Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
  • A01N37/02—Saturated carboxylic acids or thio analogues thereof; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
  • A61Q17/005—Antimicrobial preparations
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/50—Isolated enzymes; Isolated proteins
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K30/00—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
  • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
  • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
  • A23L3/3463—Organic compounds; Microorganisms; Enzymes
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
  • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
  • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
  • A23L3/3463—Organic compounds; Microorganisms; Enzymes
  • A23L3/3481—Organic compounds containing oxygen
  • A23L3/3508—Organic compounds containing oxygen containing carboxyl groups
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
  • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
  • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
  • A23L3/3463—Organic compounds; Microorganisms; Enzymes
  • A23L3/3526—Organic compounds containing nitrogen
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
  • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
  • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
  • A23L3/3463—Organic compounds; Microorganisms; Enzymes
  • A23L3/3535—Organic compounds containing sulfur
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
  • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
  • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
  • A23L3/358—Inorganic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
  • A61K8/20—Halogens; Compounds thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
  • A61K8/22—Peroxides; Oxygen; Ozone
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
  • A61K8/23—Sulfur; Selenium; Tellurium; Compounds thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
  • A61K8/36—Carboxylic acids; Salts or anhydrides thereof
  • A61K8/361—Carboxylic acids having more than seven carbon atoms in an unbroken chain; Salts or anhydrides thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
  • A61K8/37—Esters of carboxylic acids
  • A61K8/375—Esters of carboxylic acids the alcohol moiety containing more than one hydroxy group
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/46—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing sulfur
  • A61K8/463—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing sulfur containing sulfuric acid derivatives, e.g. sodium lauryl sulfate
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
  • A61K8/66—Enzymes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
  • A61K2800/51—Chelating agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
  • A61K2800/52—Stabilizers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
  • A61K2800/59—Mixtures
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
  • A61K2800/88—Two- or multipart kits
  • A61K2800/882—Mixing prior to application

Definitions

• This invention relates to microbially resistant compositions and methods for their preparation. It further relates to methods of use of such compositions and to products which are made microbially resistant due to the addition or application of such compositions.
• Lactoperoxidase is an enzyme that is part of the natural non-immune defence systems in milk and mucous secretions (such as saliva, tears and intestinal secretions). LP, together with various naturally occurring cofactors, forms the Lactoperoxidase system (LPS) that has pronounced anti-microbial activity.
• the LPS incorporates LP (extracted from bovine milk), a source of peroxide and a cofactor (generally thiocyanate) .
• LP extracted from bovine milk
• a source of peroxide and a cofactor (generally thiocyanate) .
• glucose oxidase from a microbial source
• glucose are incorporated to provide a source of hydrogen peroxide.
• the use of an enzyme system is often preferred as this ensures that the delivery of peroxide is sustained. However this requires aerobic conditions so that in anaerobic conditions a direct source of peroxide may be required.
• Reactions catalysed by LP and where the cofactor is thiocyanate yield short-lived intermediary oxidation products of thiocyanate that show the anti-microbial activity.
• LP utilises peroxide to catalyse the oxidation of the thiocyanate ion in the presence of water to generate the hypothiocyanite ion (OSCN-).
• OSCN- hypothiocyanite ion
• the hypothiocyanite then is believed to react with the sulphydryl groups on the bacterial membranes with catastrophic effects on the bacteria. Much of the thiocyanate is regenerated in the process.
• the LPS is regarded as being bactericidal against Gram-negatives (eg. E. coli, Yersinia entercolitica, Pseudomonas spp., Salmonella spp, Campylobacter spp) and bacteriostatic against Gram-positive bacteria (Listeria monocytogenes, Staphylococcus aureus, Streptococcus spp).
• Gram-negatives eg. E. coli, Yersinia entercolitica, Pseudomonas spp., Salmonella spp, Campylobacter spp
• bacteriostatic against Gram-positive bacteria Listeria monocytogenes, Staphylococcus aureus, Streptococcus spp.
• the LPS is also suggested to have anti- viral activity in some situations.
• the anti-microbial effects of a number of fatty acids are also well-documented.
• the most active are the medium chain fatty acids lauric (dodecanoic) acid and myristic (tetradecanoic) acid.
• the fatty acids are regarded as especially effective against the Gram-positive bacteria and the fatty acid derivative monolaurin (1-monododecanyol- rac-glycerol) is generally regarded as the most active.
• anti- viral activity (against the enveloped viruses) has been claimed for the fatty acid derivatives, including sodium dodecyl sulphate.
• Monolaurin has GRAS status as an emulsifying agent and is used mostly in vegetable shortenings and to some extent in ice creams and baked goods.
• Lauricidin E Monolaurin is marketed as Lauricidin E for use as an anti-microbial in food systems.
• Lauricidin E Monolaurin is marketed as Lauricidin E for use as an anti-microbial in food systems.
• it has not found wide acceptance as an anti-microbial because of the concentrations required and the resultant effects on organoleptic quality of the treated food products.
• PS such as the LPS
• fatty acids/fatty acid derivatives such as monolaurin
• the present invention provides a method of preparing a microbially resistant composition which comprises forming a mixture of the following components:
• PS peroxidase system
• said fatty acid or derivative is present in an amount effective to interact with said PS to produce an enhanced anti-microbial effect.
• enhanced anti-microbial effect means an anti-microbial effect which is more microbiocidal against at least one type of microorganism than would be predicted from the known properties of the individual components.
• microorganism means microbial pathogens, ineffective particles and spoilage organisms, including those of bacterial, viral, fungal or protozoal origin.
• said fatty acid is an anti-microbial fatty acid, or an anti-microbial derivative of a fatty acid.
• the peroxidase is a lactoperoxidase.
• the peroxide is hydrogen peroxide.
• the cofactor is selected from thiocyanate or iodide, and is most preferably thiocyanate.
• component (b) is or includes an anti-microbial fatty acid selected from C ⁇ , Cio, C12, C ⁇ and Ci6 fatty acids or their derivatives.
• component (b) is or includes an anti-microbial ester of a fatty acid or a salt thereof.
• component (b) is or includes monolaurin (1-monododecanoyl-rac- glycerol) or its salt, sodium dodecyl sulphate.
• component (b) is monolaurin.
• the anti-microbial fatty acid or derivative of a fatty acid is present in an amount which is at least 5% by weight of the total lipid present in the composition.
• said composition is formed by the addition of one or more of the components to a pre-formed mixture which already contains the remaining component(s) .
• the pre-formed mixture is a food, cosmetic or healthcare product.
• the food product may be a dietary supplement or nutraceutical. It may also be a dairy product, meat product or fish product.
• the food product may also be an animal feed, which in its simplest form may be water.
• said composition when formed, consists of said components in admixture.
• the invention provides a preparative composition suitable for use in preparing a microbially resistant composition which comprises at least two components selected from:
• the peroxidase is a lactoperoxidase.
• the invention provides a preparative pack suitable for use in preparing a microbially resistant composition which comprises, in separate containers, a peroxidase and at least one anti-microbial fatty acid or anti-microbial derivative of a fatty acid.
• a peroxidase is a lactoperoxidase.
• said pack further includes a source of peroxide and/ or a cofactor which is capable of yielding anti-microbial oxidation products.
• the source of peroxide and/or said cofactor are in separate containers.
• the invention provides an anti-microbial composition which comprises a peroxidase, a cofactor which is capable of yielding anti-microbial oxidation products and at least one anti-microbial fatty acid or anti-microbial derivative of a fatty acid, wherein said fatty acid or derivative thereof is present in an amount effective to synergistically interact with said peroxidase and said cofactor, in the presence of peroxide, to produce an enhanced anti-microbial effect.
• the peroxidase is a lactoperoxidase.
• said anti-microbial composition further includes a source of peroxide.
• composition may also include a further anti-microbial agent or a chelating agent.
• the further anti-microbial agent is selected from phenols, organic acids, bacteriocins, derivatives of these and mixtures of these, or anti-microbial components or mixtures of components extracted from or found in milk, such as lactoferrin.
• the chelating agent is EDTA.
• the invention provides a microbially resistant product which is prepared by a method as defined above.
• the invention provides a product which includes the following components:
• the peroxidase is a lactoperoxidase.
• said product is resistant to the growth of both Gram positive and Gram negative bacteria.
• said product is a food product, such as a dietary supplement, nutraceutical dairy product, meat product, fish product or animal feed.
• said product is a cosmetic product.
• said product is a healthcare product.
• the invention provides a method of treating a surface which comprises the step of applying to said surface an effective amount of an anti-microbial composition as defined above.
• the surface is a surface which is used in the preparation and/ or handling of food products.
• the invention provides a method of treating a product for the purpose of rendering that product microbially resistant which comprises the step of adding to said product an effective amount of an anti-microbial composition as defined above.
• Figure 1 is a graph showing the effect of monolaurin concentration on the growth of bacteria in a broth culture inoculated with S. aureus at a rate of 8 x 10 6 cfu per ml (equivalent to a 1% inoculum of the specially prepared stock culture).
• the Optical Density (OD) measured at 600 nm was used as an index of bacterial cell numbers.
• Figure 2 is a graph showing the inhibition of the growth of bacteria by various compositions in a broth culture inoculated with E. coli at a rate of 1.5 x 10 6 cfu per ml (equivalent to a 0.05% inoculum of the specially prepared stock culture).
• Figure 3 is a graph showing the inhibition of the growth of bacteria by various compositions in a broth culture inoculated with S. aureus at a rate of 4 x 10 5 cfu per ml (equivalent to a 0.05% inoculum of the specially prepared stock culture).
• Figure 4 is a graph showing the inhibition of the growth of bacteria by various compositions in a broth culture inoculated with S. aureus at a rate of 8 x 10 6 cfu per ml (equivalent to a 1% inoculum of the specially prepared stock culture).
• Figure 5 is a graph showing the inhibition of the growth of bacteria by various compositions in a broth culture inoculated with L. monocytogenes at a rate of 1.5 x 10 6 cfu per ml (equivalent to a 0.05% inoculum of the specially prepared stock culture).
• Figure 6 is a graph showing the inhibition of the growth of bacteria by various compositions in a broth culture inoculated with L. monocytogenes at a rate of 3 x 10 7 cfu per ml (equivalent to a 1% inoculum of the specially prepared stock culture).
• compositions exert an anti-microbial effect through the synergistic combination of the PS and an anti-microbial fatty acid or fatty acid derivative component.
• the surprising finding made by the applicants is that the anti-microbial efficacy of the PS can be supplemented markedly by addition of an anti-microbial fatty acid component.
• the improvement in anti-microbial efficacy is enhanced or synergistic in character. This synergism is particularly evident against Gram-negative microorganisms such as E. coli.
• the PS requires three components. These are in turn a peroxidase, a source of peroxide and a cofactor.
• the cofactor is one which yields intermediary anti-microbial oxidation products. Examples of suitable cofactors include thiocyanate and halides (particularly iodide) .
• the peroxidase itself can be any of those which are commercially available. GRAS status peroxidases are preferred, with a lactoperoxidase being particularly preferred.
• the peroxide can be directly added (for example as hydrogen peroxide) or can be the product of enzymic digestion of an appropriate substrate.
• a combination of glucose oxidase and glucose can provide the source of hydrogen peroxide.
• Sodium percarbonate-based systems can also be used.
• the components of the PS will be provided in art standard amounts.
• the peroxidase is lactoperoxidase
• the peroxide source is glucose/glucose oxidase
• the cofactor is thiocyanate
• the components can be included in a liquid medium in the following amounts ( g per litre) :
• the same components can be included, for example, in the following amounts (mg per kg):
• the fatty acid or fatty acid derivative component will generally be a C ⁇ -Ci ⁇ fatty acid or derivative.
• Cio, C12 and C ⁇ fatty acids are generally regarded as the most anti-microbial and therefore these are preferred if the fatty acid component is to be added in the form of a fatty acid per se.
• anti-microbial derivatives includes the use of esters for fatty acids or their salts.
• One particular ester which the applicants found to be useful is monolaurin (Lauricidin /E).
• the sodium dodecyl sulphate salt can also be used as a suitable derivative.
• the fatty acid component need not be in pure form.
• the useful fatty acid/ fatty acid derivative can be included in a mixture such as an extract of bovine milk fat or coconut oil in which the lipid has been treated to ensure that the requisite proportion of anti-microbial components are present.
• a synergisticaUy effective amount of fatty acid/fatty acid derivative must be present. This amount is greater than the levels at which anti-microbial fatty acids are present in standard bovine milk (in which the lipid is predominantly in the form of triglycerides), and reflects the applicants finding that when the PS plus fatty acid component is applied to milk, significantly enhanced and effective anti-microbial result is achieved.
• the applicants have determined that where the synergistic anti-microbial effect is to be produced in a composition which contains lipid, the anti-microbial fatty acid/ fatty acid derivative must be present in an amount which is at least 5% by weight of the total lipid in the composition.
• L. monocytogenes strain L45 and S. aureus strain R37 were obtained from Dr Roger Cook, the Meat Industries Research Institute of New Zealand strain culture collection and E. coli 0157:57 strain NCTC 12900 was obtained from Dr Heather Brooks, the Department of Microbiology, University of Otago strain culture collection. Stock cultures of all strains were stored in skim milk at -70°C and when required were subcultured onto Plate Count Agar (PCA) (Difco Laboratories, Detroit, Michigan, USA) or blood agar (BA) (Columbia Agar Base (GIBCO BRL, Life Tech Ltd, Paisly UK) supplemented with 5% whole human blood (Dunedin Public Hospital, Dunedin, NZ)).
• PCA Plate Count Agar
• BA Blood agar Base
• Glucose oxidase (GOX) and glucose oxidase were sourced from Sigma; lactoperoxidase was sourced from Tatua Biologies, Morrinsville, NZ; and the sodium or potassium thiocyanate was sourced from Bio Serae SA Limited, Montolieu, France.
• THB Todd-Hewitt broth
• monolaurin was added to each tube prior to autoclaving of tubes at 121°C for 15 min.
• components were added to autoclaved tubes in the following order, cell inoculum, 16 LPX stock, glucose stock, thiocyanate stock and glucose oxidase stock. In all cases tubes were inoculated with 0.05% and 1.0% (v/v) of an overnight THB culture of the appropriate bacterial strain.
• the tubes were incubated at 37°C for 48 hours and their ODeoonm read by use of a spectrophoto eter (Spectronic 20D+, Milton Roy Company, USA) at intervals as appropriate. Viable counts of each bacterial strain were determined by dilution in saline of five overnight cultures of each strain and plating of each dilution onto PCA by use of a spiral plating machine (Spiral Systems, Cincinnati, USA).
• the concentration (mg/litre) of the components of the LPS in the culture solution was as follows: 6.85 for lactoperoxidase, 3.17 for glucose oxidase, 31.7 for glucose and 29.0 for thiocyanate.
• the test system The viable count of overnight broths for each test strain used in the experiments was 3 x 109, 8 x 108 ⁇ d 3 x 10 ⁇ cfu per ml for E coli 0157:57 strain NCTC 12900 ("E. coif), S aureus strain R37 (“S. aureus”) and L monocytogenes strain L45 (“L. monocytogenes”) respectively.
• the degree of inhibition of monolaurin against both Gram-positive strains appeared to be proportional to the concentration of monolaurin used, as is illustrated by the results shown for a 1% inoculum of S. aureus grown in the presence of 50 - 150 ppm monolaurin ( Figure 1).
• the degree of inhibition observed was inversely proportional to the bacterial load imposed on the system; that is, the greater the starting inoculum the lesser the degree of inhibition observed.
• L. monocytogenes was the strain most sensitive to inhibition and E. coli the strain least sensitive to inhibition.
• Monolaurin did not inhibit the growth of E. coli when tubes were inoculated at either 0.05% or 1.0%.
• the LPS slightly inhibited the growth of E. coli when tubes were inoculated at 0.05% but not when inoculated at 1.0%. Growth of the E. coli was strongly inhibited by combinations of the monolaurin and LPS ( Figure 2).
• S. aureus is a bacterium with an intermediate sensitivity to inhibition of growth by monolaurin as it has been reported to be sensitive to inhibition at concentrations of about 200 ppm (Kabara et al, 1977), a result which compares well with the sensitivity of the strain used in this study.
• the LPS has been reported to inhibit the growth of a wide range of bacteria including E. coli, S. aureus and L. monocytogenes (Wolfson et al, 1993).
• the wide range of component (glucose, H2O2, GOX, LPX & SCN ) concentrations, incubation media, and incubation temperatures used in these studies, make direct comparisons with the present study difficult.
• the degree of relative sensitivity of the strains to LPS alone seen in this study is consistent with that reported by others (Gaya et al, 1991; Kamau et al, 1990; Bjork et al, 1975; Siragu et al, 1989).
• a broth culture system (Todd-Hewitt Broth, THB) inoculated with either S. aureus R37 or E. coli 0157:H7 (-vt) was used as a screening system to evaluate different lipid components.
• the strains were cultured in THB overnight at 37 °C and dispensed to tubes. The initial loadings were 1 x 10 5 per ml for both the S. aureus and the E. coli.
• lactoperoxidase system LPS
• lipid components exhibit anti-microbial effects when combined with the lactoperoxidase system as evidenced in the experiments summarised in Table 1.
• Monolaurin was the most effective lipid component against S. aureus and in this simple system, it was just as effective whether or not the LPS was included.
• the Table 1 data indicate that synergy is apparent for both the Gram positive S. aureus and the Gram negative E. coli but the effect is greater with the S. aureus.
• the effect of monopalmitoleate was very similar to that of monolaurin against E. coli alone or in the presence of the LPS. However sodium lauryl sulphate was very effective against E. coli, whether or not the LPS was present.
• Table 1 Inhibition of bacterial growth in broth culture by a combination of a lipid component and the lactoperoxidase system.
• the data are expressed as the concentration of the lipid component when bacterial growth is inhibited by about 50% or 100% after 48 hours; eg. 100/250 means that growth is about 50% inhibited at lOOppm and completely inhibited at 250ppm of the lipid component; the * indicates that the organism was not 100% inhibited at any concentration up to lOOOppm.
• Tables 2 and 3 present the results of experiments in which milk and mince were inoculated with S. aureus R37. The synergistic effects of the LPS /monolaurin combination are apparent.
• Tables 4 and 5 present the results of experiments in which milk was inoculated with E. coli 0157:H7 (a Gram negative organism) or S. aureus R37 (a Gram positive organism).
• Table 4 Evaluation of the monolaurin + lactoperoxidase system in milk against a Gram negative organism (E. coli 0157:H7, at 12°C; cfu per ml).
• the ratio of LPX to GOX (Units of enzyme activity) was 9: 1, with the thiocyanate ion and glucose each present at 12mg/l.
• Table 5 Evaluation of the monolaurin + lactoperoxidase system in milk against a Gram positive organism (S. aureus R37 at 37°C; cfu per ml; component concentrations as for Table 4).
• Table 6 presents the results of two experiments that show in certain circumstances, the presence of the milk itself actually has an inhibitory effect on the efficacy of the monolaurin treatment. That is, the anti-microbial effect of the monolaurin is adversely affected by the normal levels of lipid (generally in the form of triglycerides) naturally present in milk. Similarly, the efficacy of the combination of monolaurin plus the LPS is also affected by the concentration of milk (and hence the lipid) present in the culture medium (Table 7). However the effect of the (competing) lipid content is less with the combination of monolaurin + LPS than with monolaurin alone; in other words, the synergistic effect is greater.
• Table 6 Comparison of different levels of milk (ie lipid) on the efficacy of the monolaurin system against S. aureus R37 (cfu per ml) in a Todd-Hewitt Broth (THB)/milk mixture at 37°C.
• Table 7 Comparison of different levels of milk (ie lipid) on the efficacy of the monolaurin + lactoperoxidase system (with 5 ppm of LPX) against S. aureus R37 (cfu per ml) in a THB/milk mixture at 37°C.
• Table 8 The synergistic effect of the components through comparison of the effect of the efficacy of the LPS or monolaurin systems alone and the monolaurin + LPS against the Gram-positive bacteria, S. aureus R37 in milk at 37°C.
• Table 9 The synergistic effect of the components through comparison of the effect of different levels of milk (ie fat) on the efficacy of the monolaurin system alone and the monolaurin + LPS against the Gram-positive bacteria, S. aureus R37 at 37°C.
• Table 10 presents the results of an experiment in which the effect of the concentration of monolaurin alone on S. aureus in THB was evaluated. In this experiment, a concentration of only 25 or 50ppm was required to have a significant effect on the population of S. aureus. The efficacy of the 50ppm monolaurin treatment was equivalent to that achieved with 500ppm in Table 6. As the experiment reported in Table 10 was conducted in a fat-free medium, the comparison provides further evidence of the compromising effect of the presence of other lipids on the efficacy of the monolaurin.
• Table 10 Comparison of different levels of monolaurin on the population of S. aureus R37 in THB (cfu per ml) at 37°C.
• a solution of 100% milk contains around 3% milk lipid.
• Monolaurin at a concentration of 500 ppm represents 0.05 grams per 100ml (0.05%). From the above it is clear that a certain threshold concentration of selected anti-microbial lipid components (expressed as a percentage of the total lipid) must be exceeded in order to ensure a significant anti-microbial effect of the total composition.
• Table 1 1 provides a summary of the relevant data classified according to the level of added anti-microbial lipid (in this case monolaurin) both in the actual amount present and as a proportion of the total hpid.
• Table 11 Summary of data from Tables 2, 8, 9 & 10: Evidence for the effectiveness of, and the synergistic effect of, the components of the anti-microbial composition as affected by the quantity of monolaurin present and the proportion of the total lipid present as monolaurin (reduction in population of S. aureus after 5 or 6 hours at 37°C) .
• the concentration of the selected anti-microbial lipid components must be equal to or greater than 5% of the total lipid present in order to include the synergistic anti-microbial effect.
• the free fatty acids with anti-microbial properties must therefore constitute more than 5% of the total lipids present for the milk to be transformed into anti-microbial composition to achieve a substantial and consistent anti-microbial effect.
• Such a composition will be effective against both Gram positive (as exemplified by S. aureus) and Gram negative organisms (as exemplified by E. coli, see Table 5).
• Such an amount of anti-microbial Hpid can only be achieved by addition of the selected anti-microbial Hpid or derivative in accordance with the invention.
• a simple medium of the foUowing composition was prepared:
• PBS/T/Y 195 ml of 0.2 M NaH P0 4 , 305 ml of 0.2 M Na 2 HP0 4 and 8.994 g NaCl, 10 g bactotryptone, 5 g yeast extract made up to 1 Htre with MilHQ water and autoclaved in screw-capped tubes at 121 °C for 15 min. The tubes were held at incubation temperature (37 °C) until used.
• S. aureus R37 or E. coli 0157:H7 were used in the experiments (as for the experiments in Table 1), with the following base combinations of monolaurin and the LPS selected for the two strains based on titrations of the organisms against monolaurin + LPS to define the sensitivity: S. aureus (lOOmg LPX (lactoperoxidase) per Htre in the LPS and 20mg/Htre of monolaurin) and E. coli (lOOmg LPX per Htre in the LPS and 50mg/Htre of monolaurin).
• the LPX to glucose oxidase (GOX) ratio was 9: 1 and the thiocyanate and glucose were incorporated at 12mg/Htre.
• Table 12 The importance of the individual components in ensuring the efficacy of the anti-microbial system.
• the degree of inhibition is defined by the time (hours of incubation 37 °C) at initiation of the logarithmic growth phase of the organism and the time at plateau (maximum absorbance).
• the present invention has particular benefit where the fatty acid component is monolaurin. This reflects the fact that monolaurin has GRAS status and is already included in some food products as a emulsifying agent.
• Food products in which the components can be included are any foodstuffs subject to spoilage as weU as dietary supplements and nutraceuticals.
• the invention has particular appHcation to dairy products (such as yoghurts), fish products (particularly shellfish) and meat products (including both ground meats and carcasses), as weU as to animal feeds.
• dairy products such as yoghurts
• fish products particularly shellfish
• meat products including both ground meats and carcasses
• feed is intended in its most general sense, and can include water which is fed to farmed animals including but not limited to bovines, ovines, pigs, caprines, equines and avians (such as poultry).
• the individual components can be added together or independently.
• the remaining components only need be added.
• the components can also be added to form a mixture as part of the product (such as a dairy product), or can be appHed to at least partiaUy coat the products (such as shellfish).
• a preparative pack can be provided with at least the peroxidase and fatty acid/fatty acid derivatives in different containers.
• the invention can also be appHed in the formation of an anti-microbial composition for general use.
• Such a composition will include aU four components in appropriate amounts.
• the composition can then be used to treat surfaces (for example, surfaces used in the preparation or handling of foodstuffs or in healthcare) to ensure that the microbial population is at least reduced if not eliminated.
• anti-microbial compositions of the invention can also be used to supplement the action of other agents.
• the additional agent can be used separately or, more usually, as part of a mixture with the present components.
• composition may also include any of the foUowing:
• a chelating agent such as EDTA
• a phenol such as the esters if p ⁇ r ⁇ -hydroxybenzoic acid (the parabens) including the methyl, ethyl, propyl, butyl or heptyl esters of tert-butyl hydroxyanisole (BHA); an organic acid, (which is recognised as a preservative), such as formic acid, acetic acid, propionic acid, lactic acid, sorbic acid, benzoic acid, citric acid or derivatives of any of these acids; a bacteriocin, such as nisin; lyzozyme; extracts of milk.
• a chelating agent such as EDTA
• a phenol such as the esters if p ⁇ r ⁇ -hydroxybenzoic acid (the parabens) including the methyl, ethyl, propyl, butyl or heptyl esters of tert-butyl hydroxyanisole (BHA);

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