USOO6818212B2 (12) United States Patent (10) Patent No.: US 6,818,212 B2 Johansen (45) Date of Patent: Nov. 16, 2004 (54) ANTIMICROBIAL COMPOSITION (58) Field of Search .................... 422/28, 29; 424/94.1, CONTAINING A HALOPEROXIDASE, A 424/94.2, 401; 435/189, 264, 190, 192, HYDROGEN PEROXIDE SOURCE, A 263; 8/107 HALIDE SOURCE AND ANAMMONUM SOURCE (56) References Cited (75) Inventor: Charlotte Johansen, Holte (DK) U.S. PATENT DOCUMENTS 3,539,520 A 11/1970 Cantor ....................... 252/106 (73) Assignee: Novozymes A/S, Bagsvaerd (DK) 5,227,161 A 7/1993 Kessler 5,389,369 A 2/1995 Allen ........................ 424/94.4 (*) Notice: Subject to any disclaimer, the term of this 5,928,380 A * 7/1999 Winkler ......................... 8/107 patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days. FOREIGN PATENT DOCUMENTS EP O SOO 387 8/1992 WO WO 94/04127 3/1994 (21) Appl. No.: 09/768,803 WO WO 94/05252 3/1994 (22) Filed: Jan. 24, 2001 WO WO95/27046 10/1995 WO WO 96/38548 12/1996 (65) Prior Publication Data WO WO 97/42825 11/1997 US 2001/0009664 A1 Jul. 26, 2001 * cited by examiner Related U.S. Application Data Primary Examiner Irene Marx (74) Attorney, Agent, or Firm-Elias J. Lambiris (63) Continuation of application No. 09/133,149, filed on Aug. 12, 1998. (57) ABSTRACT (60) Provisional application No. 60/056,622, filed on Aug. 20, An enzymatic antimicrobial composition comprising a 1997. haloperoxidase, a hydrogen peroxide Source, a halide (30) Foreign Application Priority Data Source, and an ammonium Source, in particular an ammo nium Salt or an aminoalcohol, in which there is a hitherto Aug. 14, 1997 (DK) .............................................. O940/97 unknown Synergistic effect between the halide and the (51) Int. Cl. ................................................ A61K 38/43 ammonium Source. (52) U.S. Cl. ..................... 424/94.4; 424/94.1; 424/401; 422/28; 422/29; 435/189; 435/264; 435/192 12 Claims, 5 Drawing Sheets U.S. Patent Nov. 16, 2004 Sheet 1 of 5 US 6,818,212 B2 Figure 1 log cfulmi = -0.25"DT + 10.10 t r’=0.99 234.5678 O 5 10 15 20 25 30 Detection time (h) US 6,818,212 B2 Figure 2 24/722 U.S. Patent Nov. 16, 2004 Sheet 3 of 5 US 6,818,212 B2 Figure 3 100 s 80 e is 60 U N "O 's 40 " SB - Kland (NH)SO, A 20 -- Kl U.S. Patent Nov. 16, 2004 Sheet 4 of 5 US 6,818,212 B2 Figure 4 100 80 60 - KBrand (NH),so -- KBr 40 U.S. Patent Nov. 16, 2004 Sheet 5 of 5 US 6,818,212 B2 Fig. 5 No enzyme rCVP + CVP -- CVP + Chloride ethanolamine chloride + ethanolamine US 6,818,212 B2 1 2 ANTMICROBAL COMPOSITION example, for the preservation of food, beverages, cosmetics, CONTAINING A HALOPEROXIDASE, A deodorants, contact lens products, food ingredients or HYDROGEN PEROXIDE SOURCE, A enzyme compositions, as a disinfectant for use, e.g., on HALIDE SOURCE AND ANAMMONUM human or animal skin, hair, oral cavity, mucous membranes, SOURCE wounds, bruises or in the eye; for killing microbial cells in laundry; and for incorporation in cleaning compositions or CROSS-REFERENCE TO RELATED disinfectants for hard Surface cleaning, for water treatment, APPLICATIONS or for disinfection. This application is a continuation of U.S. application Ser. Accordingly, in further aspects, the present invention No. 09/133,149 filed Aug. 12, 1998 and claims priority of provides a method of inhibiting microorganisms present in Danish application no. 0940/97 filed Aug. 14, 1997 and U.S. laundry, wherein the laundry is treated with a Soaking, provisional application No. 60/056,622 filed Aug. 20, 1997, Washing or rinsing liquor comprising this composition; a the contents of which are fully incorporated herein by method of inhibiting microbial growth on a hard Surface, reference. wherein the Surface is contacted with this composition; a 15 method of inhibiting microbial cells present in industrial BACKGROUND OF THE INVENTION water lines, and a method of killing microbial cells present 1. Field of the Invention on human or animal skin, mucous membranes, teeth, The present invention relates to a very effective enzymatic wounds, bruises or in the eye or inhibiting the growth composition capable of killing or inhibiting microbial cells thereof, wherein the cells to be killed or inhibited on the or microorganisms, more specifically microbial cells or skin, mucous membrane, teeth, wound or bruise are/is microorganisms present in laundry, on hard Surfaces, in contacted with this composition. water Systems, on Skin, teeth or mucous membranes, and for BRIEF DESCRIPTION OF DRAWINGS preserving food products, cosmetics, paints, coatings, etc. The present invention is further illustrated by reference to 2. Description of Related Art 25 the accompanying drawings, in which Various enzymatic antimicrobial compositions are known in the art. For instance, WO 94/04127 discloses stabilized FIG. 1 shows a calibration curve for growth of Pseudomo dentifrice compositions which are capable of producing nas aeruginosa ATCC 10146 in TSB at 25 C.; see Example antimicrobially effective concentrations of hypothiocyanite 1. ions. The compositions contain an oxidoreductase capable FIG. 2 shows a response surface plot for the antibacterial of producing hydrogen peroxide and a peroxidase enzyme activity against StaphylococcuS epidermidis; when Sodium capable of oxidizing thiocyanate ions, which are normally chloride and/or ammonium are added to the reaction present in Saliva, to antimicrobial hypothiocyanite ions. medium; See Example 1. Suitable peroxidase S include lactop eroxidase, FIG. 3 shows the bactericidal activity of the Curvularia myeloperoxidase, Salivary peroxidase and chloroperoxidase. 35 haloperOxidase combined with iodide and ammonium Sul In EP-A-0 500 387 enzymatic antimicrobial compositions phate ( : KI and (NH4)2SO4; - - - : KI); see Example are disclosed comprising a haloperoxidase, e.g., myelo 2. peroxidase, eosinophil oxidase, lactoperoxidase and FIG. 4 shows the bactericidal activity of the Curvularia chloroperoxidase, which selectively binds to and inhibits the haloperOxidase combined with bromide and ammonium growth of target microorganisms in the presence of peroxide 40 Sulphate; ( : KBr and (NH4)2SO4; - - - : KBr); see and halide. Example 2. WO 95/27046 discloses an antimicrobial composition FIG. 5 shows the bactericidal activity of the Curvularia comprising a Vanadium chloroperoxidase, halide ions, and haloperoxidase (rCvP) combined with chloride and ethano hydrogen peroxide or a hydrogen peroxide-generating agent. lamine; See Example 4. 45 WO 96/38548 discloses an antimicrobial composition DETAILED DESCRIPTION OF THE comprising a haloperoxidase, a halide ion, a peroxide gen INVENTION erating agent and an amino acid type. The object of the present invention is to provide a In the context of the present invention the term “antimi composition for killing or inhibiting microbial cells, i.e., for crobial' is intended to mean that there is a bactericidal disinfection or preservation, which is easy to use and an 50 and/or a bacterioStatic and/or fungicidal and/or fungistatic effective alternative to the known disinfecting and preserv effect and/or a virucidal effect, wherein ing compositions and methods. The term “bactericidal' is to be understood as capable of killing bacterial cells. SUMMARY OF THE INVENTION 55 The term “bacteriostatic' is to be understood as capable of Surprisingly, it has been found that the combined action inhibiting bacterial growth, i.e. inhibiting growing bacterial of a haloperoxidase, a hydrogen peroxide Source, a halide cells. Source and an ammonium Source results in a hitherto The term “fungicidal' is to be understood as capable of unknown Synergistic antimicrobial effect. killing fungal cells. Thus, based on these findings, the present invention 60 The term “fungistatic' is to be understood as capable of provides, in a first aspect, an enzymatic antimicrobial com inhibiting fungal growth, i.e. inhibiting growing fungal position comprising a haloperoxidase, a hydrogen peroxide cells. Source, a halide Source and an ammonium Source wherein The term “virucidal' is to be understood as capable of Said composition results in a hitherto unknown Synergistic inactivating virus. antimicrobial effect. 65 The term “microbial cells' denotes bacterial or fungal The composition of the invention is useful as antimicro cells, and the term “microorganism' denotes a fungus bial ingredient wherever Such an ingredient is needed, for (including yeasts) or a bacterium. US 6,818,212 B2 3 4 In the context of the present invention the term “inhibiting Bromide peroxidase has been isolated from algae (see growth of microbial cells” is intended to mean that the cells U.S. Pat. No. 4.937,192). are in the non-growing State, i.e., that they are not able to In use, the concentration of the haloperoxidase may be progate. varied in order to achieve the desired antimicrobial effect in The term “hard surface” as used herein relates to any the desired time frame. However, according to the invention Surface which is essentially non-permeable for microorgan the haloperoxidase will normally be added in a concentra isms. Examples of hard Surfaces are Surfaces made from tion of 0.01-100 mg enzyme protein per liter, preferably in metal, e.g., StainleSS Steel, plastics, rubber, board, glass, a concentration of 0.05–50 mg enzyme protein per liter, Wood, paper, textile, concrete, rock, marble, gypsum and more preferably in a concentration of 0.5-10 mg enzyme ceramic materials which optionally may be coated, e.g., with protein per liter. paint, enamel and the like. The hard Surface can also be a Hydrogen Peroxide Sources proceSS equipment, e.g., a cooling tower, an osmotic According to the invention the hydrogen peroxide needed membrane, a water treatment plant, a dairy, a food proceSS for the reaction with the haloperoxidase may be achieved in ing plant, a chemical or pharmaceutical process plant.