WO2017192417A1

WO2017192417A1 - Enzymatic cleaning compositions and methods

Info

Publication number: WO2017192417A1
Application number: PCT/US2017/030337
Authority: WO
Inventors: Ranjani V. Parthasarathy; Catherine D. HEAPY
Original assignee: 3M Innovative Properties Company
Priority date: 2016-05-04
Filing date: 2017-05-01
Publication date: 2017-11-09

Abstract

Enzymatic cleaning compositions and methods of use, wherein the cleaning compositions include an enzyme and a surfactant, or an enzyme, a surfactant, and a cationic antimicrobial, or an enzyme, a surfactant, a cationic antimicrobial, and an antimicrobial lipid.

Description

ENZYMATIC CLEANING COMPOSITIONS AND METHODS Background

After a surgical procedure or other medical procedure, medical instruments used in the procedure are wiped to remove large or loosely held bone, tissue, and/or blood and washed to remove any gross blood and/or tissue residuals. The instruments are then placed in a surgical tray and loaded into a case or cart for transport to a sterile processing department for further cleaning and sterilization. All of the instruments are manually inspected and hand washed in wash sinks before the surgical trays are placed in automatic dishwashers for continued processing through the department.

The consequences of the use of contaminated medical instruments, particularly endoscopes, are a recurrent topic in the literature. For example, flexible endoscopes may become heavily contaminated with blood, secretions, and microorganisms in the planktonic state as well as in the form of a biofilm during use. These instruments are difficult to clean and disinfect and easy to damage because of their complex design, with narrow lumens and multiple internal channels. If the instruments are not properly cleaned, the disinfection and drying procedures can fail and increase the possibility of transmission of infection from one patient to another. In addition, the ability of bacteria to form biofilms in the endoscope channels, especially when these become damaged, can contribute to failure of the

decontamination process.

Accurate reprocessing of flexible endoscopes is a multistep procedure involving cleaning, followed by high-level disinfection (HLD), with further rinsing and drying before storage. Endoscope reprocessing can be performed with the use of automated endoscope reprocessors (AERs) and manual methods. Since almost all outbreaks are related to breaches in reprocessing techniques, it is crucial that endoscope cleaning, disinfection, and drying are performed.

Even when adhering to a strict protocol, however, control of endoscope reprocessing does not guarantee prevention of settlement of biofilm during endoscopy. The most common factors associated with microbial transmission involve inadequate cleaning, disinfection, and drying procedures, the use of contaminated AERs, and flaws in instrument design or the use

of damaged endoscopes.

Conventional cleaning products used for washing medical instruments, including endoscopes, typically include enzyme solutions and preparations that are provided in a concentrated form that can be diluted for use during manual cleaning, as well as in the washers and the AER's. Commercially available enzyme solutions, both in concentrated and dilute forms, have one or more disadvantages. For example, conventional enzyme solutions may be adversely affected by storage temperatures that may destroy or greatly reduce the effectiveness of the enzyme solutions, thereby limiting shelf life. During use of conventional enzyme solutions, it may be necessary to control the water temperature so that the effectiveness of the enzymes is not reduced. Directions for use of the enzymes suggest relatively long soak times for the enzymes to work on the organic materials on the instruments; however, throughput requirements during sterile processing may result in insufficient soak times for effective cleaning.

Accordingly, there is a need for improved enzymatic cleaning compositions for use in sterile processing of medical instruments, for example.

Summary

The present disclosure is directed to enzymatic cleaning compositions and methods of use.

Cleaning compositions (i.e., enzymatic cleaning compositions) of the present disclosure include an enzyme and a surfactant. In certain embodiments, cleaning compositions of the present disclosure include an enzyme, a surfactant, and a cationic antimicrobial. In certain embodiments, cleaning compositions of the present disclosure include an enzyme, a surfactant, a cationic antimicrobial, and an antimicrobial lipid.

In certain embodiments, enzymatic cleaning compositions of the disclosure are effective to remove at least a portion of a biofilm from a substrate. In certain embodiments, enzymatic cleaning compositions of the disclosure are effective to remove at least a portion of biological residue from a substrate. In certain embodiments, enzymatic cleaning compositions of the disclosure are effective to kill at least a portion of bacteria on a substrate.

In one embodiment of the disclosure, there is provided a method of removing at least a portion of a biofilm from a surface of a non-biological article. Such method includes contacting the (biofilm- coated) surface with an enzymatic cleaning composition under conditions effective to remove at least a portion of a biofilm, wherein the enzymatic cleaning composition includes a cationic antimicrobial, a surfactant, an enzyme, and optionally an antimicrobial lipid.

In one embodiment of the disclosure, there is provided a method of cleaning a surface of a non- biological article. Such method includes contacting the surface with an enzymatic cleaning composition under conditions effective to remove at least a portion of a biofilm, at least a portion of a biological residue, and at least a portion of bacteria, wherein the enzymatic cleaning composition includes a surfactant and an enzyme.

In certain embodiments, methods of the present disclosure remove biofilm by at least 2 log reduction (i.e., 99% reduction) according to the Biofilm Generation and Removal Test Method Two. In certain embodiments, methods of the present disclosure remove biofilm by at least 3 log reduction (i.e., 99.9% reduction) according to the Biofilm Generation and Removal Test Method One. In certain embodiments, methods of the present disclosure remove bacteria by at least 3 log reduction. In certain embodiments, methods of the present disclosure provide a cleaning efficiency of at least 7 on a scale of 1 to 10 (per the Cleaning Ability test described in Example 6).

In certain embodiments, the enzyme includes a protease. In certain embodiments, the composition further includes an amylase, a lipase, a cellulase, or combinations thereof. In certain embodiments, the surfactant includes a nonionic surfactant, a zwitterionic surfactant, or combinations thereof. In certain embodiments, the composition includes a nonionic surfactant. In certain embodiments, the composition further includes an anionic surfactant.

In certain embodiments, the cationic antimicrobial includes a biguanide, a bisbiguanide, a quaternary ammonium compound (including polymeric and small molecule quaternary ammonium compounds), or combinations thereof.

In certain embodiments, the antimicrobial lipid includes a compound that has a solubility in water of no greater than 1.0 gram per 100 grams (1.0 g/ 100 g) deionized water. In certain embodiments, the antimicrobial lipid includes a (C7-C12)saturated fatty acid ester of a polyhydric alcohol, a (C12- C22)unsaturated fatty acid ester of a polyhydric alcohol, a (C7-C12)saturated fatty ether of a polyhydric alcohol, a (C6-C12)alkyl or (C8-C18)alkylene 1,2-diol, a (C12-C22)unsaturated fatty ether of a polyhydric alcohol, an alkoxylated derivative thereof, or combinations thereof.

Definitions

A cleaning composition of the present disclosure may be in a "ready-to-use" form or a

"concentrated" form. Herein, a "ready-to-use" composition is one that is not diluted before cleaning a surface. In contrast, a "concentrated" composition is one that is diluted before cleaning a surface.

Dilutions typically seen are 1 : 1 to 1:500, wherein 1 part of concentrate is added to 1 part water (or 500 parts water).

In the context of a composition, "solids" or "total solids" refers to the amount of solids, without a volatile liquid carrier, unless specified otherwise. In certain embodiments, an enzymatic cleaning composition includes up to 30 wt-% solids.

The term "biofilm" refers to microbiologically generated polysaccharide-containing (and often protein- and DNA-containing) matrices that form when bacteria adhere to surfaces in aqueous environments, multiply, and begin to excrete an extracellular polymeric substance (i.e., a slimy, glue-like substance that includes polysaccharide and protein) that can anchor to various materials such as those found in medical instruments.

The term "biological residue" refers to soil from blood, mucus, saliva, bone, feces, and/or other biological fluids and/or tissue that is distinct from biofilm. Biological residue may contain, e.g., red blood cells, hemoglobin, iron, fibrin, blood-containing proteins, saliva-containing proteins, prions, etc.

The terms "comprises" and "includes" and variations thereof do not have a limiting meaning where these terms appear in the description and claims. Such terms will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By "consisting of is meant including, and limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

The words "preferred" and "preferably" refer to embodiments of the disclosure that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

As used herein, "a," "an," "the," "at least one," and "one or more" are used interchangeably. For example, a composition that includes "a" surfactant may include "one or more" surfactants.

As used herein, the term "or" is generally employed in its sense including "and/or" unless the content clearly dictates otherwise. The term "and/or" means one or all of the listed elements or a combination of any two or more of the listed elements.

As used herein, all numbers are assumed to be modified by the term "about" and in certain embodiments by the term "exactly." Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Herein, "up to" a number (e.g., up to 50) includes the number (e.g., 50).

The term "in the range" or "within a range" (and similar statements) includes the endpoints of the stated range.

Groupings of alternative elements or embodiments disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found therein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

When a group is present more than once in a formula described herein, each group is

"independently" selected, whether specifically stated or not. For example, when more than one Y group is present in a formula, each Y group is independently selected. Furthermore, subgroups contained within these groups are also independently selected. For example, when each Y group contains an R, then each R is also independently selected.

Reference throughout this specification to "one embodiment," "an embodiment," "certain embodiments," or "some embodiments," etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the invention. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

The above summary of the present disclosure is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples may be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

Detailed Description of Illustrative Embodiments

The present disclosure is directed to enzymatic cleaning compositions and methods of use. Such enzymatic cleaning compositions may be used for cleaning various articles, particularly medical instruments such as endoscopes and other instruments used for surgery. These can include both metallic and plastic instruments. Such enzymatic cleaning compositions may be used in manual cleaning methods or in automatic cleaning methods and equipment (e.g., in dishwashers and automated endoscope reprocessors).

Cleaning compositions of the present disclosure include an enzyme and a surfactant. In certain embodiments, cleaning compositions also include a cationic antimicrobial. In certain embodiments, cleaning compositions also include an antimicrobial lipid.

In certain embodiments, enzymatic cleaning compositions of the present disclosure are stable. In this context, "stable" means physically stable and/or chemically stable and/or enzymatically stable. In certain embodiments, enzymatic cleaning compositions of the present disclosure are chemically, physically, and enzymatically stable.

As defined herein, "physically stable" compositions are those that do not significantly change due to substantial precipitation, crystallization, phase separation, or the like, from their original ready-to-use condition until used. The amount of time will depend upon whether the composition is provided as a ready-to-use one-part composition or as a multiple part composition, the parts of which are combined just prior to application. The compositions are preferably physically stable for at least two weeks, more preferably at least 3 months, or at least 6 months as a concentrate. Particularly preferred compositions are completely physically stable if a 10-milliliter (10-mL) sample of the composition when placed in a 15-mL conical-shaped graduated plastic centrifuge tube (Corning) and centrifuged at about 2275 x g (e.g., 3,000 revolutions per minute (rpm) for 10 minutes using a Labofuge B, model 2650 manufactured by Heraeus Sepatech GmbH, Osterode, West Germany) or similar centrifuge at a centrifugal force of 2275 x g has no visible phase separation in the bottom or top of the tube. Phase separation of less than 0.5 mL is also considered stable as long as there is no other sign of physical separation in the sample.

As defined herein, "chemically stable" means an average of at least 97 wt-% of the cationic antimicrobial and/or antimicrobial lipid is retained after aging for 4 weeks at 40°C in a sealed container beyond the initial 5 -day equilibration period at 23 °C. This is determined by comparing the amount remaining in a sample aged (i.e., aged beyond the initial 5 -day equilibration period) in a sealed container that does not cause degradation, to the actual measured level in an identically prepared sample (preferably from the same batch) and allowed to sit at 23 °C for five days. The level of cationic antimicrobial and/or antimicrobial lipid is preferably determined using gas chromatography or high performance liquid chromatography.

As defined herein, "enzymatically stable" means an average of at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, or at least 90%, of the enzyme activity is retained after aging for 4 weeks at 40°C in a sealed container beyond the initial 5- day equilibration period at 23°C. The percent retention is understood to mean the enzyme activity retained. This is determined by diluting the composition to the use concentration. The enzyme activity retention applies to at least one of the enzymes present in the composition. In certain embodiments, the enzyme activity retention applies to at least protease, and preferably to each enzyme in the compositions of the present disclosure. Enzymatic activity can be determined by the following assays: PIERCE Colorimetric Protease Assay Kit and PIERCE Fluorescent Protease Assay Kit, both available from Thermo Fisher Scientific of Waltham, MA; Amylase/Lipase/Protease Assay Kits available from Sigma Aldrich; and Amylase/Lipase/Cellulase/Protease Assay Kits available from Creative Proteomics of Shirley, NY.

A significant problem in cleaning medical instruments, particularly those that are flexible (e.g., flexible endoscopes), is related to a class of materials called "biofilms." Biofilms are microbiologically generated polysaccharide -containing (and often protein- and DNA-containing) matrices that form when bacteria adhere to surfaces in aqueous environments and begin to multiply and excrete a slimy, glue-like substance that can anchor to various materials (metal, plastic, glass) such as those found in medical instruments. A biofilm may be formed by a single bacterial species, but more likely will consist of multiple species of bacteria, as well as fungi, algae, protozoa, and inorganic products. Biofilms can form on a wide variety of surfaces exposed to bacteria, nutrients, and water under the right conditions. Many species of bacteria are becoming recognized as capable of existing in a free suspended state called the planktonic state or in a biofilm matrix referred to as the biofilm state. It is a characteristic of biofilms that the planktonic and nonplanktonic states may be interchanged under the right conditions. Once anchored to a surface, biofilm microorganisms can colonize and grow into a complex colony that tenaciously adheres to the surface encasing and protecting the microorganisms from outside attack. It is important to have a solution that can kill/remove bacteria in the planktonic state as well as in the form of a biofilm. In certain embodiments, enzymatic cleaning compositions of the disclosure are effective to remove at least a portion of biofilm from a substrate. In certain embodiments, according to the Biofilm Generation and Removal Test Method One, methods of the present disclosure remove biofilm by at least 3 log reduction (i.e., 99.9%), at least 4 log reduction, at least 5 log reduction, or at least 6 log reduction (i.e., 99.9999%). In certain embodiments, according to the Biofilm Generation and Removal Test

Method Two, methods of the present disclosure remove biofilm by at least 2 log reduction, at least 3 log reduction, at least 4 log reduction, at least 5 log reduction, or at least 6 log reduction.

In certain embodiments, enzymatic cleaning compositions of the disclosure are effective to remove and/or kill at least a portion of bacteria (e.g., those associated with a biofilm). In certain embodiments, methods of the present disclosure remove and/or kill bacteria (e.g., associated with a biofilm) by at least 3 logs.

In certain embodiments, enzymatic cleaning compositions of the disclosure are generally nonspecific toward bacteria, including gram-positive and gram-negative microbes. In addition, they are generally active against lipid-enveloped viruses and fungi. At least one mechanism of action is believed to be membrane disruption. Examples of relevant microorganisms against which the cleaning compositions are active include Staphylococcus spp., Streptococcus spp., Pseudomonas spp.,

Enterococcus spp., Esherichia spp., Aspergillus spp., Fusarium spp., Acinetobacter spp., Candida spp. Particularly virulent organisms include Staphylococcus aureus, including resistant strains such as Methicillin Resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Streptococcus mutans, Streptococcus pneumoniae, Enterococcus faecalis, Vancomycin Resistant Enterococcus (VRE), Pseudomonas aeruginosa, Escherichia coli, Aspergillus niger, Aspergillus fumigatus, Aspergillus clavatus, Fusarium solani, Fusarium oxysporum, Fusarium chlamydosporum, Candida albicans, Candida glabrata, and Candida krusei.

Another problem in cleaning medical instruments is related to the presence of biological residue, particularly blood-containing soil. Dried blood on medical instruments is hazardous to the employees of the hospital and to the next surgical patient upon which the instruments are used. The danger of handling instruments contaminated with blood is obvious in this age of hepatitis and HIV. Cleaning dried blood is much more difficult than cleaning other soils. Blood-containing soils are particularly tenacious and difficult to remove for at least three reasons. First, red blood cell surfaces are hydrophobic and therefore difficult to wet with aqueous use solutions of detergents. Second, blood-containing soils reside with hemoglobin that have limited water solubility and also contain iron. Iron is particularly difficult to remove from surfaces. Third, blood-containing soils contain fibrin. Fibrin is a protein involved in the clotting of blood. It is a fibrillar protein that is polymerized to form a "mesh" that forms a hemostatic plug or clot (in conjunction with platelets) over a wound site. This "mesh" formation is a result of intermolecular crosslinking of cysteine in the protein. While it is desirable and necessary for clot formation, it also acts to make blood-containing stains all the more tenacious. The fibrin jams itself into microscopic irregularities in the surface of instrumentation. This is a physical attachment to the surface through mechanical means, not just chemical means as with traditional adhesives. The action is similar to the roots of plants growing into cracks in rocks, anchoring themselves to the surface.

During the use of surgical instruments, such as an endoscope, it is possible to come into contact with other biological residue, such as feces, mucus, saliva, bone, prions, etc., as well as non-biological residue, such as fibers, lint, etc. If present, these should also be removed during enzymatic cleaning.

In certain embodiments, enzymatic cleaning compositions of the disclosure are effective to remove at least a portion of biological residue from a substrate. In certain embodiments, methods of the present disclosure provide a cleaning efficiency (particularly of blood-containing soils) of at least 7 on a scale of 1 to 10 (per the Cleaning Ability test in Example 6).

Enzymes

Compositions, and methods of use thereof, of the present disclosure include one or more enzymes.

In certain embodiments, the enzyme includes a protease.

In certain embodiments, the composition further includes an amylase, a lipase, a cellulase, or combinations thereof.

Suitable enzymes may be vegetable, animal, bacterial, fungal, or yeast enzymes, or genetic variations thereof. An enzyme is typically selected based on factors like pH, stability, temperature, and compatibility with materials found in cleaning compositions and cleaning applications. Preferred enzymes have activity in the pH range of 2 to 14 (or 6 to 12) and at temperatures from 20°C to 80°C. The enzyme may be a wild type enzyme or a recombinant enzyme. Preferred enzymes have a broad spectrum of activity and a high tolerance for materials found in cleaning compositions like alkalinity, acidity, chelating agents, sequestering agents, and surfactants.

One or more enzymes may be incorporated in a composition of the present disclosure in any suitable form, e.g., as granulates, marumes, prills, etc., but are more conveniently added to liquid compositions in a fluid form such as in a liquid or slurry.

An exemplary protease includes serine protease, such as, for example, subtilisins. Serine protease is an enzyme that catalyzes the hydrolysis of peptide bonds, and includes an essential serine residue at the active site. Other proteases that may be used include neutral proteases including, for example, aspartate and metallo-proteases. Neutral proteases have optimal proteolytic activity in a neutral pH range of 6 to 8, and may be isolated from bacterial, fungal, yeast, plant, or animal sources.

Exemplary proteases may be isolated from Bacillus lentus, Bacillus licheniformis, Bacillus amyloliquefaciens , and the like. Other commercial proteases include serine-proteases from the species Nocardiopsis, Aspergillus, Rhizopus, Bacillus alcalophilus, B. cereus, N. natto, B. vulgatus, and B.

myocoide. Subtilins from Bacillus may also be used, including proteases from the species Nocardiopsis spe and Nocardiopsis dassonvillei. Trypsin and pepsin also may be useful.

Suitable conventional fermented commercial proteases may include, for example, those available under the trade names ALCALASE (produced by submerged fermentation of a strain of Bacillus licheniformis), ESPERASE (produced by submerged fermentation of an alkalophilic species of Bacillus), RENNILASE (produced by submerged fermentation of a nonpathogenic strain of Mucor miehei),

SAVINASE (produced by submerged fermentation of a genetically modified strain of Bacillus), and DURAZYME (a protein-engineered variant of SAVINASE protease). Metallo-proteases may include those of microbial origin, including, for example, that available under the trade name NEUTRASE (produced by submerged fermentation of a strain of Bacillus subtilis). Other suitable proteases are those available under the trade names MAXACAL, OPTICLEAN, PROPERASE, MAXATASE, and

PRJMASE. Sources of exemplary proteases include Novo Industries A/S (Denmark), Solvay Enzymes, Genencor International, and Gist-Brocades (the Netherlands). Amylases may include those from a strain of Bacillus sp. For example, the amylase may include those obtained from Bacillus stearothermophilus, Bacillus amyloliquefaciens, Bacillus subtilis, or Bacillus licheniformis . Suitable Aspergillus amylases may include, for example, Aspergillus niger or Aspergillus oryzae. Commercially suitable amylases include those available under the trade names TERMAMYL, STAINZYME, DURAMYL, BIOMYLASE D(G), KEMZYM AT 9000, PURASTAR StL, PURASTAR HPAmL, PURAFECT OxAm, RAPIDASE TEX, KAM, and FUNGAMYL. Sources of exemplary proteases include Novo Industries A/S (Denmark), Solvay Enzymes, Genencor

International, and Gist-Brocades (the Netherlands).

Lipases may include a microbial lipase obtained from yeast, for example Candida, from bacteria, for example, Pseudomonas or Bacillus, or from filamentous fungi, for example, Humicola or Rhizomucor. Suitable lipases include, but are not limited to, those obtained from Rhizomucor miehei, Thermomyces lanuginosa, Humicola insolens, Pseudomonas stutzeri, Pseudomonas cepacia, Candida antartica, Absidia blakesleena, Absidia corymbifera, Fusarium solani, Fusarium oxysporum, Penicillum expansum, Rhodotorula glutinis, Thiarosporella phaseolina, Rhizopus microsporus, Sporobplomyces shibatanus, Aureobasidiurn puliulans, Hansenula anomala, Geotricum penicillatum, Lactobacillus curvatus,

Brochothris thermosohata, Coprinus cinerius, Trichoderma harzanium, Trichoderma reesei, Rhizopus japonicas, and/or Pseudomonas plantari.

Cellulases may include any enzyme capable of degrading cellulose to glucose, cellobiose, triose, and other cellooligosaccharides. For example, the cellulose may include an endoglucanase including, but not limited to, bacterial and/or fungal endoglucanase. Examples of endoglucanases may include those isolated from the bacteria Pseudomonas or Bacillus lautus. Additionally, the cellulase may include those isolated from the bacteria Aspergillus niger, Aspergillus oryzae, Botrytis cinerea, Myrothecium verrucaria, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride, Acremonium, Aspergillus, Chaetomium, Cephalosporium, Fusarium, Gliodadium, Humicola (e.g., Humicola insolens, Humicola strain DSM 1800), Irpex, Myceliophthora, Mycogone, Myrothecium, Papulospora, Penicillium, Scopulariopsis, Stachybotrys , and/or Verticillium. Other cellulases include those isolated from cellulase 212-producing fungus of the genus Aeromonas, and the hepatopancrease of the marine mollusk Dorabella Auricula Solander. Commercially suitable cellulases include those available under the trade names CAREZYME and CELLUZYME. Sources of examplary cellulases include Novo Industries A/S

(Denmark).

In certain embodiments, an enzyme is present in a total amount of at least 0.0001 weight percent (wt-%), based on the total weight of a ready-to-use composition. In certain embodiments, an enzyme is present in a total amount of up to 0.3 wt-%, based on the total weight of a ready-to-use composition.

In certain embodiments, an enzyme is present in a total amount of at least 0.05 wt-%, based on the total weight of a concentrated composition. In certain embodiments, an enzyme is present in a total amount of up to 15 wt-%, based on the total weight of a concentrated composition. In certain embodiments, a protease is present in a total amount of at least 0.05 wt-%, at least 0.1 wt-%, at least 0.5 wt-%, at least 1 wt-%, or at least 1.5 wt-%, based on the total weight of a concentrated composition. In certain embodiments, a protease is present in a total amount of up to 10 wt-%, up to 5 wt-%, or up to 2 wt-%, based on the total weight of a concentrated composition. Herein, the weight percent of protease is based on enzyme activity of 18 knpu/g.

In certain embodiments, an amylase is present in a total amount of at least 0.05 wt-%, at least 0.1 wt-%, or at least 0.5 wt-%, based on the total weight of a concentrated composition. In certain embodiments, an amylase is present in a total amount of up to 7.5 wt-%, up to 5 wt-%, or up to 1 wt-%, based on the total weight of a concentrated composition. Herein, the weight percent of amylase is based on the enzyme activity of 56041 units/mL.

In certain embodiments, a lipase is present in a total amount of at least 0.05 wt-%, or at least 0.1 wt-%, based on the total weight of a concentrated composition. In certain embodiments, a lipase is present in a total amount of up to 7.5 wt-%, up to 5 wt-%, or up to 1 wt-%, based on the total weight of a concentrated composition. The weight percent of lipase is based on the enzyme activity of 130000 units/g,

In certain embodiments, a cellulase is present in a total amount of at least 0.05 wt-%, or at least 0.1 wt-%, based on the total weight of a concentrated composition. In certain embodiments, a cellulase is present in a total amount of up to 7.5 wt-%, up to 5 wt-%, or up to 1 wt-%, based on the total weight of a concentrated composition. Herein, the weight percent of cellulase is based on the enzyme activity of 5380 units/g.

Surfactants

Compositions, and methods of use thereof, of the present disclosure include one or more surfactants.

In certain embodiments, a surfactant has an HLB (i.e., hydrophile to lipophile balance) of at least

8, at least 10, or at least 12. In certain embodiments, a surfactant has an HLB of up to 18.

For nonionic ethoxylated surfactants, the HLB values may be calculated using the method of W.C. Griffin, J. Soc. of Cosmetic Chemists, 5, 259 (1954) (the "HLB Method"). In this method, HLB = (E+P)/5, where E is the wt-% of oxyethylene content and P is the wt-% of polyhydric alcohol content (glycerol, sorbitol, etc). For the compounds herein, glycerol segments with two hydroxyl groups, glycerol segments with one hydroxyl group, and hydroxyl-containing segments of any additional polyhydric molecules are included in the definition of P.

The group contribution method is used for calculating HLB are available and may be required when determining the HLB value for compounds lacking both E and P groups, as defined above. See, e.g., Surfactant Systems-Their Chemistry. Pharmacy, and Biology. Attwood and Florence, Chapman and Hall, 1983, pp 471-479. While the calculated value of HLB may vary depending on the method used, the trends and relative hydrophobicity of materials are expected to be similar. In certain embodiments, the surfactant includes a nonionic surfactant, a zwitterionic surfactant, or combinations thereof. In certain embodiments, the composition includes a nonionic surfactant.

In certain embodiments, the composition further includes an anionic surfactant in addition to one of a nonionic surfactant, zwitterionic surfactant, or combinations thereof.

Nonionic surfactants include a hydrophobic group and a hydrophilic group. They are typically produced by the reaction of an organic aliphatic, alkyl aromatic, or polyoxyalkylene hydrophobic compound with a hydrophilic alkaline oxide group such as ethylene oxide. The length of the hydrophilic group may be adjusted to influence the hydrophobic/hydrophilic balance of the molecule. In certain embodiments, a nonionic surfactant is a linear alcohol ethoxylate.

Exemplary nonionic surfactants are listed in the treatise Nonionic Surfactants, edited by M.J.

Schick, Vol. 1 of the Surfactant Science Series, Marcel Dekker, Inc., New York, 1983. Also a typical listing of nonionic surfactant classes, and species of these surfactants, is given in U.S. Pat. No. 3,929,678 (Laughlin et al.). Further examples are given in Surface Active Agents and Detergents (Vol. I and II by Schwartz, Perry and Berch).

The following list is also exemplary of classes on nonionic surfactants.

(1) Block polyoxypropylene-polyoxyethylene polymeric compounds based upon propylene glycol, ethylene glycol, glycerol, trimethylolpropane, and ethylenediamine as the initiator reactive hydrogen compound such as difunctional block copolymers (e.g., those available under the trade name PLURONIC from BASF Corp.) and tetra-functional block copolymers (e.g., those available under the trade name TETRONCI from BASF Corp .) .

(2) Reaction products of one mole of alkyl phenol wherein the alkyl chain, of straight chain or branched chain configuration, or of single or dual alkyl constituent, contains from 8 carbon atoms to 18 carbon atoms with from 3 moles to 50 moles of ethylene oxide. The alkyl group can, for example, be represented by diisobutylene, di-amyl, polymerized propylene, iso-octyl, nonyl, and di-nonyl. These surfactants may be polyethylene, polypropylene, and polybutylene oxide condensates of alkyl phenols (e.g., those available under the trade name IGEPAL from Rhone -Poulenc).

(3) Reaction products of one mole of a saturated or unsaturated, straight or branched chain, alcohol having from 6 carbon atoms to 24 carbon atoms with from 3 moles to 50 moles of ethylene oxide (e.g., such as NEODOL from Shell Chemical Co. and ALFONIC from Vista Chemical Co.). The alcohol group can consist of mixtures of alcohols in the above delineated carbon range or it can consist of an alcohol having a specific number of carbon atoms within this range.

(4) Reaction products of one mole of saturated or unsaturated, straight or branched chain, carboxylic acid having from 8 to 18 carbon atoms with from 6 to 50 moles of ethylene oxide (e.g., such as NOPALCOL from Henkel Corp. and LIPOPEG from Lipo Chemicals, Inc.). The acid may be a mixture of acids in the above defined carbon atoms range or it may be an acid having a specific number of carbon atoms within the range. (5) Alkanoic acid esters formed by reaction with glycerides, glycerin, and polyhydric (saccharide or sorbitan/sorbitol) alcohols. All of these ester moieties have one or more reactive hydrogen sites on their molecule which can undergo further acylation or ethylene oxide (alkoxide) addition to control the hydrophilicity of these substances.

(6) Reverse block copolymers which are block copolymers, essentially reversed, by adding ethylene oxide to ethylene glycol to provide a hydrophile of designated molecular weight, and then adding propylene oxide to obtain hydrophobic blocks on the outside (ends) of the molecule. The hydrophobic portion of the molecule weighs from 1,000 to 3, 100 with the central hydrophile including 10 wt-% to 80 wt-% of the final molecule. Also included are difunctional reverse block copolymers (e.g., such as those available under the trade name PLURONIC from BASF Corp.) and tetra-functional reverse block copolymers (e.g., such as those available under the trade name TETRONIC from BASF Corp.).

(7) Capped nonionic surfactants which are modified by "capping" or "end blocking" the terminal hydroxy group or groups (of multifunctional moieties) to reduce foaming by reaction with a small hydrophobic molecule such as propylene oxide, butylene oxide, benzyl chloride; and, short chain fatty acids, alcohols or alkyl halides containing from 1 to 5 carbon atoms, and mixtures thereof. Also included are reactants such as thionyl chloride which convert terminal hydroxy groups to a chloride group. Such modifications to the terminal hydroxy group may lead to all-block, block-heteric, heteric -block or all- heteric nonionics.

(8) Alkylphenoxypolyethoxyalkanols of U.S. Pat. No. 2,903,486 (Brown et al.) represented by the formula (I):

wherein:

R represents a (C8-C9)alkyl group;

A represents an (C3-C4)alkylene group;

n is 7 to 16; and

m is 1 to 10.

(9) Polyalkylene glycol condensates of U.S. Pat. No. 3,048,548 (Martin et al.) having alternating hydrophilic oxyethylene chains and hydrophobic oxypropylene chains where the weight of the terminal hydrophobic chains, the weight of the middle hydrophobic unit, and the weight of the linking hydrophilic units each represent about one-third of the condensate.

(10) Defoaming nonionic surfactants disclosed in U.S. Pat. No. 3,382, 178 (Lissant et al.), which are represented by the general formula (II):

wherein: Z¹ represents an alkoxylatable material;

R¹ represents a group derived from an alkaline oxide which may be ethylene and propylene;

n is at least 10 (preferably from 10 to 2,000); and

z is a value determined by the number of reactive oxyalkylatable groups.

(11) Conjugated polyoxyalkylene compounds described in U.S. Pat. No. 2,677,700 (Jackson et al.), which are represented by the general formula (III):

wherein:

Y¹ represents the residue of organic compound having from 1 carbon atom to 6 carbon atoms and one reactive hydrogen atom;

n is an average value of at least 6.4, as determined by hydroxyl number; and

m is a value such that the oxyethylene portion constitutes 10% to 90% by weight of the molecule.

(12) Conjugated polyoxyalkylene compounds described in U.S. Pat. No. 2,674,619 (Lundsted et al.), which are represented by the general formula (IV):

wherein:

Y² represents the residue of an organic compound having from 2 carbon atoms to 6 carbon atoms and containing x reactive hydrogen atoms where x has a value of at least 2;

n is a value such that the molecular weight of the polyoxypropylene hydrophobic base is at least 900; and

m is a value such that the oxyethylene content of the molecule is selected such that the PEG content is from 10 wt-% to 90 wt-% of the surfactant molecule.

Compounds falling within the scope of the definition for Y include, for example, propylene glycol, glycerine, pentaerythritol, trimethylolpropane, ethylenediamine, and the like. The oxypropylene chains optionally, but advantageously, contain small amounts of ethylene oxide and the oxyethylene chains also optionally, but advantageously, contain small amounts of propylene oxide.

(13) Conjugated polyoxyalkylene surface-active agents represented by the general formula (V):

P[(C3H₆0)„(C2H₄0)_mH]_x

wherein:

P represents the residue of an organic compound having from 8 carbon atoms to 18 carbon atoms and containing x reactive hydrogen atoms where x has a value of 1 or 2;

n is a value such that the molecular weight of the polyoxyethylene portion is at least 44; and

m is a value such that the oxypropylene content of the molecule is from 10 wt- % to 90 wt-%. The oxypropylene chains in formula (V) may optionally contain small amounts of ethylene oxide and the oxyethylene chains may also optionally contain small amounts of propylene oxide.

(14) Polyhydroxy fatty acid amide surfactants represented by the general formula (VI):

R²CONR³Z²

wherein:

R² represents a (C5-C31)hydrocarbyl, which may be straight-chain;

R³ represents H, (Cl-C4)hydrocarbyl, 2-hydroxy ethyl, 2-hydroxy propyl, ethoxy, propoxy group, or a mixture thereof; and

Z² represents a polyhydroxyhydrocarbyl having a linear hydrocarbyl chain with at least 3 hydroxyls directly connected to the chain, or an alkoxylated derivative (preferably ethoxylated or propoxylated) thereof.

In formula (VI), Z² may be derived from a reducing sugar in a reductive animation reaction; such as a glycityl group.

(15) Alkyl ethoxylate condensation products of aliphatic alcohols with from 0 to 25 moles of ethylene oxide. The alkyl chain of the aliphatic alcohol can either be straight or branched, primary or secondary, and generally contains 6 carbon atoms to 22 carbon atoms.

(16) Ethoxylated (C6-C18)fatty alcohols and (C6-C18)mixed ethoxylated and propoxylated fatty alcohols. Suitable ethoxylated fatty alcohols include the (C10-C18)ethoxylated fatty alcohols with a degree of ethoxylation of 3 to 50.

(17) Nonionic alkylpolysaccharide surfactants disclosed in U.S. Pat. No. 4,565,647 (Llenado).

These surfactants include a hydrophobic group containing from 6 to 30 carbon atoms and a

polysaccharide (e.g., a polyglycoside), and a hydrophilic group containing from 1.3 saccharide units to 10 saccharide units. Any reducing saccharide containing 5 or 6 carbon atoms may be used (e.g., glucose, galactose and galactosyl moieties may be substituted for the glucosyl moieties). (Optionally the hydrophobic group is attached at the 2-, 3-, 4-, etc. positions thus giving a glucose or galactose as opposed to a glucoside or galactoside.) The intersaccharide bonds may be, e.g., between the one position of the additional saccharide units and the 2-, 3-, 4-, and/or 6-positions on the preceding saccharide units.

(18) Fatty acid amide surfactants including those represented by the following general formula

(VII):

R⁴CON(R⁵)₂

wherein:

R⁴ represents a (C7-C21)alkyl group; and

each R⁵ independently represents hydrogen, (Cl-C4)alkyl, (Cl-C4)hydroxyalkyl, or - (C2H40)x, where x is 1 to 3.

(19) Alkoxylated amines or, most particularly, alcohol alkoxylated/aminated/alkoxylated surfactants. These nonionic surfactants may be at least in part represented by the general formulae (VIII, IX, and X, in order):

R⁷-(PO)_sN-(EO)_tH(EO)_uH, and

R⁸-N(EO)_tH

wherein:

each R⁶, R⁷, and R⁸ independently represents an alkyl, alkenyl or other aliphatic group, or an alkyl -aryl group of 8 to 20, preferably 12 to 14, carbon atoms;

EO represents oxyethylene;

PO represents oxypropylene;

s is 1 to 20 (preferably 2 to 5);

t is 1 to 10 (preferably 2 to 5); and

u is 1 to 10 (preferably 2 to 5).

Other variations on the scope of these compounds may be represented by the alternative formula

(XI) :

R⁹-(PO)v-N[(EO)_wH] [(EO)_zH]

wherein:

R⁹ represents an alkyl, alkenyl or other aliphatic group, or an alkyl-aryl group of 8 to 20, preferably 12 to 14, carbon atoms;

v is 1 to 20 (e.g., 1, 2, 3, or 4 (preferably 2)); and

w and z are independently 1 to 10 (preferably 2 to 5).

These compounds are represented commercially by a line of products sold by Huntsman

Chemicals as nonionic surfactants. A preferred chemical of this class includes that available under the trade name SURFONIC PEA 25 Amine Alkoxylate.

(20) Amine oxides that are tertiary amine oxides represented by the following general formula

(XII) :

R¹⁰-(OR^u)n-N(R¹²)(R¹³)— >0

wherein:

the arrow is a conventional representation of a semi-polar bond; and

R¹⁰, R¹², and R¹³ of formula (XII) each independently represents aliphatic, aromatic, heterocyclic, alicyclic groups, or combinations thereof;

R¹¹ represents an alkylene or a hydroxyalkylene group containing 2 to 3 carbon atoms; and

n is 0 to 20.

Generally, for amine oxides of formula (XII), R¹⁰ represents an alkyl group having 8 to 24 carbon atoms; R¹² and R¹³ each independently represent an alkyl or hydroxyalkyl group of 1 to 3 carbon atoms, or a mixture thereof; R¹² and R¹³ may be attached to each other, e.g., through an oxygen or nitrogen atom, to form a ring structure. Useful water-soluble amine oxide surfactants are selected from the coconut or tallow alkyl di- (lower alkyl) amine oxides, specific examples of which are dodecyldimethylamine oxide,

tridecyldimethylamine oxide, etradecyldimethylamine oxide, pentadecyldimethylamine oxide, hexadecyldimethylamine oxide, heptadecyldimethylamine oxide, octadecyldimethylaine oxide, dodecyldipropylamine oxide, tetradecyldipropylamine oxide, hexadecyldipropylamine oxide, tetradecyldibutylamine oxide, octadecyldibutylamine oxide, bis(2-hydroxyethyl)dodecylamine oxide, bis(2-hydroxyethyl)-3-dodecoxy-l-hydroxypropylamine oxide, dimethyl-(2-hydroxydodecyl)amine oxide, 3,6,9-trioctadecyldimethylamine oxide and 3-dodecoxy-2-hydroxypropyldi-(2-hydroxyethyl)amine oxide.

(21) Water-soluble phosphine oxides represented by the following general formula (XIII):

(R¹⁴)(R¹⁵)(R¹⁶)P— >0

wherein:

the arrow is a conventional representation of a semi-polar bond;

R¹⁴ represents an alkyl, alkenyl, or hydroxyalkyl group having 10 to 24 carbon atoms; and

R¹⁵ and R¹⁶ each independently represents an alkyl or hydroxyalkyl group having 1 to 3 carbon atoms.

Examples of useful phosphine oxides include dimethyldecylphosphine oxide,

dimethyltetradecylphosphine oxide, methylethyltetradecylphosphone oxide, dimethylhexadecylphosphine oxide, diethyl-2-hydroxyoctyldecylphosphine oxide, bis(2-hydroxyethyl)dodecylphosphine oxide, and bis(hydroxymethyl)tetradecylphosphine oxide .

(22) Water-soluble sulfoxide compounds which have the formula (XIV):

(R¹⁷)(R¹⁸)S— >0

wherein:

the arrow is a conventional representation of a semi-polar bond;

R¹⁷ represents an alkyl or hydroxyalkyl group having 8 to 28 carbon atoms, with 0 to 5 ether linkages and 0 to 2 hydroxyl substituents; and

R¹⁸ represents an alkyl or hydroxyalkyl group having 1 to 3 carbon atoms. Useful examples of these sulfoxides include dodecyl methyl sulfoxide, 3-hydroxy tridecyl methyl sulfoxide, 3-methoxy tridecyl methyl sulfoxide, and 3-hydroxy-4-dodecoxybutyl methyl sulfoxide.

In certain embodiments, the nonionic surfactant includes alkyl glucosides, alkyl polyglucosides, polyhydroxy fatty acid amides, sucrose esters, esters of fatty acids and polyhydric alcohols, fatty acid alkanolamides, ethoxylated fatty acids, ethoxylated aliphatic acids, ethoxylated fatty alcohols (e.g., octyl phenoxy polyethoxyethanol available under the trade name TRITON X-100 and nonyl phenoxy poly(ethyleneoxy) ethanol available under the trade name NONIDET P-40, both from Sigma, St. Louis, MO), ethoxylated and/or propoxylated aliphatic alcohols (e.g., that available under the trade name BRIJ from ICI), ethoxylated glycerides, ethoxylated/propoxylated block copolymers (e.g., such as those available under the trade names PLURONIC and TETRONIC from BASF), ethoxylated cyclic ether adducts, ethoxylated amide and imidazoline adducts, ethoxylated amine adducts, ethoxylated mercaptan adducts, ethoxylated condensates with alkyl phenols, ethoxylated nitrogen-based hydrophobes, ethoxylated polyoxypropylenes, polymeric silicones, and polymerizable (reactive) surfactants (e.g., SAM 211 (alkylene polyalkoxy sulfate) surfactant available under the trade name MAZON from PPG

Industries, Inc., Pittsburgh, PA), poloxamers (such as those available under the trade name PLURONIC from BASF), sorbitan fatty acid esters, or combinations thereof.

In certain embodiments, the nonionic surfactant includes alkyl polyglucoside (such as that available under the trade name GLUCOPON 225 DK), ethoxylated fatty alcohol (such as that available under the trade name TRITON X-100), nonylphenol ethoxylate (such as that available under the trade name TERGITOL NP 10), ethoxylated acetylenic diol (SURFYNOL 485), secondary alcohol ethoxylate (such as that available under the trade name TERGITOL 15-S-9), (C9-C1 l)ethoxylated alcohol (such as that available under the trade name TOMAKLEEN G12), peg 35 castor oil (such as that available under the trade name KOLLIPHOR EL), peg 5 cocamide (such as that available under the trade name

HETOXAMIDE C4), laureth-7 (such as that available under the trade name HEXOTOL LA-7) or combinations thereof.

(23) Nonionic surfactants of the following formula (XV):

R R

R C I C≡C— C I— R

(O— CH₂CH₂)„OR

(O CH₂CH₂)_mOR wherein:

each R independently represents a (C2-C20)alkyl or (C2-C20)aryl group that may be straight-chained, branched, or, if sufficiently large, cyclic, or any combination thereof, optionally including one or more catenary heteroatoms such as oxygen, hexavalent sulfur, and trivalent nitrogen atoms bonded to the carbon atoms; and

n and m each independently represents 1 to 100 (preferably 1 to 20) chosen such that the weight percent of polyoxyethylene in the surfactant is on average 5 to 80 wt-% (preferably 10 and 60 wt-%).

In calculating the weight persent, the average amount of polyoxyethylene is determined by gas or liquid chromatography together with mass spectrometry and is taken as the weight average.

Examples of this class of nonionic surfactants include those available under the trade names SURFYNOL AND DYNOL surfactants available from Air Products, Allentown, PA.

The following list is exemplary of classes of zwitterionic (i.e., amphoteric) surfactants. (1) Ammonium carboxylate amphoterics is a class of surfactants that may be represented by the following formula (XVI):

R²¹-(C(0)-NH)_a-R²²-N⁺(R²³)₂-R²⁴-COO- wherein:

a is 0 or 1;

R²¹ represents a (C7-C21)alkyl group (saturated straight, branched, or cyclic group), a (C6-C22)aryl group, or a (C6-C22)aralkyl or alkaryl group (saturated straight, branched, or cyclic alkyl group), wherein R²¹ may be optionally substituted with one or more N, O, or S atoms, or one or more hydroxyl, carboxyl, amido, or amino groups;

R²³ is H, a (CI- C8)alkyl group (saturated straight, branched, or cyclic group), optionally substituted with one or more N, O, or S atoms, or one or more hydroxyl, carboxyl, or amino groups, a (C6-C9)aryl group, or a (C6-C9)aralkyl or alkaryl group; and

R²² and R²⁴ are each independently a (Cl-ClO)alkylene group that may be the same or different and may be optionally substituted with one or more N, O, or S atoms, or one or more hydroxyl or amino groups.

In certain embodiments of formula (XVI), R²¹ is a (C10-C18)alkyl group and R²³ is a (Cl- C2)alkyl group or benzyl group (preferably a methyl group). When R²³ is H it is understood that the surfactant at higher pH values could exist as a tertiary amine with a cationic counterion such as Na, K, Li, or an organic amine.

Examples of such amphoteric surfactants of formula (XVI) include, but are not limited to: certain betaines such as cocobetaine and cocamidopropyl betaine (commercially available under the trade designations MACKAM CB-35 and MACKAM L from Mclntyre Group Ltd., University Park, IL); monoacetates such as sodium lauroamphoacetate; diacetates such as disodium lauroamphoacetate; amino- and alkylamino-propionates such as lauraminopropionic acid (commercially available under the trade designations MACKAM IL, MACKAM 2L, and MACKAM 15 IL, respectively, from Mclntyre Group Ltd.).

(2) Ammonium sulfonate amphoterics is a class of amphoteric surfactants that is often referred to as "sultaines" or "sulfobetaines" and may be represented by the following formula (XVII):

R²¹-(C(0)-NH)_a-R²²-N⁺(R²³)₂-R²⁴-S0₃ wherein R²¹-R²⁴ and "a" are defined as above for formula (XVI).

Examples of amphoteric surfactants of formula (XVII) include cocamidopropylhydroxysultaine (commercially available as MACKAM 50-SB from Mclntyre Group Ltd.). At low pH, the

sulfoamphoterics may be preferred over the carboxylate amphoterics since the sulfonate group will remain ionized at much lower pH values.

The following list is exemplary of classes of anionic surfactants. By "anionic surfactants," it is meant surfactants containing one or more anionic groups and having net negative charges. Illustrative anionic surfactants may be based on sulfate, sulfonate or carboxylate anions, examples of which include, but are not limited to perfluorooctanoate (PFOA or PFO); perlluorooctanesulfonate (PFOS); sodium dodecyl sulfate (SDS), ammonium lauryl sulfate, and other alkyl sulfate salts; sodium laureth sulfate, also known as sodium lauryl ether sulfate (SLES); alkyl benzene sulfonate; and fatty acid salts. Commercially available anionic surfactants include the TRITON GR series (dioctyl sulfosuccinates), TRITON H-55 surfactant (phosphate ester), and TRITON QS-15 surfactant (sulfate) from Dow Chemical Company of Midland MI; STEPANOL AM (ammonium lauryl sulfate), STEPANOL WA EXTRA (sodium lauryl sulfate), STEP AN Mild SL3-BA (disodium laureth sulfosuccinate), and POLYSTEP B-22 (ammonium lauryl ether sulfate, POE-12) from Stepan Company of Northfield, IL; and KLEARFAC AA 270 surfactant (phosphate ester), MAPHOS 60 A (alphatic phosphate ester), and MAPHOS 66 H (aromatic phosphate ester) from BASF of Ludwigshafen, Germany.

In one embodiment, the anionic surfactant is a sulfonate salt and/or a sulfate salt, each of which independently contains an organic group having 6 to 22 carbon atoms. In one embodiment, the organic group is selected from the group consisting of a fatty acid group or a salt thereof, an ester of a fatty acid group, an alkyl group, an alkenyl group, an alkyl ether group, an alkenyl ether group, and a mixture thereof. In one embodiment, the sulfonate salt and/or sulfate salt is independently an alkali metal salt, such as sodium salt, or an ammonium or amine salt.

Useful anionic surfactants may also include water-soluble salts of the higher fatty acids (i.e., "soaps") are useful anionic surfactants in the compositions herein. This includes alkali metal soaps such as the sodium, potassium, ammonium, and alkyl ammonium salts of higher fatty acids having 8 to 24 carbon atoms, and preferably 12 to 18 carbon atoms. Soaps may be made by direct saponification of fats and oils or by the neutralization of free fatty acids. Particularly useful are the sodium and potassium salts of the mixtures of fatty acids derived from coconut oil and tallow, i.e., sodium or potassium tallow and coconut soap.

Additional non-soap anionic surfactants which are suitable for use herein include the water- soluble salts, preferably the alkali metal, and ammonium salts, of organic sulfuric reaction products having in their molecular structure an alkyl group having 10 to 20 carbon atoms and a sulfonic acid or sulfuric acid ester group. (Included in the term "alkyl" is the alkyl portion of acyl groups.) Examples of this group of synthetic surfactants are a) the sodium, potassium and ammonium alkyl sulfates, especially those obtained by sulfating the higher alcohols (C8-C 18 carbon atoms) such as those produced by reducing the glycerides of tallow or coconut oil; b) the sodium, potassium and ammonium alkyl polyethoxylate sulfates, particularly those in which the alkyl group contains 10 to 22, preferably 12 to 18, carbon atoms, and wherein the polyethoxylate chain contains 1 to 15, preferably 1 to 6, ethoxylate groups; and c) the sodium and potassium alkylbenzene sulfonates in which the alkyl group contains from 9 to 15 carbon atoms, in straight chain or branched chain configuration, e.g., those of the type described in U.S. Pat. Nos. 2,220,099 and 2,477,383. Also useful are linear straight chain alkylbenzene sulfonates in which the average number of carbon atoms in the alkyl group is 11 to 13, abbreviated as (CI 1-C13)LAS.

In certain embodiments, a surfactant is present in a total amount of at least 0.06 wt-%, based on the total weight of a ready-to-use composition. In certain embodiments, a surfactant is present in a total amount of up to 1.9 wt-%, based on the total weight of a ready-to-use composition.

In certain embodiments, a surfactant is present in a total amount of at least 30 wt-%, based on the total weight of a concentrated composition. In certain embodiments, a surfactant is present in a total amount of up to 93.65 wt-%, based on the total weight of a concentrated composition. Cationic Antimicrobials

Conventional enzymatic cleaners contain enzymes and surfactants are typically not designed to kill bacteria quickly. Even when antimicrobials are introduced into the enzymatic composition, they usually are added as preservatives and are designed to prevent growth of bacteria in the stored composition. Thus, such enzymatic compositions are more bacteriostatic than they are bactericidal.

Enzymatic cleaners of the present disclosure include cationic antimicrobials for quick kill of bacteria. Typically, cationic antimicrobials are not compatible with enzymes at higher pH's and tend to precipitate out of solution, although the high pH is typically needed for the best cleaning capability. Cationic antimicrobials can also be inactivated by high concentration of surfactants.

Surprisingly, the addition of certain surfactants and/or certain amounts of certain antimicrobials result in stable solutions (physically and/or chemically) without damaging antimicrobial efficacy or cleaning. In particular, such enzymatic cleaners demonstrate excellent antimicrobial activity in short times while maintaining cleaning capability. Any reduction in bacterial load with short exposure times is desirable before medical instruments are sterilized or endoscopes are disinfected.

In certain embodiments, compositions and methods of use thereof of the present disclosure includes one or more cationic antimicrobials. Exemplary cationic antimicrobials include a biguanide, a bisbiguanide, a quaternary ammonium compound (including polymeric and small molecule quaternary ammonium compounds), or combinations thereof. In certain embodiments, the cationic antimicrobial includes a biguanide, a quaternary ammonium compound, or combinations thereof.

Biguanides and bisbiguanides are represented by the formula (XVIII):

R³⁰-NH-C(NH)-NH-C(NH)-NH(CH₂)nNHC(NH)-NH-C(NH)-NH-R wherein:

n is 3 to 10 (preferably 4 to 8, and more preferably 6); and

each R³⁰ independently represents a (C4-C18) alkyl group, which may be branched or straight chain, optionally substituted in available positions by halogen or (C6-C12)aryl or alkaryl, optionally substituted in available positions by halogen. In certain embodiments, the cationic antimicrobial includes a biguanide. Biguanide has the formula (XIX):

A preferred bisbiguanide is chlorhexidine. This may be present as the free base but is preferably present as a disalt of acetate, gluconate, lactate, methosulfate (CH3OSO3^"), or a halide or combinations thereof. Most preferred are the diacetate, digluconate, dilactate, and dimethosulfate salts since these salts all have solubility limits in excess of 1 grams per 100 milliliter (1 g/100 mL). For example, the solubility limit of the digluconate salt is 20 g/100 mL and that of the diacetate is 1.9 g/100 mL. The most preferred compound is chlorhexidine digluconate (CHG). Other anions may be useful. It is particularly important, however, with this class as well as other cationic antimicrobials to use a counter ion that ensures solubility in aqueous fluid above the minimum inhibitory concentration (MIC) of the treatment organism. If the solubility limit is less than the MIC treatment may be ineffective.

Another useful bisbiguanide is alexidine.

Care must also be taken when formulating chlorhexidine as well as other cationic antimicrobials to avoid inactivation by sequestering it in micelles which may be formed by incorporation of surfactants and/or emulsifiers.

Bisbiguanides such as chlorhexidine are very basic and capable of forming multiple ionic bonds with anionic materials. For this reason, biguanide-containing compositions are preferably free of anionic compounds that can result in precipitation of the cationic antimicrobial. For this reason, thickener systems, if present, are preferably based on nonionic and/or cationic polymers or emulsifiers. Anionic surfactants useful, for example, as wetting agents, may also need to be avoided. Certain zwitterionic, very water soluble, or non-precipitating anionic emulsifiers and surfactants may also be useful. Halide salts may need to be avoided. For example, chlorhexidine digluconate (CHG) will precipitate rapidly in the presence of halide salts above a concentration of about 0.1M. Therefore, if a system includes CHG or other cationic antimicrobials of this class, and needs to comprise salts for stability or other purposes, preferably gluconate salts such as triethanolamine gluconate or sodium gluconate, are used.

Quaternary ammonium compounds include polymeric and small molecule quaternary ammonium compounds.

Small molecule quaternary ammonium compounds typically include one or more quaternary ammonium groups wherein attached to the quaternary ammonium group is at least one C6-C18 linear or branched alkyl or aralkyl chain. Suitable compounds include those disclosed in Disinfection Sterilization and Preservation, S. Block, 4^th ed., 1991, Chapter 13, Lea & Febiger. Particularly preferred compounds of this class have one or two C8-C 18 alkyl or aralkyl chains and may be represented by the following formula (XX):

(R³¹)(R³²)N(R³³)(R³⁴)⁺X^" wherein:

R³¹ and R³² each independently represent a (C I -C I 8) linear or branched alkyl, alkaryl, or aralkyl chains that may be substituted in available positions by N, O, or S provided at least one R³¹ or R³² is a (C8-C18) linear or branched alkyl, alkaryl, or aralkyl chains that may be substituted in available positions by N, O, or S;

R³³ and R³⁴ each independently represent a (Cl-C6)alkyl, phenyl, benzyl, or (C8- C 12)alkaryl groups; R³³ and R³⁴ may also form a ring such as a pyridine ring with the nitrogen of the quaternary ammonium group; and

X is an anion, preferably a halide, and most preferably CI- or Br-. Other anions may include methosulfate, ethosulfate, phosphates, and the like.

Preferred compounds of this class include monoalyltrimethylammonium salts,

monoalkyldimethylbenzyl ammonium salts, dialkyldimethyl ammonium salts, benzethonium chloride, and octenidine.

Examples of preferred quaternary ammonium antimicrobials include benzalkonium halides having an alkyl chain length of C8-C 18, more preferably C12-C 16, and most preferably a mixture of chain lengths. For example, a typical benzalkonium chloride sample may be comprise of 40% C 12 alkyl chains, 50% C 14 alkyl chains, and 10% C 16 alkyl chains. These are commercially available from numerous sources including Lonza (Barquat MB-50); Benzalkonium halides substituted with alkyl groups on the phenyl ring. A commercially avaible example is Barquat 4250 available from Lonza;

dimethyldialkylammonium halides where the alkyl groups have chain lengths of C8-C18. A mixture of chain lengths such as mixture of dioctyl, dilauryl, and dioctadecyl may be particularly useful. Exemplary compounds are commercially available from Lonza as Bardac 2050, 205M and 2250 from Lonza;

Cetylpyridinium halides such as cetylpyridinium chloride available from Merrell labs as Cepacol Chloride; Benzethonium halides and alkyl substituted benzethonium halides such as Hyamine 1622 and Hyamine 10x available from Rohm and Haas; octenidine and the like.

Polymeric quaternary ammonium compounds (i.e., polymeric quaternary amine compounds) may also be used as the antimicrobial of the present invention. These are typically polymers having quatemary amine groups with at least one alkyl, alkylene, or aralkyl chain of at least 6 carbon atoms and preferably as least 8 carbon atoms. The polymers may be linear, branched, hyperbranched or dendrimers. Preferred antimicrobial polymeric quaternary amine polymers include those described in U.S. Pat. Nos. 6,440,405; 5,408,022; and 5,084,096; PCT Publication No. WO/02102244; and Disinfection. Sterilization and Preservation. S. Block, 4^th ed., 1991, Chapter 13, Lea & Febiger. A particularly preferred class of polymeric quaternary ammonium antimicrobial compounds are polybiguanides. Compounds of this class are represented by the formula (XXI):

X-R³⁵-NH-C(NH)-NH-C(NH)-NH-R³⁶-NHC(NH)-NH-C(NH)-NH-R³⁷-X wherein:

R³⁵, R³⁶, and R³⁷ each independently represent bridging groups such as polymethylene more preferably 4 to 8 methylene groups and most preferably 6 methylene groups. The methylene groups may be optionally substituted in available positions with halogen, hydroxyl, or phenyl groups; and

X is a terminal group and is typically an amine, amine salt, or a dicyandiamide group.

The preferred compound of this class is polyhexamethylene biguanide (PHMB) commercially available as Cosmocil CQ from Aveci, Wilmington, DE.

In certain embodiments, a cationic antimicrobial is present in a total amount of at least 0.006 wt-

%, based on the total weight of a ready-to-use composition. In certain embodiments, a cationic antimicrobial is present in a total amount of up to 0.5 wt-%, or up to 0.3 wt-%, based on the total weight of a ready-to-use composition.

In certain embodiments, a cationic antimicrobial is present in a total amount of at least 3 wt-%, based on the total weight of a concentrated composition. In certain embodiments, a cationic antimicrobial is present in a total amount of up to 15 wt-%, based on the total weight of a concentrated composition.

Antimicrobial Lipids

In certain embodiments, compositions, and methods of use thereof, of the present disclosure include one or more antimicrobial lipids. Such antimicrobial lipids are distinct from the cationic antimicrobials.

"Antimicrobial lipid" means a compound that preferably has a solubility in water of no greater than 1.0 gram per 100 grams (1.0 g/100 g) deionized water. Preferred antimicrobial lipids have a solubility in water of no greater than 0.5 g/100 g deionized water, more preferably, no greater than 0.25 g/100 g deionized water. Solubilities are determined using radiolabeled compounds as described under

"Conventional Solubility Estimations" in Solubility of Long-Chain Fatty Acids in Phosphate Buffer at pH 7.4, Henrik Vorum et al., in Biochimica et. Biophysica Acta.. 1 126, 135-142 (1992). Preferred antimicrobial lipids have a solubility in deionized water of at least 100 micrograms ^g) per 100 grams deionized water, more preferably, at least 500 μg/100 g deionized water, and even more preferably, at least 1000 μg/100 g deionized water.

In certain embodiments, the antimicrobial lipids have a hydrophile/lipophile balance (HLB) of up to 10, or up to 8. In certain embodiments, the antimicrobial lipids have an HLB of at least 3, preferably at least 3.2, and even more preferably at least 3.4. Exemplary antimicrobial lipids include a (C7-C12) saturated fatty acid ester of a polyhydric alcohol, a (C12-C22)unsaturated fatty acid ester of a polyhydric alcohol, a (C7-C12)saturated fatty ether of a polyhydric alcohol, a (C6-C12)alkyl or (C8-C18)alkylene 1,2-diol, a (C12-C22)unsaturated fatty ether of a polyhydric alcohol, an alkoxylated derivative thereof, or combinations thereof. Alkyl or alkylene chains may be straight chain or branched.

In certain embodiments, the antimicrobial lipid includes glycerol monolaurate, glycerol monocaprate, glycerol monocaprylate, propylene glycol monolaurate, propylene glycol monocaprate, propylene glycol monocaprylate, ethyl hexyl glycerin, (C6-C12)alkyl 1,2-diols, or combinations thereof.

In certain embodiments, the antimicrobial lipid includes glycerol monocaprylate, propylene glycol monolaurate, ethyl hexyl glycerin, (C6-C12)alkyl 1,2-diols, or combinations thereof.

In certain embodiments, the antimicrobial lipid includes propylene glycol monolaurate, ethyl hexyl glycerin, 1,2-hexane diol, 1,2-octane diol, or combinations thereof.

In certain embodiments the antimicrobial lipid comprises ethyl hexyl glycerin.

In certain embodiments, an antimicrobial lipid is present in a total amount of at least 0.006 wt-%, based on the total weight of a ready-to-use composition. In certain embodiments, an antimicrobial lipid is present in a total amount of up to 0.5 wt-%, based on the total weight of a ready-to-use composition.

In certain embodiments, an antimicrobial lipid is present in a total amount of at least 3 wt-%, based on the total weight of a concentrated composition. In certain embodiments, an antimicrobial lipid is present in a total amount of up to 25 wt-%, based on the total weight of a concentrated composition.

Optional Hydrotropes

Enzymatic cleaning compositions may include one or more hydrotropes. The term hydrotrope refers to an agent that will solubilize one or more of the surfactants used in an aqueous composition of the present disclosure.

Exemplary hydrotropes include the ammonium or alkali metal salts of benzene sulfonate and mono- or di -alkyl substituted benzene sulfonates wherein the alkyl chains contain 1 to 3 carbon atoms. Suitable such alkali metal salts of alkylbenzene sulfonates include alkali metal salts of compounds such as toluene, ethyl benzene, isopropyl benzene, and o-, m- and p-xylene sulfonates, and mixtures thereof. An exemplary such compound is sodium xylene sulfonate. Hydrotropes suitable for use in this invention may also include short chain alcohols containing from two to six carbon atoms and mixtures thereof.

In certain embodiments, a hydrotrope is present in a total amount of at least 0.002 wt-%, based on the total weight of a ready-to-use composition. In certain embodiments, a hydrotrope is present in a total amount of up to 0.2 wt-%, based on the total weight of a ready-to-use composition.

In certain embodiments, a hydrotrope is present in a total amount of at least 1 wt-%, based on the total weight of a concentrated composition. In certain embodiments, a hydrotrope is present in a total amount of up to 10 wt-%, or up to 5 wt-%, based on the total weight of a concentrated composition. Optional Stabilizers

Enzymatic cleaning compositions may include one or more enzyme stabilizers.

Exemplary stabilizers include alkaline earth salts, boron-containing compounds, polyols, or combinations thereof.

Exemplary alkaline earth salts that are water-soluble salts include calcium and magnesium salts such as chlorides, sulfates, and acetates.

Exemplary boron-containing compounds include boric acid, boric oxide, and alkali metal borates. Typical examples of alkali metal borates are sodium and potassium, ortho, pyro, and meta borates, polyborates, and borax. Borax is the preferred alkali metal borate and is a tetraborate that is sold commercially in either the pentahydrate or decahydrate form (e.g., sodium borate decahydrate).

Exemplary poyols (e.g., having two to six hydroxyl groups) include glycerol, ethylene glycol, propylene glycol, and diethylene glycol.

In certain embodiments, an enzyme stabilizer is present in a total amount of at least 0.0006 wt-%, based on the total weight of a ready-to-use composition. In certain embodiments, a stabilizer is present in a total amount of up to 0.56 wt-%, based on the total weight of a ready-to-use composition.

In certain embodiments, an enzyme stabilizer is present in a total amount of at least 0.3 wt-%, based on the total weight of a concentrated composition. In certain embodiments, a stabilizer is present in a total amount of up to 28 wt-%, based on the total weight of a concentrated composition.

In certain embodiments, a boron-containing stabilizer is present in a total amount of at least 0.2 wt-% and up to 10 wt-%, based on the total weight of a concentrated composition. In certain

embodiments, an alkaline earth metal stabilizer is present in a total amount of at least 0.001 wt-%, and up to 3 wt-%, based on the total weight of a concentrated composition. In certain embodiments, a polyol stabilizer is present in a total amount of at least 0.1 wt-%, and up to 15 wt-%, based on the total weight of a concentrated composition.

Other Optional Additives

Enzymatic cleaning compositions may include one or more additives selected from chelators, organic solvents, builders, alkalinity sources and other pH adjusters, defoamers (i.e., foam control agents), supplemental antimicrobial agents (other than the antimicrobial lipids and cationic

antimicrobials), preservatives (such as isothiazoline derivatives), viscosity modifiers (e.g., thickeners such as cellulose ethers), bleaching agents, corrosion inhibitors, dyes, colorants, fragrances, opacifiers (such as polystyrene latices), bluing agents, processing aids, or combinations thereof.

Enzymatic cleaning compositions may include one or more chelators (i.e., chelating agents). Examples of suitable chelators include ethanoldiglycine or a salt thereof (e.g., disodium ethanoldiglycine (Na2EDG)), methylgylcinediacetic acid or a salt thereof (e.g., trisodium methylgylcinediacetic acid such as that available under the trade name TRILON M (40% MGDA) from BASF Corp.), iminodisuccinic acid or a salt thereof (e.g., iminodisuccinic acid sodium salt (IDS) available from Lanxess, Leverkusen, Germany), N,N-bis(carboxylatomethyl)-L-glutamic acid (GLDA) or a salt thereof (e.g., iminodisuccinic acid sodium salt (GLDA-Na/i) such as that available under the trade name DISSOLVINE GL-38 (38% GLDA) from Akzo Nobel), [S— S]ethylenediaminedisuccinic acid (EDDS) or a salt thereof (e.g., a sodium salt of [S— S]-ethylenediaminedisuccinic acid), and 3-hydroxy-2,2'-iminodisuccinic acid (HIDS) or a salt thereof (e.g., tetrasodium 3-hydroxy-2,2'-iminodisuccinate (HIDS 50%) available from Innospec Performance Chemicals)), nitrilotriacetic acid (NTA) or a salt thereof. In certain embodiments, an aminocarboxylate such as MGDA, GLDA, or IDS, which is boidegradable, is used as a chelating agent. If used, a chelating agent may be present in a total amount of at least 0.01 wt-%, at least 0.1 wt-%, or at least 1 wt-%, based on the total weight of a concentrated composition. If used, a chelating agent may be present in a total amount of up to 20 wt-%, up to 15 wt-%, up to 10 wt-%, or up to 5 wt-%, based on the total weight of a concentrated composition.

Enzymatic cleaning compositions may include one or more organic solvents. An organic solvent may positively contribute to enzymatic stability when used as part of an enzyme stabilizing system. Examples of suitable organic solvents include alcohols including polyols (e.g., having two to six hydroxyl groups) and glycol ethers such as those sold under the DOWANOL and CELLOSOLVE tradenames.

Exemplary such organic solvents include methanol, ethanol, propanol, and butanol. If used, an organic solvent may be present in a total amount of at least 1 wt-%, at least 3 wt-%, or at least 5 wt-%, based on the total weight of a concentrated composition. If used, an organic solvent may be present in a total amount of up to 20 wt-%, up to 15 wt-%, or up to 10 wt-%, based on the total weight of a concentrated composition.

Enzymatic cleaning compositions may include one or more builders, which may be organic and inorganic builders. Examples of suitable inorganic builders include the alkali metal salts of ortho-, pyro-, or tri-polyphosphate, silicates, and crystalline or amorphous zeolites. Examples of suitable organic builders include the alkali salts of ethylenediamine tetraacetic acid, nitrilotracetic acid, and

polycarboxylic acids such as citric acid. Other examples of suitable organic builders include carbonates, succinates, and polymers and copolymers of maleic and acrylic acids. If used, a builder may be present in a total amount of at least 0.5 wt-%, based on the total weight of a concentrated composition. If used, a builder may be present in a total amount of up to 10 wt-%, based on the total weight of a concentrated composition.

Enzymatic cleaning compositions may include one or more alkalinity sources. Examples of suitable alkalinity sources include alkali metal hydroxides, silicates, carbonates, and alkanolamines (e.g., monoethanolamine, diethanolamine, triethanolamine, and mixtures thereof).

If used, one or more optional additives may be present in a total amount of at least 0.05 wt-%, based on the total weight of a concentrated composition. If used, one or more optional additives may be present in a total amount of up to 25 wt-%, based on the total weight of a concentrated composition.

Methods In one embodiment of the disclosure, there is provided a method of removing at least a portion of a biofilm from a surface of a substrate, particularly a non-biological article. Such non-biological articles (e.g., medical instruments) include a surface made from metals (e.g., stainless steel), plastic, glass, or ceramic.

Such method includes contacting the surface with an enzymatic cleaning composition under conditions effective to remove at least a portion of a biofilm, wherein the enzymatic cleaning composition includes a cationic antimicrobial, optionally an antimicrobial lipid, a surfactant, and an enzyme.

In certain methods, the effective conditions include a temperature from room temperature (e.g., 23°C to 27°C) to 60°C.

In certain methods, the effective conditions include a time from immediately to 24 hours.

In certain methods, the effective conditions include mechanical assistance. Examples of mechanical assistance includes scrubbing, wiping, shaking, sonicating, vortexing, brushing, water jets, turbulent water, and gas bubbles.

In certain methods, the enzymatic cleaning compositions may be used in conjunction with mechanical means, such as a wipe.

Illustrative Embodiments

Embodiment 1 is an enzymatic cleaning composition comprising:

a cationic antimicrobial comprising a biguanide, a bisbiguanide, a quaternary ammonium compound, or combinations thereof;

an antimicrobial lipid comprising a compound that has a solubility in water of no greater than 1.0 gram per 100 grams (1.0 g/100 g) deionized water;

a surfactant comprising a nonionic surfactant, a zwitterionic surfactant, or combinations thereof; and

an enzyme comprising a protease.

Embodiment 2 is the enzymatic cleaning composition of embodiment 1 wherein the cationic antimicrobial comprises a biguanide, a bisbiguanide, a quaternary ammonium compound, or

combinations thereof.

Embodiment 3 is the enzymatic cleaning composition of embodiment 2 wherein the cationic antimicrobial comprises a biguanide. Embodiment 4 is the enzymatic cleaning composition of any one of embodiments 1 through 3 wherein the cationic antimicrobial is present in a total amount of at least 0.006 wt-%, based on the total weight of a ready-to-use composition.

Embodiment 5 is the enzymatic cleaning composition of any one of embodiments 1 through 4 wherein the cationic antimicrobial is present in a total amount of up to 0.5 wt-%, based on the total weight of a ready-to-use composition.

Embodiment 6 is the enzymatic cleaning composition of any one of embodiments 1 through 5 wherein the cationic antimicrobial is present in a total amount of at least 3 wt-%, based on the total weight of a concentrated composition.

Embodiment 7 is the enzymatic cleaning composition of any one of embodiments 1 through 6 wherein the cationic antimicrobial is present in a total amount of up to 15 wt-%, based on the total weight of a concentrated composition.

Embodiment 8 is the enzymatic cleaning composition of any one of embodiments 1 through 7 wherein the antimicrobial lipid comprises a (C7-C12)saturated fatty acid ester of a polyhydric alcohol, a (C 12-C22)unsaturated fatty acid ester of a polyhydric alcohol, a (C7-C 12)saturated fatty ether of a polyhydric alcohol, (C6-C12)alkyl or (C8-C18)alkylene 1,2-diol, a (C12-C22)unsaturated fatty ether of a polyhydric alcohol, an alkoxylated derivative thereof, or combinations thereof (in certain embodiments, the antimicrobial lipid comprises glycerol monolaurate, glycerol monocaprate, glycerol monocaprylate, propylene glycol monolaurate, propylene glycol monocaprate, propylene glycol monocaprylate, ethyl hexyl glycerin, (C6-C12)alkyl 1,2-diols, or combinations thereof).

Embodiment 9 is the enzymatic cleaning composition of embodiment 8 wherein the antimicrobial lipid comprises glycerol monocaprylate, propylene glycol monolaurate, ethyl hexyl glycerin, (C6- C12)alkyl 1,2-diols, or combinations thereof.

Embodiment 10 is the enzymatic cleaning composition of embodiment 9 wherein the

antimicrobial lipid comprises propylene glycol monolaurate, ethyl hexyl glycerin, 1,2-hexane diol, 1,2- octane diol, or combinations thereof.

Embodiment 11 is the enzymatic cleaning composition of embodiment 10 wherein the antimicrobial lipid comprises ethyl hexyl glycerin.

Embodiment 12 is the enzymatic cleaning composition of any one of embodiments 1 through 11 wherein the antimicrobial lipid is present in a total amount of at least 0.006 wt-%, based on the total weight of a ready-to-use composition.

Embodiment 13 is the enzymatic cleaning composition of any one of embodiments 1 through 12 wherein the antimicrobial lipid is present in a total amount of up to 0.5 wt-%, based on the total weight of a ready-to-use composition.

Embodiment 14 is the enzymatic cleaning composition of any one of embodiments 1 through 13 wherein the antimicrobial lipid is present in a total amount of at least 3 wt-%, based on the total weight of a concentrated composition. Embodiment 15 is the enzymatic cleaning composition of any one of embodiments 1 through 14 wherein the antimicrobial lipid is present in a total amount of up to 25 wt-%, based on the total weight of a concentrated composition.

Embodiment 16 is the enzymatic cleaning composition of any one of embodiments 1 through 15 further comprising an anionic surfactant.

Embodiment 17 is the enzymatic cleaning composition of any one of embodiments 1 through 16 wherein the surfactant has an HLB (i.e., hydrophile to lipophile balance) of at least 8.

Embodiment 18 is the enzymatic cleaning composition of embodiment 17 wherein the surfactant has an HLB of at least 10.

Embodiment 19 is the enzymatic cleaning composition of embodiment 18 wherein the surfactant has an HLB of at least 12.

Embodiment 20 is the enzymatic cleaning composition of embodiment 19 wherein the surfactant has an HLB of up to 18.

Embodiment 21 is the enzymatic cleaning composition of any one of embodiments 1 through 20 wherein the surfactant comprises a nonionic surfactant.

Embodiment 22 is the enzymatic cleaning composition of embodiment 21 wherein the nonionic surfactant comprises alkyl glucosides, alkyl polyglucosides, polyhydroxy fatty acid amides, sucrose esters, esters of fatty acids and polyhydric alcohols, fatty acid alkanolamides, ethoxylated fatty acids, ethoxylated aliphatic acids, ethoxylated fatty alcohols, ethoxylated and/or propoxylated aliphatic alcohols, ethoxylated glycerides, ethoxylated/propoxylated block copolymers, ethoxylated cyclic ether adducts, ethoxylated amide and imidazoline adducts, ethoxylated amine adducts, ethoxylated mercaptan adducts, ethoxylated condensates with alkyl phenols, ethoxylated nitrogen-based hydrophobes, ethoxylated polyoxypropylenes, polymeric silicones, and polymerizable surfactants, poloxamers, sorbitan fatty acid esters, or combinations thereof.

Embodiment 23 is the enzymatic cleaning composition of embodiment 22 wherein the nonionic surfactant comprises alkyl polyglucoside, ethoxylated fatty alcohol, nonylphenol ethoxylate, ethoxylated acetylenic diol, secondary alcohol ethoxylate, (C9-C1 l)ethoxylated alcohol, peg 35 castor oil, peg 5 cocamide, laureth-7, or combinations thereof.

Embodiment 24 is the enzymatic cleaning composition of any one of embodiments 1 through 23 wherein the surfactant is present in a total amount of at least 0.06 wt-%, based on the total weight of a ready-to-use composition.

Embodiment 25 is the enzymatic cleaning composition of any one of embodiments 1 through 24 wherein the surfactant is present in a total amount of up to 1.9 wt-%, based on the total weight of a ready- to-use composition.

Embodiment 26 is the enzymatic cleaning composition of any one of embodiments 1 through 25 wherein the surfactant is present in a total amount of at least 30 wt-%, based on the total weight of a concentrated composition. Embodiment 27 is the enzymatic cleaning composition of any one of embodiments 1 through 26 wherein the surfactant is present in a total amount of up to 93.65 wt-%, based on the total weight of a concentrated composition.

Embodiment 28 is the enzymatic cleaning composition of any one of embodiments 1 through 27 further comprising an enzyme selected from an amylase, a lipase, a cellulase, or combinations thereof.

Embodiment 29 is the enzymatic cleaning composition of any one of embodiments 1 through 28 wherein the enzyme is present in a total amount of at least 0.0001 wt-%, based on the total weight of a ready-to-use composition.

Embodiment 30 is the enzymatic cleaning composition of any one of embodiments 1 through 29 wherein the enzyme is present in a total amount of up to 0.3 wt-%, based on the total weight of a ready- to-use composition.

Embodiment 31 is the enzymatic cleaning composition of any one of embodiments 1 through 30 wherein the enzyme is present in a total amount of at least 0.05 wt-%, based on the total weight of a concentrated composition.

Embodiment 32 is the enzymatic cleaning composition of any one of embodiments 1 through 31 wherein the enzyme is present in a total amount of up to 15 wt-%, based on the total weight of a concentrated composition.

Embodiment 33 is the enzymatic cleaning composition of any one of embodiments 1 through 32 further comprising a stabilizer.

Embodiment 34 is the enzymatic cleaning composition of embodiment 33 wherein the stabilizer comprises alkaline earth salts, boron-containing compounds, polyols, or combinations thereof.

Embodiment 35 is the enzymatic cleaning composition of embodiment 33 or 34 wherein the stabilizer is present in a total amount of at least 0.0006 wt-%, based on the total weight of a ready-to-use composition.

Embodiment 36 is the enzymatic cleaning composition of any one of embodiments 33 through 35 wherein the stabilizer is present in a total amount of up to 0.56 wt-%, based on the total weight of a ready- to-use composition.

Embodiment 37 is the enzymatic cleaning composition of any one of embodiments 33 through 36 wherein the stabilizer is present in a total amount of at least 0.3 wt-%, based on the total weight of a concentrated composition.

Embodiment 38 is the enzymatic cleaning composition of any one of embodiments 33 through 37 wherein the stabilizer is present in a total amount of up to 28 wt-%, based on the total weight of a concentrated composition.

Embodiment 39 is the enzymatic cleaning composition of any one of embodiments 1 through 38 further comprising a hydrotrope .

Embodiment 40 is the enzymatic cleaning composition of embodiment 39 wherein the hydrotrope comprises an alkali metal salt of an alkylbenzene sulfonate or a combination thereof. Embodiment 41 is the enzymatic cleaning composition of embodiment 39 or 40 wherein the hydrotrope is present in a total amount of at least 0.002 wt-%, based on the total weight of a ready-to-use composition.

Embodiment 42 is the enzymatic cleaning composition of any one of embodiments 39 through 41 wherein the hydrotrope is present in a total amount of up to 0.2 wt-%, based on the total weight of a ready-to-use composition.

Embodiment 43 is the enzymatic cleaning composition of any one of embodiments 39 through 42 wherein the hydrotrope is present in a total amount of at least 1 wt-%, based on the total weight of a concentrated composition.

Embodiment 44 is the enzymatic cleaning composition of any one of embodiments 1 through 43 wherein the hydrotrope is present in a total amount of up to 10 wt-%, based on the total weight of a concentrated composition.

Embodiment 45 is the enzymatic cleaning composition of any one of embodiments 1 through 44 further comprising one or more additives selected from chelators, organic solvents, builders, alkalinity sources and other pH adjusters, defoamers, supplemental antimicrobial agents other than the

antimicrobial lipids and cationic antimicrobials, preservatives, viscosity modifiers, bleaching agents, corrosion inhibitors, dyes, colorants, fragrances, opacifiers, bluing agents, preservatives, processing aids, and combinations thereof.

Embodiment 46 is the enzymatic cleaning composition of any one of embodiments 1 through 45 comprising up to 30 wt-% solids.

Embodiment 47 is the enzymatic cleaning composition of any one of embodiments 1 through 46 which is effective to remove at least a portion of biofilm from a substrate.

Embodiment 48 is the enzymatic cleaning composition of embodiment 47 which is effective to remove at least a portion of biological residue from a substrate.

Embodiment 49 is the enzymatic cleaning composition of embodiment 47 or 48 which is effective to kill at least a portion of bacteria on a substrate.

Embodiment 50 is a method of removing at least a portion of a biofilm from a surface of a non- biological article, the method comprising:

contacting the surface with an enzymatic cleaning composition under conditions effective to remove at least a portion of a biofilm, the enzymatic cleaning composition comprises:

an antimicrobial lipid comprising a compound that has a solubility in water of no greater than 1.0 gram per 100 grams (1.0 g/100 g) deionized water (in certain embodiments, an antimicrobial lipid comprising a (C7-C 12)saturated fatty acid ester of a polyhydric alcohol, a

(C 12-C22)unsaturated fatty acid ester of a polyhydric alcohol, a (C7-C12)saturated fatty ether of a polyhydric alcohol, a (C6-C12)alkyl or (C8-C18)alkylene 1,2-diol, a (C12-C22)unsaturated fatty ether of a polyhydric alcohol, an alkoxylated derivative thereof, or combinations thereof); a surfactant comprising a nonionic surfactant, a zwitterionic surfactant, or combinations thereof; and

an enzyme comprising a protease.

Embodiment 51 is the method of embodiment 50 wherein the contacting step occurs under conditions effective to remove at least a portion of biological residue.

Embodiment 52 is the method of embodiment 50 or 51 wherein the contacting step occurs under conditions effective to kill at least a portion of bacteria.

Embodiment 53 is a method of removing at least a portion of a biofilm from a surface of a non- biological article, the method comprising:

an enzyme comprising a protease;

wherein the biofilm is removed by at least 3 log reduction according to the Biofilm Generation and Removal Test Method One.

Embodiment 54 is a method of cleaning a surface of a non-biological article, the method comprising:

contacting the surface with an enzymatic cleaning composition under conditions effective to remove at least a portion of a biofilm, at least a portion of a the biological residue, and at least a portion of the bacteria, wherein the enzymatic cleaning composition comprises:

an enzyme comprising a protease;

wherein the biofilm is removed by at least 3 log reduction according to the Biofilm Generation and Removal Test Method One;

wherein the bacteria is removed by at least 3 log reduction; and

wherein the cleaning efficiency is at least 7 on a scale of 1 to 10. Embodiment 55 is the method of embodiment 54 wherein the enzymatic cleaning composition further comprises a cationic antimicrobial comprising a biguanide, a bisbiguanide, a quaternary ammonium compound, or combinations thereof.

Embodiment 56 is the method of embodiment 53 or 54 wherein the enzymatic cleaning composition further comprises an antimicrobial lipid comprising a compound that has a solubility in water of no greater than 1.0 gram per 100 grams (1.0 g/100 g) deionized water (in certain embodiments, an antimicrobial lipid comprising a (C7-C 12)saturated fatty acid ester of a polyhydric alcohol, a (C12- C22)unsaturated fatty acid ester of a polyhydric alcohol, a (C7-C12)saturated fatty ether of a polyhydric alcohol, a (C6-C12)alkyl or (C8-C18)alkylene 1,2-diol, a (C12-C22)unsaturated fatty ether of a polyhydric alcohol, an alkoxylated derivative thereof, or combinations thereof).

Embodiment 57 is the method of any one of embodiments 50 through 56 wherein the conditions comprise a temperature from room temperature to 60°C.

Embodiment 58 is the method of any one of embodiments 50 through 57 wherein the conditions comprise a time from immediately to 24 hours.

Embodiment 59 is the method of any one of embodiments 50 through 57 wherein the conditions comprise mechanical assistance.

Embodiment 60 is the method of any one of embodiments 50 through 59 wherein non-biological article comprises a surface comprising stainless steel, plastic, ceramic, or glass.

Embodiment 61 is the method of any one of embodiments 50 through 60 wherein the log reduction of the biofilm is at least 4 according to the Biofilm Generation and Removal Test Method One.

Embodiment 62 is the method of embodiment 61 wherein the log reduction of the biofilm is at least 5 according to the Biofilm Generation and Removal Test Method One.

Embodiment 63 is the method of embodiment 62 wherein the log reduction of the biofilm is at least 6 according to the Biofilm Generation and Removal Test Method One.

Embodiment 64 is the method of any one of embodiments 50 through 53 and 55 through 63 wherein the cationic antimicrobial is present in the enzymatic cleaning composition in a total amount of at least 0.006 wt-%, based on the total weight of a ready-to-use composition.

Embodiment 65 is the method of any one of embodiments 50 through 53 and 55 through 64 wherein the cationic antimicrobial is present in the enzymatic cleaning composition in a total amount of up to 0.5 wt-%, based on the total weight of a ready-to-use composition.

Embodiment 66 is the method of any one of embodiments 50 through 53 and 55 through 65 wherein the cationic antimicrobial is present in the enzymatic cleaning composition in a total amount of at least 3 wt-%, based on the total weight of a concentrated composition.

Embodiment 67 is the method of any one of embodiments 50 through 53 and 55 through 66 wherein the cationic antimicrobial is present in the enzymatic cleaning composition in a total amount of up to 15 wt-%, based on the total weight of a concentrated composition. Embodiment 68 is the method of any one of embodiments 50 through 52 and 56 through 67 wherein the antimicrobial lipid is present in the enzymatic cleaning composition in a total amount of at least 0.006 wt-%, based on the total weight of a ready-to-use composition.

Embodiment 69 is the method of any one of embodiments 50 through 52 and 56 through 68 wherein the antimicrobial lipid is present in the enzymatic cleaning composition in a total amount of up to 0.5 wt-%, based on the total weight of a ready-to-use composition.

Embodiment 70 is the method of any one of embodiments 50 through 52 and 56 through 69 wherein the antimicrobial lipid is present in the enzymatic cleaning composition in a total amount of at least 3 wt-%, based on the total weight of a concentrated composition.

Embodiment 71 is the method of any one of embodiments 50 through 52 and 56 through 70 wherein the antimicrobial lipid is present in the enzymatic cleaning composition in a total amount of up to 25 wt-%, based on the total weight of a concentrated composition.

Embodiment 72 is the method of any one of embodiments 50 through 71 wherein the antimicrobial cleaning composition further comprises an anionic surfactant.

Embodiment 73 is the method of any one of embodiments 50 through 72 wherein the surfactant has an HLB of at least 8.

Embodiment 74 is the method of embodiment 73 wherein the surfactant has an HLB of at least

10.

Embodiment 75 is the method of embodiment 74 wherein the surfactant has an HLB of at least 12.

Embodiment 76 is the method of any one of embodiments 50 through 75 wherein the surfactant has an HLB of up to 18.

Embodiment 77 is the method of any one of embodiments 50 through 76 wherein the surfactant comprises a nonionic surfactant.

Embodiment 78 is the method of any one of embodiments 50 through 77 wherein the surfactant is present in the enzymatic cleaning composition in a total amount of at least 0.06 wt-%, based on the total weight of a ready-to-use composition.

Embodiment 79 is the method of any one of embodiments 50 through 78 wherein the surfactant is present in the enzymatic cleaning composition in a total amount of up to 1.9 wt-%, based on the total weight of a ready-to-use composition.

Embodiment 80 is the method of any one of embodiments 50 through 79 wherein the surfactant is present in the enzymatic cleaning composition in a total amount of at least 30 wt-%, based on the total weight of a concentrated composition.

Embodiment 81 is the method of any one of embodiments 50 through 80 wherein the surfactant is present in the enzymatic cleaning composition in a total amount of up to 93.65 wt-%, based on the total weight of a concentrated composition. Embodiment 82 is the method of any one of embodiments 50 through 81 wherein the enzymatic cleaning composition further comprises an enzyme selected from an amylase, a lipase, a cellulase, or combinations thereof.

Embodiment 83 is the method of any one of embodiments 50 through 82 wherein the enzyme is present in the enzymatic cleaning composition in a total amount of at least 0.0001 wt-%, based on the total weight of a ready-to-use composition.

Embodiment 84 is the method of any one of embodiments 50 through 83 wherein the enzyme is present in the enzymatic cleaning composition in a total amount of up to 0.3 wt-%, based on the total weight of a ready-to-use composition.

Embodiment 85 is the method of any one of embodiments 50 through 84 wherein the surfactant is present in the enzymatic cleaning composition in a total amount of at least 0.05 wt-%, based on the total weight of a concentrated composition.

Embodiment 86 is the method of any one of embodiments 50 through 85 wherein the surfactant is present in the enzymatic cleaning composition in a total amount of up to 15 wt-%, based on the total weight of a concentrated composition.

Embodiment 87 is the method of any one of embodiments 50 through 86 wherein the enzymatic cleaning composition further comprises a stabilizer.

Embodiment 88 is the method of any one of embodiments 50 through 87 wherein the enzymatic cleaning composition further comprises a hydrotrope.

Embodiment 89 is the method of any one of embodiments 50 through 88 wherein the enzymatic cleaning composition further comprises one or more additives selected from chelators, organic solvents, builders, alkalinity sources and other pH adjustors, defoamers, supplemental antimicrobial agents other than the antimicrobial lipids and cationic antimicrobials, preservatives, viscosity modifiers, bleaching agents, corrosion inhibitors, dyes, colorants, fragrances, opacifiers, bluing agents, preservatives, processing aids, and combinations thereof.

Embodiment 90 is the method of any one of embodiments 50 through 89 wherein the enzymatic cleaning composition comprises up to 30 wt-% solids.

Embodiment 91 is the composition or method of any one of the preceding embodiments wherein the composition is physically stable.

Embodiment 92 is the composition or method of any one of the preceding embodiments wherein the composition is chemically stable.

Embodiment 93 is the composition or method of any one of the preceding embodiments wherein the composition is enzymatically stable. Examples

Objects and advantages of various embodiments of this invention are further illustrated by the following examples, but the particular materials and amounts thereof recited in these examples, as well as other conditions and details, should not be construed to unduly limit this invention. These examples are merely for illustrative purposes only and are not meant to be limiting on the scope of the appended claims.

Table la: Surfactants

Trade Percent

Chemical Name Lot Number Supplier Location

Name Active

Surfactants

Nonionics

Ludwig-

Glucopon

Caprylyl/decyl glucoside 70% 8558986 BASF shafen,

225DK

Germany

Polyethylene glycol p-

Dow

(1, 1,3,3- Triton X- 614006

97% Chemical Midland, MI tetramethylbuty 1) -phenyl 100 (89521)

Company

ether

Poly(ethylene glycol)-

Ludwig- Woc -poly(propylene Pluronic

100% 537G BASF shafen, g\yco\)-block- L64

Germany poly(ethylene glycol)

Tergitol NP- Saint Louis,

Nonylphenol ethoxylate 100% 92H0575 Sigma

10 MO ethoxylated acetylenic

diol (ethoxylated Surfynol Air Allentown,

100% 969267

2,4,7,9-tetramethyl 5 485 Products PA decyn-4,7-diol)

Ludwig-

Kolliphor

PEG-35 castor oil 97% 29209724U0 BASF shafen,

EL

Germany

Hetoxamide Global

PEG-5 Cocamide 100% G30532 Franklin, NJ

C4 Seven

Mclntyre

University cocamidopropyl betaine Mackam L 35% 53407 Group

Park, IL LTD

Ludwig-

PPG-l-PEG-9 lauryl

Eumulgin L 99% 13332646 BASF shafen, glycol ether

Germany

Dow

Ecosurf EH-

2-ethyl hexanol EO-PO 99% D678E8LD02 Chemical Midland, MI

3

Company

alcohols, CI 2- 14, Tergitol Dow

ethoxylated Min-foam 97% D678F3AD02 Chemical Midland, MI propoxylated lx Company

Dow

alcohols, C 12-14- Tergitol 15-

97% D358F7PIC1 Chemical Midland, MI secondary, ethoxylated S-9

Company

C9-11, ethoxylated Tomakleen Air Allentown,

100% 2008364

alcohol G-12 Products PA

Octylphenoxy

Igepal CA- Saint Louis, poly(ethyleneoxy)ethano 100% 032K0130 Sigma

630 MO 1, branched b-Alanine, N-(2-

Tomamine

carboxyethyl)-N-(2- Air Allentown,

Amphoteric 50% 2123525

ethylhexyl)-, Products PA

400

monosodium salt

C8-C14 alkyl Glucopon Cincinnati,

50% UR9F02N003 Cognis

polyglucoside 425N OH

Hetoxol LA- Global

Laureth-7 100% G31 108 Franklin, NJ

7 Seven

Anionics

ammonium lauryl ether Polystep B- Stepan Northfield,

30% 7648777

sulfate, POE- 12 22 Company IL disodium laureth Stepan Mild Stepan Northfield,

30% 732651 1

sulfosuccinate SL3-BA Company IL

Table lb: Enzymes, antimicrobial lipids and antimicrobials

Chemical Name Trade Name Lot Number Supplier Location

Enzymes

Protease from Bacillus Sigma Life Saint Louis,

Savinase 16L SLBN5637V

sp. Sciences MO a-Amylase from Termamyl Sigma Life Saint Louis,

SLBK7549V

Bacillus lichenformis 300L Sciences MO

Lipase from

Sigma Life Saint Louis,

Termomyces Lipolase 100L SLBK0930V

Sciences MO lanuginosus

Cellulase from Carezyme Sigma Life Saint Louis,

SLBN9759V

Aspergillus sp. 1000L Sciences MO

Protease from Bacillus Sigma Life Saint Louis,

Protamex 1 19K1454V

sp. Sciences MO

Protease from Bacillus Sigma Life Saint Louis,

Alcalase 2.4L SLBL2953V

licheniformis Sciences MO

Antimicrobial Lipids (also called FAMEs)

SENSIVA Schiilke And Mayr Norderstedt,

Ethylhexylglycerin 1155888

SC50 GmbH Germany

Glyceryl Janesville,

Capmul MCM 091019-TMC Abitec Corporation

monocaprylate WI

Glyceryl

Janesville, monocaprylate and Capmul 708G 120803-8 Abitec Corporation

WI

glyceryl monocaprate

Propylene glycol Alzo International Sayreville,

Dermol PGML 990435

laurate Inc NJ

1,2-hexanediol and Symdiol 68 10300017 Symrise Teterboro, NJ 1,2-octanediol Corporation I

Antimicrobials

Table lc: Biological testing materials and miscellaneous

Scientific)

Butyl diglycol FGL01 TCI Tokyo, Japan

TO SI washer 701821 Healthmark Fraser, MI

test strips Industries

Preparatory Example 1 : Preparation of Enzymatic Cleaner Solution

Preparatory Example 1, the Enzymatic Cleaner Solution was prepared by the step-wise addition of the components listed in Table 2 into a 100-mL jar in the order they are listed. After the sodium xylene sulfonate was added but before the Lipolase 100L was added the jar was capped and set to roll at 60 revolutions per minute (rpm) until all components had completely dissolved. After the solution was visibly clear, the Lipolase 100L and other enzymes were added and set to roll at 30 rpm until the solution was again clear. The final solution was a clear orange.

Table 2: Enzymatic Cleaner Solution

Preparatory Example 2:

Preparatory Examples p2- 1 to p2-21 (which could be used as exemplary enzymatic cleaning compositions in certain methods of the present disclosure) were prepared by first adding the Enzymatic Cleaner Solution (Preparatory Example 1) to a 20-mL vial. To the same vial a calculated amount of water and surfactant were added and vortexed until the solution was homogeneous. After the solution was homogeneous the solid antimicrobial was added to the vial and the vial was left to roll at 60 rpm overnight to dissolve the antimicrobial. The next morning the vials were taken off the rollers and left to sit for at least 5 minutes. After this each vial was visibly evaluated for any signs of instability such as precipitate, cloudy solution or phase separation.

Table 3: Preparatory Examples p2-l to p2-21

Working Example 1: Concentrate

Antimicrobial lipids, were added to portions of the Preparatory Example 2. These mixtures briefly vortexed and additional surfactant was added slowly as needed to stabilize the solution.

Table 4a: Working Examples la-1 to la-29 (Concentrate)

la-6 p2-6 84.45 15.55 Sensiva SC50 0 Hetoxamide C4 la-7 p2-7 76.82 13.98 Sensiva SC50 9.20 Mackam L la-8 p2-8 84.81 15.19 Sensiva SC50 0 Igepal CA-630 la-9 p2-9 83.51 16.49 Sensiva SC50 0 Ecosurf EH-3 la-10 p2-10 84.75 15.25 Sensiva SC50 0 Tergitol 15-S-9 la-11 p2-l l 84.42 15.58 Sensiva SC50 0 Tomakleen G-12

Tomamine la-12 p2-12 84.39 15.61 Sensiva SC50 0

Amphoteric 400

Tergitol Min Foam la-13 p2-13 86.80 13.20 Sensiva SC50 0

lx la-14 p2-14 73.04 11.48 Sensiva SC50 15.48 Glucopon 425N la-15 p2-l 86.58 6.06 Sensiva SC50 7.36 Glucopon 225DK la-16 p2-l 67.57 18.58 Sensiva SC50 13.85 Glucopon 225DK la-17 p2-l 84.57 15.43 Capmul 708G 0 Glucopon 225DK la-18 p2-l 70.28 12.94 PGML 16.78 Glucopon 225DK la-19 p2-l 83.92 16.08 Symdiol 68 0 Glucopon 225DK la-20 p2-l 84.39 15.61 Capmul MCM 0 Glucopon 225DK la-21 p2-18 68.61 11.32 Sensiva SC50 20.07 Glucopon 225DK la-22 p2-17 78.62 14.88 Sensiva SC50 6.50 Glucopon 225DK la-23 p2-20 78.45 12.82 Capmul 708G 8.73 Hetoxol LA-7 la-24 p2-20 80.85 13.71 Sensiva SC50 5.44 Hetoxol LA-7 la-25 p2-19 81.12 14.86 Sensiva SC50 4.02 Glucopon 225DK la-26 p2-21 78.21 14.40 Sensiva SC50 7.39 Hetoxol LA-7 la-27 p2-21 80.68 15.09 Capmul 708G 4.23 Hetoxol LA-7 la-28 p2-l 35.04 32.92 Capmul 708G 32.04 Glucopon 225DK la-29 p2-l 68.37 31.63 Capmul 708G 0 Glucopon 225DK

Table 4b: Working Examples lb-1 to lb-3 (Concentrate)

Comparative Example 1

Comparative Example 1, shown in Table 5, was made by the following procedure. Part A was made by mixing 3.05 grams of Terric GN9, 5.01 grams dipropylene glycol methyl ether and 15.01 grams water together in a 100-mL jar. The jar containing Part A was then capped and heated to 70°C in an oven. Part B was made by mixing 9.01 grams sodium tetraborate decahydrate, 6.03 grams glycerol and 7.52 grams water in a 50 mL beaker. After all three components in Part B had been combined, a stir bar was added and the solution was mixed at 300 revolutions per minute (rpm) on a heater until it was approximately 80° C. Part C was made by combining 5.02 grams ethylene glycol and 3.04 grams Alcalase 2.4L in a 20-mL vial. Part D was made by mixing 24.02 grams benzalkonium chloride and 30.06 grams of water in a 100-mLjar. The jars of Part C and Part D were each capped and set on a roller at 30 rpm.

An amount of 18.40 grams of Part A was mixed together with 12.09 grams of Part B in a new 100-mL jar. The pH of the Part A + Part B mixture solution was adjusted to approximately 7.2 with acetic acid added dropwise. After the pH of the solution was lowered, 4.43 grams of Part C was added to the solution and shaken briefly to mix. Next, 43.03 grams of solution D was added to the solution. The final mixture of Part A, Part B, Part C and Part D was set to roll at 30 rpm for approximately 20 minutes to make sure it was well mixed. After rolling, the solution was noted to be a clear, yellow solution.

Table 5: Com arative Exam le 1

Comparative Example 2

To make Comparative Example 2, all of the components in Table 6 were added to a 100-mL jar. Components were added in the order they are listed and after each addition (besides the first addition) the jar was swirled briefly to mix all of the components. After all of the components were added, the jar was set to roll at 30 rpm overnight as the solution appeared to be yellow and cloudy. The next morning the solution remained cloudy and separated into two phases after sitting. Before being diluted the solution was shaken to try and ensure that all components were proportionally contained in the dilution.

Table 6: Com arative Exam le 2

Eumulgin L 3.1

Propylene Glycol 15.0

Termamyl 300L 5.1

Dioctyl dimethyl ammonium chloride 7.9

Protamex 2.0

Comparative Example 3

Comparative Example 3, shown in Table 7, was made in the same way as Comparative Example 2. After rolling, Comparative Example 3 was clear, yellow and one phase.

Table 7: Com arative Exam le 3

Working Example 2: "Use" Diluted Working Examples

In order to test the cleaning ability of the Working Example concentrates, they were diluted to "use" concentrations. The Examples were prepared as concentrates as described above and then diluted so that the final concentration of enzymatic solution would be 0.5%. For example, if pure Enzymatic Cleaner Solution was used to make a 20 gram total dilution, an amount of 0.1 gram of pure Enzymatic Cleaner Solution was added to 19.9 grams water. For example, for Working Example la-6 (45.23 multiplied by 84.45 = 38.19 wt%) this solution would have to be diluted 76.38 times to get a final dilution of 0.5 wt%. This would result in a final concentration of 0.5% enzymatic solution (and also containing 0.1% PHMB and 0.2% SENSIVA SC50). Some solutions that included extra excipients were cloudy when diluted even though they came from a clear concentrate. Additional surfactant was added to these dilute solutions until the solution became clear, as shown in Table 8, below.

Table 8: "Use" Diluted Working Examples

2-1 la-1 1.4 0.6 98.0 Glucopon 225DK

2-2 la-2 1.4 0 98.6 Tergitol NP-10

2-3 la-3 1.3 0 98.7 Triton X-100

2-4 la-4 1.3 0.4 98.2 Surfynol 485

2-5 la-5 1.5 0 98.5 Kolliphor EL

2-6 la-6 1.3 0 98.7 Hetoxamide C4

2-7 la-7 1.5 0 98.6 Mackam L

2-8 la-10 1.3 0 98.7 Tergitol 15-S-9

2-9 la-1 1 1.3 0 98.7 Tomakleen G-12

2-10 la-15 1.2 0 98.8 Glucopon 225DK

2-1 1 la-16 1.6 1.3 97.1 Glucopon 225DK

2-12 la-17 1.3 0 98.7 Glucopon 225DK

2-13 la-18 1.5 3.0 95.5 Glucopon 225DK

2-14 la-19 1.3 0 98.7 Glucopon 225DK

2-15 la-20 1.3 1.3 97.4 Glucopon 225DK

2-16 la-21 1.8 0 98.2 Glucopon 225DK

2-17 la-22 1.3 1.8 96.9 Glucopon 225DK

2-18 la-23 1.5 0 98.5 Hetoxol LA-7

2-19 la-24 1.5 0 98.5 Hetoxol LA-7

2-20 la-25 1.4 0.7 97.9 Glucopon 225DK

2-21 la-26 1.4 0 98.6 Hetoxol LA-7

2-22 la-27 1.4 0 98.6 Hetoxol LA-7

2-26 la-28 2.6 2.3 95.1 Glucopon 225DK

2-27 la-29 1.6 1.4 97.0 Glucopon 225DK

2-23 lb-1 1.6 0.3 98.1 Glucopon 225DK

2-24 lb-2 1.5 1.0 97.5 Glucopon 225DK

2-25 lb-3 1.6 3.2 95.2 Glucopon 225DK

Preparatory

2-28 0.5 0 99.5 N/A

Example 1

Comparative Example 4: "Use" Diluted Comparative Examples 1-3

Comparative Example 1 was diluted 1 :200 and Comparative Examples 2 and 3 were diluted 1 : 100. These dilution ratios left them with approximately 0.1% antimicrobial which is the final target concentration of antimicrobial in the "use" diluted Working Examples. Comparative Examples 1-3, which were concentrates, were designated Comparative Examples 4-1, 4-2, 4-3, respectively, after dilution.

Example 6: Cleaning Ability of Diluted Examples TOSI blood soil washer test strips were used to test the cleaning ability of the Examples and Comparative Examples. The example to be tested was diluted to 0.5% Enzymatic Cleaning Solution and a final weight of 20 grams, as described above. One TOSI blood soil test strip was separated from the plastic cover and inserted into the solution vial and the bottle was inverted and returned 50 times. It has been found that 50 (inversions + returns) was the average number for 0.5% of enzymatic solution to nearly completely clean the TOSI test strip. Cleanliness of the TOSI strip was then visually evaluated. Cleanliness values were assigned based on a scale of 1-10 with "1" being no difference from an untested TOSI strip and "10" being a completely clean TOSI strip.

Table 9a shows that cleaning of blood-containing soils is often negatively impacted with the addition of the antimicrobial lipid. Table 9b show that the Comparative Examples have some cleaning ability. Table 9c shows that individual components can have some cleaning ability. Cleaning ability of blood-containing soils, however, does not equate to biofilm removal ability. Biofilm removal is illustrated in Tables 10 and 1 1. Surprisingly, preferred compositions of the present disclosure demonstrate good biofilm removal and cleaning ability of biological residue, particularly blood- containing soil, and are stable.

Table 9a: Cleanin Abilit of Examples

Table 9b: Cleanin Abilit of Com arative Exam les

Biofilm Generation and Removal Test Method One

Pseudomonas aeruginosa biofilm generation was performed with CDC Bioreactor

ASTM Method E2562-12 for Biofilm Testing with Enzymatic Cleaners. The CDC Biofilm Reactor used consisted of multiple coupon holders (rods) suspended from a UHMW-polyethylene ported lid. The coupon holders (rods) accommodated three 1/2 inch (12.7 mm) diameter coupons each. The lid with coupon holders (rods) and coupons were mounted in a 1-L glass vessel with side-arm discharge port. The liquid growth media was circulated through the vessel while mixing and shear was generated by a magnetic stir bar rotated by a magnetic stir plate. Sampling of the coupons was conducted by aseptically removing individual coupon holders with accompanying coupons. The CDC Biofilm Reactor was autoclavable and re-useable. The total liquid volume was approximately 350 mL. The CDC Biofilm Reactor provided a controlled and reproducible environment for growing biofilm on test surfaces. The CDC Biofilm Reactor is part of the ASTM Standard Method E2562-12: Standard Test Method for Quantification of Pseudomonas aeruginosa Biofilm Grown with High Shear and Continuous Flow using CDC Biofilm Reactor. The biofilm generation and removal test method took several days.

Day 1 : A tryptic soy broth (TSB) growth media 1 was prepared by dissolving 6 grams of TSB in 10 liters of Milli-Q purified water for each of two 10-L carboys. The caps were loosely attached to the carboys, but secured with steam indicator tape, and the carboys were autoclaved at 121°C for 120 minutes on a liquid cycle. The growth media 1 was then allowed to cool overnight.

A growth media 2 was prepared by dissolving 30g TSB into 1 liter of Milli-Q purified water. The tops of both flasks were covered with aluminum foil, and autoclaved at 121 °C for 30 minutes on a liquid cycle. The growth media 2 was then allowed to cool overnight.

A single TSA plate was placed into a 37°C incubator to warm before plating. P. aeruginosa (ATCC 15442) glycerol stock was removed from a -80°C deep freeze storage and allowed to slightly thaw, just enough for the top to unfreeze. The thawed top was aseptically scraped with a sterile loop and transferred to the warmed TSA plate. This was immediately streaked for isolation with a new sterile loop for each streak. The plate was then wrapped in parafilm and placed into a 37°C incubator for

approximately 24 hours.

Day 2: The reactor was prepared by putting the test coupons in the rods and attaching the rods to the inside of the lid of the reactor chamber. The reaction chamber was then autoclaved at 121°C for 20 minutes on a liquid cycle. The reactor was then cooled overnight Using the TSA plate prepared the previous day, an overnight culture was made by aseptically pulling a swab through the culture plate and adding all collected colonies into 100 mL of growth media 2. This was placed in a 37°C shaker-incubator at 175 rpm for at least 18 hours.

Day 3: The reactor was set up for batch operation. This was done by first setting the reactor on a stir plate, then connecting tubing from the carboys filled with TSB to the reactor and from the reactor to an additional 20-L waste carboy. The tubing was connected to a peristaltic pump which filled the reactor with the TSB media. When the media flowed out into the waste carboy the pump was turned off. With this method the batch operation had a concentration of 600 mg/L TSB. The stir plate was set to 120 rpm. The reactor was inoculated with 1.00 mL of the overnight culture of P. aeruginosa. This was left with stirring but no flow for 24 hours.

Day 4: The pump was turned on to a flow rate of 11.5 mL/min and operated (with both stirring and flow) for 24 hours.

Dey-Engley (D/E) neutralizing broth was prepared according to the manufacturer's instructions. Day 5: The samples were tested on the fifth day. The pump and the stir plate were turned off. Each rod was removed from the reactor cap and separately dipped into 30 mL of sterile water to remove any unattached bacteria. Each coupon, now having P. aeruginosa biofilm, was aseptically removed from the rod and, using tweezers, lowered into an empty 50-mL tube without touching the sides. Concurrently, 10 mL of each example to be tested (at its appropriate dilution) was warmed up in a 40°C water bath for 15 minutes. A sterile water control was also prepared in the same fashion. After 15 minutes of warming, each example was poured into the tube containing the P. aeruginosa biofilm coupon and put back into the 40°C water bath for an additional 30 minutes of exposure time. After the 30-minute exposure time, the coupons were aseptically removed using a pair of sterile tweezers and transferred into 50-mL tubes containing 10 mL of D/E neutralizing broth.

To recover the P. aeruginosa biofilm remaining on the coupon, the tubes were first vortexed for 30 seconds, sonicated for 30 seconds, vortexed for another 30 seconds, sonicated for 30 seconds, and vortexed for a final 30 seconds. The next step was to remove 200 microliters (μί) of the solution from the tube and transfer it to sterile Butterfields Buffer; followed by serial dilutions from 10^"1 to 10^"7. Examples were then plated onto 3M PETRIFILM Aerobic Count Plates and incubated for 96 hours at 25°C. After incubation, the plates were read and the log reduction (versus the saline control) was reported. 3M PETRIFILM Aerobic Count Plates and the PETRIFILM Plate Reader (PPR) were obtained from 3M Company (St. Paul, MN).

Biofilm Generation and Removal Test Method Two

Pseudomonas aeruginosa biofilm generation was performed with CDC Bioreactor

Day 1 : A tryptic soy broth (TSB) growth media 1 was prepared by dissolving 1 gram of TSB in 10 liters of Milli-Q purified water for each of two 10-L carboys. The caps were loosely attached to the carboys, but secured with steam indicator tape, and the carboys were autoclaved at 121°C for 120 minutes on a liquid cycle. The growth media 1 was then allowed to cool overnight.

A growth media 2 was prepared by dissolving 0.3g TSB into 1 liter of Milli-Q purified water. This was separated into two portions—100 mL in one flask to be used for the overnight culture and 500 mL in a separate flask to be used as batch media. Extra growth media 2 was discarded. The tops of both flasks were covered with aluminum foil, and autoclaved at 121°C for 30 minutes on a liquid cycle. The growth media 2 was then allowed to cool overnight.

A single TSA plate was placed into a 35°C incubator to warm before plating. P. aeruginosa (ATCC 15442) glycerol stock was removed from a -80°C deep freeze storage and allowed to slightly thaw, just enough for the top to unfreeze. The thawed top was aseptically scraped with a sterile loop and transferred to the warmed TSA plate. This was immediately streaked for isolation with a new sterile loop for each streak. The plate was then wrapped in parafilm and placed into a 35°C incubator for

approximately 24 hours.

Day 2: The reactor was prepared by putting the test coupons in the rods and attaching the rods to the inside of the lid of the reactor chamber. The reaction chamber was then autoclaved at 121°C for 20 minutes on a liquid cycle. The reactor was then cooled overnight Using the TSA plate prepared the previous day, an overnight culture was made by aseptically picking and transferring a single colony into the 100 mL of growth media 2. This was placed in a 35°C shaker-incubator at 175 rpm for at least 18 hours.

Day 3 : The reactor was set up for batch operation. This was done by first setting the reactor on a stir plate, and clamping the waste tube shut. The reactor was then filled with 500 mL of growth media 2. With this method the batch operation had a concentration of 300 mg/L TSB. The stir plate was set to 120 rpm. The reactor was inoculated with 1.00 mL of the overnight culture of P. aeruginosa. This was left with stirring but no flow for 24 hours.

Day 4: The reactor was set up for continuous flow operation by first connecting tubing from the carboys filled with TSB to the reactor and from the reactor to an additional 20-L waste carboy. The tubing was then connected to a peristaltic pump. The pump was turned on to a flow rate of 1 1.5 mL/min and operated (with both stirring and flow) for 24 hours.

Dey-Engley (D/E) neutralizing broth was prepared according to the manufacturer's instructions. Day 5 : The samples were tested on the fifth day. The pump and the stir plate were turned off.

Each rod was removed from the reactor cap and separately dipped into 30 mL of sterile water to remove any unattached bacteria. Each coupon, now having P. aeruginosa biofilm, was aseptically removed from the rod and, using tweezers, lowered into an empty 50-mL tube without touching the sides. Concurrently, 10 mL of each example to be tested (at its appropriate dilution) was warmed up in a 40°C water bath for 15 minutes. A sterile water control was also prepared in the same fashion. After 15 minutes of warming, each example was poured into the tube containing the P. aeruginosa biofilm coupon and put back into the 40°C water bath for an additional 30 minutes of exposure time. After the 30-minute exposure time, the coupons were aseptically removed using a pair of sterile tweezers and transferred into 50-mL tubes containing 10 mL of D/E neutralizing broth.

Table 10 shows both the cleaning results and the biofilm removal results for diluted Working Examples and diluted Comparative Examples.

Table 10: Cleaning and Biofilm Removal from Biofilm Removal Method 1

Example Log Standard Cleaning Anti¬

Ex. Excipient

Source Reduction Deviation Rating microbial

Comp Comp. Ex.

2.60 0.05 8 0.12% BZK none

7-1 4-1

Comp Comp. Ex.

0.70 0.04 7 0.08% DDAC none

7-2 4-2

0.05% DDAC

Comp Comp.

1.33 0.06 8 and 0.08% none

7-3 Ex.4-3

PHMB

0.2%

7-1 2-1 5.20 0.09 8 0.1% PHMB SENSIVA

SC50

0.2%

7-2 2-2 5.9 0.5 9 0.1% PHMB SENSIVA

SC50

0.2%

7-3 2-3 6.1 0.3 6 0.1% PHMB SENSIVA

SC50

0.2%

7-4 2-4 7.8 0.3 9 0.1% PHMB SENSIVA

SC50

0.2%

7-5 2-5 3.5 0.3 8 0.1% PHMB SENSIVA

SC50

0.2%

7-6 2-6 3.2 0.1 7 0.1% PHMB SENSIVA 0.2%

7-7 2-7 4.5 0.5 5 0.1% PHMB SENSIVA

SC50

7-8* p2- l 3.96 0.09 9 0.1% PHMB none

Example 7-8 is an example of an effective composition that does not include an antimicrobial lipid.

Example 8: Biofilm Removal and Cleaning Results from Biofilm Removal Method 2

Table 1 1 shows both the cleaning results and the biofilm removal results for diluted Working Examples and diluted Comparative Examples.

Table 11: Cleaning and Biofilm Removal with biofilm method 2

Example Log Standard Cleaning Anti¬

Ex. Excipient Source Reduction Deviation Rating microbial

Comp.

2-28 0.1 0.3 10 none none 8-1

0.2% Sensiva

8- 1 2-1 3.4 0.5 8 0.1% PHMB

SC50

0.2% Sensiva

8-2 2-4 3.4 0.3 9 0.1% PHMB

SC50

0.075%

8-3 2-10 3.9 0.3 9 0.1% PHMB

Sensiva SC50

0.3% Sensiva

8-4 2-1 1 3.6 0.3 2 0.1% PHMB

SC50

0.2% Capmul

8-5 2-12 2.4 0.3 9 0.1% PHMB

708G

8-6 2-13 2.5 0.5 5 0.1% PHMB 0.2% PGML

0.2% Symdiol

8-7 2-14 3.5 0.9 8 0.1% PHMB

68

0.2% Capmul

8-8 2-15 2.4 0.3 7 0.1% PHMB

MCM

0.2% Sensiva

8-9 2-16 3.9 0.4 5 0.2% PHMB

SC50

0.2% Sensiva

8-10 2-17 3.6 0.3 5 0.05% PHMB

SC50

0.2% Capmul

8-1 1 2-18 2.4 0.3 7 0.2% BZK

708G

0.2% Sensiva

8-12 2-19 4.1 0.7 7 0.2% BZK

SC50

0.2% Sensiva

8-13 2-20 2.4 0.7 7 0.1% BZK

SC50

0.2% Sensiva

8-14 2-21 3.1 0.6 8 0.1% CHG

SC50

0.2% Capmul

8-15 2-22 3.2 0.3 5 0.1% CHG

708G

0.2% Sensiva

8-16 2-23 2.8 0.1 9 0.1% PHMB SC50 & 0.3%

Polystep B-22

0.2% Sensiva

8-17 2-24 2.6 0.3 8 0.1% PHMB SC50 & 0.1%

Capmul 708G 0.2% Sensiva

8-18 2-25 2.8 0.3 4 0.1% PHMB SC50 & 0.3%

Stepan Mild

1% Capmul

8-19 2-26 2.6 0.6 9 0.1% PHMB

708G

0.5% Capmul

8-20 2-27 2.5 0.3 8 0.1% PHMB

708G

The complete disclosures of the patents, patent documents, and publications cited herein are incorporated by reference in their entirety as if each were individually incorporated. Various modifications and alterations to this invention will become apparent to those skilled in the art without departing from the scope and spirit of this invention. It should be understood that this invention is not intended to be unduly limited by the illustrative embodiments and examples set forth herein and that such examples and embodiments are presented by way of example only with the scope of the invention intended to be limited only by the claims set forth herein as follows.

Claims

What is claimed is:

1. An enzymatic cleaning composition comprising:

an enzyme comprising a protease.

2. The enzymatic cleaning composition of claim 1 wherein the cationic antimicrobial comprises a biguanide, a bisbiguanide, a quaternary ammonium compound, or combinations thereof.

3. The enzymatic cleaning composition of claim 1 or 2 wherein the antimicrobial lipid comprises a (C7-C12)saturated fatty acid ester of a polyhydric alcohol, a (C12-C22)unsaturated fatty acid ester of a polyhydric alcohol, a (C7-C12)saturated fatty ether of a polyhydric alcohol, a (C6-C12)alkyl or (C8- C18)alkylene 1,2-diol, a (C12-C22)unsaturated fatty ether of a polyhydric alcohol, an alkoxylated derivative thereof, or combinations thereof.

4. The enzymatic cleaning composition of any one of claims 1 through 3 wherein the surfactant has an HLB of at least 8.

5. The enzymatic cleaning composition of any one of claims 1 through 4 wherein the surfactant comprises a nonionic surfactant.

6. The enzymatic cleaning composition of any one of claims 1 through 5 further comprising an anionic surfactant.

7. The enzymatic cleaning composition of any one of claims 1 through 6 further comprising an enzyme selected from an amylase, a lipase, a cellulase, or combinations thereof.

8. The enzymatic cleaning composition of any one of claims 1 through 7 further comprising a stabilizer.

9. The enzymatic cleaning composition of any one of claims 1 through 8 further comprising a hydrotrope.

10. The enzymatic cleaning composition of any one of claims 1 through 9 further comprising one or more additives selected from chelators, organic solvents, builders, alkalinity sources and other pH adjustors, defoamers, supplemental antimicrobial agents other than the antimicrobial lipids and cationic antimicrobials, preservatives, viscosity modifiers, bleaching agents, corrosion inhibitors, dyes, colorants, fragrances, opacifiers, bluing agents, preservatives, processing aids, and combinations thereof.

11. The enzymatic cleaning composition of any one of claims 1 through 10 comprising up to 30 wt-% solids.

12. The enzymatic cleaning composition of any one of claims 1 through 11 which is effective to remove at least a portion of biofilm from a substrate.

13. The enzymatic cleaning composition of claim 12 which is effective to remove at least a portion of biological residue from a substrate.

14. The enzymatic cleaning composition of claim 12 or 13 which is effective to kill at least a portion of bacteria on a substrate.

15. A method of removing at least a portion of a biofilm from a surface of a non-biological article, the method comprising:

an enzyme comprising a protease.

16. A method of removing at least a portion of a biofilm from a surface of a non-biological article, the method comprising:

a cationic antimicrobial comprising a biguanide, a bisbiguanide, a quaternary ammonium compound, or combinations thereof; a surfactant comprising a nonionic surfactant, a zwitterionic surfactant, or combinations thereof; and

an enzyme comprising a protease;

17. A method of cleaning a surface of a non-biological article, the method comprising:

contacting the surface with an enzymatic cleaning composition under conditions effective to remove at least a portion of a biofilm, at least a portion of the bacteria, and at least a portion of a the biological residue, wherein the enzymatic cleaning composition comprises:

an enzyme comprising a protease;

wherein the bacteria is removed by at least 3 log reduction; and

wherein the cleaning efficiency is at least 7 on a scale of 1 to 10.

18. The method of claim 17 wherein the enzymatic cleaning composition further comprises a cationic antimicrobial comprising a biguanide, a bisbiguanide, a quaternary ammonium compound, or combinations thereof.

19. The method of claim 17 or 18 wherein the enzymatic cleaning composition further comprises an antimicrobial lipid comprising a compound that has a solubility in water of no greater than 1.0 gram per 100 grams (1.0 g/100 g) deionized water.

20. The method of any one of claims 17 through 19 wherein the conditions comprise a temperature from room temperature to 60°C.

21. The method of any one of claims 17 through 20 wherein the conditions comprise a time from immediately to 24 hours.

22. The method of any one of claims 17 through 21 wherein the conditions comprise mechanical assistance.

23. The method of any one of claims 17 through 22 wherein the non-biological article comprises a surface comprising stainless steel, plastic, ceramic, or glass.

24. The method of any one of claims 17 through 23 wherein the log reduction of the biofilm is at least 4 according to the Biofilm Generation and Removal Test Method One.

25. The method of any one of claims 17 through 24 wherein the antimicrobial cleaning composition further comprises an anionic surfactant.