AU697046B2 - Method of killing microorganisms - Google Patents

Description

U~Y I II WO 95/26137 PCT/EP95/01037 1 METHOD OF KILLING MICROORGANISMS The present invention relates to a method of killing microorganisms such as bacteria, protozoa, fungi and viruses and to a packaged chemical composition suitable for use in such a method.

International Patent Publication No. W091/11105 (The Boots Company PLC) discloses anti-microbial compositions comprising iodide anions and thiocyanate anions in a weight weight ratio of from 0.1:1 to 50:1 and having a combined anion weight concentration of at least 5 mg/kg, D-glucose in a weight concentration of at least 0.2 g/kg and an effective amount of the oxidoreductase enzyme glucose oxidase.

The present invention provides a method of killing microorganisms by mixing effective amounts of D-glucose and glucose oxidase, incubating the resulting mixture at a temperature of from -10 to 50 0 C and at a pH of from 1 to 8 for a period of at least about 30 minutes and then applying the incubated mixture to the microorganisms to be killed. Remarkably, the method of the invention provides improved speed of kill.

Preferably, the resulting mixture is incubated for at least 1 hour, preferably at least 2 hours, more preferably at least 4 hours. The inventors have found that the fast-kill activity increases rapidly on incubation from 30 minutes to 48 hours.

A favoured incubation period is 12 to 48 hours. A particularly favoured incubation period is 24 hours, as the mixture can be made up a day in advance and the incubation step carried out during transport to an end user.

L-b WO 95/26137 PCT/EP95/01037 2 The incubated mixture of the invention has been found to retain its fast-kill properties for at least a 2 year test period.

Preferably, the concentration of glucose oxidase in the.resulting mixture is at least 25 U/kg, suitably at least 150 U/kg.

Preferably, the resulting mixture comprises iodide anions and/or thiocyanate anions.

Suitably, the resulting mixture comprises both iodide anions and thiocyanate anions, suitably in a weight weight ratio within the range 0.1:1 to 50:1 and suitably to a combined anion weight concentration of at least 5 mg/kg, preferably at least 10 mg/kg.

Iod'de and thiocyanate anions are generally included in the compositions according to the invention in the form of salts. Suitable iodide salts include alkali metal salts such as potassium iodide and sodium iodide and mixtures thereof. Suitable thiocyanate salts include, for example, potassium, sodium, ammonium, ferric and cuprous salts of thiocyanate and mixtures thereof. Preferably the weight concentration of iodide anions is at least 5 mg/kg and the weight concentration of thiocyanate anions is at least 2 mg/kg. The weight:weight ratio of iodide:thiocyanate anions is preferably in the range 0.2:1 to 20:1, more preferably 0.5:1 to 15:1, particularly 1:1 to 5:1.

All units of enzyme activity referred to herein relate to International Units of activity defined as the amount of enzyme required to catalyse the transformation of 1.0 micromole of substrate per minute at 25 0 C under optimal conditions. All concentrations referred to c- b~RIII&i~BPP~R~BPIIL~na~lanrrs~---- WO 95/26137 PCT/EP95/01037 3 herein relate to amounts per kilogram of the total composition.

The method may be used to kill most if not all types of microorganisms, for example gram negative bacteria such as Escherichia coli and Pseudomonas aeruqinosa, gram positive bacteria such as StaDhylococcus aureus and Propionibacterium acnes, moulds such as Asoerqillus niger and Penicillium funiculosum, yeasts such as Candida albicans, Saccharomvces cerevisiae and PityrosDorum ovale, dermatophytic fungi such as Trichoohvton rubrum, microalgae such as Chlorella spp.

and Svroqvra spp. and viruses such as Herpes virus and Picornavirus.

The oxidoreductase enzyme, glucose oxidase, catalyses the production of H 2 0 2 by oxidation of Dglucose in the presence of water and oxygen. It is classified as E.C.1.1.3.4. (IUPAC) and is defined herein in International Units (amount of enzyme required to catalyze the oxidation of 1.0 micromole P-D-glucose per minute at pH 7.0 and 25 0 Glucose oxidase is available commercially from a number of sources, for example from Sturge-ABM under the trade designations "Glucox P200" (2000 U/ml) and "Glucox PS" (75 U/mg).

Glucose oxidase concentrations in excess of 150 U/kg provide excellent protection against bacterial, mould and yeast growth.

The oxidisable suhtsnate for glucose oxidase, namely D-glucose, is generally included ~t a concentration (in the resulting mixture, before incubation) of at least 0.2 g/kg, preferably az least 0.5 g/kg, preferably at least 1 g/kg, ar.1 more particularly at least 2 g/kg. It will be appreciated by those skilled in the art that D-glucose may be provided oer se or may r g W WO 95/26137 PCT/EP95/01037 4 be formed in situ from suitable precursors, for example, as a result of the breakdown of an oligomer or polymer containing D-glucose. Suitable precursors such as sucrose or starch may be used alone or in admixture with D-glucose and may advantageously support more sustained anti-microbial activity than obtained with Dglucose alone.

The efficiency of iodide and thiocyanate anion oxidation in the presence of H 2 0 2 may be enhanced by the addition of small amounts of a peroxidase enzyme such as lactoperoxidase, preferably at least 100 U/kg lactoperoxidase. Lactoperoxidase is classified as E.C.1.17.1.7 (IUPAC) and is defined herein in International Units (amount of enzyme required to catalyse the reduction of 1.0 micromole H 2 0 2 per minute at pH 7.0 and 25 0 Lactoperoxidase is available commercially from a number of sources, for example from DMV (De Melkindustrie Veghel bv) (275 U/mg). It may be supplied, for example, in the form of a freeze-dried powder or in an aqueous salt solution e.g. 1.8% NaCl or 12% NaCl. The compositions according to the invention which further comprise lactoperoxidase exhibit effective killing activity against the organisms listed above.

The method of the invention may, if desired, incorporate further agents which may supplement or enhance the anti-microbial activity thereof, for example other enzymes such as lactoferrin or salts such as calcium chloride. Anti-microbial activity may be enhanced by the addition of agents having antioxidant activity. Typical antioxidants include, for example, butylated hydroxyanisole, butylated hydroxytoluene, a-tocopherol and esters thereof, ascorbic acid, salts and esters thereof, gallic acid, salts and esters thereof e.g. propyl gallate, quinones such as ditertiary butylhydroquinone, propolis, flavenoidla au "s~rrra~r~a~i-n~-n~ l- l WO 95/26137 PCT/EP95/01037 5 containing materials such as quercetin, sulphurcontaining materials such as dilauryl-3,3thiodipropionate and distearyl-3,3-thiodipropionate, and mixtures thereof. Preferred antioxidants are selected from butylated hydroxyanisole, butylated hydroxytoluene, a-tocopherol and esters thereof and ascorbic acid, salts and esters thereof, preferably in a weight concentration of at least 1 mg/kg, more preferably at least 50 mg/kg.

The use of a-tocopherol and esters thereof as "natural" antioxidants is particularly preferred.

There is also provided apparatus in the form of a packaged chemical composition for use in the method defined above, said apparatus comprising: a) a first reservoir comprising a source of D-glucose; b) a second reservoir comprising a source of glucose oxidase; c) an incubation chamber connected to said first and second reservoirs; and d) means for introducing controlled quantities of said D-glucose and glucose oxidase into said incubation chamber to prepare and incubate a biocidal mixture of glucose and glucose oxidase ready for use.

Preferably, the first reservoir further comprises a source of thiocyanate and/or iodide anions.

Preferably, the second reservoir further comprises a source of lactoperoxidase.

Suitably, the effective concentrations of Dglucose, glucose oxidase, lactoperoxidase and thiocyanate and iodide are such as to provide a biocidal mixture having the following composition on mixing (but before incubation): i I- 1- 1- -4 WO 95/26137 PCT/EP95/01037 6 10 to 500 mg/kg iodide anions; (ii) 5 to 200 mg/kg thiocyanate anions; (iii)0.2 to 100 g/kg D-glucose; and (iv) 150 to 20000 U/kg glucose oxidase; wherein the weight weight ratio of iodide thiocyanate anions is 0.2:1 to 20:1 and the combined anion weight concentration _s at least 25 mg/kg, in combination with a suitable carrier or excipient.

One aspect of the invention provides concentrated biphasic compositions in packaged and substantially nonreacting form which may be stored for prolonged periods prior to use. Concentrated compositions according to the invention will maintain physical separation of the glucose oxidase and its substrate, namely D-glucose, such that H 2 0 2 production is substantially prevented during storage. However, it will be understood that prior to storage concentrated compositions may contain a low level of at least one such substrate sufficient to support an initial reaction but insufficient to sustain activity under the desired storage conditions. The initial reaction may advantageously provide adequate self-preservation of the concentrated compositions according to the invention. Self-preservation is of particular benefit in aqueous concentrates according to the invention which may otherwise require the use of conventional chemical preservatives to avoid microbial spoilage during storage. The substantially non-reacting concentrated compositions according to the invention are intended to be mixed or diluted and activated immediately prior to use by bringing the glucose oxidase and subszrates thereof into intimate admixture to produce compositions having the desired anti-microbial properties.

IIIP~1~8 WO 95/26137 PCT/EP95/01037 7 The concentrated compositions according to the invention optionally further comprise suitable carriers and/or excipients. Advantageously the compositions may incorporate at least one buffering agent to minimise the fall of pH which may otherwise occur after activation of the concentrated composition. The concentrated compositions may be provided in the form of packs containing one or more discrete units of an appropriate weight or volume for batch or unit dosing.

Concentrated compositions according to the invention may comprise substantially anhydrous mixtures of each of the essential components mentioned hereinbefore, optionally combined with suitable nonaqueous carriers or excipients.

Concentrated water-containing compositions, optionally combined with suitable carriers or excipients, may be packaged and maintained prior to use.

They may be in the form of, for example, solutions, suspensions, pastes or gels.

Compositions useful in the present invention may take the form of two or more powders, liquids, pastes or gels which prevent the glucose oxidase and D-glucose from reacting until the two are combined prior to use.

For example, the glucose and D-glucose might be formulated as dry granules to be activated by addition of liquid prior to use, allowing the reagents to react.

Examples of such products include a) deodorants e.g. for topical administration in the form of roll-on or stick formulations; b) antibacterial skin washes e.g. in the form of lotions; c) anti-acne preparations e.g. in the form of lotions or creams; USQ ~F~srau~ i~sll~-na u~arP--~ WO 95126137 PCTIEP95/01037 8 d) anti-athlete's foot preparations e.g. in the form of lotions; e) anti-dandruff preparations e.g. in the form of shampoos or lotions; f) dental preparations e.g. mouth washes suitable for general oral hygiene and in particular having antiplaque properties, and dentifrices such as toothpastes, toothpowders, chewing gums and lozenges; g) impregnated materials e.g. wound dressings, sutures and dental floss; h) pharmaceuticals e.g. wound irrigants and burn treatments, anti-diarrhoeal agents and medicaments suitable for the treatment of infections such as Candida and Tinea infections; i) ophthalmic preparations e.g. eye washes and solutions for rinsing and/or sterilising contact lenses; j) sterilants e.g. for baby bottles and surgical or dental instruments.

k) sterilants for use in healthcare/manufacturing environments to decontaminate for example surgical instruments or food processing equipment; and 1) surface cleaning agents for use in food preparation/handling or healthcare environments to effectively decontaminate working surfaces.

In another aspect, it is preferred that the incubated mixture is prcvided as a concentrate for dilution to produce a solution for killing microorganisms. In this aspect, the incubation step to activate the mixture for fast-kill activity is carried out during manufacture and/or distribution to an end user.

A range of oral hygiene preparations may be envisaged which incorporate the anti-microbial It WO 95/26137 PCTIEP95/01037 9 compositions of the invention into conventional dental preparations such as mouthwashes, gargles and dentifrices as an anti-plaque agent and/or as a general antiseptic agent, for example in denture cleansing tablets or solutions. The oril hygiene compositions of the present inventic may, if desired, contain one or more active ingredients conventionally used in the art.

These include, for example, other anti-plaque agents such as bromochlorophene, triclosan, cetylpyridinium chloride and chlorhexidine salts; fluoride ion sources such as sodium fluoride, sodium monofluorophosphate and amine fluorides; anti-tartar agents such as zinc salts, preferably zinc citrate, and water soluble pyrophosphate salts, preferably alkali metal pyrophosphates; and agents which reduce tooth sensitivity including potassium salts such as potassium nitrate and potassium chloride and strontium salts such as strontium chloride and strontium acetate.

It will be appreciated that the compositions and methods of the invention are suitable for a whole host of anti-microbial applications in areas such as agriculture, horticulture, veterinary medicine, and aquaculture, such as trout farming.

The invention will be understood with reference to the following non-limiting Tests and Examples: Comoarative Test A Two phases of an antimicrobial composition were prepared as follows: WO 95/26137 PCTIEP95/01037 10 Phase A Component Concentracion D-Glucose Sodium Thiocyanate Potassium Iodide 45 to 0.42 to 0.52 0.66 to 0.80 Phase B Comonent Concentration Lactoperoxidase Glucose Oxidase Both phases were adjusted to with buffer solutions.

5500 Units/ml 2250 Units/ml between pH 5.5 and A test solution was prepared by mixing Phases A and B together with water t concentrations of 0.9% and 0.05% respectively. An aliquot of the resulting solution was removed immediately and inoculated with Pseudomonas aeruginosa.

The inoculated solution was incubated at room temperature. Samples were taken at regular intervals (viz at 0, 5, 10, 15, 30 and 60 minutes after mixing) and subjected to serial dilution and agar plating in known manner (normal aseptic technique being used throughout) to determine the numbers of any surviving organisms.

At each serial dilution point a parallel sample was taken, inoculated into a nutrient broth and incubated and then checked at an appropriate time for visual signs of growth. These parallel samples acted as so-called "broth controls" to check that the inoculation had been

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WO *5'26137 PCT/EP95/01037 11 performed correctly and to check whether viable organisms were present which might be missed by the serial dilution process. These controls show uL; as counts of "less than 10" on the agar plates.

The original mixture of Phases A and B and water was stored at room temperatire for three months.

Aliquots were taken at regular intervals (viz at 4 hours, 24 hours, 48 hours, 7 days, 13 days, 21 days, 27 days, 2 months and 3 months) and inoculated with Pseudomonas aeruginosa as described above to determine any change in the speed of kill.

Results are set out below in Table 1. In each case the "broth controls" are recorded as or "-ve" above the corresponding plate counts.

Plate counts are given in "colony forming units per ml" m- 1 In each case denotes the test solution and a control (the buffer solution without Phases A and B above).

Comparative Test B The procedure of Compk .tive T'est A above was repeated with the modifications that the test and control solutions were inoculated with Staphylococcus aureus rather than Pseudomonas aeruginosa and that the storage times were varied. Results are set out in Table 2.

Comparative Test C The procedure of Comparative Test A above was repeated with the modifications that the test and control solutions were inoculated with Escherichia coll -4 WO 95/26137 PCT/EP95/01037 12 rather than Pseudomonas aeruginosa and that the storage times were varied. Results are set out in Table 3.

Comoarative Test D The procedure of Comparative Test A above was repeated with the modifications that the test and control solutions were inoculated with Enterobacter cloacae rather than Pseudomonas aeruginosa and that the storage times were varied. Results are set out in Table 4.

Comoarative Test E The procedure of Comparative Test A above was repeated with the modification that the test and control solutions were inoculated with Corynebacterium xerosis rather than Pseudomonas aeruginosa and that the storage times were varied. Results are set out in Table Comoarative Test F The procedure of Comparative Test A above was repeated with the modifications that the test and control solutions were inoculated with Candida albicans rather than Pseudomonas aeruginosa and that the storage times were varied. Results are set out in Table 6.

Comparative Test G The procedure of Comparative Test A above was repeated with the modifications that the test anr control solutions were inoculated with Aspergillus niger rather than Pseudomonas aeruginosa and that the storage times were varied. Results are set out in Table 7.

WO 95/26137 PCT/EP95/01037 13 Comparative Test H The procedure of Comparative Test A above was repeated with the modifications that the test and control solutions were inoculated with Micrococcus luteus rather than Pseudomonas aeruginosa and that the storage times were varied. Results are set out in Table 8.

Comoarative Test I The procedure of Comparative Test A above was repeated with the modifications that the test and control solutions were inoculated with Staphylococcus albus rather than Pseudomonas aeruginosa and that the storage times were varied. Results are set out in Table 9.

Comoarative Test J The procedure of Comparative Test A above was repeated with the modifications that the test and control solutions were inoculated with Pityrosporum orale rather than Pseudomonas aeruginosa and that the storage times were varied. Results are set out in Table Comoarative Test K The procedure of Comparative Test A above was repeated with the modifications that the test and control solutions were inoculated with Streptococcus mutans rather than Pseudomonas aeruginosa and that the storage times were varied. Results are set out in Table 11.

Table 1 Pseudomonas aeruginosa c.f.u. mJJ 1 Storage 0 0 time I

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Sampling time (minutes) 110 15 30 1 t-1 I I 0 hours +ve 2. lx10 6 +ve 1. 1X10 6 +ve 5 8xl0 4 1. 7xl0 6 I

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4 hours +ve 1. 5xl0 5 +ve <10 -ye <10 i 2 x10 6 24 T houirs 48 T hoursI +ve <10 9x10 +ve <10 2 .3-x0 6 1 -ye <10 -ye <10 -ye <10 -ye <10 -ye <10 -ye <10 -ye <10 -ye <10 -ye ye -ye -ye ye <10 -ye <10 -ye <10 -ye <10 J

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Table 1 continued Sampling time (minutes) Storage 0 5 10 15 30 time 7 clays T +ve -ye -ire -ye -ye <10 <10 <10 C l.3xl0 6 2. 2x10 6 1.3 days T +ve -ye -ye -ye -ye <10 <.10 <10 C 2.6xl0 6 2. 0x10 6 21 days T +ve ye -ye -ye -ye <10 <10 <10 C 3.6x10 6 3. 2xl0 6 27 days T +-ve -ye -ye -ye -ye <10 <10 <10 C 3.1x10 6 3. 0x,0 6 Table 1 continued Sampling time (minutes) Storage 0 5 10 15 30 time 2 T +ve -ye -ye, -ye months <10 <10 <10 C 3.3xl0 6 7. 8x10 6 3 T +ve -we -we -ve <10 <10 C 6.0x,0 6 7. 0xl0 6

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Table 2 Staphylococcus aureus c.fLu. ml- Sampling time (minutes) Storage 0 5 10 15 30 time 0 hours T +ve +ve +ve +vcj 2.7x10 6 2.7xl0 6 2.2xl0 6 2.2x10 6 2.5xl0 5 C 2.9X10 6 4 hours T 26,6.515+ve +ve +ve -ye 2.x1 6 2.x1 5 2.4x10 4 2.Ox1O1 <10 C 4.7x10 6 24 T +ve -tve +ve +ve -ye hours 2.8x,0 6 2.4x107 2..0x10 1 2.OxlO' C 2.8x10 6 48 T -f-e -ye -ye -ye -ye hours 2.8x10 4 I <10 <10 <10 C 4.1x10 6 Table 2 continued Sampling time (minutes) Storage 0 5 10 15 30 time days T i-ye +ve +ve -ye -ye 2.7xl0 6 3.2x10 3 <10 <10 C 3.2xl0 6 7 days T +ve +ve +ve -ye -ye 6..3xl0 5 5.OxlO' 1.0XI10 3 7.0xl0 1 C 3.9x,0 6 14 days T +ve +ve -ye -ye -ye 1.2x10 4 <10 <10 <10 C 3.2x10 6 28 days T +ve -ye -ye -ye -ye 6.6xl0 2 <10 <10 <10 C 4.1x10 6 Table 2 continued Sampling time (minutes) Storage J 0 5 10 15 30 timeI 2%4 T 1+ve -ye -ye -ye -ye months .7xl0 4 <10 <10 <10 Cj 2.6x10 6 4 T +ve +ve +ve -ye -ye months 4.8X10 5 <10 <10 <10 4.4x10 6 160 6 6 +ve +ve +ve +ve months 1.3x10 6 3.4x10 3 <10 C 2.3x10 6 2. 0xl0 6 Table 3 Escherichia coli c.f.u. ml~-l Sampling time (minutes) Storage 0 5 10 15 30 time 0 hours T +ve +ve +ve +ve -ye 5..6x,0 6 5.2xl0 6 4.8x10 6 5.5x10 6 8.4xl0 4 C 4.2x10 6 4.6xl0 6 4 hours T +ve -ye -ye -ye -ye -ye 2.7xl0 6 <10 <10 <10 <10 C 2.6xl0 6 6. 8xl0 6 24 T +ve -ye -ye -ye -ye ye hours <10 <10 <10 <10 <10 C 4.7xl0 6 4. 5x10 6 48 T +ve +ve -ye -ye -ye -ye hours 3.3x10 5 <10 <10 <10 <10 CI 4.9xl0 6 LII3. 4x10 6 Table 3 continued Sampling time (minutes) Storage 0 5 10 15 30 time 6 days T +ve -ye -v ee -ve <10 <10 <10 C 4.8x10 6 A.0x10 6 9 days T +ve -ye -ye -ye -ye <10 <10 <10 C 3.2x10 6 5.2x10 6 14 days T +ve -ye -ye -ye -ye <10 <10 <10 C 3.1x10 6 3. 5x10 6 23 days T +ve -ye -ye -ye <10 <10 C 3.3x10 6 3. 6x10 6

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Table 3 continued Sampling time (minutas) Storage 0 5 10 15 30 time 28 days T +ve -xre -ye -xre <10 <10 C 2.3x10 6 3. 0x10 6 2 T +ve -ye -ye -ye months 3.7x10 4 <10 <10 C 4.9x10 6 2. 6xl0 6 3 T +ve -ye -ye -ye month <10 <10 <10 Ii .2x10 6 T 2.5x,0 6 j Table 4 Enterobacter cloacae c.f.u. ml1- Sampling time (minutes) Storage 0 5 10 15 30 I time 0 hours T +ve +ve -ye -ye 2.4x10 6 3.5xl0 6 1.1X10 6 3..9x10 6 <10 C 5.7x10 6 4 hours T +ve -ye -ye -ye -ye -ye ,.4x10 5 <10 <10 <10 <10 C 4.6X10 6 24 T -ye -ye -ye -ye -ye v hours <10 <10 <10 <10 <10 C 2.4xlOG 48 T i-ye +ve i-ye -ye -ye -ye hours 2.7xIO 4 <10 <10 <10 <10 C 2. 5x10 6 Table 4 continued Sampling time (minutes) Storage 0 5 10 15 30 t i%' 3 days T +ev -ye -ye -ye -ye 6.c3XI0 5 <10 <10 <10 <10 C 2.8x10 6 8 days T +ve -ye -ye -ye -ye <10 <10 <10 C 3.lx10 6 2. 6x10 6 14 clays T ve -ye -ve -ye -ye <10 <10 <10 <10 C 4.7x10 6 2.lxO 4- 23 days +ve -ye -ye <10 -ve <1 0 -ye SI i I I I OxlOO 5 7xlO6 5.7x10 6 I I- I I Table 4 continued Sampling time (minutes) Storage 0 5 10 1530 time 28 days T +ve -ye -ye -ye -ye 1. 0x10 1 <10 <10 <10 5.8x,0 6 4.2x,0 6 -I 2 months +ve -ye <10 -ye <10 -ye I I I I I 3. 2x10 0 3. 4x10 6 1-t I I I I 3 months -ye <10 -ye <10 -ye -1 1 1 6.7xI0 6 4. 2xl0 6 I

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Tabln Corynebacterium xero.r;is c.f.u. m1 1 l Samnpling time (minutes) Storage 0 5 10 15 -r 30 time 0 hours T +ve +ve +ve -+ve +ve I +ve 1.4xl0 6 9.3x!0 4 1.2x"0 4 5.lx10 3 <10 C 1.3xl0 6 A1x2.06 4 hours T +ve +ve +ve +ve -ye -ye 3.4xl0 4 <10 <10 <10 <10 C 8.7x,0 5 3 .5xl0 24 T +ve -ye -ye -ye -ye -ye hours <10 <10 <10 <10 <10 C 7.3xl0 5 7 .5xl0 48 T +ve -ve -ye -ye -ye -ye hours 2.7x103 <10 <10 <10 <10 c 6.3x,0 5 j8.2x10 Table 5 continued Sampling time (minutes) Storage 0 5 10 15 30 time 6 days T +ve -ye -ye -ye -ye <10 <10 <10 C 7.6x,0 5 9. 4x10 9 days T +ve -ye -ye -ye -ye C l.2x10 5 9. 5x10 14 days T +ve -ye -ye -ye -ye <10 <10 <10 6.1x10 6 4.21 6 23 days T -i-e -ye -ve -ye <10 <10 l.4x10 6 1.8x,0 6 Table 5 continued Sampling time (minutes) Storage 0 5 10 15 30 time 28 days T +ve -ye -ye -ye <10 <10 C 8.5x10 5 9.3x10 2 T +ve -ye -ye -ye months <10 <10 <10 C 1,9X10 6 8.2xl0 3 T +ve -ye -ve -ye months <10 <10 <10 C 6.9x,0 5 1 6. 1x10 Table 6 Candida albicans c.f.u. ml-l time (minutes) Storage 0 5 10 1530 J 6 time 0 hours 4 hours 24 hours 48 hours +ve 2 .8x10 6 2. 6x,0 6 +ve 2. lx10 6 2.5X10 6 +ve 2 0xl0 2 8. 2x10 6 +ve 1. 6x10 6 1. 8x,0 6 +ve +ve +ve 3.1x10 6 J 3.2x10 6 3. 2x1 6 2.4x10 6 +ve 3. 1x10 6 +ve +ve +ve +ve 12xi 6 f2.5xl0 <10 1. OX10 6 2.3xl0 6 -ye 2. 2x10 6 -ye 2.7x10 6 -ye -ve <10 j <10 <10 -ye <10 -ye <10 -ye <10 -ye <10 -ye <10 I Ix1I Table 6 continued Sampling time (minutes) Storage 0 5 10 15 30 time 6 days T +ve -ye -ye -ye -ye <10 <10 C 2.6x10 6 2.2x10 6 days T +ve -ve -ye -ye -ye 8.6X10 4 <10 <10 <10 C 1.5x10 6 1.3X10 6 23 days T +ve -ye -ye -ve -ye 5.5x10 5 <10 <10 <10 C 1.2x10 6 1.5x,0 6 27 days T +ve -ye -ye -ye -ye 4.9X10 5 <10 <10 <10 C 3. 3x1 6 3. >c,0 6 Table 6 continued Sampling time (minutes) Storage 0 5 10 15 30 tine 2 T ±ve -ye -ye -ye -ye months 6.0x10 4 <10 <10 <10 C 5.9x10 6 6. 6x10 6 3 T -4-e -ye -ye -ye -ye months 2.5xl0 5 <10 <10 <10 C 1.8 x10 6 -2.0xl0 6 Table 7 Aspergillus niger c.f.u. m1 1 l Sampling time (minutes) Storage 0 5 10 15 30 60 time 0 hours T +ve +ve +ve +ve +ve ±ve 1.2x,0 6 5.0xl0 6 1.6xl0 6 1.4xl0 6 1.0X10 6 1.2x10 6 C 2.2x10 7 1.3xl0 6 hours T +ve +ve +ve +ve +ve +ve I1.2x10 6 1.2x10 6 1.3xl0 6 5.0xl0 5 9.0x10 5 4.OxlO' C 1.3x10 6 1.0X10 6 24 T +ve ve +ve -ye -ye -ye hours 5.0X10 6 1.0x10 1 <10 <10 <10 C 3.7x10 6 1. 2x10 6 4 days T -ye -ye -ye -ye -ye -ye 1.2x10 5 <10 <10 <10 <10 <10 C 2.4x10 6 1. 4x10 6 Table 7 continued Sampling time (minutes) Storage 0 5 10 15 30 60 time 7 days T -ye -ye, -ye -ye -ye -ye 4.0x10 6 <10 <10 <10 <10 <10 C 1.2x10 6 9. 0X1.0 14 days T -ye -ye -ye -ye -ye 1.1x10 6 <10 <10 <10 <10 C 2.3x10 6 106 22 days T -ye -ye -ye -ye -ye 1.0X10 5 <10 <10 <10 <10 C 4.0x10 6 2. 9X0 6 26 days T -we -ye -ye -ye -ye 1.OXlO6 <10 <10 <10 <10 C :3.0x10 6 7 2-6xl0 6 Table 7 continued Sampling time (minutes) Storage 40 5 10 15 30 60 time4 2 months 3. 0X10 6 -ye <10 <10 -ye, <10 -ye <10 -ve <10 I I I 1 I IC I 4.0xl~' I 6.X 0x1 6 3 montChs 9. 0X10 5 -ye <10 -ye <10 -ye <10 Ci 3.0OX106j -ye -ye <10 2. 0x,0 6 _I Table 8 Nicrococcus luteus c.f.u. ml71 ~~~apln Storage time 30 1 I-I I I I I I 0 hours +ve 1. 7x10 5 +ve 1. 8xl0 5 ve 1 .7x10 5 +ve 1.3x10 5 +ve 1. 2x10 5 +ve 2.2X10 1. 4xl0 c 2.0OX105 i 1-I I 4 hours +ve 1. 5x10 5 +ve 8. OX10 5 +ve 2 .5x10 4 +ve 7. 5x10 2 +ve 4. 0x10 1 +ve 771 1 t I I I 1. 2x1 0~ 24 hours +ve 1. 5x10 5 +ve 1. 4xl0 4 +ve 3 .8x,0 2 -ye <10 -ye <10 8.7xI0 4 -ve -t I 1- 1 4 I C-I 1.7x101 -A 48 hours +ve 4.7x,0 5 ve 1. 4xI0 2 +ve <10 -ye <10 -ye <10 1.7xl0 -ye 4. 5x10

I

CI

4~X10, L I L I Table 8 continued Samnpling time (minutes) Storrage 0 5 10 15 30 time 6 T +ve +ve +ve +ve -ye days 1.2x10 5 3.3x10 4 1.0x10 2 <10 C l.6x10 5 1 .3x10 13 days T +ve +ve +ve +ve -,-ve 1.8x,05 <10 <10 <10 C 2.2x10 5 2. 0x10 days T +ve ve -ye +ve -ye 2.5x10 4 <10 <10 <10 C 7.9x10 4 8.4x10 4 27 days T +ve +ve -ye -ye -ve 1.3x10 5 <10 <10 <10 C 1.8xl0 5 2. 0x10 5 Table 9 Staphylococcus albus c.f.u. ml-1 Sampling time (minutes) Storage 0 5 10 f 15 30 time 0 hours T +ve +ve +ve +ve +ve +ve 2.3x10 6 1.1x,0 6 2.0x10 6 2.0x10 6 1.6x10 6 9.4xI0 6 C 1.9X10 6 2..5xl0 6 4 hours Tf +ve +ve +ve ±ve -ye -ye 2.0x10 6 8.2xl0 5 6.7x1-0 4 1.5X10 4 2.0xl0 1 C 2.4x10 6 2. 0x10 6 24 T +ve +ve +ve +ve -ye -ye hours 2.0X10 6 2.0xl0 5 1.8X10 4 <10 <10 C 2.3x10 6 1.5x10 6 48 T +ve -ye -ye -ye -ye -ye hours 1.3x10 6 1.5X10 2 <10 <10 <10 C l.4x10 6 iI1.6xl0 6 Table 9 continued Sampling time (minutes) Storage 0 5 10 15 30 time 6 T +ve -+ve +ve -ye -yea days 2.1X10 7 1..1x10 5 8.7x10 2 <10 C 3.6x,0 6 1.8x,0 6 13 days T +ve -ye -ye -ye -ye 2.8x,0 5 7.0x10'1 -<10 <10 C l.9xl0 6 2 .3x,0 6 days T +ve -ye -ye -Fve -ye 2.7xl0 5 <10 <10 <10 C 2.3xl0 6 2.5x10 6 27 days T +ve -ye -ye -ye -ye 1.8x,0 5 <10 <10 <10 C 9.6x105 1 l.X106 Table Pityrosporum ovale c.f.u. ml1 Sampling time (minutes) Storage 0 5 10 15 30 t imle

III

0 hours T j v +ve +ve I6.1xl0 4 4.8xl0 4 5.5x10 4 3.7x10 4 4.3xlO 4 9.0X10 1 C 4..7x10 4 6.4xO 4 hour-, T +ve -ye -ye -ye -ye -ye I 5.1x10 3 4.OxlO' <10 <10 <10 C 6.2xl0 4 2..8xl0 4 24 T +ve -ye -ye -ye -ye -ve hours 9.8X10 3 <10 <10 <10 <10 C 1.3xl0 4 1.2x10 4 48 -ye -ye -ye -ye -ye hours <10 <20 <10 j <10 <10 8.0x10 5.4xl0 4 Table 10 continued Sampling time (minutes)] Storage 0 b 10 159 30 timeI 8 T +ve -ye samnpling -ye -e days Ll 4 <10 interrupted <10 5 1. 8x10 5 1.6x'0 days T -ye -ve -ye -ye -ye <10 <10 <10 C 2A.x10 3 Z.0x10 3 22 days T +ve -ye -ye -ye -ye 6.0x10 1 <10 <10 <10 C 2AX10 5 2. 2x10 29 days T -ye -ye -ye -ve 9.OxlOl <10 <10 <10 [C 4.0x10 5 3. 9x107 Table 11 Streptococcus mutans c.f.u. ml-I Sampling time (minutes) Storage 0 5 10 15 30 time 0 hours T +ve +ve +ve -i-e +ve +ve 3.5xl0 5 4.7x10 5 3.6X10 5 3..4x10 5 3.4xl0 5 3.9x,0 4 C 3.8x10 5 2 .0X10 4 hours T +ve +ve -i-e -ye ve -ve 2.2xl0 5 1.1x10 5 3.1xl0 4 1.3xl0 4 <10 c 3.3ix10 5 2. 1x10 24 T i-ye +ve +ve i-ye +ve -ye hours 2.1x10 5 7.4xl0 4 8.4x10 2 5.QxlOl <10 C 2.9xl0 5 2. 1x10 48 T +ve -we -ye -ve -ye I -ye hours 1.OXl0b <10 <10 <10 <10 C 1.6X10 6 1. 0x10 6 Table 11 c-ontinuea Sampling time (minutes) Storage 0 5 10 15 30 7 T +ve -v~e +ve ve-ye days 1.4x10 5 2.0x10 3 <10 <10 C l.7x10 5 1.3x10 13 days T i-ye -ye -ye -ve -ye 3.9xl0 <10 <10 <10 C 4.1x10 5 3. 1xl0 WO 95/26137 PCT/EP95/01037 43 EFFECT OF pH The following tests were carried out to assess the effect of pH on anti-microbial activity.

Test Procedure Two phases of an antimicrobial composition were prepared as follows: Phase A Comoonent D-glucose Sodium thiocyanate Potassium iodide Concentration (w/v 45 to 0.42 to 0.52 0.66 to 0.80 Phase B Component Lactoperoxidase Glucose O::idase Concentration 5,500 Units/ml 2,250 Units/ml Both phases were adjusted to between pH 5.5 and with buffer solutions.

Test solutions were prepared by mixing Phases A and B together with citrate phosphate buffer at pH 3, 4, 6, 7, 8 to concentrations of 0.9% and 0.05% respectively. The test solutions were then stored at room temperature for 48 hours.

Aliquots of the stored test solutions were removed and inoculated with a single organism from the following organisms: E.coli, S.aureus, C.albicans, A.niger.

The inoculated solution was incubated at room temperature for 15 minutes serially diluted and agar c -~L WO 95/26137 WO 9526137PCTIE P95/0 1037 44 plated to organisms.

determine the numbers of any surviving organism cfu ml-1 E.coli S.aureus C.albicans A.niger initial 8.2x10 5 2.4x10 6 1.2x10 6 l.0x10 Inocu lum count pH 3 <10 <10 1.9x10 6 pH 4 <10 <10 <10 pH 5 <10 5.0X10 3 <10 pH 6 <10 5.0Ox10 5 <10 pH 7 l.2xl0 2 6.4xl0 5 <10 3.0x10 1 pH 8 <10 2.1X10 6 1.7x10 5 1.7x10 3 The above results demonstrate that pH can be chosen so as to kill a particular micro-organism selectively.

For example, at pH 8 only E.coli is killed. Broad spectrum anti-microbial activity is obtained when the pH is around 4 in the above tests.

WO 95/26137 PCT/EP95/01037 45 Examole 1 Glycol paint for athlete's foot or acne A biphasic glycol paint is prepared to the following composition: Comoonent A Propylene glycol D-Glucose 1g Sodium thiocyanate 8.4mg Potassium iodide 13.2mg Water to Component B Glucose oxidase (available under the Trade Designation "Glucox P200") 112U Lactoperoxidase 275U Water to Using a suitably designed dispenser 1 part of Component A is combined with 1 part of Component B and allowed to stand for the required activation time. After this period the mixed components can be applied to control two strains of inter alia Staphylococcus aureus and also Propionibacter acnes, Candida albicans, Trichoderma rubrum, Trichoderma mentagrophytes and Trichoderma interdigitale.

-I I- ~1~LA~ ICYIX- I WO 95/26137 PCTIEP95/01037 46 Example 2 Roll-on Deodorant A biphasic roll-on deodorant is prepared to the following composition: Component A Tetrasodium EDTA 0.125g Mixture of stearates (available under the Trade Designation "Cithrol GMS 3.75g Ethoxylated fatty alcohol (available under the Trade Designation "Cromul EM 0685") 3.125g Light liquid paraffin 3.75g D-Glucose 0.625g Sodium thiocyanate 5.25mg Potassium iodide 8.25mg Water to Component B Glucose oxidase (available under the Trade Designation "Glucox P200") 280U (37.5ppm) Lactoperoxidase 687.5U Water to Using a suitably designed dispenser, 4 parts of Component A are combined with 1 part of Component B in a mixing chamber and allowed to stand for the required activation time. After this period the mixed components can be applied to provide the necessary killing activity against inter alia two strains of Staphylococcus aureus.

i WO 95/26137 PCT/EP95/01037 47 Example 3 Anti-dandruff shampoo A biphasic antibacterial anti-dandruff prepared to the following composition: shampoo is Component A Sodium laureth-2-sulphate (23% soln) Zinc sulphate Mixture of diethanolamides (available under the Trade Designation "Empilan CDE") Stearic acid toilet Mixture of mono and distearates (available under the Trade Designation "Empilan EGMS") D-Glucose Sodium thiocyanate Potassium iodide Water 68.75g 0.125g 6.25g 1.25g 3.75g 0.625g 5.25mg 8.25mg to l00g Comoonent B Glucose oxidase (available under the Trade Designation "Glucox P200") Lactoperoxidase Water 280U (37.5ppm) 687.5U to Using a suitably designed dispenser 4 parts of Component A are combined with 1 part of Component B and allowed to stand for the required activation time. After this period the mixed components can be applied to achieve the desired killing activity against inter alia S.

aureus and Pityrosporum ovale.

WO 95/26137 PCTIEP95/01037 48 CONCENTRATED FORMULATION Example Sterilising Concentrate for Dilution Glucose Sodium Thiocyanate Potassium Iodide Potassium Dihydrogen Phosphate Sodium Hydroxide Glucose Oxidase Lactoperoxidase Water q.s to Concentration 45% w/v 0.42% w/v 0.66% w/v 0.62% w/v 0,02% w/v 5600 U 13750 U 100% volume In the above concentrated formulation the components are pre-activated by incubation during manufacture and distribution. The concentration is then diluted 1 part in 100 parts of water to yield a steriling solution with fast-kill activity.

The concentrated formulation is advantageous because the weight of product is greatly reduced compared to the liquid solution, making the concentrated product relatively cheap to distribute. The concentrate formulation is also convenient for the end user because it is easier to transport and takes up less storage space than the equivalent liquid solution.

WO 95/26137 PCT/EP95/01037 49 Example 4 Sterilant solution (eq for contact lenses) A biphasic sterilant solution is prepared to the following composition: Component A D-Glucose ig Sodium thiocyante 8.4mg Potassium iodide 13.2mg Water to Component B Glucose oxidase (available under the Trade Designation "Glucox P200") 112U Lactoperoxidase 275U Water to Using a suitably designed dispenser 1 part of Component A is combined with 1 part of Component B and allowed to stand for the required activation time. After this period the mixed components can be applied to give effective control of Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Streptococcus mutans, Enterobacter cloacae and Candida albicans.

An example of packaging suitable for use in the method of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which: LI-- WO 95/26137 PCT/EP95/01037 50 Figure 1 shows a side section view of a packaged chemical composition according to the present invention, along the line I-I of Figure 2; and Figure 2 shows a plan section view of the packaged chemical composition of Figure 1 along the line II- II of Figure 1.

The packaged chemical composition, generally indicated as 1, comprises a first reservoir 2 and a second reservoir 3. The reservoirs are of approximately equal size and are generally tubular in shape. The reservoir walls 4,5 are made from resilient polyethylene but it will be appreciated that other resilient materials, such as polyvinylchloride (PVC) or polyethyleneterephthalate (PET) may also be used if desired. The walls are strong enough to avoid the risk of breakage but flexible enough to permit "squeezing" of the contents in use.

One of the reservoirs, 2, houses Component A from Example 1 above and the other reservoir, 3, houses Component B therefrom. The reservoirs 2,3 are substantially sea? I so that no leakage of contents can occur from one to the other. For convenience they are welded together at one point by a polythene web 6 which is continuous with the reservoir walls 4,5. The walls of the reservoirs are independently deformable such that it is possible to deform one reservoir, for example by "squeezing", without deforming the other, if desired.

The lower end of the reservoirs provide a base 7 to allow the packaging to be rested in an upright position.

The upper ends of the reservoirs 2,3 are constricted to form a neck 8 which is blocked by a neck-piece 9 made of stiff non-resilient polyethylene. The neck 8 and neckpiece 9 support a bulbous incubation chamber 10. The incubation chamber 10 is constricted at its upper end to WO 95/26137 63rC/P19/01037 51 provide a second neck 11 which is threaded in the manner of a screw-top bottle neck to connect with a scre-j-cap 12 which may be used to close off the packaging when not in use.

Polythene tubes 13,14 extend substantially from the bases of the two reservoirs 2,3 respectively, through holes in the neck-piece 9, with which they form an interference fit, to the base of the incubation chamber The tubes 13,14 are narrow enough to prevent significant leakage of the contents of the reservoirs 2,3 into the incubation chamber 10 during normal storage. Such leakage as may occur is contained by the cap 12. However, when the cap 12 is removed and one or both of the reservoir walls is deformed, for example by "squeezing", a portion of the reservoir contents is forced up the tube 13 or 14 by the pressure created and into the incubation chamber In use, controlled mixing of the reservoir contents (Components A and B of Example 1 above) is achieved by squeezing each of the reservoirs in turn and/or simultaneously by roughly equal amounts to introduce equal quantities of the reservoir solutions into the incubation chamber 10. Indicia 15 are provided on the walls of the incubation chamber 10 to assist in measuring the quanitities to be mixed. The walls of the incubation chamber are made from transport/translucent polyethylene to assist in the measuring process but this is not essei .ial _nd it will be appreciated that opaque plastics materials may be used if desired. The cap 12 may then be replaced and the apparatus 1 may be shaken gently to complete the mixing process. The resulting mixture is left to incubate for the desired period, typically from 5 to 10 minutes.

After incubation the cap 12 is removed and the incubated anti-microbial mixture is used in the desired I ~II WO 95/26137 PCT/EP95/01037 52 manner, by application to the feet or other areas affected by so-called "athlete's foot".

It will be appreciated that the packaging could house any of the other Examples given above and/or numerous variants thereof. Where the components are not mixed in equal proportions (eg Examples 2 and 3 above) the reservoirs may be made of correspondingly unequal size and corresponding modifications may be made to the other parts of the apparatus as appropriate. Thus, if four parts of component A are needed to each part of component B, then the reservoir housing component A may be squeezed four times as often as the reservoir housing component B (as in Example 3 above), until the desired quantity of mixture has been generated in the incubation chamber 10. Alternatively or additionally, the indicia may be used to measure the required proportions.

Instructions for mixing the components in the appropriate proportions will normally be supplied with the apparatus, preferably by printing onto the packaging itself.

-1 1.

Claims (7)

1. A method of killing microorganisms with an improved speed of killing by mixing effective amounts of D-glucose and glucose oxidase, incubating the resulting mixture which includes both iodide ions and thiocyanate io, a wherein the weight ratio of iodide ions to thiocyanate ions is from 0.1:1 to 50:1 and the combined anion weight concentration is at least 5 mg/kg wherein the concentration of glucose oxidase in the resulting mixture is at least 150U/kg at a temperature of from -10 to 500C and at a pH of Trom 1 to 8 for a period of from minutes to 48 hours and then applying the incubated mixture to the microorganisms to be killed.

2. A method as claimed in claim 1 wherein the resulting mixture is incubated for 12 to 48 hours.

3. A method as claimed in claim 1 or claim 2 wherein the resulting mixture further includes a peroxidase enzyme.

4. A method as claimed ir claim 3 wherein the peroxidase enzyme is lactoperoxidase.

A method as claimed in any one of claims 1 to 4 wherein the incubated mixture is provided as a concentrate for dilution to produce a solution for killing microorganisms.

6. A method as claimed in any one of claims 1 to 5 wherein the components are provided as a concentrated biphasic composition in packaged and substantially non-reacting form. 0 (Y iL ron

7. Apparatus in the form of a packaged chemical composition when used in the method defined in claim 1 said apparatus including: a) a first reservoir including a source of D-glucose; b) a second reservoir including a source of glucose oxidase; c) an incubation chamber connected to said first and second reservoirs; and d) means for introducing controlled quantities of said D-glucose and glucose oxidase into said incubation chamber to prepare and incubate a biocidal mixture of glu.ose and glucose oxidase ready for use. DATED this 31st day of July, 1998 KNOLL AKTIENGESELLSCHAFT WATERMARK PATENT TRADEMARK ATTORNEYS S290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA CJH:DM doc 22: AU2 10395.WPC 'r YB