soap, 2%glycerol, 2%sodium hydroxide, 2%sodium citrate, 1%sodium formiate, 0.2%DTMPA and 0.2%PCA (all percentages are w/w) .
Persil Biological Tablets
Sodium carbonate, Sodium Carbonate Peroxide, Sodium bicarbonate, Zeolite, Aqua, Sodium Silicate, Sodium Lauryl Sulfate, Cellulose, TAED, Sodium Dodecylbenzenesulfonate, Hemicellulose, Lignin, Lauryl Glucoside, Sodium Acrylic Acid/MA Copolymer, Bentonite, Sodium chloride, Perfume, Tetrasodium Etidronate, Sodium sulfate, Sodium Polyacrylate, Dimethicone, Disodium Anilinomorpholinotriazinylaminostilbenesulfonate, Dodecylbenzene Sulfonic Acid, Trimethylsiloxysilicate, Calcium carbonate, Cellulose, PEG-75, Titanium dioxide, Dextrin, Protease, Corn Starch Modified, Sucrose, CI 12490, Sodium Polyaryl Sulphonate, Sodium Thiosulfate, Amylase, Kaolin.
Composition of Ariel Actilift (powder)
Ingredients: 5-15%Anionic surfactants, Oxygen-based bleaching agents, <5%Non-ionic surfactants, Phosphonates, Polycarboxylates, Zeolites, Optical brighteners, Enzymes, Perfumes, Butylphenyl Methylpropional, Coumarin, Hexyl Cinnamal.
The present invention can also be described in the following paragraphs:
1. Use of one or more enzymes in preventing, inhibiting or reducing microbe growth on a surface, wherein after use of said enzymes, said microbe on said surface have an  increase in TTC detectable time of at least 20%, at least 30%, at least 50%, at least 70%, at least 80%, or at least 100%.
2. The use according to paragraph 1, wherein the microbe is a fungi, and the fungi has an increase in the spore forming time of at least 20%, and/or an decrease in spore density score of at least 1 in a spore density scale of from 0 to 7, compared to those on a surface not treated with enzymes.
3. The use of paragraph 1 or 2 in inhibiting or reducing malodor caused by microbe on a textile surface.
4. The use of paragraph 1 or 2 in preventing or reducing microbe attachment onto a textile surface.
5. The use according to any of paragraphs 1-4, wherein the microbe is a fungi or bacteria selected from a group consisting of Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Pseudomonas putida, Micrococcus luteus, Staphylococcus epidermidis, Enhydrobacter aerosaccus, Corynebacterium jeikeium, Propionibacterium acnes, Brevundimonas vesicularis, Streptomyces griseus, Streptomyces odorifer, Exophiala phaeomuriformis, Fusarium oxysporum, Alternaria alternate, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aureobasidium pullulans, Chaetomium globosum, Cladosporium sphaerospermum, Gliocladium virens, Mucor plumbeus, Penicillium chrysogenum, Penicillium commune, Penicillium crustosum, Penicillium rubens, Penicillium varians, Penicillium citrinum, Stachybotrys chartarum, Trichoderma viride, Trichophyton rubrum, Candida parapsilosis, Rhodotorula mucilaginosa, Debaryomyces hansenii, Meyerozyma guilliermondii, Candida albicans, and combinations thereof.
6. The use according to paragraph 1-5, wherein said microbe growth is on the residual stain area of said surface.
7. The use according to any of paragraphs 1-6, wherein said one or more enzymes are selected from a group consisting of amylase, protease, lipase, mannase, cellulase, pectinase, and combinations thereof.
8. The use according to paragraph any of paragraphs 1-7, wherein the enzyme is an amylase, protease, cellulase or combinations thereof.
9. The use according to paragraph 3 or 4, wherein the enzyme is a cellulase.
10. The use according to any of the above paragraphs, wherein the protease is selected from a group consisting of:
i) a protease comprising a substitution compared to the protease shown in SEQ ID NO 1 or SEQ ID NO 2 in one or more of the following positions: 3, 4, 9, 15, 24, 27, 42, 55, 59, 60, 66, 74, 85, 96, 97, 98, 99, 100, 101, 102, 104, 116, 118, 121, 126, 127, 128, 154, 156, 157, 158, 161, 164, 176, 179, 182, 185, 188, 189, 193, 198, 199, 200, 203, 206, 211, 212, 216, 218, 226, 229, 230, 239, 246, 255, 256, 268 and 269, wherein the positions correspond to the positions of the protease shown in SEQ ID NO 1, or
ii) one or more protease variant of a protease parent, wherein the protease variant comprises one or more mutation selected from the group consisting of S3T, V4I, S9R, S9E, A15T, S24G, S24R, K27R, N42R, S55P, G59E, G59D, N60D, N60E, V66A, N74D, N85S, N85R, G96S, G96A, S97G, S97D, S97A, S97SD, S99E, S99D, S99G, S99M, S99N, S99R, S99H, S101A, V102I, V102Y, V102N, S104A, G116V, G116R, H118D, H118N, N120S, S126L, P127Q, S128A, S154D, A156E, G157D, G157P, S158E, Y161A, R164S, Q176E, N179E, S182E, Q185N, A188P, G189E, V193M, N198D, V199I, Y203W, S206G, L211Q, L211D, N212D, N212S, M216S, A226V, K229L, Q230H, Q239R, N246K, N255W, N255D, N255E, L256E, L256D T268A and R269H, wherein the positions correspond to the positions of the protease shown in SEQ ID NO 1, wherein the protease parent is selected from the protease shown in SEQ ID NO 1 and the Bacillus amylolichenifaciens protease (BPN’) shown in SEQ ID NO 2 and wherein the protease variant has at least 80%sequence identity to SEQ ID NO 1 or 80%sequence identity to SEQ ID NO 2,
iii) a protease comprising a substitution at one or more positions corresponding to positions 171, 173, 175, 179, or 180 of SEQ ID NO: 14, compared to the protease shown in SEQ ID NO 14, wherein the protease variant has a sequence identity of at least 75%but less than 100%to SEQ ID NO 14,
iv) a protease comprising the amino acid sequence shown in SEQ ID NO 1 or 2 or a protease having at least 80%sequence identity to; the polypeptide comprising amino acids 1-269 of SEQ ID NO 1 or the polypeptide comprising amino acids 1-275 of SEQ ID NO 2,
v) One or more of the following protease variants selected from the group:
SEQ ID NO 1 with the alterations T22R+S99G+S101A+V102I+A226V+Q239R,
SEQ ID NO 2 with the alterations S24G+S53G+S78N+S101N+G128A+Y217Q,
SEQ ID NO 2 with the alterations S24G+S53G+S78N+S101N+G128S+Y217Q,
SEQ ID NO 1 with the alterations S9E + N42R + N74D + V199I + Q200L + Y203W + S253D + N255W + L256E,
SEQ ID NO 1 with S9E+N42R+N74D+H118V+Q176E+A188P+V199I+Q200L Y203W+S250D+S253D+N255W+L256E,
SEQ ID NO 1 with S9E+N42R+N74D+Q176E+A188P+V199I+Q200L+Y203W S250D+S253D+N255W+L256E,
SEQ ID NO 1 with S3V+N74D+H118V+Q176E+N179E+S182E+V199I+Q200L Y203W+S210V+S250D+S253D+N255W+L256E,
SEQ ID NO 1 with the alterations T22A+N60D+S99G+S101A+V102I+N114L+G157D +S182D+T207A+A226V+Q239R+N242D+E265F,
SEQ ID NO 1 with the alterations S9E + N42R + N74D + H118V + Q176E + A188P + V199I + Q200L + Y203W + S250D + S253D + N255W + L256E,
SEQ ID NO 1 with the alterations S9E + N42R + N74D + Q176E + A188P + V199I + Q200L + Y203W + S250D + S253D + N255W + L256E,
SEQ ID NO 1 with the alterations S9E + N42R + N74D + H118V + Q176E + A188P + V199I + Q200L + Y203W + S250D + N255W + L256E + *269aH + *269bH,
SEQ ID NO 1 with the alterations S3V + N74D + H118V + Q176E + N179E + S182E + V199I + Q200L + Y203W + S210V + S250D + N255W + L256E,
SEQ ID NO 1 with the alterations S9E + N74D + G113W + G157P + Q176E + V199I + Q200L + Y203W + S250D + T254E + N255W + L256E,
SEQ ID NO 1 with the alterations S3V + S9R + N74D + H118V + Q176E + N179E + S182E + V199I + Q200L + Y203W + S212V + S250D + N255W + L256E,
SEQ ID NO1 with the alteration S99E, and
SEQ ID NO 2 with the alteration L217D,
and combinations thereof.
11. The use of any of the above paragraphs, wherein the amylase is selected from the group consisting of:
i) variants comprising one or more substitutions in the following positions: 9, 26, 30, 33, 82, 37, 106, 118, 128, 133, 149, 150, 160, 178, 182, 186, 193, 195, 202, 203, 214, 231, 256, 257, 258, 269, 270, 272, 283, 295, 296, 298, 299, 303, 304, 305, 311, 314, 315, 318, 319, 320, 323, 339, 345, 361, 378, 383, 419, 421, 437, 441, 444, 445, 446, 447, 450, 458, 461, 471, 482, 484, wherein the positions corresponds to positions of SEQ ID NO 11;
ii) variants exhibiting at least 90 percent identity with SEQ ID NO 12, with deletions in the 183 and 184 positions,
iii) variants exhibiting at least 95 percent identity with SEQ ID NO 13, comprising mutations in one or more of the following positions M202, M208, S255, R172 and/or M261,
iv) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 9 or 10,
v) a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at leaset 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%sequence identity to SEQ ID NO: 9 or 10, and combinations thereof.
12. The use according to any of the above paragraphs, wherein the cellulase is selected from the group consisting of :
a. a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at leaset 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%sequence identity to the polypeptide shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and combinations thereof.
13. The use according to any of the above paragraphs, wherein said surface is a textile made of nature fiber such as cotton, synthetic fiber such as polyester, or a mixture thereof.
14. The use according to any of the above paragraphs, wherein the enzyme (s) is incorporated in a detergent composition comprising essentially no biocide.
15. The use according to any of the above paragraphs, wherein the enzyme (s) is incorporated in a powder detergent or liquid deterget.
16. A method of preventing, inhibiting or reducing microbe growth on surface, comprising the steps of :
a. treating the surface with enzymes as defined in any of paragraphs 10-12 to the extent that at least one microbe has a TTC detectable time on said surface after the treatment which increases at least 20%.
17. A method of demonstrating the microbe growth inhibition or deep cleansing benefit of an enzyme or enzyme combination as defined in paragraphs 10-12 on a surface, comprising the steps of:
a. Providing two surfaces A and A’,
b. Applying a soil comprising a microbe growth indicator onto each of the surfaces A and A’,
c. Washing A with a detergent composition comprising no enzyme, and washing A’with said detergent composition comprising one or more enzymes,
d. Preparing an inoculation of microbe and apply it onto the surfaces A and A’,
e. Incubating A and A’under suitable conditions to allow microbe growth for a predetermined period of time,
f. Comparing the growth of microbe on A and A’.
18. The method according to paragraph 17, wherein the microbe growth indicator is triphenyltetrazolium chloride (TTC) .
19. The method according to paragraph 16 or 17, wherein the surfaces A and A’provided in step a. of paragraph 14 are made of essentially the same material.
Assays
Wash Assay
Terg-O-to-meter (TOM) wash assay
The Tergo-To-Meter (TOM) is a medium scale model wash system that can be applied to test 16 different wash conditions simultaneously. A TOM is basically a large temperature  controlled water bath with up to 16 open metal beakers submerged into it. Each beaker constitutes one small top loader style washing machine and during an experiment, each of them will contain a solution of a specific detergent/enzyme system and the soiled and unsoiled fabrics its performance is tested on. Mechanical stress is achieved by a rotating stirring arm, which stirs the liquid within each beaker. Because the TOM beakers have no lid, it is possible to withdraw samples during a TOM experiment and assay for information on-line during wash.
The TOM model wash system is mainly used in medium scale testing of detergents and enzymes at US or LA/AP wash conditions. In a TOM experiment, factors such as the ballast to soil ratio and the fabric to wash liquor ratio can be varied. Therefore, the TOM provides the link between small scale experiments, such as AMSA and mini-wash, and the more time consuming full scale experiments in top loader washing machines.
Equipment: The water bath with 16 steel beakers and 1 rotating arm per beaker with capacity of 500 to 1200 mL of detergent solution. Temperature ranges from 3.5 to 60℃. The water bath has to be filled up with deionised water. Rotational speed can be set up to 40 to 200 rpm/min.
Set temperature in the Terg-O-Tometer and start the rotation in the water bath. Wait for the temperature to adjust (tolerance is +/-0.5℃) . All beakers, the stirring arms and the strainer shall be clean in a dish washer and without traces of prior test material.
The wash solution with desired amount of detergent and water hardness are prepared in a bucket. The detergent is allowed to dissolve during magnet stirring for 10 min, and measure pH of detergent solution after 10 min stirring. Wash solution shall be used within 30 to 60 min after preparation.
1000 ml wash solution is added into a TOM beaker. The wash solution is agitated at 120 rpm and let rotate until the temperature is correct. The swatches are sprinkled into the beaker and the ballast load and then optionally one or more enzymes are added to the beaker. Time measurement starts when the swatches and ballast are added to the beaker. The swatches are washed for 20 minutes after which agitation is terminated. The wash load is subsequently transferred from the TOM beaker to a container and rinse with cold tap water. The soiled swatches are separated from the ballast load. The soil swatches are transferred to a 5 L beaker with cold tap water under running water for 5 minutes. The ballast load is kept separately for the coming inactivation. The water is gently pressed out of the swatches by hand and placed on a tray covered with a paper. Another paper is placed on top of the swatches. The swatches are allowed to dry overnight before subjecting the swatches to analysis, such as measuring the color intensity using a Color Eye as described herein.
Full scale wash (FSW) assay
FSW, i.e. to test product performance in washing machines under scientifically designed conditions, is a household care application assay that is in the largest scale, at the end of the screening flow and with the most consumer relevance. The FSW system can be used at US or LA, AP and EU wash conditions according to different regional machines. Test swatches and ballast are added to each wash together with detergent and enzyme.
Equipment: AP Washing Machine –Panasonic XQB65-Q680U automatic washing machine. The water used in these machines is all deionized. The Normal wash program is the default program for mixed clothes and is used herein. Other suitable washing machine can be used as well.
Enzyme: The enzymes can be added on basis of e.g. Molar Concentration, Protein amount, or on Activity basis.
Ballast: Clean white cloth (without optical whitener) made of cotton, polyester or cotton/polyester. The composition of the ballast is a mix of different items at a cotton/polyester ratio of 65/35 based on weight. The ballast weight, dryness and item composition must be the same in each wash. After each wash the ballast is inactivated in an industrial washer at 85℃ /15 min or in a 90℃ wash (EU machine) without detergent. An example of standard AP ballast composition, co/pe ratio at 65/35 (1.5 kg ± 50 g) : 1 T-shirt (100%cotton) + 5 shirts w. short sleeves (55%cotton/45%polyester) + 2 pillow cases (35%cotton/65%polyester, 110x75cm) + 3 tea towels (100%cotton) . The ballast weight is adjusted with shirts, short sleeves to the right ballast.
Test swatches: The test swatches, i.e. technical and natural stains, are either commercial or NZ-produced. The same batch must be used in all washes in a trial. Different swatch sizes can be used. The swatches are attached to tea towels by a stapler; the same swatch Type (stain) is placed on different towels or in different positions on the towel. Each swatch is marked individually for identification and to indicate the front side. It is important to keep the swatches in the dark and limit the exposure to light at all times as many stains are sensitive to light. The total weight of textiles include the weight of ballast and the tesed swatches.
Water hardness: Natural water contains different levels of metal ions (mainly Ca 2+ and Mg 2+) depending on geographical area. The metal ions considered to make up the water hardness are the ones that precipitate fatty acid soaps (mainly Ca 2+ and Mg 2+, but not for example Na +) . Natural water also contains hydrogencarbonate, HCO 3 -, at varying levels, on  average 1.5 times more than the sum of Ca 2+ and Mg 2+ on a molar basis, and may contain a number of other anions, including Cl -and SO 4 2-. Presence of HCO 3 -is important for the buffer capacity of the water and affects the pH of wash solutions prepared by adding a detergent. Artificial hard water is prepared by adding CaCl 2, MgCl 2 and NaHCO 3 to Milli-Q water or deionized water.
Wash conditions: Standard AP washing conditions are described below
Figure PCTCN2018078160-appb-000038
Other wash conditions may apply. Wash conditions in various regions for normal heavy duty wash are exemplified in below table.
Figure PCTCN2018078160-appb-000039
*°dH: adjusted by adding CaCl 2*2H 2O, MgCl 2*6H 2O and NaHCO 3 to Milli-Q water. Note (this is estimations and not absolutes, there is speciel conditions for different regions and also with different machines)
Wash Procedure
a. Prepare the ballast and test swatches (staple to tea towels) .
b. Select parameters for the wash: Program and Water level. Please note when using the automatic water injection, the declared water level is NOT the true water level. For 32 L program, the actual water intake is 33 L. The amount of enzyme and detergent added should be adjusted to reflect such water level.
c. Program and start automatic water meter system.
d. Press Start/Pause button to start water filling, and adjust water temperature during the water intake. Water consumption is registered automatically during this time.
e. Add Ca/Mg according to water consumption
a. Press the button to stir water.
b. Stop and check the water hardness.
f. The detergent and powder NaHCO 3 are added to the machine –short mix (10 sec) .
g. The enzyme solution or granulate is added –short mix (10 sec) .
h. Ballast and swatches are added to the washing machine.
i. Quickly Re-start the washing machine, select the wash program and water level, start the wash by pressing the Start/Pause button. If soaking, stir 30 s, then stay for the soaking time. The machine will automatically inject water (0.2 –1 L) during main wash if low water level is detected.
j. Measure pH after 8 or 10 min in wash.
k. The washing machine by default gives 2 rinses. Rinsing with water having the same hardness . The machine will automatically add 1 more rinse if over foaming is detected. Water consumption is registered automatically during this time.
l. After the wash is completed, the test swatches are removed from the tea towels and placed on trays for drying -make sure the swatches are dried in completely darkness, as many stains are sensitive to light. The swatches are dried overnight and must be completely dry before measurement.
Mini Launder-O-Meter (MiniLOM) Model Wash System
MiniLOM is a modified mini wash system of the Launder-O-Meter (LOM) , which is a medium scale model wash system that can be applied to test up to 32 different wash conditions  simultaneously. MiniLOM model wash system is mainly used to wash hygiene experiment. A MiniLOM is a rotator with free 50 ml x 16 (usually 6-32) tube rotisserie accessory included, with capacity of 10 to 20 mL of detergent solution in each tube. The water bath has to be filled up with sterilized deionised water. Each tube will contain a solution of a specific detergent/enzyme system and the soiled and unsoiled fabrics its performance is tested on. Mechanical stress is achieved by a rotating axis mix them horizontally or vertically. It can be used in ambient temperatures from 4C to 55C, and the rotation range is from 10 to 70 rpm. In a MiniLOM experiment, factors such as the microorganisms loading and the fabric pieces can be varied. The tubes shall be discarded after wash.
The wash solution with desired amount of detergent and water hardness are prepared in a 1 L Beaker. The detergent is allowed to dissolve during magnet stirring for 10 min, and measure pH of detergent solution after 10 min stirring. Wash solution shall be used within 30 to 60 min after preparation.
20 ml wash solution is added into a sterilized miniLOM tube. The wash solution is agitated at 20 rpm. The swatches are sprinkled into the beaker and microbial broth load. All the above operations should be carried out in clean bench. Time measurement starts when the swatches and microbial broth are added to the beaker and MiniLOM machine are moved to a chamber with the targeting temperature. The swatches are washed for 60 minutes after which agitation is terminated. After wash, transfer swatches from tube to a beaker with 400 ml sterilized water and rinse for 10 min. Then the swatches are treated according to the reduce attachment assay.
Stain recipes
Figure PCTCN2018078160-appb-000040
Figure PCTCN2018078160-appb-000041
Preparation of stained swatches:
Put clean swatch (6×6cm) on the cylindrical bottom of the 25 mL round flask (the diameter of the bottom is about 3.8 mm) , and fix the swatch with one hand. Load the prepared soil on the centre of swatch with a pitette, and then spread the stain solution evenly on the swatch surface with fingers. Three different types of swatches mentioned below from the Center For Testmaterials BV, the Netherlands are used.
Figure PCTCN2018078160-appb-000042
Evaluation of stains
Wash performance is expressed as a delta remission value (ΔRem) . After washing and rinsing the swatches were spread out flat and allowed to air dry at room temperature over night. All washes are evaluated the day after the wash. Light reflectance evaluations of the swatches were done using a Macbeth Color Eye 7000 reflectance spectrophotometer with very small aperture. The measurements were made without UV in the incident light and remission at 460  nm was extracted. Measurements were made on unwashed and washed swatches. The test swatch to be measured was placed on top of another swatch of same type and colour (twin swatch) . With only one swatch of each kind per beaker, a swatch from a replicate wash was used in this way. Remission values for individual swatches were calculated by subtracting the remission value of the unwashed swatch from the remission value of the washed swatch. The total wash performance for each stained swatch set was calculated as the sum of individual ΔRem.
Calculating the enzyme effect is done by taking the measurements from washed swatches with enzymes and subtract with the measurements from washed without enzyme for each stain. The total enzyme performance is calculated as the sum of individual ΔRem enzyme.
TTC Assay (In situ detection of microbe growth)
Triphenyltetrazolium Chloride (TTC) is a reduction–oxidation reaction indicator, and it can be used to differentiate metabolically active and inactive tissues/organisms. It is white in color, and the color will turn to red when TTC is enzymatically reduced to water-insoluble TPF (1, 3, 5-triphenylformazan) in living cells due to the activity of various dehydrogenases (enzymes important in oxidation of organic compounds and thus cellular metabolism) . Scheme I below show the reaction from TTC to TPF. For this reason, TTC can be used for detecting microbial growth.
Figure PCTCN2018078160-appb-000043
Scheme I
Other microbe growth indicators having TTC-like microbial growth indication function can be used in the present study. They can include the redox indicator, living cell dye, the colored metabolites and metabolites detecting assay. The redox indicators include all redox assay, such as INT (2- (4-iodophenyl) -3- (4-nitrophenyl) -5-phenyl-2H-tetrazolium) , MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) , XTT (2, 3-bis- (2-methoxy-4-nitro-5-sulfophenyl) -2H-tetrazolium-5-carboxanilide) , MTS (3- (4, 5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium) , WSTs (Water-soluble  Tetrazolium salts) and CCK-8 (Cell Counting Kit-8) . The living cell dye can include acridine orange, Calcein-AM and Hoechst. All the colored metabolites assays/microorganisms should be included, such as all the carotenoid, luciferin, fluorescent protein/fluorescent producing microorganisms. Metabolites detecting assays include all the metabolites and enzyme detecting assay, such as ATP detect assay, using X-Gal or MUG to detect β-Galactosidase.
Material:
a. TTC solution.
b. 0.1 g 2, 3, 5-Triphenyl tetrazolium chloride (TTC) (Sigma, cas: 298-96-4) is dissolved in 2 mL sterilized Mili-Q water as a 1000× stock solution (50 mg/mL) .
c. Culture medium
· Nutrient agar medium: 5 g Peptone, 30 g Beef extract, 5 g NaCl, 15 g Agar, in 1 liter distilled water, adjust pH to 7.0-7.2. This medium is used for cultivating P. putida
· Potato Dextrose Agar medium (PDA) : Boil 200 g sliced, unpeeled potatoes in 1 liter distilled water for 30 min. Filter through cheesecloth, saving effluent, which is potato infusion (or use commercial dehydrated form) . Mix with 20 g dextrose and add water to dilute it to 1 L. Distribute it to flasks, and add 15 g/L agar to each flask, then autoclave 15 min at 121℃. Then dispense 20-25 ml portions into sterile 15 × 100 mm petri dishes. This medium is used for cultivating fungi.
d. PBS buffer (0.01 M, pH 7.4) , sterilize at 121℃, 15 min, and stock at 4℃.
Procedure:
1. Preparation of the microbe/TTC solution
a) Incubate bacteria or fungi on each of the corresponding solid medium till colonies are formed or spores are seen.
b) Inoculate a single colony to liquid medium and incubate overnight till the OD600 reaches about 0.6-0.8) at each of the microbe’s optimum growth temperature according to the specification of the microbe, then take a portion of broth into a fresh liquid medium at the volume ratio of 0.1%-0.5%, incubate at 28-37℃ for about 8-12 hours, then collect the pellet of the culture and resuspend in sterilized PBS buffer twice, and prepare a solution with OD600 of about 0.10 –0.13.
c) Add TTC solution (50 mg/ml) into the microbe solution from step 2, to make the concentration of TTC reach 50 mg/L, mixed well and use immediately.
2. Prepartion of stained swatches
a) The swatches which have finished the wash steps following the wash procedure described in the Wash Assay section are rinsed with water for 7-10 min, put the swatches on the plates. The plates are sterilized by immersing in 75%ethanol and irradiate with UV light for 0.5-1 hrs before use.
b) Inject 300 μL bacteria or fungi spore solution evenly on each swatch by using a pipette
c) Incubate thus prepared swatches in a sterilized culture chamber at 34℃, 90%-98%RH, regardless of the microbe applied onto the swatches are bacterial or fungi.
3. TTC in situ dection of microbe growth on the swatches.
The swatches are taken out from incubator at a 6 hour interval, and the evaluation can be conducted on scanner. First, scanning the swatches with a scanner (e.g., Epson Expression 10000XL) under a predetermined setting (e.g., the software of Silverfast can be used and is set to scan in 200dpi and 48→24 bit color) and then analyse the scanned picture of the swatch. The software of Color vector program for RGB reading can be used, and a circled area with a diameter of 1.2 fold of the diameter of the stain are choosen for the color analysis purpose. The intensity of the color on swatches is calculated according to the following equation. Then compare the intensity of the tested swatches with the intensity of the similarly stained and washed (but without enzyme useful in the present invention) swatch, if the change of the intensity is a decrease of 4, then record the time as TTC detectiable time.
Figure PCTCN2018078160-appb-000044
When the surface is of a type other than the swatches, essentialy the same procedure as described above will applie for the TTC assay purpose, except that the wash condition will need be adjusted to fit for the particular surface wash purpose.
Fungi Spore/Mycelium Formation Time Assay (mainly for in situ detection of fungi growth)
Procedure:
The above steps described above in the TTC Assay section applies here, with only difference in that no TTC is added into the culture solutions as in step 1c) , and the inoculated microbe is fungi rather than bacteria.
Following step 2 c) , the swatches are taken out for microscope detection at an interval of 12 hrs to check the spore formation. Olympus SZX16 microscope can be used, and a 10 fold magnification can be used by randomly selecting 3 visual field under the microscope for any visible conidia. Depending on different species of fungi, their spore/mycelium may show a color of black, green, or red and so on. The color can be predetermined through studying the specification of the fungi.
Once the spore (conidia, sporangia or other type of spores) or mycelium is dected under 10 fold stereo microscopre screening, then proceed to higher magnification detection of 50 fold to confirm the size of the conidial head, if the conidial head has a diameter of no less than 20 μm (for Aspegillus niger) or the mycelium has a diameter of no less than 2 μm, then record the time as fungi spore formation time.
Spore Density Score Assay
Spore Density Score is used to determines the growth of fungi as represented by visible marks on household surfaces caused by the spores/conidia of the fungi which grow on said surface.
The swatches in the TTC Assay section described above are taken out for measuring the spore density score after being cultivated for 7 days.
A scale of 0 to 7 is set for measuring the spore density value, where 0 represents no fungi growth on the surface, and 7 being the surface are overgrown with fungi. For different fungi, the color of the spore can be different, but the same 0-7 scale can apply for different fungi growth and spore density measurement. A group of panellists is first trained to understand the scale and corresponding spore density value, and then score the spore dentisty by comparing each surfaces (swatches) as tested with the scale.
Screening of Aspergillus niger used in the Examples
A filamentous fungi Aspergillus niger (Novozymes internal strain number 57825) is used. This strain is isolated from the moldy corn in Liaoning province of China in the year of 2014. It was isolated and cultured in Potato Dextrose Agar medium (PDA) medium at 25℃ for 3 days. As an alternative, other commercially available Aspergillus niger can be used interchangeably with this strain in the Examples.
Examples
The following examples further describe and demonstrate aspects within the scope of  the present invention. The examples are given solely for the purpose of illustration and are not to be construed as limitations of the present invention, as many variations thereof are possible without departing from the spirit and scope of the invention.
Example 1 Prevention of fungi growth on soils on chopping board treated with enzymatic Hand Dish Wash (HDW) detergent
Material
1. HDW Soil Recipe:
35%potato starch (food grade, Hua’ou company, China) + 2%semi-skimmed milk (Oldenburger, Germany) + 2%egg yolk (from fresh egg) + 1.3%soy sauce (Lijinji company, China)
2. Fungi -Aspergillus niger
A filamentous fungi Aspergillus niger (Novozymes internal strain number 57825) , is used. This strain is isolated from the moldy corn in Liaoning province of China in the year of 2014. It was isolated and cultured in Potato Dextrose Agar medium (PDA) medium at 25℃ for 3 days. It is believed that other commercially available Aspergillus niger can be used interchangeably with this strain in the Examples.
3. Chopping board
wooden chopping board from supermarket (Taixing, ginkgo wood)
4. Detergent: SEA model detergent, wash liquor 0.75g/L
5. Enzymes: Protease (SEQ ID NO: 3) , Amylase (SEQ ID NO: 9)
6. Grouping:
Figure PCTCN2018078160-appb-000045
Procedure:
1. Procedure to prepare soil solution
5%potato starches are gelatinized in tap water at 85℃ for 5 min, which forms a hot paste, and then add 2%semi-skimmed milk and 2%egg yolk, and further add 1.3%soy sauce to make a homogeneous solution.
2. Preparation of the fungi spores
Incubate Aspergillus niger on petri dish (PDA or czapek's medium) in incubator at 30℃ for 3 days. Collect the spores by flushing the plate with sterilized PBS buffer (0.01 nM, pH7.4) ; vortexing thus prepared spore solution vigorously for 5 min; then dilute the spore solution to reach a final OD 600 of between 0.10 –0.13.
3. Enzymatic treatment of the chopping board and fungi growth comparison
0.5g of the freshly made soil solution according to the step 1 above in the present Example were respectively applied onto each of the boards and the boards were then left dry at 25℃ for 2 hour.
Then, each group was added with its corresponding wash solution as listed in the Grouping table shown above. The boards were then left standing still for 10 mins. Afterwards, the boards were sponge-scrubbed under flowing tap water with 5 stokes of scrubbing.
1 ml of Aspergillus niger spore solution prepared according to the procedure described above in the TTC Assay section of the Assay part at the concerntration of OD = 0.124 was inoculated by using a pipette onto each board, respectively. The chopping boards are then incubated at 34℃, 90%-98%RH, where the incubator has been sterilized with UV before the incubation of the board.
The growth of the fungi was observed every 12 hours. Record the timing when the visible black color sporange/spore is identified by naked eye by following the procedure described in the Fungi Spore formation Assay section in the Assays part.
Results:
1. Spore forming time
Table 1. Visible conidia forming time of the Aspergillus niger on chopping board swatches
Figure PCTCN2018078160-appb-000046
It can be seen from Table 1 that the chopping boards from all the Test groups 1-3 which were enzymatically treated has much longer spore forming time than the blank group and the negative control group which are stained and respectively washed with the water only or  washed liquid detergent itself comprising no such protease and amylase. In particular, the test group has a spore forming time which is at least 1.5 fold, i.e., an increase of at least 50%compared with that of the negative control group.
This result clearly suggests the effect of protease and amylase used in water or liquid detergent in inhibiting the growth of Aspergillus niger on the chopping board.
2. Spore Density Score Measurement
Each of the chopping board was observed by a panel for spore density score evaluation after being cultivated for 7 days, by following the procedure described in the Spore Density Score Assay section of the Assay part in previous paragraphs.
Table 2. Spore Density Score –Panel score and the average
Figure PCTCN2018078160-appb-000047
As shown in Table 2, the chopping boards from all the test groups 1-3 which were enzymatically treated has much lower Spore Density Score than the blank group (score 7) and at least one score lower than the negative control group (score 3) which are stained and washed with the water only or liquid detergent itself comprising no such protease and amylase.
This clearly suggests the effect of protease and amylase used in water or liquid detergent help inhibiting the growth of the fungi, Aspergillus niger on the chopping board.
Example 2. Inhibition of bacterial growth by washing with enzymatic liquid detergent comprising enzymes at different dosage
Materials
1. Bacterial: Pseudomonas. putida. (China General Microbiological Culture Collection Center (CGMCC) , the strain number 1.3096)
2. Detergent: SEA Model detergent
3. Enzyme: Protease (SEQ ID NO: 3) , Amylase (SEQ ID NO: 9)
4. Surfaces: plastic chopping board (Jie neng brand purchased from supermarket, polypropylene) which has been sliced slightly with a saw to make a few lines on its surface to mimic the board in real life use.
5. Stain recipe: Baby food #2
6. Grouping:
Groups Wash condition
Blank Water only
Test 1 Water with 0.51 ppm SEQ ID NO: 3+ 0.26 ppm SEQ ID NO: 9
Test 2 Water with 1.03 ppm SEQ ID NO: 3+ 0.52 ppm SEQ ID NO: 9
Negative Control detergent only
Test 3 detergent with 1.03 ppm SEQ ID NO: 3+ 0.52 ppm SEQ ID NO: 9
Procedure:
1. Procedure to prepare soil solution
The Baby food #2 soil was prepared according to the description in the Stain Recipe section in the Assays part. 1 mL of each soil solution was applied onto each respective chopping board. Then the boards were left for air dry for 3hrs at room temperature.
2. Enzymatic treatment of the chopping board and bacterial growth comparison
Then, the board from each group was added with 1 mL its corresponding wash solution as listed in the Grouping table shown above. The boards were then left standing still for 10 mins. Afterwards, the boards were sponge-scrubbed under flowing tap water with 5 stokes of scrubbing.
700 uL of bacterial solution prepared according to the procedure described above in the TTC Assay section of the Assay part at the concerntration of OD = 0.124 was inoculated by using a pipette onto each boards, respectively. The chopping boards are then incubated at 34℃, 90%-98%RH, where the incubator has been sterilized with UV before the incubation of the board. The boards were observed for TTC detectable time by following the procedure described in the TTC Assay section in the Assays part.
Results
1. TTC Detectable Time
TTC detectable time was measured by following the procedure in the TTC Assay section in the Assay part.
Table 3
Blank Test 1 Test 2 Negative Control Test 3 Test 3/Negative
24 hr >48 hr >48 hr 24 hr >48 hr >2
Results in Table 3 show that for boards from all the test groups 1-3 which were respectively washed with water or detergent comprising blend of protease and amylase, the TTC detectable time that was were significantly prolonged (ratio>2) as compared to the  negative control group. This suggests the bacterial growth was inhibited by using these enzymes in water or liquid detergent for plastic chopping board surface.
Example 3. Inhibition of fungi growth by washing with enzymatic liquid detergent comprising combination of amylase and protease
Materials
1. Microorganism-Aspergillus niger (Same as in Example 2)
2. Stain: Baby food #3
3. Detergent: BlueMoon Deepclean liquid detergent
4. Swatch type: PCN01, W30A and CN42
5. Enzyme: Protease (SEQ ID NO: 3) , Amylase (SEQ ID NO: 9)
6. Grouping:
Figure PCTCN2018078160-appb-000048
Procedure:
1. Stained swatches are prepared according to the procedure described in the Wash Assay part above.
2. The TOM wash procedure was used. Wash condition: TOM, 30℃, 120 rpm, 14°dH (Ca 2+: Mg 2+ = 3: 2) , 10/20 min (when the textile of tested swatches are CN42, wash for 20 min, when the when the textile of tested swatches are PCN01 or W30A, wash for 10 min) .
3. The preparation of the fungi spore inoculation and the application of such onto the washed swatches, and the observation of the growth of the fungi on the swatches from different groups follow the description in the Spore Formation Assay section in the Assay Part.
Results
1. Spore forming time
Table 4. Visible conidia forming time of the Aspergillus niger on swatches
Figure PCTCN2018078160-appb-000049
It can be seen from the result in Table 4 that the test group swatches, which was washed with detergent comprising protease and amylase, at least the PCN01 and W30A showed the same result as the unstained and unwashed swatches. The results from the test group and the blank control group are siginificantly longer than the two negative control groups, which are either stained but unwashed, or stained and washed with the liquid detergent itself comprising no such protease and amylase. This clearly suggests protease and amylase can be useful in inhibiting the growth of the fungi, Aspergillus niger.
2. Spore Density Score Measurement
Each of the swatches are observed by a panel, and given a score of the spore conidia size and density. The measurement refers to the “Spore Density Score” section under the Assay part.
Table 5. Spore Density Score-Panel score and the average
Figure PCTCN2018078160-appb-000050
As the result shown in Table 5 indicates, the test group swatches, which was washed with detergent comprising protease and amylase, at least the PCN01 and W30A swatches showed the same result as the unstained and unwashed swatches.
Besides, the results from both the test group and the blank control group are siginificantly smaller than the two negative control groups, which are either stained but unwashed, or stained and washed with the detergent itself comprising no such protease and amylase.
Both aspects clearly suggests protease and amylase can be useful in preventing, inhibiting, or reducing the growth of the fungi, Aspergillus niger.
Example 4. Inhibition of fungi growth by washing with enzymatic powder detergent comprising combination of amylase and protease
Materials
1. Microorganism-Aspergillus niger (same as in Example 2)
2. Stain: Baby food #3
3. Detergent: Model X
4. Swatch type: W30A and CN42
5. Enzyme: blend of protease (SEQ ID NO: 1) and amylase (SEQ ID NO: 10) .
6. Grouping:
Figure PCTCN2018078160-appb-000051
Procedure:
1. Stained swatches are prepared according to the procedure described in the Wash Assay part above.
2. The TOM wash procedure was used. Wash condition: TOM, 30℃, 120 rpm, 14°dH (Ca 2+: Mg 2+ = 3: 2) , 20 min.
3. The preparation of the fungi spore inoculation and the application of such onto the washed swatches, and the observation of the growth of the fungi on the swatches from  different groups follow the description in the Spore Formation Assay section in the Assay Part.
Results
1. Spore forming time
Aspergillus niger is a black-spored filamentous fungus that forms asexual spores called conidospores (conidia) during growing. The Aspergillus niger conidial head can be observed by naked eye. Record when can see the visible black conidia.
Table 6
Figure PCTCN2018078160-appb-000052
It can be seen from the result in Table 6 that the test group swatches, which was washed with detergent comprising protease and amylase, at least the W30A swatch showed the same result as the unstained and unwashed swatches. Besides, the above results show that the test group and the blank control group have siginificantly longer spore formation time than the two negative control groups, which are either stained but unwashed, or stained and washed with the powder detergent itself comprising no such protease and amylase. Both aspects of the results clearly suggests protease and amylase can be useful in inhibiting the growth of the fungi, Aspergillus niger.
2. Spore Density Score Measurement
Each of the swatches are observed by a panel, and given a score of the spore conidia size and density. The measurement refers to the “Spore Density Score” section under the Assay part.
Table 7. Spore Density Score
Figure PCTCN2018078160-appb-000053
The results in above Table 7 shows the test group swatches, which was washed with detergent comprising protease and amylase showed essentially the same result as the unstained and unwashed swatches from the Blank Control group. The results from both the test group and the blank control group are siginificantly smaller than the two negative control groups, which are either stained but unwashed, or stained and washed with the detergent itself comprising no such protease and amylase. This clearly suggests the effect of protease and amylase being useful in preventing, inhibiting, or reducing the growth of the fungi, Aspergillus niger.
Example 5. Inhibition of fungi growth by washing with enzymatic liquid detergent comprising enzymes at different dosage
Materials
1. Microorganism-Aspergillus niger. (same as in Example 2)
2. Stain: Baby food #1
3. Detergent: Model O
4. Swatch type: PCN01 and CN42
5. Different Enzymes groups :
A: single protease (SEQ ID NO: 3) ,
B: single amylase (SEQ ID NO: 9) ,
C: blend of Protease (SEQ ID NO: 3) , Amylase (SEQ ID NO: 9) in weight ratio of 4: 1.
6. Grouping:
Figure PCTCN2018078160-appb-000054
Figure PCTCN2018078160-appb-000055
Procedure:
1. Stained swatches are prepared according to the procedure described in the Wash Assay part above.
2. The TOM wash procedure was used. Wash condition: TOM, 30℃, 120 rpm, 14°dH (Ca 2+: Mg 2+ = 3: 2) , 20 min.
3. The preparation of the fungi spore inoculation and the application of such onto the washed swatches, and the observation of the growth of the fungi on the swatches from different groups follow the description in the Spore Formation Assay section in the Assay Part.
Results
1. Spore forming time
The Aspergillus niger conidia were observed following the same procedure in the previous Examples. can be observed by naked eye. Record when can see the visible black conidia.
Table 8. Visible conidia forming time
Figure PCTCN2018078160-appb-000056
Figure PCTCN2018078160-appb-000057
It can be seen from the result in Table 8 that for the test group C and group B which were respectively washed with detergent comprising blend of protease and amylase and amylase itself. For both the PCN01 and W30A swatches, the above results show that at lower enzyme dosage the fungi spore forming time were significantly prolonged as compared to that of the negative control group, and at higher dosagethe fungi spore forming time was even the same as the blank control, which were unstained and unwashed.
This result suggests the fungi growth inhibition benefit of use of enzymes in powder detergent. It is also seen that W30A was a bit better in retarding fungi spore formation than PCN01.
2. Spore Density Score Measurement
Each of the swatches are observed by a panel, and given a score of the spore conidia size and density. The measurement refers to the “Spore Density Score” section under the Assay part.
Table 9
Figure PCTCN2018078160-appb-000058
The rusults in Table 9 shows the swatches from all three test groups A-C, and for both the PCN01 and W30A swatches, it showed at lower enzyme dosage the fungi density score were much smaller as compared to the negative control group, and at higher dosagethe fungi spore forming time was even the same as the blank control, which were unstained and unwashed.
This clearly suggests protease and amylase can be useful in preventing, inhibiting, or reducing the growth of the fungi, Aspergillus niger.
3. CFU counting
Further, CFU counting is conducted to quantify the fungi on swatches. The PCN01 swatches from Test Group C was used for this purpose. The spore amount is calculated from the CFU by this equation: ( (Average CFU with the dosage of 10 -n) × 10 n /0.2 mL) × 10 Ml.
Table 10
Figure PCTCN2018078160-appb-000059
The result in Table 10 clearly shows that the test group has significantly lower amount of fungi left on the swatches which are treated with enzymatic detergent comprising protease and amylase.
Example 6. Inhibition of bacterial growth by washing with with enzymatic liquid detergent comprising enzymes at different dosage
Materials
1. Bacterial: P. putida. (China General Microbiological Culture Collection Center (CGMCC) , the strain number 1.3096)
2. Detergent: Model O, wash liquor concerntration 2g/L
3. Enzyme: Protease (SEQ ID NO: 3) , Amylase (SEQ ID NO: 9)
4. Swatches: CN42 and W30A
5. Stain recipe: Baby food #2
6. Grouping:
Figure PCTCN2018078160-appb-000060
Figure PCTCN2018078160-appb-000061
Procedure:
1. Stained swatches are prepared according to the procedure described in the Wash Assay part above.
2. The TOM wash procedure was used. Wash condition: TOM, 30℃, 120 rpm, 14°dH (Ca 2+: Mg 2+ = 3: 2) , for 10/20 min (when the textile of tested swatches are CN42, wash for 20 min, when the when the textile of tested swatches are PCN01 or W30A, wash for 10 min) .
3. The preparation of the bacterial inoculation and the application of such onto the washed swatches, and the observation of the growth of the TTC color change on the swatches from different groups follow the description in the TTC Assay section in the Assay Part.
Results
1. TTC Detectable Time
TTC detectable time was measured by following the procedure in the TTC Assay section in the Assay part.
Table 11
Figure PCTCN2018078160-appb-000062
It can be seen from the results in Table 11 that for the test group swatches washed with detergent comprising blend of protease and amylase, both the W30A and the CN42 showed TTC detectable time that was were significantly prolonged as compared to the negative control 1 group, and the Test/Negative 1 ratio are respectively 2 and 1.5 for W30A swatch and the CN42 swatch.
This suggests the bacterial growth inhibition benefit by using these enzymes in liquid detergent for cleaning the fabric surfaces.
Example 7. Use of cellulase in reducing attachment of bacterial on textile
Materials:
1. Detergents: Liquid Detergent: Model A; Powder detergent: Model X
2. Microorganism: Pseudomonas putida (China General Microbiological Culture Collection Center (CGMCC) , the strain number 1.3096)
3. Pigment soil: WFK09V from Center For Testmaterials BV, CFT. It is a standard pigment mixture in all soiled/stained test fabrics containing 'Pigment' ) . Addition of pigment soil is to mimic the real wash situation where there are relatively dirty laundry releasing soil into the wash liquor.
4. Washing machine:
a. miniLOM, 25℃, 60 min, 20 rpm, 20 ml washing liquor, 10 min rinses in 400 ml washing beaker, when liquid detergent Model N was used
b. TOM, 25C, 30 or 20 min, 120 rpm, 500 ml washing liquor, 10 min rinses in 500 ml washing beaker, when powder detergent Model X was used
5. Swatch: Emp252 (kinnted striped cotton textile)
Procedures:
1. Prewash of the swatch: The swatches were first aged in Wascator machine, program 151, age time: 10 h. Then the test swatches were washed with model detergents either liquid dertergent Model A comprising 0.075 ppm Cellulase, SEQ ID NO: 15; or powder detergent X comprising 0.05 ppm Cellulase, SEQ ID NO: 15; and the control swatches were washed with respective Model Detergent only, in FSW. After wash for 20 cycles, the swatches were cut to round shape with a diameter of 1.9 cm and then sterilize in autoclave under 121℃, 15 min and dry at oven with 60℃.
Table 12. FSW wash condition for Model A:
EU washing machine Wash conditions
Wash program Standard
Volume 15L
Washing time (main wash)  51 min
Temperature 40℃
Water hardness 15°dH (2: 1: 7.5)
Rinse 2
Ballast 3 Kg ballast (mixed 60%cotton + 40%polyester)
Table 13. FSW wash condition for Model X:
AP washing machine Wash conditions
Wash program Standard
Volume 33 L
Washing time (main wash) 15 min
Temperature 25℃
Water hardness 14°dH (2: 1: 4.5)
Rinse 2
Ballast 1.5 Kg ballast (mixed 60%cotton + 40%polyester)
2. Second wash of the above prewashed swatch together with pigment soil and bacterial innoculation
1) A fresh Pseudomonas putida broth to reach OD 600=0.3 was prepared according to normal procedure. 
2) A 20 ml wash liquor was prepared by loading the detergent and pigment soil into a miniLOM beaker (or 500 mL wash liquor for TOM beaker) by adding water (water hardness 14°dH, Ca 2+ : Mg 2+ : Na +=2 : 1 : 4.5) to reach pigment soil concentration of 0.7 g/L, and detergent concerntation of 2 g/L. Then a 100μL for miniLOM wash or 1900 ul for TOM wash of fresh bacteria broth (OD 600=0.3) was added into the wash liquor.
3) 3 pieces of the prewashed swatches was put into each of the miniLOM beaker or TOM beaker, wash for 60 min, 20 rpm at 25℃ for miniLOM, wash for 30 min, 120 rpm at 30℃ for TOM.
4) After wash, take swatches out from beakers and put into 400 mL sterilized water after washed in miniLOM or 500 ml sterilized water after washed in TOM. Rinse for 10 min.
3. Measure the amount of bacterial remained on the swatches: Transfer different swatches onto Nutrient agar plate and let it stay for 20 seconds. Incubate the agar plates in incubator at 28℃ for about 12-24 hours till colonies are formed, determine CFU.
Results:
Table 14. Bacterial CFU attached to Emp252 swatches after the prewash with liquid detergent
Groups Average CFU
Swatches prewashed without cellulase 142
Swatches pre-washed with cellulase (SEQ ID NO: 15) , 108
Reduction percentage 23.9%
From the result in above table, it can be seen that for those swatches pre-washed with liquid detergents comprising cellulase (SEQ ID NO: 15) , when it further underwent co-wash with relatively dirty laundries as mimiced by the addition of pigment soil and bacterial for those released from such relatively dirty laundry to the wash liquor, there are 23.9%less of bacterial CFUs remained on the surface of the prewashed swatches than those swatches which were pre-washed with detergents only comprising no cellulase. This result shows cellulase can be effectively used in reducing the attachment of bacteria onto textiles.
Table 15. Bacterial CFU residue on Emp252 swatches after prewash with powder detergent
Groups Average
Swatches prewashed without cellulase 27
Swatches washed with cellulase (SEQ ID NO: 15) , 18
Reduction percentage 33.0%
From the result in above table, it can be seen that for those swatches pre-washed with liquid detergents comprising cellulase (SEQ ID NO: 15) , when it further underwent co-wash with relatively dirty laundries as mimiced by the addition of pigment soil and bacterial for those released from such relatively dirty laundry to the wash liquor, there are 33.0%less of bacterial CFUs remained on the surface of the prewashed swatches than those swatches which were  pre-washed with detergents only comprising no cellulase. This result shows cellulase can be effectively used in reducing the attachment of bacteria onto textiles.
Example 8 Use of cellulase in reducing attachment of fungi on textile
Example 8 was conducted essentially the same as the previous example with the exception in that the fungi Candida parapsilosis (China General Microbiological Culture Collection Center (strain number: 2.1846) ) replaced the bacterial Pseudomonas putida, the amount of Candida parapsilosis and the cultivation condition was accordingly different as well from previous Example.
1) The amount of yeast load is: 100 μL yeast fresh broth (OD 600=0.3) into the wash liquor for miniLOM, load 1000 μL yeast fresh broth (OD 600=0.3) into the wash liquor for TOM.
2) The washing time in TOM is 20 min.
3) The cultivation medium for Candida parapsilosis is Yeast Extract–Peptone–Dextrose (YPD) Medium: 10 g/L Yeast extract, 20 g/L Peptone, 20 g/L Dectrose (sterilize Dectrose separately with others, mix together after sterilization) , 20 g/L agar for solid medium.
Independent from the CFU assay, the swatches were subject to the fluorescene detection from remaining microbes on the swatches using the PrestoBlue TM Cell Viability Reagent to compare the viability of microbes remained on it after the second wash.
PrestoBlue TM Cell Viability Reagent (Thermo Fisher Scientific, Catalog number: A13261) was used. PrestoBlue is a cell permeable resazurin-based solution that functions as a cell viability indicator by using the reducing power of living cells to quantitatively measure the proliferation of cells. When added to cells, the PrestoBlue reagent is modified by the reducing environment of the viable cell and turns red in color, becoming highly fluorescent. This color change can be detected using fluorescence or absorbance measurements. PrestoBlue solution is prepared with the ratio of PrestoBlue TM Cell Viability Reagent : YPD medium=1: 9.
After wash in TOM or miniLOM, the swatches in different beakers are rinsed respectively with 500 mL sterilized water for 10 min, then carefully remove the excessive water from swatches. Swatches are transferred into 12-well plate. Add 2 ml PrestoBlue solution in each well, then cover the plate and detect Fluorescence kinetics (Excitation: 544 nm, Emission: 590 nm;interval: 30 min, duration: 22 hours) .
Results:
Table 16 Fungi CFU residue on Emp252 swatches after prewash with liquid detergent.
Groups Average
Swatches prewashed without cellulase 22.3
Swatches prewashed with cellulase (SEQ ID NO: 15) , 7.3
Reduction percentage 67.3%
From the result in above table, it can be seen that for those swatches pre-washed with liquid detergents comprising cellulase (SEQ ID NO: 15) , when it further underwent co-wash with relatively dirty laundries as mimiced by the addition of pigment soil and bacteria for those released from suchrelatively dirty laundry to the wash liquor, there are 67.3%less of fungi CFUs remained on the surface of the prewashed swatches than those swatches which were pre-washed with detergents only comprising no cellulase. This result shows cellulase can be effectively used in reducing the attachment of fungi onto textiles.
Table 17. Fungi CFU residue on Emp252 swatches after prewash with powder detergent
Figure PCTCN2018078160-appb-000063
From the result in above table, it can be seen that for those swatches pre-washed with liquid detergents comprising cellulase (SEQ ID NO: 15) , when it further underwent co-wash with relatively dirty laundries as mimiced by the addition of pigment soil and bacteria for those released from such relatively dirty laundry to the wash liquor, there are 85%less of fungi CFUs remained on the surface of the prewashed swatches than those swatches which were pre-washed with detergents only comprising no cellulase. This result shows cellulase can be effectively used in reducing the attachment of fungi onto textiles.
Table 18 PrestoBlue fluorescence emission results
Figure PCTCN2018078160-appb-000064
Figure PCTCN2018078160-appb-000065
The same results corresponding to Table 18 is also shown in Figure 1.
From the PestoBlue fluorescence assay results in above Table and the Figure 1, it can be seen that for those swatches pre-washed with liquid detergents comprising cellulase (SEQ ID NO: 15) , when it further underwent co-wash with relatively dirty laundries as mimiced by the addition of pigment soil and bacteria for those released from such relatively dirty laundry to the wash liquor, the fluroscence kinetics chart indicates a much lower viability of the fungi remained on the surface of the prewashed swatches than those swatches which were pre-washed with detergents only comprising no cellulase. Specifically, the swatches enzymatically prewashed showed a much slower flatter rise of the fluorescene unit valuem and this is significantwhen the swatches had been incubated for a period of time of more than 900 mins. This result shows cellulase can be effectively used in reducing the attachment of fungi onto textiles.
Example 9. Use of enzyme in inhibiting bacteria generated with enzymatic liquid detergent
Materials
1. Bacterial: Pseudomonas putida. (China General Microbiological Culture Collection Center (CGMCC) , the strain number 1.3096)
2. Detergent: BlueMoon Deepclean liquid detergent, wash liquor concentration 2 g/L
3. Enzyme:
a. Protease, SEQ ID NO: 3
b. Enzyme mixture 1: 0.41 ppm protease (SEQ ID NO: 3) , 0.052 ppm amylase (SEQ ID NO: 9)
c. Enzyme mixture 2: 0.62 ppm protease (SEQ ID NO: 3) , 0.052 ppm amylase (SEQ ID NO: 9) and 0.16 ppm celluclase (SEQ ID No: 17)
4. Swatches: pre-aged PCN01: swatches are aged in Wascator machine, program 151, age time: 10 h.
5. Stains: Baby food #2, braised beef stain
Procedure:
1. The baby food stained swatches or the braised beef stained swatches were prepared according to the procedure described in the Assays section: Preparation of stained swatches.
2. FSW wash procedure was used to wash the above stained swatches. Wash condition: AP top loader machine, 25℃, 14°dH (Ca 2+: Mg 2+ = 3: 2) , for 15 min main washing time, 2 rinses. For different groups, the swatches were washed with detergents only (BlueMoon Deepclean) , or detergent comprising various enzyme or enzyme combinations.
3. The swatches from step 2 were put into sterilized petri dish (Diameter=9 cm) , one piece swatch one petri dish, for receiving an inoculation of the Pseudomonas putida. The preparation of the bacterial inoculation and the application of such onto the washed swatches, and the observation of the growth of the TTC color change on the swatches follow the relevant description in the TTC Assay section in the Assay Part.
4. After incubating the swatches for 30±4 hours, all petri dishes were taken out from incubator to undergo odor panel evaluation by the description mentioned above.
5. Odor panel evaluation:
The swatches taken out from the incubator were allowed to balance at room temperature and room humidity for 30 min before moving into the panel room for evaluation. The panelists are selected from experienced and qualified pool of candidates who. The panelists performed the evaluation without being able to see the swatches. Two samples in two petri dishes which need to be evaluated were provided, the panellist smelled each sample for 2 second and then took some breath of fresh air for more than 10 s before smell another sample. After breathing fresh air for more than 10s, they can evaluate the next pair of samples following the same procedure.
6. In parallel to the panel evaluation, the swatches from step 4 also went through GS-MS measurement for malodor:
Take out of the microbe stained swatches from incubator. Using sterilized scissor to cut off a small piece of stain with 1.5 cm × 1.5 cm from the stain area. Each cut off swatch was then transferred to a 20 mL GC headspace vial, using clean, sterile tweezers, and the headspace from the capped vial was then analysed.
The headspace from the capped vials is analysed by GC-MS:
GCMS Agilent 7890 GC with split/splitless injector and 5977 MS with extractor ion source coupled to a Gerstel MPS2 sampler with HS/SPME, SPME needle heater.
Gerstel MPS SPME Incubator: Agitator. Incubation Temperature: 60℃.
Incubation Time: 10.00 min. Agitator Speed: 250 rpm. Sample parameters:
Extraction Time: 20 min; Inj. Desorption Time: 120 s.
SPME (Solid Phase Micro Extraction) Fiber type: Carboxen/Polydimethylsiloxane (CAR/PDMS)
The method used was: GC Oven Temperature: Initial 40 ℃; hold 2 min; Rate 5 ℃/min until 150 ℃; Rate 35 ℃/min until 240 ℃; Hold 3 min. Front SS Inlet He: Mode Split; T a 230 ℃, Split Ratio 10 : 1; Split Flow 15 mL/min. Column: Agilent 19091F-433: FFAP-01 HP-FFAP 30 m x 250 μm x 0.25 μm
MS Information: Acquisition Mode: Scan. Solvent Delay (minutes) : 1. Scan Parameters: Start Time: 1. Low Mass: 35. High Mass: 350. Threshold: 100. A/D Samples: 4. MS Zones: MS Source: 230 ℃. MS Quad: 150 ℃
Results
1. Odor panel
Table 19. 5 panelists odor evaluation on baby food and Pseudomonas putida stained swatches:
Figure PCTCN2018078160-appb-000066
Figure PCTCN2018078160-appb-000067
The results of Table 19 above show that for those Baby food stained swatches which were washed with detergent comprising different enzyme or enzyme mixtures and subsequently exposed to bacterial (Pseudomonas putida) , they all show significantly better malodor inhibition result as indicated by the panel evaluation than the parallel group of swatches which were washed with detergents only.
Table 20. 11 panelists odor evaluation on baby food and Pseudomonas putida stained swatches:
Figure PCTCN2018078160-appb-000068
The results of Table 20 above show that for those Baby food stained swatches which were washed with detergent comprising different enzyme or enzyme mixtures and subsequently exposed to bacterial (Pseudomonas putida) , they all show significantly better malodor inhibition result as indicated by the panel evaluation than the parallel group of swatches which were washed with detergents only.
2. GCMS results
Table 21. GCMS malodor detection results for baby food and Pseudomonas putida stained swatches:
Figure PCTCN2018078160-appb-000069
Figure PCTCN2018078160-appb-000070
The four VOC molecules are representative malodor molecules.
The results of Table 21 above show that for those Baby food stained swatches which were washed with detergent comprising different enzyme or enzyme mixtures and subsequently exposed to bacterial (Pseudomonas putida) , shows significantly lower VOC levels as compared to those parallel group of swatches washed with detergents only comprising no enzymes.
Table 22. GCMS malodor detection results for braised beef stain and Pseudomonas putida stained swatches:
Figure PCTCN2018078160-appb-000071
The seven VOC molecules can be representative malodor molecules, particularly 3-octanone has musty and moldy smell, 2, 3-Butanedione has skink smell.
The results of Table 22 above show that for those braised beef stained swatches which were washed with detergent comprising different enzyme or enzyme mixtures and subsequently exposed to bacterial (Pseudomonas putida) , shows significantly lower VOC levels as compared to those parallel group of swatches washed with detergents only comprising no enzymes.
Example 10 Inhibition of fungal malodor generation by washing with multi enzymatic liquid detergent comprising enzymes
Example 9 was conducted essentially the same as the previous example with the exception in that the fungi Aspergillus niger (stain information is same as in Example 2) replaced the bacterial Pseudomonas putida. The preparation of the fungal inoculation and the application of such onto the washed swatches, and the observation of the growth of the fungi on the swatches follow the relevant description in the Fungi Spore/Mycelium Formation Time Assay section in the Assay Part. was accordingly different as well from previous Example. The swatches were only evaluated with GS-MS measurement for malodor.
Results
Table 23. GCMS malodor results for baby food and Aspergillus niger stained swatches:
Figure PCTCN2018078160-appb-000072
Figure PCTCN2018078160-appb-000073
The results of Table 23 above show that for those baby food stained swatches which were washed with detergent comprising different enzyme or enzyme mixtures and subsequently exposed to Aspergillus niger, shows significantly lower VOC levels as compared to those parallel group of swatches washed with detergents only comprising no enzymes.
Table 24. GCMS malodor detection results for braised beef stain and Aspergillus niger stained swatches:
Figure PCTCN2018078160-appb-000074
Figure PCTCN2018078160-appb-000075
Figure PCTCN2018078160-appb-000076
The results of Table 24 above show that for those braised beef stained swatches which were washed with detergent comprising different enzyme or enzyme mixtures and subsequently exposed to Aspergillus niger, shows significantly lower VOC levels as compared to those parallel group of swatches washed with detergents only comprising no enzymes.

Claims (19)

  1. Use of one or more enzymes in preventing, inhibiting or reducing microbe growth on a surface, wherein after use of said enzymes, said microbe on said surface have an increase in its TTC detectable time of at least 20%, at least 30%, at least 50%, at least 70%, at least 80%, or at least 100%.
  2. The use according to claim 1, wherein the microbe is a fungi, and the fungi has an increase in the spore forming time of at least 20%, and/or an decrease in spore density score of at least 1 in a spore density scale of from 0 to 7, compared to those on a surface not treated with enzymes.
  3. The use of claim 1 or 2 in inhibiting or reducing malodor caused by microbe on a textile surface.
  4. The use of claim 1 or 2 in preventing or reducing microbe attachment onto a textile surface.
  5. The use according to any of claims 1-4, wherein the microbe is a fungi or bacteria selected from a group consisting of Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Pseudomonas putida, Micrococcus luteus, Staphylococcus epidermidis, Enhydrobacter aerosaccus, Corynebacterium jeikeium, Propionibacterium acnes, Brevundimonas vesicularis, Streptomyces griseus, Streptomyces odorifer, Exophiala phaeomuriformis, Fusarium oxysporum, Alternaria alternate, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aureobasidium pullulans, Chaetomium globosum, Cladosporium sphaerospermum, Gliocladium virens, Mucor plumbeus, Penicillium chrysogenum, Penicillium commune, Penicillium crustosum, Penicillium rubens, Penicillium varians, Penicillium citrinum, Stachybotrys chartarum, Trichoderma viride, Trichophyton rubrum, Candida parapsilosis, Rhodotorula mucilaginosa, Debaryomyces hansenii, Meyerozyma guilliermondii, Candida albicans, and combinations thereof.
  6. The use according to claim 1-5, wherein said microbe growth is on the residual stain area of said surface.
  7. The use according to any of claims 1-6, wherein said one or more enzymes are selected from a group consisting of amylase, protease, lipase, mannase, cellulase, pectinase, and combinations thereof.
  8. The use according to claim any of claims 1-7, wherein the enzyme is an amylase, protease, cellulase or combinations thereof.
  9. The use according to claim 3 or 4, wherein the enzyme is a cellulase.
  10. The use according to any of the above claims, wherein the protease is selected from a group consisting of:
    i) a protease comprising a substitution compared to the protease shown in SEQ ID NO 1 or SEQ ID NO 2 in one or more of the following positions: 3, 4, 9, 15, 24, 27, 42, 55, 59, 60, 66, 74, 85, 96, 97, 98, 99, 100, 101, 102, 104, 116, 118, 121, 126, 127, 128, 154, 156, 157, 158, 161, 164, 176, 179, 182, 185, 188, 189, 193, 198, 199, 200, 203, 206, 211, 212, 216, 218, 226, 229, 230, 239, 246, 255, 256, 268 and 269, wherein the positions correspond to the positions of the protease shown in SEQ ID NO 1, or
    ii) one or more protease variant of a protease parent, wherein the protease variant comprises one or more mutation selected from the group consisting of S3T, V4I, S9R, S9E, A15T, S24G, S24R, K27R, N42R, S55P, G59E, G59D, N60D, N60E, V66A, N74D, N85S, N85R, G96S, G96A, S97G, S97D, S97A, S97SD, S99E, S99D, S99G, S99M, S99N, S99R, S99H, S101A, V102I, V102Y, V102N, S104A, G116V, G116R, H118D, H118N, N120S, S126L, P127Q, S128A, S154D, A156E, G157D, G157P, S158E, Y161A, R164S, Q176E, N179E, S182E, Q185N, A188P, G189E, V193M, N198D, V199I, Y203W, S206G, L211Q, L211 D, N212D, N212S, M216S, A226V, K229L, Q230H, Q239R, N246K, N255W, N255D, N255E, L256E, L256D T268A and R269H, wherein the positions correspond to the positions of the protease shown in SEQ ID NO 1, wherein the protease parent is selected from the protease shown in SEQ ID NO 1 and the Bacillus amylolichenifaciens protease (BPN’ ) shown in SEQ ID NO 2 and wherein the protease variant has at least 80%sequence identity to SEQ ID NO 1 or 80%sequence identity to SEQ ID NO 2,
    iii) a protease comprising a substitution at one or more positions corresponding to  positions 171, 173, 175, 179, or 180 of SEQ ID NO: 14, compared to the protease shown in SEQ ID NO 14, wherein the protease variant has a sequence identity of at least 75%but less than 100%to SEQ ID NO 14,
    iv) a protease comprising the amino acid sequence shown in SEQ ID NO 1 or 2 or a protease having at least 80%sequence identity to; the polypeptide comprising amino acids 1-269 of SEQ ID NO 1 or the polypeptide comprising amino acids 1-275 of SEQ ID NO 2,
    v) One or more of the following protease variants selected from the group: SEQ ID NO 1 with the alterations T22R+S99G+S101A+V102I+A226V+Q239R, SEQ ID NO 2 with the alterations S24G+S53G+S78N+S101N+G128A+Y217Q, SEQ ID NO 2 with the alterations S24G+S53G+S78N+S101N+G128S+Y217Q, SEQ ID NO 1 with the alterations S9E + N42R + N74D + V199I + Q200L +Y203W + S253D + N255W + L256E,
    SEQ ID NO 1 with S9E+N42R+N74D+H118V+Q176E+A188P+V199I+Q200L Y203W+S250D+S253D+N255W+L256E
    SEQ ID NO 1 with S9E+N42R+N74D+Q176E+A188P+V199I+Q200L+Y203W S250D+S253D+N255W+L256E
    SEQ ID NO 1 with S3V+N74D+H118V+Q176E+N179E+S182E+V199I+Q200L Y203W+S210V+S250D+S253D+N255W+L256E
    SEQ ID NO 1 with the alterations T22A+N60D+S99G+S101A+V102I+N114L+G157D +S182D+T207A+A226V+Q239R+N242D+E265F,
    SEQ ID NO 1 with the alterations S9E + N42R + N74D + H118V + Q176E +A188P + V199I + Q200L + Y203W + S250D + S253D + N255W + L256E,
    SEQ ID NO 1 with the alterations S9E + N42R + N74D + Q176E + A188P +V199I + Q200L + Y203W + S250D + S253D + N255W + L256E,
    SEQ ID NO 1 with the alterations S9E + N42R + N74D + H118V + Q176E +A188P + V199I + Q200L + Y203W + S250D + N255W + L256E + *269aH +*269bH,
    SEQ ID NO 1 with the alterations S3V + N74D + H118V + Q176E + N179E +S182E + V199I + Q200L + Y203W + S210V + S250D + N255W + L256E,
    SEQ ID NO 1 with the alterations S9E + N74D + G113W + G157P + Q176E +V199I + Q200L + Y203W + S250D + T254E + N255W + L256E,
    SEQ ID NO 1 with the alterations S3V + S9R + N74D + H118V + Q176E +N179E + S182E + V199I + Q200L + Y203W + S212V + S250D + N255W +L256E,
    SEQ ID NO1 with the alteration S99E, and
    SEQ ID NO 2 with the alteration L217D,
    and combinations thereof.
  11. The use of any of the above claims, wherein the amylase is selected from the group consisting of:
    i) variants comprising one or more substitutions in the following positions: 9, 26, 30, 33, 82, 37, 106, 118, 128, 133, 149, 150, 160, 178, 182, 186, 193, 195, 202, 203, 214, 231, 256, 257, 258, 269, 270, 272, 283, 295, 296, 298, 299, 303, 304, 305, 311, 314, 315, 318, 319, 320, 323, 339, 345, 361, 378, 383, 419, 421, 437, 441, 444, 445, 446, 447, 450, 458, 461, 471, 482, 484, wherein the positions corresponds to positions of SEQ ID NO 11;
    ii) variants exhibiting at least 90 percent identity with SEQ ID NO 12, with deletions in the 183 and 184 positions,
    iii) variants exhibiting at least 95 percent identity with SEQ ID NO 13, comprising mutations in one or more of the following positions M202, M208, S255, R172 and/or M261,
    iv) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 9 or 10,
    v) a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at leaset 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%sequence identity to SEQ ID NO: 9 or 10, and combinations thereof.
  12. The use according to any of the above claims, wherein the cellulase is selected from the group consisting of :
    a. a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at leaset 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%sequence identity to the polypeptide shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and combinations thereof.
  13. The use according to any of the above claims, wherein said surface is a textile made of  nature fiber such as cotton, synthetic fiber such as polyester, or a mixture thereof.
  14. The use according to any of the above claims, wherein the enzyme (s) is incorporated in a detergent composition comprising essentially no biocide.
  15. The use according to any of the above claims, wherein the enzyme (s) is incorporated in a powder detergent or liquid deterget.
  16. A method of preventing, inhibiting or reducing microbe growth on surface, comprising the steps of :
    a. treating the surface with enzymes as defined in any of claims 10-12 to the extent that at least one microbe has a TTC detectable time on said surface after the treatment which increases at least 20%.
  17. A method of demonstrating the microbe growth inhibition or deep cleansing benefit of an enzyme or enzyme combination as defined in claims 10-12 on a surface, comprising the steps of:
    a. Providing two surfaces A and A’ ,
    b. Applying a soil comprising a microbe growth indicator onto each of the surfaces A and A’ ,
    c. Washing A with a detergent composition comprising no enzyme, and washing A’ with said detergent composition comprising one or more enzymes,
    d. Preparing an inoculation of microbe and apply it onto the surfaces A and A’ ,
    e. Incubating A and A’ under suitable conditions to allow microbe growth for a predetermined period of time,
    f. Comparing the growth of microbe on A and A’ .
  18. The method according to claim 17, wherein the microbe growth indicator is triphenyltetrazolium chloride (TTC) .
  19. The method according to claim 16 or 17, wherein the surfaces A and A’ provided in step a. of claim 14 are made of essentially the same material.
PCT/CN2018/078160 2017-03-06 2018-03-06 Use of one or more enzymes in preventing, inhibiting or reducing microbe growth on a surface WO2018161899A1 (en)

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CN201880006185.3A CN110381973A (en) 2017-03-06 2018-03-06 One or more enzymes are preventing, inhibiting or are reducing the purposes in microbial growth on surface
PH12019501420A PH12019501420A1 (en) 2017-03-06 2019-06-20 Use of one or more enzymes in preventing, inhibiting or reducing microbe growth on a surface

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CN116694486A (en) * 2023-02-24 2023-09-05 天津科技大学 Rhodotorula mucilaginosa strain and application thereof in preparation of soy sauce
WO2023225459A2 (en) 2022-05-14 2023-11-23 Novozymes A/S Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections
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CN116694486B (en) * 2023-02-24 2024-05-28 天津科技大学 Rhodotorula mucilaginosa strain and application thereof in preparation of soy sauce

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