WO2012054391A2

WO2012054391A2 - Compositions and methods for reducing microbial levels on a surface

Info

Publication number: WO2012054391A2
Application number: PCT/US2011/056548
Authority: WO
Inventors: Dale A. Grinstead; Kathleen J. Bixler; Ying Zhou
Original assignee: Diversey, Inc.
Priority date: 2010-10-18
Filing date: 2011-10-17
Publication date: 2012-04-26
Also published as: WO2012054391A3

Abstract

Provided are compositions for reducing microbial levels on a surface, the compositions comprising a biocide and a zwitterionic surfactant present in the composition at a ratio of about 4:1 to about 8:1. Also provided are methods and compositions that comprise a decreased amount of a biocide to reduce effective microbial levels on a surface.

Description

COMPOSITIONS AND METHODS FOR REDUCING

MICROBIAL LEVELS ON A SURFACE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.

61/455,288, filed October 18, 2010, which is incorporated herein by reference in its entirety.

FIELD

[0002] The disclosure relates to compositions and methods for reducing microbial activity on a surface.

BACKGROUND

[0003] Various compounds exhibit antimicrobial or biocidal activity. Some compounds exhibit biocidal activity only at high concentrations. Compositions that have a low use concentration of active ingredient and meet performance standards for food contact surfaces sanitizers are desired.

SUMMARY

[0004] in an aspect, the disclosure provides a composition for reducing microbial levels on a surface, the composition comprising a biocide and a surfactant comprising a zwitterion. In some embodiments the composition comprises a ratio of biocide to zwitterion of about 4:1 to about 8:1. in further embodiments, the surfactant comprises a betaine.

[0005] In another aspect, the disclosure provides a biocidal composition comprising a biocide and a surfactant comprising a zwitterion, wherein the composition is substantially free of an alky! amine.

[0006] In a further aspect, the disclosure provides an antimicrobial composition comprising about 30 to about 2000 ppm peracetic acid, and a surfactant comprising a zwitterion, wherein the composition is substantially free of an alkyl amine, and wherein the peracetic acid and the zwitterion are present in the composition at a ratio of about 4:1 to about 8:1.

[0007] Another aspect of the disclosure provides a composition having synergistic biocidal activity, wherein the composition comprises cocobetaine and a biocide in a ratio of about 4:1 to about 8:1, and in an amount effective to reduce the amount of biocide required for biocidal activity by at least about 20% to about 90% relative to the same composition without the cocobetaine. Embodiments also provide for a method of making a synergistic biocidal composition.

[0008] In an aspect, the disclosure provides a method of reducing microbial levels or microbial activity on a surface or airborne microbial levels or microbial activity, the method comprising contacting the surface the air with an effective amount of a composition described herein.

[0009] The disclosure relates to other aspects and embodiments which will become apparent in view of the following detailed description and accompanying Figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 is a graph of the number of carriers with no growth after 10 min contact time for various surfactant blends.

[0011] Figure 2 is a graph of the number of carriers with no growth after 1 min contact time for various surfactant blends with 50 ppm peracetic acid (PAA).

[0012] Figure 3 is a graph of the mean log reduction in Salmonella choleraesuis with 25 ppm and 50 ppm PAA.

[0013] Figure 4 is a graph of the mean log reduction in Pseudomonas aeruginosa with 25 ppm and 50 ppm PAA.

[0014] Figure 5 is a graph of the mean log reduction in Staphylococcus aureus with 25 ppm and 50 ppm PAA.

[0015] Figure 6 is a graph of the mean log reduction in Staphylococcus aureus or Pseudomonas aeruginosa with 0 and 0.3 mUL cocobetaine.

[0016] Figure 7 is a quadratic fit to the log reduction in Staphylococcus aureus (7A) or Pseudomonas aeruginosa (7B) with various molar ratios of betaine to PAA. (A) Polynomial fit degree = 2, log reduction S aureus = 4.6879315 + 0.0561347*moles of betaine per mole of PAA - 0.0205156*(moies of betaine per molve of PAA-5.6)². Analysis of variance: Model, DF = 2, sum of squares = 1.0358770, mean square = 0.517938; Error, DF = 12, sum of squares = 2.1326830, mean square = 0.177724; C. Total, DF = 14, sum of squares = 3.1685600; F Ratio = 2.9143; Prob > F = 0.0930. (B) Polynomial fit degree = 2, log reduction P. aeruginosa = 6.9659862 - 0.1433417*moles of betaine per mole of PAA - 0.0149692*(moies of betaine per molve of PAA-5.6)². Analysis of variance: Model, DF = 2, sum of squares = 3.3008201 , mean square = 1.65041 ; Error, DF = 12, sum of squares = 1.5571394, mean square = 0.12976; C. Total, DF = 14, sum of squares = 4.8579600; F Ratio = 12.7188; Prob > F = 0.0011.

DETAILED DESCRIPTION

[0017] While some surfactants individually may exhibit a minimal amount of biocidal activity against an organism (e.g., microbes such as bacteria), some surfactants have synergy with and enhance the biocidal efficacy of other materials. As described herein, and as shown in the Examples, the inventors have identified biocidal (e.g., antimicrobial) compositions comprising a biocide in combination with a surfactant that can potentiate the efficacy of the biocide.

[0018] In an aspect, the disclosure provides a composition comprising a surfactant and a biocide. Suitably, some embodiments provide a composition that is effective for reducing microbial levels or microbial activity on a surface and/or airborne microbial levels or microbial activity. In some embodiments the composition comprises a biocide and a surfactant in amounts that provide for a synergistic biocidal (e.g., antimicrobial) effect (i.e., provide effective biocidal activity at lower amounts/concentration of the biocide, relative to the biocide without the surfactant). In some embodiments, the compositions meet efficacy standards for a food contact surface sanitizer and/or disinfectant. In various embodiments relating to the compositions described herein, the composition can comprise, consist essentially of, or consist of a biocide and a surfactant, as well as any other recited component.

[0019] The surfactant can be classified generally as an amphoteric surfactant (e.g., a surfactant comprising a zwitterion). In some embodiments, the zwitterion comprises one or more compounds generally known as, or that can be classified as, a betaine such as, for example, glycine betaine, proline betaine, cocobetaine, and cocoamidopropyl betaine. In certain embodiments, the surfactant comprises cocobetaine. In some embodiments the surfactant can comprise a blend of more than one surfactant.

[0020] The surfactant may be present in the composition in an amount of at least about 5 ppm, at least about 10 ppm, at least about 20 ppm, at least about 40 ppm, at least about 60 ppm, at least about 80 ppm, at least about 100 ppm, at least about 200 ppm, at least about 500 ppm, at least about 800 ppm, at least about 1000 ppm, at least about 1500 ppm, at least about 2000 ppm, at least about 2500 ppm, at least about 3000 ppm, at least about 3500 ppm, at least about 4000 ppm, or at least about 4500 ppm. The surfactant may be present in the composition in an amount of less than about 5000 ppm, less than about 4500 ppm, less than about 4000 ppm, less than about 3500 ppm, less than about 3000 ppm, less than about 2500 ppm, less than about 2000 ppm, less than about 1500 ppm, less than about 1000 ppm, less than about 800 ppm, less than about 500 ppm, less than about 200 ppm, less than about 100 ppm, or less than about 80 ppm. As can be taken from the above description, the composition may comprise a range of surfactant, suitably from about 5 ppm to about 5000 ppm. In some embodiments, the composition comprises surfactant having a range of about 20 or 25 ppm to about 200 ppm (e.g., 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 ppm) or within a range of about 50 ppm to about 100 ppm surfactant. In some embodiments, the surfactant is present in the composition in amount effective to provide an additive biocidal effect (i.e., the surfactant can contribute to the biocidal activity of the composition). In some embodiments, the surfactant is present in the composition in amount effective to provide a synergistic biocidal effect (i.e., the biocidal activity of the composition is greater than expected based on the amount of biocide and surfactant).

[0021] The biocide, in some embodiments, can be selected from any variety compound classes that exhibit a general reduction in the activity of a target organism such as, for example, a microbe, plant, rodent, or insect. Some non-limiting examples of a biocide include compounds typically classified as a pesticide and/or an antimicrobial. In some embodiments the biocide can comprise one or more pesticides such as, for example, a fungicide, an herbicide, an insecticide, an algicide, a molluscicide, a miticide, and/or a rodenticide. Additionally or alternatively a biocide can comprise an antimicrobial agent such as, for example, a germicide, an antibiotic, an antibacterial, an antiviral, an antifungal, an antiprotozoal, and/or an antiparasitic.

[0022] Additional non-limiting examples of biocides include, but are not limited to, compounds comprising one or more halogens (e.g., chlorine, chlorite, chlorine dioxide, iodine, and bromine); organic acids (e.g., lactic, acetic, and propionic acid); peroxides (e.g., hydrogen peroxide) and peracids comprising at least 2 carbon atoms (e.g., peracetic acid); surfactants (e.g., cationic, anionic, non-ionic, and zwitterionic surfactants); metals (e.g., silver, copper, and zinc); essential oils; and protein/amino acid based agents (e.g., bacteriocins, lysozyme, and bacteriophage). In some embodiments, the biocide can be selected from the non-limiting group of, quaternary ammonium compounds (e.g., ammonium chloride), peroxides (e.g., hydrogen peroxide), and peracids (e.g., peracetic acid (PAA)). In certain embodiments, the biocide comprises peracetic acid. [0023] The biocide may be present in the composition in an amount that provides the desired biocidal effect. Suitably, the composition comprises an amount of biocide of at least about 1 ppm, at least about 10 ppm, at least about 20 ppm, at least about 30 ppm, at least about 35 ppm, at least about 40 ppm, at least about 50 ppm, at least about 100 ppm, at least about 50 ppm, at least about 200 ppm, at least about 250 ppm, at least about 300 ppm, at least about 350 ppm, at least about 400 ppm, at least about 450 ppm, at least about 500 ppm, at least about 1000 ppm, at least about 2000 ppm, at least about 5000 ppm, at least about 8000 ppm, or at least about 10,000 ppm. The biocide may be present in the composition in an amount of less than about 10,000 ppm, less than about 8000 ppm, less than about 5000 ppm, less than about 2000 ppm, less than about 1500 ppm, less than about 1000 ppm, less than about 800 ppm, or less than about 500 ppm.

[0024] Thus, the compositions may comprise a range of biocide that is typically from about 1 ppm to about 10,000 ppm, about 30 ppm to about 2000 ppm, about 1000 ppm to about 2000 ppm. Suitable amounts of biocide can be determined by one of skill in the art using any appropriate method, including those described herein. In some non-limiting embodiments, the biocide comprises peracetic acid in the above ranges, including a range of about 35 ppm to about 45 ppm.

[0025] The biocide and the surfactant may be present in the composition at various ratios. In certain embodiments, the biocide and the surfactant may be present in the composition at a ratio of at least about 2.5:1 , at least about 2.8:1 , at least about 3:1 , at least about 3.2:1, at least about 3.5:1 , or at least about 4:1. In certain embodiments, the biocide and the surfactant may be present in the composition at a ratio of less than about 8:1, less than about 7.8:1, less than about 7.5:1, less than about 7.2:1 , less than about 7:1 , less than about 6.8:1 , less than about 6.5:1 , less than about 6.2:1 , less than about 6:1 , less than about 5.8:1 , less than about 5.5:1, less than about 5.2:1 , or less than about 5:1. In certain embodiments, the biocide and the surfactant may be present in the composition at a ratio of about 2.5:1 to about 8:1 , or about 3:1 to about 7:1. Some embodiments provide for a ratio of biocide and surfactant that provide a synergistic composition (e.g., a composition that requires less biocide for same relative efficacy when compared to the biocide alone).

Suitably a synergistic composition exhibits a biocidal effect that is greater than an expected additive effect provided by the surfactant or another active agent or composition component.

[0026] The composition can have a broad pH range of about 2 to about 12. Suitably, the composition has a phi that is neutral to slightly acidic. In some embodiments, the

composition has a pH of about 4 to about 6 (e.g., about 4.0, about 4.2, about 4.5, about 4.8, about 5.0, about 5.2, about 5.5, about 5.8, or about 6.0), inclusive of any intervening pH value. The composition may comprise an acid or buffer to maintain pH. Acids may include inorganic acids and organic acids such as, for example, citric acid, nitric acid, phosphoric acid, octanoic acid, methyl sulfonic acid, and sulfuric acid.

[0027] In certain embodiments, the composition comprises, consists essentially of, or consists of peracetic acid and cocobetaine.

[0028] The composition may contain additional agents including, but not limited to, standard formulary agents known in the art. In some embodiments the compositions described herein can further comprise one or more chelating agents (chelants). Chelating agents may include, but are not limited to, L-glutamate-N-N-diacetic acid (GLDA), ethylenediaminetetraacetic acid (EDTA), methyl glycine diacetic acid ( GDA), hydroxy iminodiacetic acid (HIDA), nitrilotriacetic acid (NTA), and citrate. In certain embodiments, the compositions may comprise chelant in an amount of at least about 1%, at least about 2%, at least about 3%, at least about 4%, or at least about 5% by weight. In certain embodiments, the compositions may comprise chelant in an amount of less than about 15%, les than about 14%, less than about 13%, or less than about 12% by weight. In certain embodiments, compositions may comprise about 0-15% chelating agent, or about 5% to about 12% chelating agent by weight. In certain embodiments, the chelating agent comprises GLDA.

[0029] Suitably, the compositions may have reduced odor compared to other compositions. The compositions may have reduced corrosion compared to other compositions. The compositions may be substantially free of alkyl amine. As used herein, "substantially free" means having less than about 3%, less than about 1%, less than about 0.5%, or less than about 0.2%, and more particularly about 0.2% to 0% of component.

[0030] The compositions may be formulated for a particular route of delivery or application. The compositions may be in the form of a spray, aerosol, gel, film, wipe, solid (e.g., particulate or powder), suspension, or solution, and applied using any suitable route, apparatus, and/or mechanism for the delivery or application of the compositions and formulations. Such formulation and application strategies can be determined based on the intended or desired characteristics or use of the composition/formulation and are within the ability of one or ordinary skill in the art.

[0031] As described herein, embodiments of the disclosure provide compositions that exhibit biocidal activity against an organism. As used herein, "biocidal" activity can reduce, slow, or stop growth and/or proliferation, reduce, slow, or stop the rate of growth and/or proliferation, or stun, inactivate, or kill an organism such as, for example, a microbe. The term can also relate to reducing, inhibiting, or preventing cellular differentiation in an organism. Thus, biocidal activity includes, for example, pesticidal, antiparasitic, larvicidal, antimicrobial, antibacterial, antiviral, antifungal, antiseptic, anti-mold, antibiotic, disinfectant, or sanitization activity. In some embodiments, the biocide can reduce the proliferation of an organism. In some embodiments, the biocide can slow proliferation of an organism. In some embodiments, the biocide can stop the proliferation of an organism (e.g., act as a bacteriostatic or fungistatic agent). In some embodiments, the biocide can kill an organism. In embodiments, the disclosure provides a composition that is effective to reduce microbial levels on a surface. For example, microbial levels can be reduced by at least about 2 log units to as much as 10 log units or more (2, 3, 4, 5, 6, 7, 8, 9, 10 log units or more). In some embodiments the compositions have biocidal activity against pathogenic organisms including, but not limited to, bacteria, viruses, protists, helminths, mold, yeast, and spore- forming organisms. Certain non-limiting examples of bacteria include, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella choleraesuis, and Escherichia coli.

[0032] The biocidal activity of a composition or component may be determined using various methods known by those of skill in the art. Suitable methods are described below in the Examples. For example, the AOAC Germicidal and Detergent Sanitizer test may be used (AOAC Official Method 960.09. Germicidal and Detergent Sanitizing Action of Disinfectants, AOAC Official Methods of Analysis, 995, Chapter 6, page 9, incorporated herein by reference in its entirety). As another example, Use Dilution tests may be used (AOAC Official Method 955.14. Testing Disinfectants Against Salmonella choleraesuis, Use- Dilution Method, 1998, Chapter 6, page 4, incorporated herein by reference in its entirety). As another example, the EN 1276 test may be used (British Standard BS EN 1276:1977. Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic, and institutional areas - test method and requirements, phase 2, stepl , incorporated herein by reference in its entirety).

[0033] In another aspect, the disclosure provides a method of reducing microbial levels and/or microbial activity on a surface or reducing airborne microbial levels and/or microbial activity, wherein the method comprises contacting the surface or the air with an effective amount of the composition described herein.

[0034] In another aspect, the disclosure provides a method of disinfecting a surface or air wherein the method comprises contacting the surface or the air with an effective amount of the composition described herein. [0035] In some embodiments, the methods can comprise contacting a surface such as, for example, the surface of a floor, counter, wall, or other hard surface. The surface can be a porous or non-porous organic or inorganic material. In some embodiments the surface can comprise materials such as, for example, plastics (e.g., polymers comprising

polyethylene, polypropylene, vinyl, etc.), ceramics, glass, stone, (e.g., limestone, marble, granite, quartz, onyx, etc.), terrazzo, linoleum, concrete, rubber, or combinations thereof. In some embodiments at least a portion of the surface can comprise a food contact surface. In other embodiments at least a portion of the surface can comprise a non-food contact surfaces. In some embodiments, the surface can comprise organic material such as, for example, water, food or foodstuffs (e.g., meat, dairy, fish, bread, cereal, fruit, vegetable, grain, flour, sugar, salt, spices, herbs, processed foods, etc.), plants, and animals, including skin, such as human and/or animal skin.

[0036] The compositions may be applied to a surface and exposed for a contact time that is sufficient to provide the desired level of biocidal activity. In some embodiments a surface may be contacted for a contact time of at least about 20 seconds, at least about 30 seconds, at least about 45 seconds, at least about 1 min, at least about 2 min, at least about 4 min, at least about 6 min, at least about 8 min, at least about 10 min, at least about 15 min, or at least about 20 min. Surfaces may be contacted for a contact time of less than about 20 min, less than about 15 min, less than about 10 min, less than about 8 min, less than about 6 min, less than about 4 min, less than about 2 min, less than about 1 min, less than about 45 seconds, or less than about 30 seconds. Surfaces may be contacted for a contact time of about 30 seconds. Surfaces may be contacted for a contact time of about 1 min.

[0037] In another aspect, the disclosure provides a composition comprising synergistic amounts of a biocide and a surfactant. In embodiments the biocide comprises peracetic acid. I some embodiments the surfactant comprises cocobetaine. Other embodiments relate to a method for preparing a synergistic composition. In some embodiments the biocide comprises peracetic acid and the surfactant comprises betaine. In such

embodiments, the peracetic acid and the cocobetaine may be present in a ratio of about 4:1 to about 8:1 (peracetic acid:cocobetaine). Suitably, the composition has a pH in a range of slightly acidic to about neurtral (e.g., about 4 to about 6). In some embodiments the addition of cocobetaine to the composition comprising peracetic can provide for a lesser amount (from about 20% to about 50% less) of peracetic acid in order to reduce microbial levels on a surface by at least 3 log units relative to the same peracetic composition in the absence of cocobetaine. [0038] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including but not limited to") unless otherwise noted. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to illustrate aspects and embodiments of the disclosure and does not limit the scope of the claims.

EXAMPLES

[0039] Reference Example 1. Use-Dilution (UD) Method

[0040] The Use-Dilution (UD) Method (AOAC Official Method 955.14) was used to test disinfectants against microorganisms, such as Salmonella choleraesuis, Staphylococcus aureus, and Pseudomonas aeruginosa.

[0041] Salmonella choleraesuis

[0042] Ring carriers were soaked overnight in 1 N NaOH, rinsed with tap water until rinse water was neutral to phenolphthalein, and then rinsed twice with distilled water. Clean ring carriers were placed in multiples of 10 in cotton-plugged Erlenmeyer flasks or 25 x 150 mm cotton plugged Pyrex test tubes, covered with asparagine solution, sterilized 20 min at 121°C, cooled, and held at room temperature. For the asparagine solution, a 0.1% solution in water was made in an Erlenmeyer flask of convenient size, plugged with cotton, and sterilized for 20 min at 121 °C. Nutrient broth test culture was vortex-mixed for 3-4 sec and stood for 10 min at room temperature before continuing. 20 sterile ring carriers were transferred using a flamed nichrome wire hook into 20 mL of 38-54 h nutrient broth test culture in sterile 25 x 15 mm medication tubes. One or two additional carriers were optionally added at the same inoculum rate to serve as reserves. Carriers that fell over in Petri dishes were not used in the test. After a 15 min contact period, the cylinders were removed using a flamed nichrome wire hook, the carrier was shaken vigorously against the side of the tube to remove excess culture, and the carrier was placed on end in the vertical position in a sterile Petri dish matted with two layers of S&S No. 597 or Whatman No. 2, 9 cm filter paper, making sure that the carriers did not touch to prevent improper drying. They were covered and placed in an incubator at 37°C and dried for 40 min. The broth culture was held for determination of its resistance to phenol by the phenol coefficient method. [0043] From 5% stock phenol solution, 1-90 and 1-100 dilutions were made directly into medication tubes. A tube for each dilution was placed in a 20°C water bath for 10 min. A stock solution of germicide to be tested was made in a sterile glass-stoppered cylinder. From this solution, 10 ml_ dilutions to be tested were made, depending upon phenol coefficient found and/or claimed against S. typhi at 20°C, directly into each of ten 25 x 150 mm medication tubes. The 10 tubes were placed in a 20°C water bath and allowed to come to temperature. A dilution of germicide to be tested was prepared by dilution in sterile water. The sterile water had been prepared by sterilizing for 20 min at 121 °C in flasks. Dilutions of sample were made using≥ 1.0 ml_ of sample. Volume/volume dilutions were used for liquid products and weight/volume dilutions were used for solids. Results were rounded to two decimal places toward a stronger product. To ensure stable product, a solution was prepared < 3 h prior to use. Tubes were placed in a 20°C water bath for at least 10 min. The dilution to be tested was determined by multiplying the phenol coefficient number found and/or claimed by 20 to determine the number of parts of water in which one part germicide was to be incorporated. This determination was not required when the disinfectant under test yielded a phenol coefficient that could not be converted validly to presumptive use- dilution, or when an analyst determined that a use-dilution range could be found without resort to the phenol coefficient test.

[0044] 0.5 mL of test culture suspension was added to 1-90 dilution of phenol control. After a 30 sec interval, 0.5 mL was added to 1-100 dilution of control, using sterile cotton- plugged pipettes. After adding culture, the tubes were agitated gently but thoroughly to distribute bacteria evenly, and replaced in the water bath. 5 min after seeding the first medication tube, one loopful of mixture of culture and diluted phenol was transferred from the medication tube to the corresponding subculture tube. After 30 sec, a loopful was transferred from the second medication tube. 5 min after making the first set of transfers, the second set of transfers for a 10 min period was begun. It was repeated for a 15 min period. The technique of loop sampling, flaming the loop and mouths of tubes, and agitating the medication and subculture tubes was used. The subcultures were incubated for 48 h at 37°C and results were recorded. Resistance in 48-54 h of a culture of S. choleraesuis should fall within a range period specified for a 24 h culture of S. tphi in the phenol coefficient method.

[0045] Without touching the sides of the tube with a contaminated carrier or hook, either when placing the carrier in a tube or when withdrawing the hook, one contaminated dried cylinder carrier was added at 1 min intervals to each of the 10 tubes of use-dilution of germicide to be tested. Thus, by the time 10 tubes had been seeded, 9 min had elapsed, plus a 1 min interval before transfer of the first carrier in a series to an individual tube of subculture broth. This interval was constant for each tube with a prescribed exposure period of 10 min. The 1 min intervaf between transfers allowed adequate time for flaming and cooling the nichrome wire hook and making the transfer in a manner so as to drain all excess medication from the carrier by shaking the carrier against the side of the tube.

Shorter intervals may have been used in adding and removing carriers if two alternately flamed and cooled hooks were used. Individual manipulation of carriers was required; use of semi-automated ring carrier was prohibited. The lips of the medication and subculture tubes were flamed in a conventional manner. Immediately after placing a carrier in a medication tube, the tube was swirled 3 times before placing it back in the water bath. The subculture tubes were thoroughly shaken and incubated for 48 h at 37°C, and the results were reported as + (growth) or - (no growth) values. Growth in at least 20% of the positive tubes was optionally checked by a gram stain to ensure that no contamination was present. AN positive results were confirmed by duplicate testing to assure against false positive tests.

[0046] Lack of growth at the conclusion of the incubation period may be due to bacteriostatic action of medicament adsorbed on the carrier that has not been neutralized by subculture medium used, and so each ring was optionally transferred to new tube of sterile medium and re-incubated for an additional 48 h at 37°C. Where the solution under test was such that material adsorbed on ring carriers and transferred into subculture medium made it unsuitable for growth of the test organism, as may be case with concentrated acids and alkalies, products carrying antibiotics, and wax emulsions, each ring was transferred to a new tube of sterile medium 30 min after the initial transfer and both primary and secondary subculture tubes were incubated for 48 h at 37°C. Results showing no growth on all 10 carriers confirmed the phenol coefficient number found. Results showing growth on any of the 10 carriers indicated the phenol coefficient number was an unsafe guide to dilution for use. In the latter case, the test was repeated using lower dilutions (higher concentrations) of germicide under study. The maximum dilution of germicide which killed the test organism on 10 carriers in a 10 min interval represented a presumed maximum safe use-di!ution for practical disinfection.

[0047] Staphylococcus aureus

[0048] The same procedure was used for Staphylococcus aureus, except the phenol dilutions and test organisms were as in AOAC Official Method 955.12. A 48-54 h culture of S. aureus FDA 209, ATCC No. 6538, having at least resistance specified for 24 h culture at 20°C in phenol coefficient method, was used. Prior to beginning the use-dilution test, a nutrient broth culture was vortex-mixed. Results showing growth on any of 10 carriers indicated that the dilution was too high for use in disinfecting where pyogenic bacteria must be killed. In such cases, the tests were repeated using lower dilutions (higher

concentrations). The maximum dilution of germicide which killed both this test organism and S. choleraesuis on 10 carriers in a 10 min interval represented the maximum presumed safe use-dilution for disinfecting in hospitals, clinics, and other places where pyogenic bacteria have special significance, it is noted that while killing in 10 of 10 replicates specified provided a reasonably reliable index in most cases, killing in 50 of 60 replicates provided a confidence level of 95%.

[0049] Pseudomonas aeruginosa

[0050] The same procedure was used for Pseudomonas aeruginosa. A 48-54 h nutrient broth culture of P. Aeruginosa PRD 10 (ATCC 15442) was used. A stock culture was carried on BBL CTA (cysteine trypticase agar) in stab culture incubated 48 h at 37°C and stored at 5°C with transfer every 30 days. Nutrient broth cultures were transferred daily for 3-day intervals with incubation at 37°C. A fresh transfer from stock culture was made every 30 days. The 48-54 h test culture was not shaken. The liquid culture was decanted aseptically, leaving pellicle behind, to obtain 20 mL culture for inoculating 20 carriers in medication tubes. Vortex-mixing was done as described above prior to use of culture. Alternatively, the pellicle may be carefully suctioned off, and culture can be poured into a clean, sterile tube before vortex-mixing. Any disruption of pellicle resulting in dropping, breaking up, or stringing of pellicle in culture before or during its removal rendered that culture unusable in use-dilution test. Any pellicle fragments remaining may have resulted in uneven clumping and layering of the organism on the cylinder, allowing unfair exposure to disinfectant and causing false positive results.

[0051] Example 1. Preliminary disinfectant testing.

[0052] Use Dilution (UD) tests, as described in Reference Example 1 , were conducted with various combinations of surfactants and biocides. Biocides and surfactants were mixed in a beaker. The biocides and the surfactants tested are shown in Table 1. Table 1. Samples examined in UD screening tests.

All percents are of the active, and not as the supplied solution.

[0053] A surfactant blend and a biocide were mixed together. The surfactant blend listed in Table 1 was diluted 1 :256 in 400 ppm hard water and biocide to yield the final biocide concentrations listed in Table 1. In this series of UD tests only 10 carriers were used. Although the test normally calls for 60 carriers to be used in each test, ten carriers are frequently used for screening of multiple samples. This testing was conducted against Pseudomonas aeruginosa ATCC 15442. Samples were tested at 1 min or 10 min contact times. Results are summarized in Figure 1 (10 min contact time). The tests were repeated for solutions comprising 40 ppm PAA and various surfactant blends with a 1 min contact time, and results are presented in Figure 2. [0054] The tests indicated that surfactant blend 1 and 2 were able to increase the performance of PAA. That is, a disinfectant would be expected to kill all the test organisms on all 10 carriers tested, and 200-250 ppm of PAA would be expected to be required to disinfect with a 10 in exposure time. However, it was observed that surfactant blends 1 and 2 met or very nearly met that standard with only 40 ppm PAA. The 5 fold reduction in PAA required to achieve a high level of efficacy suggested that the surfactants tested could be used to synergize the performance of PAA.

[0055] Example 2. Additional disinfectant testing.

[0056] As shown in Example 1 , the solutions with the greatest biocidal activity contained cocobetaine, PAA, and GLDA. Additional tests were conducted to further investigate the potential of cocobetaine surfactant (and GLDA) to synergize the biocida! efficacy of PAA against P. aeruginosa and Staphylococcus aureus in UD screening tests each employing 10 carriers. A full factorial design was used and the data were analyzed with the JMP statistical analysis software. Table 2 describes the full factorial design used in these experiments and provides the results of that testing. The exposure time used was reduced to 5 min, to ensure that the PAA would not have sufficient activity on its own to kill the test organisms. All samples were prepared in 400 ppm hard water. These data were fit to a least squares model and results of that analysis are presented in Table 3 and Table 4.

Table 2. Experimental design and results of UD screening test against P. aeruginosa and S. aureus.

Sample PAA Cocobetaine GLDA # of P. aeruginosa # of S. aureus # (ppm) (ppm) (ppm) carriers with no carriers with no growth growth

1 0 0 0 0 0

2 25 200 50 0 0

3 0 0 0 0 0

4 25 200 0 0 10

5 50 0 0 3 8

6 25 0 50 0 0

7 25 200 0 1 10

8 25 100 25 0 0 Sample PAA Cocobetaine GLDA # of P. aeruginosa # of S, aureus

# (ppm) (ppm) (ppm) carriers with no carriers with no growth growth

9 50 200 50 5 0

10 0 0 50 0 0

11 0 200 0 0 0

12 0 200 50 0 0

13 25 200 50 0 0

14 0 200 50 0 0

15 50 0 50 7 9

16 25 0 0 0 6

17 50 0 0 6 9

18 50 200 0 7 9

19 0 200 0 0 0

20 25 0 50 0 0

21 50 200 50 1 0

22 0 0 50 0 0

23 50 200 0 8 9

24 25 0 0 0 9

25 25 100 25 0 0

26 50 0 50 2 0

Table 3. Analysis of results against P. aeruginosa presented in Table 2.

Table 4. Analysis of results against S. aureus presented in Table 2.

[0057] As shown in Table 2, the parameter that the most significant effect on the number of negative carriers that had been inoculated with P. aeruginosa was the level of PAA.

Neither the cocobetaine nor the GLDA had a significant effect. Similarly, the S. aureus data (Table 4) indicated that the number of carriers having no survivors was strongly influenced by the level of PAA. Unlike P. aeruginosa, however, the levei of GLDA had a significant effect on the biocidal activity against S. aureus. In this case, the GLDA appeared to strongly inhibit the biocidal performance of the PAA against S. aureus. [0058] Example s. EPA sanitizer tests.

[0059] A series of tests were undertaken using the quantitative method that is employed for non-halogen based food contact surface sanitizers in the U.S. and examining a similar set of samples. The sanitizer testing was conducted using the AOAC Germicidal and

Detergent Sanitizer test. That test is the one that must be used to demonstrate the

performance of non-halogen based sanitizers registered in the U.S. This test method is different from the tests presented in Examples 1 and 2, with the primary difference being that the organisms are not on carriers in this test. Rather, they are suspended in a solution of biocide and the number of surviving organisms is determined after a specific exposure time. Tests in which the organisms are suspended in a solution of biocide are collectively referred to as suspension tests. The exposure time in this test was 30 seconds (the U.S. EPA

performance standard requires a 5 log reduction in 30 seconds). All dilutions were

conducted using 400 ppm hard water. Test organisms were P. aeruginosa, S. aureus, and Salmonella choleraesuis. Table 5 describes the samples tested and the reduction in log

CFU/mL vs. organisms exposed to just water.

Table 5. Results of germicidal and detergent sanitizer testing.

Sam le PAA Cocobetaine GLDA Log reduction Log Log reduction # (ppm) (ppm) (ppm) P. aeruginosa reduction S,

aureus choleraesuis

1 25 0 0 0.13 -0.15 2.78

2 25 0 25 0.34 -0.01 1.47

3 25 0 50 1.4 -0.15 1.15

4 25 100 0 -2.28 -0.53 1.29

5 25 100 25 0.23 0 0.72

6 25 100 50 0.33 0.07 0.18

7 25 200 0 0.91 0.11 1 .58

8 25 200 25 -0.35 0.09 0.65 g 25 200 50 -0.36 -0.03 0.05

10 50 0 0 5.91 0.66 3.46

11 50 0 25 5.91 0.62 3.22

12 50 0 50 0.73 0.53 1.61 Sample PAA Cocobetaine GLDA Log reduction Log Log reduction

# (ppm) (PPm) (PPm) P. aeruginosa reduction S. S.

aureus choleraesuis

13 50 100 0 5.91 1.33 1.9

14 50 100 25 0.84 0.52 1.75

15 50 100 50 0.44 0.53 0.52

16 50 200 0 5.91 1.69 2.17

17 50 200 25 1.42 0.91 2.4

18 50 200 50 0.54 0.78 0.73

1 25 0 0 0.08 0.25 0.49

2 25 0 25 0.27 0.44 0.81

3 25 0 50 0.31 0.37 1.51

4 25 100 0 0.48 0.53 1.56

5 25 100 25 0.21 0.45 1.43

6 25 100 50 -0.06 0.36 -0.01

7 25 200 0 0.57 0.85 2.77

8 25 200 25 0.16 0.85 1.61

9 25 200 50 0.1 0.57 1.36

10 50 0 0 0.1 3.46

11 50 0 25 0.43 0.52 2.72

12 50 0 50 0.22 0.49 3.46

13 50 100 0 0.49 0.71 5.76

14 50 100 25 0.26 0.56 2.86

15 50 00 50 0.09 0.55 0.69

16 50 200 0 0.4 1.07 3.28

17 50 200 25 0.22 1.1 2.7

18 50 200 50 -0.02 0.89 1.04 [0060] The samples without PAA were not tested because preliminary testing (data not shown) indicated that the GLDA and cocobetaine alone had no biocidal activity. Each condition was tested twice. The data was analyzed as described in Example 2. The results of the tests and the analyses are presented in Table 6, Table 7, and Table 8.

Table 6. Analysis of results reported in Table 5 against S. choleraesuis.

Table 7. Analysis of results reported in Table 5 against P. aeruginosa.

Term Estimate Std Error t Ratio Prob>jt|

Intercept 3.3596296 0.323065 10.40 < 0001*

PAA level(25,50) 1.4475926 0.161533 8.96 < 0001*

Cocobetaine level -0.004358 0.001978 -2.20 0.0357^*

GLDA level -0.052144 0.007913 -6.59 < 0001^*

PAA level*(Cocobetaine level-100) -0.003442 0.001978 -1.74 0.0925

PAA level*(GLDA level-25) -0.054656 0.007913 -6.91 < 0001^*

(Cocobetaine level-100)*(GLDA level- -9.925e-5 0.000084 -1.18 0.2466 25) Table 8. Analysis of results reported in Table 5 against S. aureus.

[0061] PAA affected the biocidal activity of the solutions. Specifically, more PAA in the sample resulted in a greater microbial reduction. The presence of a biocide significantly further enhanced the microbial reduction. Unexpectedly, cocobetaine had a significant effect for P. aeruginosa for which it reduced the biocidal performance of samples in which it was used. The cocobetaine level was also significant for the S. aureus for which it increased the biocidal performance. The GLDA was also significant for both S. choleraesuis and P.

aeruginosa. For both of those organisms the presence of GLDA greatly reduced biocidal performance. In the case of S. aureus the GLDA also reduced the biocidal performance of test samples although in the case of that organism the reduction was not statistically significant.

[0062] Additionally, it was observed that although the use of cocobetaine and GLDA did impact the biocidal performance of the PAA, none of the samples had particularly high biocidal activity. Presented in Figure 3, Figure 4, and Figure 5 is the mean log reduction of the two levels of PAA tested against S. choleraesuis, P. aeruginosa, and S. aureus, respectively.

[0063] Note that for the graphs shown in Figure 3, Figure 4, and Figure 5, the log reduction at each PAA level was a mean of the log reductions observed with all of the other test conditions, i.e., the averages used include data from tests using various levels of chelant and surfactant. In order for a sanitizer to be approved in the U.S., the biocide must achieve a 5 log reduction in the test organisms. Even at 50 ppm PAA, a 5 log reduction was not reliably achieved against any of the test organisms. However, 80 ppm PAA alone would be expected to achieve a 5 log reduction.

[0064] Example 4. European disinfection tests.

[0065] Additional testing was conducted using the European EN 1276 disinfectant efficacy test. This method is a suspension test similar to the Germicidal and Detergent Sanitizer test but provides quantitative data and uses a 5 min exposure time.

[0066] Various combinations of PAA, cocobetaine, coco amidopropyl betaine, octanoic acid, and a nonionic blend (as described above in Example 1) were evaluated for their efficacy against P. aeruginosa, S. aureus, and Escherichia coli in the EN 1276 test under clean and soiled conditions. The samples tested are listed in Table 9, and the results are shown in Table 10.

Table 9. Samples examined in the initial round of EN 1275 tests.

Sample # Formulation

1 3300 ppm cocobetaine in 400 ppm hard water

2 3300 ppm cocobetaine; 20 ppm PAA in 400 ppm hard water

1344 ppm cocobetaine; 40 ppm PAA; 300 ppm GLDA; in 400 ppm

3

hard water

3300 ppm cocobetaine; 0.8 g/L nonionic blend; 20 ppm PAA; 60 ppm

4

octanoic acid; in 400 ppm hard water

3300 ppm cocobetaine; 0.8 g/L nonionic blend; 20 ppm PAA; in 400

5

ppm hard water

3300 ppm cocobetaine; 20 ppm PAA; 60 ppm octanoic acid; in 400

6

ppm hard water

4630 ppm coco amidopropyl betaine; 0.8 g/L nonionic blend; 20 ppm

7

PAA; 60 ppm octanoic acid; in 400 ppm hard water

4630 ppm coco amidopropyl betaine; 0.8 g/L nonionic blend; 20 ppm

8

PAA; in 400 ppm hard water

4630 ppm coco amidopropyl betaine; 20 ppm PAA; 60 ppm octanoic

9

acid in 400 ppm hard water Table 10. Results of EN1276 testing of samples listed in Table 9.

[0067] Samples 7 through 9 contained the coco amidopropyl betaine but did not show significant biocidal activity, which suggested that not all betaines would function in this system. Further, pH may have a significant effect on this system.

[0068] The effects of low pH on biocidal activity were examined using various acids with the EN 276 method, and the results are shown in Table 11. The exposure time was 5 min, and all testing was done under low soil conditions. Each sample was tested in triplicate.

Table 11. Experimental design and results of the effects of pH and acid types on biocidal efficacy.

Acid PAA Coco pH Log reduction Log reduction pH level betaine level target S. aureus. P. aeruginosa Actual (PPWI) (PPM)

Citric 0 0 4 -0.03 0.15 5.14/p.a

4.15/s.a

Citric 0 0 4 0.08 -0.08

Citric 0 0 4 0 0.03

Citric 0 126 4 0.81 -0.02 5.1/p.a Acid PAA Coco pH Log reduction Log reduction pH level betaine level target S. aureus. P. aeruginosa Actual (PPM) (PPM)

Citric 4.23/s.a

0 126 4 0.95 -0.02

Citric 0 126 4 0.95 0.02

Citric 40 0 4 2 4.17 5.18/p.a

4.26/s.a

Citric 40 0 4 0.91 4.09

Citric 40 0 4 1.01 2.98

Citric 40 126 4 2.58 4.85 5.17/p.a

Citric 40 126 4 2.77 4.20/s.a

5.07

Citric 40 126 4 2.71 4.64

Nitric 0 0 5 1.7 0.69 4.90

Nitric 0 0 5 1.66 0.06

Nitric 0 0 5 1.59 0

Nitric 0 126 5 1.59 1 4.90

Nitric 0 126 5 1.51 1

Nitric 0 126 5 1.6 1

Nitric 40 0 5 1.54 4.19 5.24

Nitric 40 0 5 1.17 5.25

Nitric 40 0 5 1.27 5.83

Nitric 40 126 5 4.5 4.71 5.01

Nitric 40 126 5 4.06 5.42

Nitric 40 126 5 3.09 6.18

Phosphoric 0 0 5 0.14 0.02 4.9

Phosphoric 0 0 5 -0.13 0.12

Phosphoric 0 0 5 0.16 0.39

Phosphoric 0 126 5 1.6 0.46 4.69

Phosphoric 0 126 5 1.51 0.33 Acid PAA Coco pH Log reduction Log reduction pH level betaine level target S. aureus. P. aeruginosa Actual (PPM) (PPM)

Phosphoric 0 126 5 1.64 0.47

Phosphoric 40 0 5 0.57 5.2 5.28

Phosphoric 40 0 5 0.8 5.09

Phosphoric 40 0 5 0.78 5.2

Phosphoric 40 126 5 4.93 5.42 5.12

Phosphoric 40 126 5 3.87 4.26

Phosphoric 40 126 5 3.23 5.8

Sulfuric 0 0 5 0.16 0.03 5.00

Sulfuric 0 0 5 0.11 0.03

Sulfuric 0 0 5 0.18 0.06

Sulfuric 0 126 5 1.52 0.09 5.28

Sulfuric 0 126 5 1.4 0.16

Sulfuric 0 126 5 1.27 0.01

Sulfuric 40 0 5 1.17 3.46 5.00

Sulfuric 40 0 5 1.13 3.76

Sulfuric 40 0 5 1.22 3.71

Sulfuric 40 126 5 5.38 4.71 5.10

Sulfuric 40 126 5 6.08 4.58

Sulfuric 40 126 5 3.25 4.83

None 0 0 7 0.25 0.32 7.23

None 0 0 7 0.19 0.27

None 0 0 7 0.24 0.42

None 0 126 7 0.93 0.3 7.0

None 0 126 7 0.64 0.5

None 0 126 7 0.79 0.32 Acid PAA Coco pH Log reduction Log reduction pH level betaine level target S. aureus. P. aeruginosa Actual {PPM) (PPM)

None 40 0 7 0.8 2.72 6.99

None 40 0 7 1.06 2.68

None 40 0 7 0.72 2.59

None 40 126 7 2.46 3.11 7.10

None 40 126 7 3.27 4.18

None 40 126 7 2.77 4.54

[0069] The data from Table 11 were analyzed using JMP. The addition of acid was found to be a significant contributor to biocidal activity against both microorganisms tested. The analysis results are shown in Table 12 and Table 13 for S. aureus and P. aeruginosa, respectively.

Table 12. Analyses of results against S. aureus according to EN 1276 testing, as reported in Table 11.

Source Nparm DF Sum of Squares F Ratio Prob > F

Acid 4 4 8.011740 8.3541 <.0001*

PAA level (mUL)(0,0.769) 1 1 35.404802 147.6714 <.0001*

Cocobetaine level (mlJL)(0,0.3) 1 1 43.707735 182.3025 <.0001*

Acid*PAA level (mlJL) 4 4 2.582373 2.6927 0.0431^*

Acid*Cocobetaine level (mL/L) 4 4 5.546673 5.7837 0.0008*

PAA level (mL/L)*Cocobetaine 1 1 11.607202 48.4130 <.ooor level (mLJL) Table 13. Analyses of results against P. aeruginosa according to EN 1276 testing, as reported in Table 11.

[0070] According to the results, acid, PAA, and cocobetaine all had significant effects on the log reduction of the test organisms. The effect of cocobetaine was much greater on the biocidal activity against S. aureus than against P aeruginosa. This finding was supported by the data presented in Table 10 wherein Sample 1 that contained cocobetaine and water demonstrated greater biocidal activity against S. aureus than P. aeruginosa. The results are further presented in Figure 6 as the mean log reduction of S. aureus and P. aeruginosa with different levels of cocobetaine according to the EN 1276 test. Further, cocobetaine had an even greater effect on the biocidal activity against S. aureus than PAA did.

[0071] The overall reductions were relatively low. For example, the 0.8-2.5 log reductions against S. aureus seen in these tests with the much higher reductions for some test conditions reported in Table 10, notably for Samples 1-6. This is particularly striking in that the PAA levels used in the studies reported in Table 10 were half that used in the studies reported in Table 11 (20 vs. 40 ppm). Despite this difference the efficacy at the lower concentration of PAA was often more than double what was seen at the higher level used in the testing reported in Table 11. One notable difference is the relative levels of cocobetaine used in the testing. The cocobetaine to PAA molar ratio was 10:1 in the tests reported in Table 0; however the molar ratio was 1 :1 in the tests reported in Table 11. This difference may explain the disparity in log reductions observed in these experiments. [0072] Example 5. The effect of different molar ratios of betaine to PAA on efficacy.

[0073] The effect of different molar ratios of betaine to PAA on efficacy was examined. The pH was adjusted to approximately 5.0 using phosphoric acid for all samples. The PAA level was 40 ppm. Tests were performed in triplicate using the EN 1276 method under clean conditions. All dilutions were prepared in 400 ppm hard water, and the exposure time was 5 min. The solutions were only examined on S. aureus and P. aeruginosa for efficacy. All tests were conducted in triplicate. The results are shown in Table 14.

Table 14. Results of EN 1276 test examining the effect of various cocobetai ne: PAA molar ratios on biocidal performance.

Moles of Betaine per mole Log Reduction Log reduction

of PAA S. aureus P. aeruginosa

10 4.98 5.29

10 4.53 5.5

10 5.27 5.28

8 5.27 5.87

8 5.12 5.04

8 4.09 5.43

6 5.29 6.61

6 4.89 5.66

3 4.4 6.13

3 5.16 6.61

3 4.59 6.61

1 4.56 6.38

1 3.76 6.61

1 4.56 6.43 [0074] These data were analyzed using JMP and a polynomial (quadratic) was fit to the data. The results of the analysis are shown in Figure 7. The molar ratio had a significant effect on the biocidal activity on both organisms. The optimum ratio of betaine to PAA was about 6:1 for biocidal activity against S. aureus. However if the betaine to PAA molar ratio was too high, it had an inhibitory effect on efficacy against P. aeruginosa. However, a 6:1 molar ratio of betaine to PAA was still effective in reducing P. aeruginosa by over the 5 log, which met the biocidal efficacy standard for the EN 1276 test.

[0075] Example 6. Formula optimization.

[0076] Based on the results presented in the Examples above, the formulation with optima biocidal activity comprised approximately 40 ppm PAA and cocobetaine at approximately a 6:1 molar ratio, with a pH of approximately 5.0.

[0077] Several acids were further examined in the formulation, including those shown in Table 11 and methyl sulfonic acid. The choice of acid did not appear to have a significant effect on biocidal activity.

[0078] It was examined whether the optimum formulation, as stated above, had sufficient biocidal efficacy against the other organisms, including Escherichia coli and Enterobacter hirae, that are required to pass the EN 1276 test. To pass the EN 1276 test, the biocidal efficacy of the formulation against all four organisms also had to be determined under high soil conditions, whereas most of the testing described above was conducted under low soil conditions. As such, the biocidal activity of various formulations was examined against E. coli and E. hirae under high soil conditions. It was found that a formulation comprising a 6:1 molar ratio of PAA to cocobetaine with a PAA level of 40-45 ppm and a solution pH of 5.0 would pass the EN 1276 test and meet the efficacy standard against all four organisms under light and heavy soil conditions, and the results of that final testing are presented in Table 15. All testing was conducted in 400 ppm hard water.

Table 15. Results of EN 1276 testing of optimized formulation and PAA alone.

ND: Not Determined.

[0079] Thus, the compositions and methods disclosed herein are effective for reducing microbial levels on a surface.

Claims

CLAIMS What is claimed is:

1. A composition comprising:

a) a biocide; and

b) a surfactant comprising a zwitterion;

wherein the biocide and the zwitterion are present in the composition at a ratio of about 4:1 to about 8: 1 , and wherein the composition reduces microbial levels or microbial activity on a surface.

2. The composition according to claim 1 , wherein the zwitterion comprises a betaine.

3. The composition according to claim 2, wherein the betaine comprises coco betaine.

4. The composition according to any one of the previous claims, wherein the biocide comprises at least one of quaternary ammonium chloride, hydrogen peroxide, peracetic acid, and a combination thereof.

5. The composition according to claim 4, wherein the biocide comprises peracetic acid.

6. The composition according to any one of the previous claims, further comprising a chelating agent.

7. The composition according to claim 6, wherein the chelating agent comprises at least one of L-glutamate-N-N-diacetic acid (GLDA), ethylenediaminetetraacetic acid (EDTA), methyl glycine diacetic acid (MGDA), hydroxy iminodiacetic acid (HI DA), nitrilotriacetic acid (NTA), citrate, and a combination thereof.

8. The composition according to claim 7, wherein the chelating agent comprises GLDA.

9. The composition according to any one of the previous claims, wherein the biocide is present in the composition in an amount of about 1 ppm to about 10,000 ppm.

10. The composition according to any one of the previous claims, wherein the biocide is present in the composition in an amount of about 30 ppm to about 2000 ppm.

1 1. The composition according to any one of the previous claims, wherein the composition has a pH of about 1 to about 12.

12. The composition according to claim 11 , wherein the composition has a pH of about 4 to about 6.

13. The composition according to claim 12, wherein the composition has a pH of about 4.5 to about 5.5.

14. The composition according to any one of the previous claims, wherein the

composition reduces the microbial levels on the surface by at least about 3 log units.

15. A composition comprising:

a) a biocide; and

b) a surfactant comprising a zwitterion;

wherein the composition is substantially free of an alkyl amine, and wherein the composition reduces microbial levels or microbial activity on a surface.

16. The composition according to claim 15, wherein the zwitterion comprises a betaine.

17. The composition according to claim 16, wherein the betaine comprises cocobetaine.

18. The composition according to any one of claims 15-17, wherein the biocide and the zwitterion are present in the composition at a ratio of about 4:1 to about 8:1.

19. The composition according to any one of claims 15-18, wherein the biocide comprises at least one of quaternary ammonium chloride, hydrogen peroxide, peracetic acid, and a combination thereof.

20. The composition according to claim 19, wherein the biocide comprises peracetic acid.

21. The composition according to any one of claims 15-20, further comprising a chelating agent.

22. The composition according to claim 21 , wherein the chelating agent comprises at least one of L-glutamaie-N-N-diacetic acid (GLDA), ethylenediaminetetraacetic acid (EDTA), methyl glycine diacetic acid (MGDA), hydroxy iminodiacetic acid (HI DA), nitrilotriacetic acid (NTA), citrate, and a combination thereof.

23. The composition according to claim 22, wherein the chelating agent comprises GLDA.

24. The composition according to any one of claims 15-23, wherein the biocide is present in the composition in an amount of about 1 ppm to about 10,000 ppm.

25. The composition according to claim 24, wherein the biocide is present in the composition in an amount of about 30 ppm to about 2000 ppm

26. The composition according to any one of claims 15-25, wherein the composition has a pH of about 1 to about 12.

27. The composition according to claim 26, wherein the composition has a pH of about 4 to about 6.

28. The composition according to claim 27, wherein the composition has a pH of about 4.5 to about 5.5.

29. The composition according to any one of claims 15-28, wherein the composition reduces the microbial levels on the surface by at least about 3 log units.

30. A composition comprising:

a) about 30 to about 2000 ppm peracetic acid; and

b) a surfactant comprising a zwitterion;

wherein

the composition is substantially free of an a Iky I amine;

the peracetic acid and the zwitterion are present in the composition at a ratio of about

4:1 to about 8:1 ; and

the composition reduces microbial levels or microbial activity on a surface.

31. The composition of claim 30, wherein the zwitterion comprises a betaine.

32. The composition of claim 31 , wherein the betaine comprises cocobetaine.

33. The composition according to any one of claims 30-32, further comprising a chelating agent.

34 The composition according to claim 33, wherein the chelating agent comprises at least one of L-glutamate-N-N-diacetic acid (GLDA), ethylenediaminetetraacetic acid (EDTA), methyl glycine diacetic acid (MGDA), hydroxy iminodiacetic acid (HI DA), nitrilotriacetic acid (NTA), citrate, and a combination thereof.

35. The composition according to claim 34, wherein the chelating agent comprises GLDA.

36. The composition according to any one of claims 30-35, wherein the composition comprises about 35 to about 45 ppm peracetic acid.

37. The composition according to any one of claims 30-35, wherein the composition comprises about 1000 to about 2000 ppm peracetic acid.

38. The composition according to any one of claims 30-37, wherein the composition has a pH of about 1 to about 12.

39. The composition according to claim 38, wherein the composition has a pH of about 4 to about 6.

40. The composition according to claim 39, wherein the composition has a pH of about 4.5 to about 5.5.

41. The composition according to any one of claims 30-40, wherein the composition reduces the microbial levels on the surface by at least about 3 log units.

42. A synergistic biocidal composition comprising cocobetaine and a biocide in a ratio of about 4: 1 to about 8:1 (cocobetaine:biocide) and in an amount effective to reduce the amount of biocide required for biocidal activity by about 20% to about 90% relative to the biocidal composition in the absence the cocobetaine.

43. The synergistic biocidal composition according to claim 42, wherein the cocobetaine reduces the amount of biocide required by at least about 20% to about 50% relative to the same composition without the cocobetaine, and reduces microbial levels on the surface by at least 3 log units.

44. The synergistic biocidal composition according to claim 42 or 43, wherein the biocide comprises at least one of quaternary ammonium chloride, hydrogen peroxide, peracetic acid, and a combination thereof.

45. The synergistic biocidal composition according to claim 44, wherein the biocide comprises peracetic acid.

46. The synergistic biocidal composition according to any one of claims 42-45, further comprising a chelating agent.

47. The synergistic biocidal composition according to claim 46, wherein the chelating agent comprises at least one of L-glutamate-N-N-diacetic acid (GLDA),

ethylenediaminetetraacetic acid (EDTA), methyl glycine diacetic acid (MGDA). hydroxy iminodiacetic acid (HI DA), nitrilotriacetic acid (NTA), citrate, and a combination thereof.

48. The synergistic biocidal composition according to claim 47, wherein the chelating agent comprises GLDA.

49. The synergistic biocidal composition according to any one of claims 42-48, wherein the composition has a pH of about 4.5 to about 5.5.

50. A method of reducing microbial levels on a surface or in air comprising applying to the surface or to the air the composition according to any one of claims 1-49.

51. A method of disinfecting a surface or air comprising applying to the surface or air the composition according to any one of claims 1-49.