WO2005122800A1 - Anti-microbial composition for food products - Google Patents

Anti-microbial composition for food products Download PDF

Info

Publication number
WO2005122800A1
WO2005122800A1 PCT/EP2005/052785 EP2005052785W WO2005122800A1 WO 2005122800 A1 WO2005122800 A1 WO 2005122800A1 EP 2005052785 W EP2005052785 W EP 2005052785W WO 2005122800 A1 WO2005122800 A1 WO 2005122800A1
Authority
WO
WIPO (PCT)
Prior art keywords
microbial
ppm
inhibitor
acid
natamycin
Prior art date
Application number
PCT/EP2005/052785
Other languages
French (fr)
Inventor
Hein Stam
Jacobus Stark
David Brian Archer
Andrew Plumridge
Kenneth Lowe
Caroline Chantal Patricia Pallard
Original Assignee
Dsm Ip Assets B.V.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dsm Ip Assets B.V. filed Critical Dsm Ip Assets B.V.
Publication of WO2005122800A1 publication Critical patent/WO2005122800A1/en

Links

Classifications

    • 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
    • A23L3/3472—Compounds of undetermined constitution obtained from animals or plants

Definitions

  • the present invention relates to anti-microbial compositions useful in food products susceptible to food spoilage by microorganisms.
  • Weak acid preservatives such as sorbic acid, propionic acid, benzoic acid, p- hydroxybenzoic acids, lactic acid, citric acid or an alkali metal or alkali earth metal salt thereof have been used in a wide range of products, such as dairy products, bakery products, fruit, fruit products, beverages, meat and fish.
  • potassium sorbate can be used in the range of from about 200 to more then 5000 ppm (for reviews see Antimicrobials in food, 1993, P.M. Davidson and A.L. Branen editors, pp49-95 and Encyclopedia of food microbiology, Academic Press, editors C.A. Batt and P.D. Patel, 2000, pp1769-1775).
  • Some fungal species are known to be resistant to weak acid preservatives such as sorbic acid.
  • yeast Zygosaccharomyces bailii and filamentous fungi such as Aspergillus or Penicillium species.
  • Zygosaccharomyces bailii is a common food spoilage yeast that is extremely resistant to weak acid preservatives such as sorbates or benzoates, tolerating in some cases preservative concentrations well in excess of those permitted legally (See Cole et al, "Probability of Growth of the Spoilage Yeast Zygosaccharomyces bailii in a Model Fruit Drink System," Food Microbiology, 1987, 4, pp. 115-19).
  • natamycin Another important fungicide used in food industry is natamycin. Natamycin has been used for more than 30 years, mainly to prevent growth of moulds and yeasts on cheeses and sausages. Natamycin can also be used for other applications, for example for combating yeasts in beverages. Natamycin has been found to be particularly effective against the sorbic acid tolerant yeasts Zygosaccharomyces bailii. See Shirk & Clark, "The Effect of Pimaricin in Retarding the Spoilage of Fresh Orange Juice," Food Technology, 1963, p 108.
  • Natamycin is poorly soluble in water. In neutral aqueous systems, solubilities of between 30 and 100 ppm have been reported. Under these conditions, most of the natamycin will be present in its crystalline form. Since only the dissolved fraction has antimicrobial activity, this low solubility may limit the effectiveness of natamycin in some applications.
  • the present invention relates to an anti-microbial composition
  • an anti-microbial composition comprising at least one anti-microbial agent and at least one inhibitor of a cytochrome p450 protein. It has been surprisingly found that the combination of these two components together (anti-microbial agent and inhibitor of the cytochrome p450 protein) works synergistically such that the combination is more effective against microbial growth than each agent taken separately.
  • the anti-microbial agent is selected from the group consisting of an anti-fungal agent, an anti-microbial agent and mixture thereof.
  • an anti-microbial agent is defined as an agent which is able to kill a microbe, or to inhibit microbial growth, or to prevent the germination of microbial spores in vitro in a given medium and/or in vivo in a given food product matrix, crop protection application, pharmaceutical or cosmetic product.
  • the microbial agent is a fungus or a bacterium.
  • the anti-microbial agent is an anti-fungal agent. More preferably, the anti-fungal agent is one of the following agents:
  • a weak acid preservative such as sorbic acid, propionic acid, benzoic acid, a p- hydroxybenzoic acids, lactic acid, citric acid, acetic acid or an alkali metal or alkali earth metal salt thereof;
  • a polyene anti-microbial compound preferably natamycin
  • an inorganic acid such as hydrochloric acid
  • imidazole such as imazalil
  • the anti-microbial agent is a weak organic acid preservative and/or natamycine.
  • the weak organic acid preservative may be sorbic acid, propionic acid, benzoic acid, lactic acid, citric acid or an alkali metal or alkali earth metal salt thereof, or mixtures thereof.
  • the anti-microbial agent is sorbic acid, potassium, or calcium sorbate; benzoic acid, sodium, potassium, or calcium benzoate; natamycine or mixtures thereof.
  • the anti-microbial composition comprises as an anti-microbial agent a weak acid preservative and/or natamycin. Even more preferably, the anti-microbial agent in the anti-microbial composition is sorbic acid and/or natamycin.
  • the cytochrome p450 enzymes constitute a large family of proteins involved in the metabolism of many compounds. These monooxygenase enzymes are involved in many bioconversions. They oxidize and thereby inactivate all kind of substrates by inserting an oxygen atom into these molecules.
  • inhibitors of this family of proteins are already well known (see for example www.aeqis.com/pubs/hivplus/1998/sep/p450.html and www.aeqis.com/pubs/hivplus/1998/sep/fail.html).
  • the list of inhibitors presented at these both sites is herewith included by reference in this context.
  • Inhibitors of a cytochrome p450 protein will be referred to herein as the inhibitor.
  • the inhibitor is a food grade inhibitor.
  • a non-exhaustive list of inhibitors is given below:
  • grapefruit juice grapefruit furanocoumarins, bergamottin, grapefruit peel, epoxybergamottin.sevilla orange juice, star fruit juice, citrus fruit juice, lime juice, citrus flavonoids (such as diosmin, naringenin, narengin, rutin).
  • Spices sage, thyme, cloves, St John's Wort, golgenseal.
  • Natural polyphenols resveratrol, epsilon-viniferin, tannic acid, epigallo-catechin gallate, naringenin, quercetin, oleuropein, chlorogenic acid, protocatechuic acid, silybin.
  • Flavonoids from Chinese skullcap Sellaria baicalensis: water extract or wogonin.
  • Natural flavonoids acacetin, disometin, hesperetin, homoeriodictyol, 3,5,7,trihydroxyflavone, 7-hydroxyflavone, flavone, tangeretin, flavonoids from propolis (galangin), flavonoids from hop (Humulus lupulus), xanthohumol (which is prenylated chalchone), 8-prenylnaringenin, isoxanthohumol).
  • phytochemicals phenethyl isothiocyanate, caffeic acid, diosmin, ferulic acid, indole-3-carbinol, phytochemical from natural essential oils (e.g. lemongrass, hop: beta-myrcene).
  • TMP/SMX (Bactrim, Spectra), erythromycin, clarithromycin, ketoconazole, itraconazole, flucanazole, cimetidine, lovastatin, vinblastin, proadifen.
  • the inhibitor that can be used in the anti-microbial composition of the invention is not limited to those mentioned in this list. Additionally, other inhibitors may be isolated by using high-throughput screening assay as earlier described (see Ansede JH, et al, High- throughput screening for stability and inhibitory activity of compounds towards cytochrom p450-mediated metabolism, Journal of Pharmaceutical Sciences, (2004), 93:239-255).
  • the inhibitor is a food-grade inhibitor. More preferably, the inhibitor is tannic acid.
  • Another preferred inhibitor is resveratrol.
  • Resveratrol trans-3,4',5-trihydroxystilbene
  • is a phytoalexin which can, for example, be isolated from grape skins.
  • Other preferred food grade inhibitors are grapefruit juice, citrus flavonoids, natural polyphenols, natural flavonoids, phytochemicals or milk thistle. According to a most preferred embodiment, the inhibitor is tannic acid and/or resveratrol.
  • one of the anti-microbial agents present in the antimicrobial composition is a weak organic acid.
  • Weak organic acids are applied in many food applications.
  • the weak organic acid may be sorbic acid, propionic acid, benzoic acid, a p-hydroxybenzoic acids, lactic acid, citric acid or an alkali metal or alkali earth metal salt thereof or a combination thereof.
  • Sorbates and benzoates can be applied alone or in combination in for example, nonalcoholic flavoured drinks, juices, lemonades, ice tea, beer, spirits, jams, marmalades, candies, fruit and vegetable preparations, dried fruit, fish products, shrimps, cheese, processed cheese, liquid egg, dehydrated concentrated frozen and deep frozen egg products, pre-packed sliced bread, bakery wares, chewing gum, toppings, fat emulsions, sauces, prepared salads, mustard and collagen based casings.
  • sorbates and/or benzoates may be used at a concentration of from 100 to 1000 ppm, more preferably from 200 to 500 ppm. In most other food products these preservatives may be used at a concentration of from 500 to 3000 ppm, more preferably from 1000 to 2000 ppm. In liquid egg the preferred concentration is about 5000 ppm.
  • Propionic acid may be applied in for example, pre-packed bread, pre-packed sliced bread and rye bread, pre-packed bakery wares, puddings and for the surface treatment of cheese and cheese analogues.
  • Propionic acid is preferably used at concentrations of from 1000 to 3000 ppm, more preferably from 2000 to 3000 ppm.
  • one of the anti-microbial agents present in the anti-microbial composition is a compound from the group of polyene anti-microbial compounds, preferably natamycin.
  • Natamycin is a known and commercially available yeast and mould inhibitor that has been used to prevent the growth of yeasts and moulds in various food products, such as cheese, sausage and juices. Natamycin may also be used for pharmaceutical or cosmetic applications and can be used for combating fungi on agricultural products (pre- and post-harvest). Natamycin may be referred to by other names such as pimaricin, antibiotic A 5283, tennecetin, CL 12625, Mycophyt, Myprozine, Natacyn and Pimafucin, all of which are collectively referred to as "natamycin" for the purposes of the present invention.
  • the term "natamycin” as used herein also includes any compounds having substantially the same chemical structure as natamycin, for example, compounds produced by chemical synthesis or biotechnology, provided that such compounds have essentially the same mould and yeast inhibition properties.
  • Natamycin is available under various trademarks, for example, from DSM under the trademark DELVOCID®., Delvocid®- Dip, Premi®Nat, Actistab® and from Danisco under the trademark NATAMAX®.
  • natamycin is usually applied via the plastic cheese coating. Most cheeses are treated from 3 to 5 times with a coating containing from 100 to 750 ppm of natamycin.
  • Coatings applied on dry sausages can be soaked in a suspension containing from 1000 to 2000 ppm of natamycin
  • Natamycin can also be applied by dipping or spraying.
  • the dipping or spraying suspension usually contains from 1 to 5 gram natamycin per litre of water.
  • the final concentration of natamycin on the surface of cheeses and sausages is preferably from 0.2 to 1.5, more preferably from 0.5 to1.0 mg / dm 2 .
  • one of the anti-microbial agents present in the anti-microbial composition is sulphur dioxide and/or a sulphite.
  • Preservatives belonging to this group of compounds may be used for the preservation of for example, wines, beer, juices, vegetables, processed fruits and vegetables, dried fruits, meat, sausages, dried fish, processed potatoes and cereals.
  • preservatives may be used at a concentration of from 20 to 2000 ppm. For most food products the preferred concentration is from 50 to 250 ppm.
  • one of the anti-microbial agents present in the anti-microbial composition is a nitrate and/or a nitrite, which may be ⁇ plied at concentrations of from 100 to 2000 ppm, more preferably from 150 to 500 ppm to prevent microbial spoilage of, for example meat products and cheeses.
  • one of the anti-microbial agents present in the anti-microbial composition is dimethyl dicarbonate.
  • Dimethyl dicarbonate may be applied in beverages such as lemonades, juices, alcohol-free wine and liquid tea.
  • the preferred concentration is about 250 ppm.
  • one of the anti-microbial agents present in the anti-microbial composition is biphenyl, diphenyl, orthophenyl phenol or its salt. These compounds may be applied on the surface of citrus fruits, for example.
  • the preferred concentration is from 10 to 100 mg / kg.
  • the anti-microbial composition of the invention comprises as anti-microbial agent at least one anti-fungal agent and/or at least one antibacterial agent and at least one inhibitor of a cytochrome p450 protein.
  • Preferred antibacterial agents are bacteriocins such as nisin, pediocin, reuterin and sakarin; compositions containing bacteriocins such as products obtained by fermenting milk products or dextrose using lactic acid - or propionic acid bacteria. Examples of such products are known under the commercial name of MicroGard® (Danisco) and DURA Fresh® (Kerry); products composed of living lactic acid cultures producing bacteriocins during the fermentation of e.g.
  • a prefered embodiment of the invention is an anti-microbial composition comprising a combination of natamycin and/or nisin or pediocin or lysozyme and at least one inhibitor of a cytochrome p450 protein.
  • Nisin is a peptide-like antibacterial substance produced by microorganisms such as Lactococcus lactis subsp. lactis. It is mainly active against gram-positive bacteria. Nisin is non-toxic and is free of side effects. Nisin is a Generally Recognized as Safe (GRAS) substance and is widely used in a variety of foods. Examples of such products are processed cheese, milk, clotted cream, dairy desserts, ice cream mixes, liquid egg, hot- baked flour products, dressings and beer. Nisin is heat-stable and can stand sterilization temperatures with minimal loss of activity.
  • GRAS Generally Recognized as Safe
  • nisin The World Health Organization Committee on Biological Standardization has established an international reference preparation of nisin, the International Unit (IU hereinafter). Delvoplus® and Nisaplin®, brand names for nisin concentrates are distributed respectively by DSM and Danisco. Delvoplus® and Nisaplin® contain 2.5% of pure nisin or 1 million IU per gram. Effective levels of nisin to preserve food products range from 10 to 800 lU/g or 0.25 to 20 ppm of nisin.
  • anti-microbial agents may cause quality losses such as off-flavours or decolouration to the food product treated with these anti-microbial agents.
  • anti-microbial agents which can cause off- flavours are weak organic acids, such as sorbates and benzoates.
  • levels of anti-microbial compounds, which are below the taste threshold are also typically sub-optimal levels, i.e. levels below the normally optimal range for the given anti-microbial compound.
  • each of the components in these anti-microbial compositions is present at levels below the taste threshold of the respective anti-microbial compound. This avoids off-flavours being imparted.
  • the anti-microbial composition comprises a mixture of an anti-microbial agent and an inhibitor such that (due to the presence of the inhibitor) the amount of anti-microbial agent needed to obtain an inhibition of growth of microbe of at least 10% when measured according to the following microtiterplate assay is reduced by at least 10% by comparison to the amount of anti-microbial agent needed to obtain the same inhibition of microbial growth when the inhibitor is not present.
  • the microtiterplate assay is the following, Yeast cells or fungal spore or bacterial cell suspensions are prepared using well-known methods. A known amount of these cell /spore suspensions are added to microtiterplates containing an appropriate growth medium, such as Potato Dextrose Broth. The anti-microbial agents and / or the cytochrome p450 inhibitors are added to this medium in known concentrations. After incubation at an appropriate temperature, such as 25 °C, the Minimal Inhibitory Concentration (MIC) is determined visually by observing turbidity of the growth medium. The lowest concentration at which no growth is observed is the MIC.
  • an appropriate temperature such as 25 °C
  • the amount of inhibitor used is such that the amount of anti-microbial agent needed to obtain a inhibition of growth of microbe of at least 30% (measured with the same microtiterplate assay) is reduced by at least 10%, preferably by at least 20%, preferably by at least 30%, more preferably by at least 40%, even more preferably by at least 50% and most preferably by at least 60%.
  • the anti-microbial composition comprises a weak acid preservative and/or natamycin and a food-grade inhibitor. Even more preferably, the composition comprises sorbic acid, and/or natamycin and a food-grade inhibitor of a cytochrome p450 protein. Even more preferably, the anti-microbial composition comprises:
  • the product treated with the anti-microbial composition of the invention comprises from 2 to 400 ⁇ M of the inhibitor, more preferably from 5 to 350 ⁇ M of the inhibitor, even more preferably from 7 to 330 ⁇ M of the inhibitor and most preferably from 10 to 300 ⁇ M of the inhibitor.
  • the product treated with the anti-microbial composition of the invention comprises from 30 to 3000 ppm of the anti-microbial agent and from 20 to 1000 ⁇ M of the inhibitor. All amounts, parts ratios and percentages used herein are by weight unless otherwise specified. According to a more preferred embodiment, the product treated with the anti-microbial composition of the invention comprises from 40 to 2000 ppm of the anti-microbial agent and from 25 to 300 ⁇ M of the inhibitor, even more preferably from 50 to 1500 ppm of the anti-microbial agent and from 30 to 200 ⁇ M of the inhibitor, most preferably from 50 to 1000 ppm of the anti-microbial agent and from 50 to 200 ⁇ M of the inhibitor.
  • the product is treated with the anti-microbial composition of the invention; said composition comprising from 0.5 to 200 ppm of the anti-microbial agent natamycin and from 25 to 300 ⁇ M of the inhibitor, even more preferably from 0.5 to 100 ppm of the anti-microbial agent natamycin and from 30 to 300 ⁇ M of the inhibitor, most preferably from 0.5 to 25 ppm of the anti-microbial agent natamycin and from 50 to 300 ⁇ M of the inhibitor.
  • the product is treated with the anti-microbial composition of the invention; said composition comprising from 100 to 5000 ppm of the anti-microbial agent sorbate and from 25 to 300 ⁇ M of the inhibitor, even more preferably from 100 to 2500 ppm of the anti-microbial agent sorbate and from 30 to 300 ⁇ M of the inhibitor, most preferably from 100 to 1000 ppm of the anti-microbial agent sorbate and from 50 to 300 ⁇ M of the inhibitor.
  • the product treated with the anti-microbial composition of the invention comprises from 30 to 3000 ppm of the anti-microbial agent and from 20 to 1000 ⁇ M of the added inhibitor.
  • the product treated with the anti-microbial composition of the invention comprises from 40 to 2000 ppm of the anti-microbial agent and from 25 to 300 ⁇ M of the added inhibitor, even more preferably from 50 to 1500 ppm of the anti-microbial agent and from 30 to 200 ⁇ M of the added inhibitor, most preferably from 50 to 1000 ppm of the antimicrobial agent and from 50 to 200 ⁇ M of the added inhibitor.
  • the product already containing an endogenous inhibitor is treated with the anti-microbial composition of the invention; said product comprises from 2 to 400 ⁇ M of the inhibitor, more preferably from 5 to 350 ⁇ M of the inhibitor, even more preferably from 7 to 330 ⁇ M of the inhibitor and most preferably from 10 to 300 ⁇ M of the inhibitor.
  • the product treated with the anti-microbial composition of the invention comprises from 0.25 to 5000 ppm of the antibacterial agent.
  • the product treated with nisine comprises from 0.25 to 50 ppm of nisine, more preferably from 0.50 to 30 ppm of nisine, and most preferably from 1 to 20 ppm of nisin.
  • the product is treated with the anti-microbial composition of the invention; said composition comprising from 0.25 to 50 ppm of the anti-microbial agent nisin and from 2 to 200 ⁇ M of the inhibitor, even more preferably from 0.50 to 30 ppm of the anti-microbial agent nisin and from 4 to 150 ⁇ M of the inhibitor, most preferably from 1 to 20 ppm of the anti-microbial agent nisin and from 5 to 130 ⁇ M of the inhibitor.
  • the product treated with the anti-microbial composition of the invention comprises: (a) from 0.5 ppm to 25 ppm of natamycine and/or (b) from 0.25 to 50 ppm nisine and (c) from 10 ⁇ M to 300 ⁇ M of an inhibitor of a cytochrome p450 protein.
  • the anti-microbial composition of the present invention can be used to treat a wide variety of food- and feed products such as cheese, shredded cheese, yoghurt, butter, processed meat products, sausages, cereals, vegetables, fruits, fruit products and ready to use meals.
  • the anti-microbial composition may also be used for the treatment of beverages such as fruit juices, lemonades, ice-tea, wine and beer.
  • Agricultural applications such as spraying in the field or in greenehouses or post-harvest treatment is also included in this invention. Examples of crops are cereals, fruits, vegetables, beans, nuts, herbs, flowers and plants. Also seeds, flower bulbs and seed potatoes can be treated with the anti-microbial composition of this invention.
  • pharmaceutical or cosmetic applications are compositions for topical applications, lotions, creams, ointments, shampoos and soaps.
  • Fungal species which are less susceptible to anti-microbial agents such as natamycin and organic acids such as sorbates are found among species belonging to the genus Penicillium and Aspergillus. Specific yeast strains of Saccaromyces and Zygosaccharomyces are known to be less sensitive towards organic acids such as sorbates. These species may grow on products such as cheeses, sausages, fruit products or in beverages. An example of a species which may cause spoilage problems in cheese industry is Penicillium discolor. This mould can grow on cheese if the relative humidity in a cheese factory is kept at a too high level. Also shortly after production, when the moisture content of cheese is high, this fungus may develop.
  • the anti-microbial composition of this invention can be added to the cheese coating in a concentration of 100-1000 ppm of natamycin, more preferably 200-500 ppm.
  • the cheeses can also be treated by spraying or dipping with a natamycin suspension containing 500-5000 ppm of natamycin, more preferably 1000-3000 ppm of natamycin. All compositions contain a cytochrome p450 inhibitor at a concentration of 30-400 ⁇ M, more preferably 100-300 ⁇ M.
  • fermented sausages are produced and stored at conditions favourable for fungal growth.
  • the composition according to this invention may be used to protect fermented meat products such as sausages against spoilage by moulds. Natamycin and cytochrome p450 inhibitors may be applied in casings as described for cheese coatings, alternatively also sausages can be sprayed or dipped as described for cheese.
  • the anti-microbial composition of this invention can also be used to prevent fungal spoilage of fruits. Fruits such as strawberries, raspberries and cranberries are extremely sensitive for fungal spoilage, both pre-and after harvest.
  • the beverage product is a non-alcoholic product, for example a fruit juice.
  • the juice can be any fruit juice, which is known for use in dilute juice beverages.
  • Beverages according to the present invention can also be formulated to contain milk solids. These milk solids can be derived from various sources including whole milk, skimmed milk, condensed milk, and dried milk powder. Beverages can comprise thickeners, such as xanthan gum, carboxymethylcellulose, propylene glycol alginate, gellan gum, guar gum, pectin, tragacanth gum, gum acacia, locust bean gum, gum arabic, gelatin, as well as mixtures of these thickeners.
  • the beverages of the present invention can, and typically will, contain an effective amount of one or more sweeteners, including carbohydrate sweeteners and natural and/or artificial no/low calorie sweeteners.
  • the beverages of the present invention can comprise other optional beverage ingredients, including other preservatives (e.g. organic acids), colorants and so forth.
  • Minerals that can be included in beverages of the present invention include calcium, magnesium, zinc, iodine, and copper.
  • the beverage is ice tea.
  • the beverage is a carbonated beverage.
  • This carbonated beverage can contain fruit juice such as blueberry and optionally sweeteners.
  • the anti-microbial composition is first prepared and then subsequently added to the food product at a given stage of its preparation and/or in the finished food product depending on the type of food product envisaged.
  • each component of the anti-microbial composition may be added separately at the same stage of the preparation of the food product and/or at alternative stages of preparation of the food product, depending on the type of food product envisaged.
  • a preferred method for making dilute juice beverages according to the present invention is as follows.
  • the anti-microbial agent and the inhibitor are added to a juice concentrate used to formulate the beverage in amounts sufficient to provide the above indicated levels of these anti-microbial components tiHhe single strength dilute juice beverage.
  • the juice concentrate comprising these anti-microbial components is then mixed, blended or otherwise combined with a source of water at the appropriate ratio to provide a single strength dilute juice beverage.
  • the present invention also relates to the use of the anti-microbial composition of the invention for the prevention of fungal growth in a food product.
  • Example 1 Three known inhibitors, erythromycin, proadifen and tannic acid increase the sensitivity of fungi to sorbate.
  • erythromycin Three known inhibitors, erythromycin, proadifen and tannic acid increase the sensitivity of fungi to sorbate.
  • cytochrome p450 inhibitors were tested to examine if inhibiting the cytochrome p450 enzyme system in fungi would increase the sensitivity of fungi to the anti-fungal agent sorbate.
  • the following compounds were examined: - the anti-fungal agent sorbate (potassium sorbate from Aldrich, lot nr.S04495- 084, 99+%); - the cytochrome p450 inhibitors proadifen (proadifen hydrochloride from Sigma, lot nr.053K1235, min.95%), erythromycin (from Sigma, lot nr. 062K1518, USP specifications) and tannic acid (from Riedel de Haen, tannic acid powder puriss. Lot nr. 30500, USP specification); - combinations of sorbate with each of the cytochrome p450 inhibitors. After incubation, the results were visually read on growth / no growth (turbidity).
  • Example 2 Increase of sensitivity of S.cerevisiae towards sorbate when the cytochrome p450 inhibitor proadifen was applied
  • the cytochrome p450 inhibitor proadifen was tested in PDB medium to examine if its presence in an anti-microbial composition comprising sorbate would increase the sensitivity of S. cerevisiaeXo sorbate.
  • the cells were prepared as described in example 1. In all cases the amount of colony forming units / ml in this experiment was 10 4 . The experiment was further executed as described in example 1. The MIC for sorbate of S.
  • proadifen was also determined in apple juice (Euroshopper of Albert Heijn, The Netherlands) a food product in which moulds easily develop.
  • the experiment was executed as described above, this time with apple juice as growth medium.
  • proadifen did not have any inhibiting effect at a concentration of 1500 ⁇ M. It was demonstrated that the MIC for sorbate was reduced to 800 ppm when 300 ⁇ M of proadifen was added.
  • Example 3 Increase of sensitivity of P. roqueforti towards sorbate when the cytochrome p450 inhibitors proadifen, tannic acid or resveratrol were applied
  • the cytochrome p450 inhibitors proadifen and tannic acid were tested in PDB medium to examine if this would increase the sensitivity of P. roqeforti to sorbate.
  • the spore suspension was prepared as described in example 1.
  • Example 4 Increase of sensitivity of A. niger towards sorbate when the cytochrome p450 inhibitors proadifen or tannic acid were applied
  • the cytochrome p450 inhibitors proadifen and tannic acid were tested in PDB medium to examine if this would increase the sensitivity of A. niger to sorbate. The experiment was executed as described in example 3.
  • the MIC for sorbate of A niger after 96 hours of hcubation was 5000 ppm, proadifen did not have any inhibiting effect at a concentration of 800 ⁇ M; tannic acid did not have any inhibiting effect at a concentration of 1500 ⁇ M. It was demonstrated that A niger has a MIC of 2200 ppm when 200 ⁇ M of proadifen was added. In case of adding 300 ⁇ M of proadifen the MIC was further reduced to 1400 ppm.
  • the MIC for sorbate of A niger in this food matrix after 96 hours of incubation was 1400 ppm, while both proadifen and tannic acid showed no inhibiting effect at a concentration up to 1500 ⁇ M.
  • the cytochrome p450 inhibitor tannic acid was tested in PDB medium to examine if this would increase the sensitivity of S. cerevisiae to natamycin.
  • the experiment was executed as described in example 2.
  • the MIC for natamycin of S. cerevisiae after 72 hours of incubation was 1.25 ppm, tannic acid did not have any inhibiting effect at a concentration of 400 ⁇ M. It was demonstrated that S. cerevisiae has a MIC of 0.1-0.4 ppm when 100 ⁇ M of tannic acid was added. In case of adding 300 ⁇ M of tannic acid no growth was observed during 72 hours.
  • Example 6 Increase of sensitivity of P. roqueforti towards natamycin when the cytochrome p450 inhibitors tannic acid and proadifen were applied
  • Example 7 Increase of sensitivity of A. niger towards natamycin when the cytochrome p450 inhibitors tannic acid or resveratrol were applied
  • the cytochrome p450 inhibitors tannic acid and resveratrol were tested in PDB medium to examine if this would increase the sensitivity of A niger to natamycin. The experiment was executed as described in example 4.
  • the MIC for natamycin of Aniger after 72 hours of incubation was 2.5 ppm, tannic acid did not have any inhibiting effect at a concentration of 800 ⁇ M, resveratrol at 1500 ⁇ M. It was demonstrated that A.niger has a MIC of 2.0 ppm when 300 ⁇ M of tannic acid was added. In case of adding 300 ⁇ M of resveratrol the MIC was reduced to 1.75 ppm.
  • Example 8 Increase of sensitivity of Bacillus subtilis towards nisin when the cytochrome p450 inhibitors proadifen or reveratrol were applied
  • the cytochrome p450 inhibitors proadifen and resveratrol were tested to examine if this would increase the sensitivity of Bacillus subtilis to the antibacterial agent nisin.
  • a fresh spore suspension of Bacillus subtilis ATCC32234 was prepared in Iso Sensitest Broth (from Oxoid lot nr. 369978) using well-known methods. A stock suspension containing 5.7 x 10 4 spores/ml was obtained.
  • the minimal inhibitory concentrations of the cytochrome p450 inhibitors were determined as described in example 1. However, in this experiment the microtiterplates were incubated at 30°C for 48 hours in Iso Sensitest Broth (the pH was 6.5). It was found that the cytochrome p450 inhibitors proadifen and resveratrol at concentrations of respectively >100 ⁇ M and >1000 ⁇ M could not inhibit the growth of the Bacillus. The minimum inhibitory concentration of nisin was 10 ppm. It was demonstrated that the minimal inhibitory concentration for nisin of B.
  • subtilis after 48 hours of incubation was 7.5 ppm when 10 ⁇ M of proadifen was added and 5 ppm when 100 ⁇ M of resveratrol was added.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Nutrition Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Botany (AREA)
  • Food Preservation Except Freezing, Refrigeration, And Drying (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

The present invention provides an anti-microbial composition comprising at least one anti-microbial agent and at least one inhibitor of a cytochrome p450 protein. The present invention further provides a food product comprising an anti-microbial composition of the invention.

Description

ANTI-MICROBIAL COMPOSITION FOR FOOD PRODUCTS
Field of the invention
The present invention relates to anti-microbial compositions useful in food products susceptible to food spoilage by microorganisms.
Background of the invention
The prevention of fungal growth is an important issue in the food and agricultural industry. Worldwide economic loss due to fungal spoilage is considerable. Products ripened in the open air, such as cheese and dry sausages are always susceptible to fungal contamination. However, other products, such as fresh fruits, fruit products, nuts, cereals, beverages, meat products, bakery products, crops on the field or after harvesting and many other products will also grow mould very easily. In spite of optimal hygiene and processing conditions, some products remain sensitive to fungal growth. Effective techniques, such as sterilization or modified atmosphere packaging, cannot be used to prevent spoilage of all food products. Often the -use of an anti-microbial agent is the only way to prevent spoilage. — Weak acid preservatives, such as sorbic acid, propionic acid, benzoic acid, p- hydroxybenzoic acids, lactic acid, citric acid or an alkali metal or alkali earth metal salt thereof have been used in a wide range of products, such as dairy products, bakery products, fruit, fruit products, beverages, meat and fish. For example, potassium sorbate can be used in the range of from about 200 to more then 5000 ppm (for reviews see Antimicrobials in food, 1993, P.M. Davidson and A.L. Branen editors, pp49-95 and Encyclopedia of food microbiology, Academic Press, editors C.A. Batt and P.D. Patel, 2000, pp1769-1775).
Some fungal species are known to be resistant to weak acid preservatives such as sorbic acid. Of particular concern are the yeast Zygosaccharomyces bailii and filamentous fungi such as Aspergillus or Penicillium species. Zygosaccharomyces bailii is a common food spoilage yeast that is extremely resistant to weak acid preservatives such as sorbates or benzoates, tolerating in some cases preservative concentrations well in excess of those permitted legally (See Cole et al, "Probability of Growth of the Spoilage Yeast Zygosaccharomyces bailii in a Model Fruit Drink System," Food Microbiology, 1987, 4, pp. 115-19).
Another important fungicide used in food industry is natamycin. Natamycin has been used for more than 30 years, mainly to prevent growth of moulds and yeasts on cheeses and sausages. Natamycin can also be used for other applications, for example for combating yeasts in beverages. Natamycin has been found to be particularly effective against the sorbic acid tolerant yeasts Zygosaccharomyces bailii. See Shirk & Clark, "The Effect of Pimaricin in Retarding the Spoilage of Fresh Orange Juice," Food Technology, 1963, p 108.
Natamycin is poorly soluble in water. In neutral aqueous systems, solubilities of between 30 and 100 ppm have been reported. Under these conditions, most of the natamycin will be present in its crystalline form. Since only the dissolved fraction has antimicrobial activity, this low solubility may limit the effectiveness of natamycin in some applications.
The use of most permitted preservatives is limited by regulation. Also, in general there is a need to reduce the amount of additives used in the food industry.
Due to the technological and/or regulatory limitations of the present anti-microbial agents, there is still a need for improved preservation systems for the food and agricultural industries.
Detailed description of the invention
The present invention relates to an anti-microbial composition comprising at least one anti-microbial agent and at least one inhibitor of a cytochrome p450 protein. It has been surprisingly found that the combination of these two components together (anti-microbial agent and inhibitor of the cytochrome p450 protein) works synergistically such that the combination is more effective against microbial growth than each agent taken separately. The anti-microbial agent is selected from the group consisting of an anti-fungal agent, an anti-microbial agent and mixture thereof.
In the context of this invention, an anti-microbial agent is defined as an agent which is able to kill a microbe, or to inhibit microbial growth, or to prevent the germination of microbial spores in vitro in a given medium and/or in vivo in a given food product matrix, crop protection application, pharmaceutical or cosmetic product. Preferably, the microbial agent is a fungus or a bacterium.
According to a preferred embodiment, the anti-microbial agent is an anti-fungal agent. More preferably, the anti-fungal agent is one of the following agents:
1 . a weak acid preservative such as sorbic acid, propionic acid, benzoic acid, a p- hydroxybenzoic acids, lactic acid, citric acid, acetic acid or an alkali metal or alkali earth metal salt thereof;
2. a polyene anti-microbial compound, preferably natamycin;
3. sulphur dioxide or sulphites;
4. nitrate and nitrite;
5. dimethyl dicarbonate;
6. biphenyl, diphenyl, orthophenylphenol or thiobendazole ;
7. an inorganic acid, such as hydrochloric acid;
8. an imidazole such as imazalil; and/or
9. any anti-microbial compound known in the art for use as a preservative for food products, crop protection or after-harvest treatment of fruits, vegetables or cereals, pharmaceutical or cosmetic products.
Preferably the anti-microbial agent is a weak organic acid preservative and/or natamycine. The weak organic acid preservative may be sorbic acid, propionic acid, benzoic acid, lactic acid, citric acid or an alkali metal or alkali earth metal salt thereof, or mixtures thereof. According to a more preferred embodiment, the anti-microbial agent is sorbic acid, potassium, or calcium sorbate; benzoic acid, sodium, potassium, or calcium benzoate; natamycine or mixtures thereof.
According to another more preferred embodiment, the anti-microbial composition comprises as an anti-microbial agent a weak acid preservative and/or natamycin. Even more preferably, the anti-microbial agent in the anti-microbial composition is sorbic acid and/or natamycin.
The cytochrome p450 enzymes constitute a large family of proteins involved in the metabolism of many compounds. These monooxygenase enzymes are involved in many bioconversions. They oxidize and thereby inactivate all kind of substrates by inserting an oxygen atom into these molecules.
Several inhibitors of this family of proteins are already well known (see for example www.aeqis.com/pubs/hivplus/1998/sep/p450.html and www.aeqis.com/pubs/hivplus/1998/sep/fail.html). The list of inhibitors presented at these both sites is herewith included by reference in this context. Inhibitors of a cytochrome p450 protein will be referred to herein as the inhibitor. Preferably the inhibitor is a food grade inhibitor. A non-exhaustive list of inhibitors is given below:
- «¥ uit: grapefruit juice, grapefruit furanocoumarins, bergamottin, grapefruit peel, epoxybergamottin.sevilla orange juice, star fruit juice, citrus fruit juice, lime juice, citrus flavonoids (such as diosmin, naringenin, narengin, rutin).
- Tea: black-tea theaflavins, herbal thea mixtures, green thea cathecins (epicatechin gallate, epigallocatechin gallate), tannic acid.
- Spices: sage, thyme, cloves, St John's Wort, golgenseal.
- Soybean: daidzain, genistein.
- Natural polyphenols: resveratrol, epsilon-viniferin, tannic acid, epigallo-catechin gallate, naringenin, quercetin, oleuropein, chlorogenic acid, protocatechuic acid, silybin.
- Flavonoids from Chinese skullcap (Scutellaria baicalensis) : water extract or wogonin. - Natural flavonoids: acacetin, disometin, hesperetin, homoeriodictyol, 3,5,7,trihydroxyflavone, 7-hydroxyflavone, flavone, tangeretin, flavonoids from propolis (galangin), flavonoids from hop (Humulus lupulus), xanthohumol (which is prenylated chalchone), 8-prenylnaringenin, isoxanthohumol).
- Phytochemicals from cruciferous vegetables: benzyl isothiocyanate.
- Other phytochemicals: phenethyl isothiocyanate, caffeic acid, diosmin, ferulic acid, indole-3-carbinol, phytochemical from natural essential oils (e.g. lemongrass, hop: beta-myrcene).
- Milk thistle: silymarin.
- Other inhibitors known:TMP/SMX (Bactrim, Spectra), erythromycin, clarithromycin, ketoconazole, itraconazole, flucanazole, cimetidine, lovastatin, vinblastin, proadifen.
The inhibitor that can be used in the anti-microbial composition of the invention is not limited to those mentioned in this list. Additionally, other inhibitors may be isolated by using high-throughput screening assay as earlier described (see Ansede JH, et al, High- throughput screening for stability and inhibitory activity of compounds towards cytochrom p450-mediated metabolism, Journal of Pharmaceutical Sciences, (2004), 93:239-255). Preferably the inhibitor is a food-grade inhibitor. More preferably, the inhibitor is tannic acid. Another preferred inhibitor is resveratrol. Resveratrol (trans-3,4',5-trihydroxystilbene) is a phytoalexin which can, for example, be isolated from grape skins. Other preferred food grade inhibitors are grapefruit juice, citrus flavonoids, natural polyphenols, natural flavonoids, phytochemicals or milk thistle. According to a most preferred embodiment, the inhibitor is tannic acid and/or resveratrol.
According to a preferred embodiment, one of the anti-microbial agents present in the antimicrobial composition is a weak organic acid. Weak organic acids are applied in many food applications. The weak organic acid may be sorbic acid, propionic acid, benzoic acid, a p-hydroxybenzoic acids, lactic acid, citric acid or an alkali metal or alkali earth metal salt thereof or a combination thereof.
Sorbates and benzoates can be applied alone or in combination in for example, nonalcoholic flavoured drinks, juices, lemonades, ice tea, beer, spirits, jams, marmalades, candies, fruit and vegetable preparations, dried fruit, fish products, shrimps, cheese, processed cheese, liquid egg, dehydrated concentrated frozen and deep frozen egg products, pre-packed sliced bread, bakery wares, chewing gum, toppings, fat emulsions, sauces, prepared salads, mustard and collagen based casings.
In beverages, sorbates and/or benzoates may be used at a concentration of from 100 to 1000 ppm, more preferably from 200 to 500 ppm. In most other food products these preservatives may be used at a concentration of from 500 to 3000 ppm, more preferably from 1000 to 2000 ppm. In liquid egg the preferred concentration is about 5000 ppm.
Propionic acid may be applied in for example, pre-packed bread, pre-packed sliced bread and rye bread, pre-packed bakery wares, puddings and for the surface treatment of cheese and cheese analogues. Propionic acid is preferably used at concentrations of from 1000 to 3000 ppm, more preferably from 2000 to 3000 ppm.
According to another preferred embodiment, one of the anti-microbial agents present in the anti-microbial composition is a compound from the group of polyene anti-microbial compounds, preferably natamycin.
Natamycin is a known and commercially available yeast and mould inhibitor that has been used to prevent the growth of yeasts and moulds in various food products, such as cheese, sausage and juices. Natamycin may also be used for pharmaceutical or cosmetic applications and can be used for combating fungi on agricultural products (pre- and post-harvest). Natamycin may be referred to by other names such as pimaricin, antibiotic A 5283, tennecetin, CL 12625, Mycophyt, Myprozine, Natacyn and Pimafucin, all of which are collectively referred to as "natamycin" for the purposes of the present invention. The term "natamycin" as used herein also includes any compounds having substantially the same chemical structure as natamycin, for example, compounds produced by chemical synthesis or biotechnology, provided that such compounds have essentially the same mould and yeast inhibition properties.
Natamycin is available under various trademarks, for example, from DSM under the trademark DELVOCID®., Delvocid®- Dip, Premi®Nat, Actistab® and from Danisco under the trademark NATAMAX®. On cheeses natamycin is usually applied via the plastic cheese coating. Most cheeses are treated from 3 to 5 times with a coating containing from 100 to 750 ppm of natamycin.
Coatings applied on dry sausages can be soaked in a suspension containing from 1000 to 2000 ppm of natamycin
Natamycin can also be applied by dipping or spraying. For cheeses and dry sausages, the dipping or spraying suspension usually contains from 1 to 5 gram natamycin per litre of water.
The final concentration of natamycin on the surface of cheeses and sausages is preferably from 0.2 to 1.5, more preferably from 0.5 to1.0 mg / dm2.
According to another preferred embodiment, one of the anti-microbial agents present in the anti-microbial composition is sulphur dioxide and/or a sulphite. Preservatives belonging to this group of compounds may be used for the preservation of for example, wines, beer, juices, vegetables, processed fruits and vegetables, dried fruits, meat, sausages, dried fish, processed potatoes and cereals.
These preservatives may be used at a concentration of from 20 to 2000 ppm. For most food products the preferred concentration is from 50 to 250 ppm.
According to another preferred embodiment, one of the anti-microbial agents present in the anti-microbial composition is a nitrate and/or a nitrite, which may be φplied at concentrations of from 100 to 2000 ppm, more preferably from 150 to 500 ppm to prevent microbial spoilage of, for example meat products and cheeses.
According to another preferred embodiment, one of the anti-microbial agents present in the anti-microbial composition is dimethyl dicarbonate. Dimethyl dicarbonate may be applied in beverages such as lemonades, juices, alcohol-free wine and liquid tea. The preferred concentration is about 250 ppm.
According to another preferred embodiment, one of the anti-microbial agents present in the anti-microbial composition is biphenyl, diphenyl, orthophenyl phenol or its salt. These compounds may be applied on the surface of citrus fruits, for example. The preferred concentration is from 10 to 100 mg / kg. According to another preferred embodiment, the anti-microbial composition of the invention comprises as anti-microbial agent at least one anti-fungal agent and/or at least one antibacterial agent and at least one inhibitor of a cytochrome p450 protein. Preferred antibacterial agents are bacteriocins such as nisin, pediocin, reuterin and sakarin; compositions containing bacteriocins such as products obtained by fermenting milk products or dextrose using lactic acid - or propionic acid bacteria. Examples of such products are known under the commercial name of MicroGard® (Danisco) and DURA Fresh® (Kerry); products composed of living lactic acid cultures producing bacteriocins during the fermentation of e.g. cheese, yoghurt, sausages or fermented vegetables; organic acids such as propionic acid, acetic acid, sorbic acids or their alkali metal or alkali earth metal salts; lysozyme, sulpur dioxide and sulphites; parabens or esters of p- hydroxy benzoic acids; nitrite; plant essential oils or their compounds such as e.g. thymol, carvacrol, cinnamic acid.
A prefered embodiment of the invention is an anti-microbial composition comprising a combination of natamycin and/or nisin or pediocin or lysozyme and at least one inhibitor of a cytochrome p450 protein.
Anti-microbial composition comprising a bacteriocin will be active against bacteria. Nisin is a peptide-like antibacterial substance produced by microorganisms such as Lactococcus lactis subsp. lactis. It is mainly active against gram-positive bacteria. Nisin is non-toxic and is free of side effects. Nisin is a Generally Recognized as Safe (GRAS) substance and is widely used in a variety of foods. Examples of such products are processed cheese, milk, clotted cream, dairy desserts, ice cream mixes, liquid egg, hot- baked flour products, dressings and beer. Nisin is heat-stable and can stand sterilization temperatures with minimal loss of activity. The World Health Organization Committee on Biological Standardization has established an international reference preparation of nisin, the International Unit (IU hereinafter). Delvoplus® and Nisaplin®, brand names for nisin concentrates are distributed respectively by DSM and Danisco. Delvoplus® and Nisaplin® contain 2.5% of pure nisin or 1 million IU per gram. Effective levels of nisin to preserve food products range from 10 to 800 lU/g or 0.25 to 20 ppm of nisin.
Sometimes even very high concentrations of anti-microbial agents cannot prevent microbe spoilage. This can happen in case of the presence of highly resistant species, high contamination levels and/or limitations of the use of the anti-microbial agents by regulation.
Furthermore, it is known that high concentrations of some anti-microbial agents may cause quality losses such as off-flavours or decolouration to the food product treated with these anti-microbial agents. Examples of anti-microbial agents, which can cause off- flavours are weak organic acids, such as sorbates and benzoates.
Especially, in the case of more tolerant fungi, levels of anti-microbial compounds, which are below the taste threshold are also typically sub-optimal levels, i.e. levels below the normally optimal range for the given anti-microbial compound.
Preferably, each of the components in these anti-microbial compositions is present at levels below the taste threshold of the respective anti-microbial compound. This avoids off-flavours being imparted.
According to a preferred embodiment, the anti-microbial composition comprises a mixture of an anti-microbial agent and an inhibitor such that (due to the presence of the inhibitor) the amount of anti-microbial agent needed to obtain an inhibition of growth of microbe of at least 10% when measured according to the following microtiterplate assay is reduced by at least 10% by comparison to the amount of anti-microbial agent needed to obtain the same inhibition of microbial growth when the inhibitor is not present.
The microtiterplate assay is the following, Yeast cells or fungal spore or bacterial cell suspensions are prepared using well-known methods. A known amount of these cell /spore suspensions are added to microtiterplates containing an appropriate growth medium, such as Potato Dextrose Broth. The anti-microbial agents and / or the cytochrome p450 inhibitors are added to this medium in known concentrations. After incubation at an appropriate temperature, such as 25 °C, the Minimal Inhibitory Concentration (MIC) is determined visually by observing turbidity of the growth medium. The lowest concentration at which no growth is observed is the MIC.
Preferably, the amount of inhibitor used is such that the amount of anti-microbial agent needed to obtain a inhibition of growth of microbe of at least 30% (measured with the same microtiterplate assay) is reduced by at least 10%, preferably by at least 20%, preferably by at least 30%, more preferably by at least 40%, even more preferably by at least 50% and most preferably by at least 60%. According to a most preferred embodiment, the anti-microbial composition comprises a weak acid preservative and/or natamycin and a food-grade inhibitor. Even more preferably, the composition comprises sorbic acid, and/or natamycin and a food-grade inhibitor of a cytochrome p450 protein. Even more preferably, the anti-microbial composition comprises:
- sorbic acid and tannic acid or
- sorbic acid and resveratrol or
- sorbic acid, tannic acid and resveratrol or
- natamycin and tannic acid or
- natamycin and resveratrol or
- natamycin, tannic acid and resveratrol or
- sorbic acid, natamycin and tannic acid or
- sorbic acid, natamycin and resveratrol or
- sorbic acid, natamycin, tannic acid and resveratrol or
- nisin and tannic acid or
- nisin and resveratrol or
- nisin and tannic acid and resveratrol or
- nisin and natamycin and tannic acid or
- nisin and natamycin and resveratrol or
- nisin and natamycin and tannic acid and resveratrol or
- nisin and sorbic acid and tannic acid or
- nisin and sorbic acid and resveratrol or
- nisin and sorbic acid and tannic acid and resveratrol or - nisin and sorbic acid and natamycin and tannic acid or
- nisin and sorbic acid and natamycin and resveratrol or
- nisin and sorbic acid and natamycin and tannic acid and resveratrol.
We define below the product treated with the anti-microbial composition of the invention. The skilled person will understand that depending on the identity of the anti-microbial agent and the identity of the inhibitor used, the amounts of inhibitor and of anti-microbial agent would have to be adjusted to obtain the best result.
According to a preferred embodiment, the product treated with the anti-microbial composition of the invention comprises from 2 to 400 μM of the inhibitor, more preferably from 5 to 350 μM of the inhibitor, even more preferably from 7 to 330 μM of the inhibitor and most preferably from 10 to 300 μM of the inhibitor.
According to a preferred embodiment, the product treated with the anti-microbial composition of the invention comprises from 30 to 3000 ppm of the anti-microbial agent and from 20 to 1000 μM of the inhibitor. All amounts, parts ratios and percentages used herein are by weight unless otherwise specified. According to a more preferred embodiment, the product treated with the anti-microbial composition of the invention comprises from 40 to 2000 ppm of the anti-microbial agent and from 25 to 300 μM of the inhibitor, even more preferably from 50 to 1500 ppm of the anti-microbial agent and from 30 to 200 μM of the inhibitor, most preferably from 50 to 1000 ppm of the anti-microbial agent and from 50 to 200 μM of the inhibitor.
According to another preferred embodiment, the product is treated with the anti-microbial composition of the invention; said composition comprising from 0.5 to 200 ppm of the anti-microbial agent natamycin and from 25 to 300 μM of the inhibitor, even more preferably from 0.5 to 100 ppm of the anti-microbial agent natamycin and from 30 to 300 μM of the inhibitor, most preferably from 0.5 to 25 ppm of the anti-microbial agent natamycin and from 50 to 300 μM of the inhibitor. According to another preferred embodiment, the product is treated with the anti-microbial composition of the invention; said composition comprising from 100 to 5000 ppm of the anti-microbial agent sorbate and from 25 to 300 μM of the inhibitor, even more preferably from 100 to 2500 ppm of the anti-microbial agent sorbate and from 30 to 300 μM of the inhibitor, most preferably from 100 to 1000 ppm of the anti-microbial agent sorbate and from 50 to 300 μM of the inhibitor.
According to a more preferred embodiment, when the food product already contains an endogenous inhibitor (for example wine contains tannic acid by nature), the product treated with the anti-microbial composition of the invention comprises from 30 to 3000 ppm of the anti-microbial agent and from 20 to 1000 μM of the added inhibitor. According to a most preferred embodiment, when the food product already contains an endogenous inhibitor, the product treated with the anti-microbial composition of the invention comprises from 40 to 2000 ppm of the anti-microbial agent and from 25 to 300 μM of the added inhibitor, even more preferably from 50 to 1500 ppm of the anti-microbial agent and from 30 to 200 μM of the added inhibitor, most preferably from 50 to 1000 ppm of the antimicrobial agent and from 50 to 200 μM of the added inhibitor.
According to another preferred embodiment, the product already containing an endogenous inhibitor is treated with the anti-microbial composition of the invention; said product comprises from 2 to 400 μM of the inhibitor, more preferably from 5 to 350 μM of the inhibitor, even more preferably from 7 to 330 μM of the inhibitor and most preferably from 10 to 300 μM of the inhibitor.
According to another preferred embodiment, the product treated with the anti-microbial composition of the invention comprises from 0.25 to 5000 ppm of the antibacterial agent. Preferably, if nisine is the antibacterial agent, the product treated with nisine comprises from 0.25 to 50 ppm of nisine, more preferably from 0.50 to 30 ppm of nisine, and most preferably from 1 to 20 ppm of nisin.
According to another preferred embodiment, the product is treated with the anti-microbial composition of the invention; said composition comprising from 0.25 to 50 ppm of the anti-microbial agent nisin and from 2 to 200 μM of the inhibitor, even more preferably from 0.50 to 30 ppm of the anti-microbial agent nisin and from 4 to 150 μM of the inhibitor, most preferably from 1 to 20 ppm of the anti-microbial agent nisin and from 5 to 130 μM of the inhibitor.
According to a most preferred embodiment, the product treated with the anti-microbial composition of the invention comprises: (a) from 0.5 ppm to 25 ppm of natamycine and/or (b) from 0.25 to 50 ppm nisine and (c) from 10 μM to 300 μM of an inhibitor of a cytochrome p450 protein.
The anti-microbial composition of the present invention can be used to treat a wide variety of food- and feed products such as cheese, shredded cheese, yoghurt, butter, processed meat products, sausages, cereals, vegetables, fruits, fruit products and ready to use meals. The anti-microbial composition may also be used for the treatment of beverages such as fruit juices, lemonades, ice-tea, wine and beer. Agricultural applications such as spraying in the field or in greenehouses or post-harvest treatment is also included in this invention. Examples of crops are cereals, fruits, vegetables, beans, nuts, herbs, flowers and plants. Also seeds, flower bulbs and seed potatoes can be treated with the anti-microbial composition of this invention. Examples of pharmaceutical or cosmetic applications are compositions for topical applications, lotions, creams, ointments, shampoos and soaps.
Fungal species which are less susceptible to anti-microbial agents such as natamycin and organic acids such as sorbates are found among species belonging to the genus Penicillium and Aspergillus. Specific yeast strains of Saccaromyces and Zygosaccharomyces are known to be less sensitive towards organic acids such as sorbates. These species may grow on products such as cheeses, sausages, fruit products or in beverages. An example of a species which may cause spoilage problems in cheese industry is Penicillium discolor. This mould can grow on cheese if the relative humidity in a cheese factory is kept at a too high level. Also shortly after production, when the moisture content of cheese is high, this fungus may develop. The anti-microbial composition of this invention, preferably containing natamycin, can be added to the cheese coating in a concentration of 100-1000 ppm of natamycin, more preferably 200-500 ppm. Alternatively the cheeses can also be treated by spraying or dipping with a natamycin suspension containing 500-5000 ppm of natamycin, more preferably 1000-3000 ppm of natamycin. All compositions contain a cytochrome p450 inhibitor at a concentration of 30-400 μM, more preferably 100-300 μM. Also fermented sausages are produced and stored at conditions favourable for fungal growth. During and just after fermentation fungi may easily develop, especially since at this stage of the production process both the temperature and the relative humidity are high. The composition according to this invention may be used to protect fermented meat products such as sausages against spoilage by moulds. Natamycin and cytochrome p450 inhibitors may be applied in casings as described for cheese coatings, alternatively also sausages can be sprayed or dipped as described for cheese. The anti-microbial composition of this invention can also be used to prevent fungal spoilage of fruits. Fruits such as strawberries, raspberries and cranberries are extremely sensitive for fungal spoilage, both pre-and after harvest. Less spoilage is expected when the fruit is treated with a solution of 500-5000 ppm of sorbate, more preferably 500-2000 ppm of sorbate or a suspension or solution containing 10-250 ppm of natamycin, more preferably 10-100 ppm of natamycin, each anti-microbial composition containing a cytochrome p450 inhibitor at a concentration of 30^400 μM, more preferably 100-300 μM. Also apples, pears and citrus such as oranges can be treated with the anti-microbial composition of this invention. For these fruits e.g. lecithin coatings containing 20-500 ppm of natamycin and a cytochrome p450 inhibitor at a concentration of 30→400 μM, more preferably 100-300 μM can be applied.
According to a preferred embodiment, the beverage product is a non-alcoholic product, for example a fruit juice. The juice can be any fruit juice, which is known for use in dilute juice beverages. Beverages according to the present invention can also be formulated to contain milk solids. These milk solids can be derived from various sources including whole milk, skimmed milk, condensed milk, and dried milk powder. Beverages can comprise thickeners, such as xanthan gum, carboxymethylcellulose, propylene glycol alginate, gellan gum, guar gum, pectin, tragacanth gum, gum acacia, locust bean gum, gum arabic, gelatin, as well as mixtures of these thickeners. The beverages of the present invention can, and typically will, contain an effective amount of one or more sweeteners, including carbohydrate sweeteners and natural and/or artificial no/low calorie sweeteners. The beverages of the present invention can comprise other optional beverage ingredients, including other preservatives (e.g. organic acids), colorants and so forth. Minerals that can be included in beverages of the present invention include calcium, magnesium, zinc, iodine, and copper.
According to a preferred embodiment, the beverage is ice tea. According to another preferred embodiment, the beverage is a carbonated beverage. This carbonated beverage can contain fruit juice such as blueberry and optionally sweeteners.
According to a preferred embodiment, the anti-microbial composition is first prepared and then subsequently added to the food product at a given stage of its preparation and/or in the finished food product depending on the type of food product envisaged. Alternatively, each component of the anti-microbial composition may be added separately at the same stage of the preparation of the food product and/or at alternative stages of preparation of the food product, depending on the type of food product envisaged.
Any method that ensures the anti-microbial composition is incorporated into the food or beverage such that any microorganisms present are effectively killed or their growth is inhibited is suitable. A preferred method for making dilute juice beverages according to the present invention is as follows. The anti-microbial agent and the inhibitor are added to a juice concentrate used to formulate the beverage in amounts sufficient to provide the above indicated levels of these anti-microbial components tiHhe single strength dilute juice beverage. The juice concentrate comprising these anti-microbial components is then mixed, blended or otherwise combined with a source of water at the appropriate ratio to provide a single strength dilute juice beverage.
The present invention also relates to the use of the anti-microbial composition of the invention for the prevention of fungal growth in a food product.
The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention. Examples
Example 1 : Three known inhibitors, erythromycin, proadifen and tannic acid increase the sensitivity of fungi to sorbate In this experiment several known cytochrome p450 inhibitors were tested to examine if inhibiting the cytochrome p450 enzyme system in fungi would increase the sensitivity of fungi to the anti-fungal agent sorbate.
For this experiment freshly cultured Saccharomyces cerevisiae cells (ATCC 62906) and spore suspensions of Aspergillus niger (ATCC 10581) and Penicillium roqueforti (CBS 304.97) were obtained using the following method. A stock suspension containing respectively 5.9 x 107, 4.6 x 104 and 2.6 x 107 colony forming units / ml was prepared in Potato Dextrose Broth (from Difco lot nr. 2157540). Respectively 4, 40 and 400 μl was added to 40 ml of liquid Potato Dextrose Broth medium (Suspensions A, B, C). Stock solutions of potassium sorbate and the cytochrome p450 inhibitors were also prepared in Potato Dextrose Broth. Said solutions were added to the wells of microtiter plates to the final concentrations as mentioned in Tables 1-3. Suspensions A, B and C were added to ttje microtiter plates to a final amount of 200μl in each well. The plates were incubated at 25^. The Minimal Inhibitory Concentration (MIC) was determined visually by observing turbidity of the growth medium. The following compounds were examined: - the anti-fungal agent sorbate (potassium sorbate from Aldrich, lot nr.S04495- 084, 99+%); - the cytochrome p450 inhibitors proadifen (proadifen hydrochloride from Sigma, lot nr.053K1235, min.95%), erythromycin (from Sigma, lot nr. 062K1518, USP specifications) and tannic acid (from Riedel de Haen, tannic acid powder puriss. Lot nr. 30500, USP specification); - combinations of sorbate with each of the cytochrome p450 inhibitors. After incubation, the results were visually read on growth / no growth (turbidity). The results showed that all cytochrome p450 inhibitors at a concentration of 200 μM could not inhibit the growth of the 3 fungal species (see Tables 1 -3). In combination with sorbate all fungi showed a significant increase in sensitivity to sorbate or a delay in growth with the same sorbate sensitivity (see Tables 1 -3).
Table 1 : Effect of sorbate alone or in combination with proadifen or erythromycin on growth of S. cerevisiae
Figure imgf000018_0001
Table 2: Effect of sorbate alone or in combination with proadifen on growth of Aspergillus niger
Figure imgf000018_0002
Table 3: Effect of sorbate alone or in combination with proadifen or tannic acid on growth of P.roqueforti
Figure imgf000018_0003
Example 2: Increase of sensitivity of S.cerevisiae towards sorbate when the cytochrome p450 inhibitor proadifen was applied In this experiment, the cytochrome p450 inhibitor proadifen was tested in PDB medium to examine if its presence in an anti-microbial composition comprising sorbate would increase the sensitivity of S. cerevisiaeXo sorbate. The cells were prepared as described in example 1. In all cases the amount of colony forming units / ml in this experiment was 104. The experiment was further executed as described in example 1. The MIC for sorbate of S. cerevisiae after 96 hours of incubation was 3500 ppm, proadifen did not have any inhibiting effect at a concentration of 300 μM. It was demonstrated that S. cerevisiae has a MIC of 1200 ppm when 100 μM of proadifen was added. In case of adding 300 μM of proadifen the MIC was even reduced to 400 ppm. This result confirms the result obtained in example 1 , Table 1.
The effect of proadifen was also determined in apple juice (Euroshopper of Albert Heijn, The Netherlands) a food product in which moulds easily develop. The experiment was executed as described above, this time with apple juice as growth medium. The MIC for sorbate of S. cerevisiae after 96 hours of incubation in apple juice (pH = 3.5) was 375 ppm, proadifen did not have any inhibiting effect at a concentration of 800 μM. It was demonstrated that the MIC for sorbate was reduced to 250 ppm when 200 μM of proadifen was added When the pH of the apple juice was adjusted to 5.0 the MIC for sorbate of S. cerevisiae after 96 hours of incubation in apple juice was 1400 ppm, proadifen did not have any inhibiting effect at a concentration of 1500 μM. It was demonstrated that the MIC for sorbate was reduced to 800 ppm when 300 μM of proadifen was added.
These results clearly demonstrate that the sensitivity of S. cerevisiae cells towards sorbate was increased when the cytochrome p450 inhibitor proadifen was added to an anti-microbial composition comprising sorbate. Example 3: Increase of sensitivity of P. roqueforti towards sorbate when the cytochrome p450 inhibitors proadifen, tannic acid or resveratrol were applied In this experiment, the cytochrome p450 inhibitors proadifen and tannic acid were tested in PDB medium to examine if this would increase the sensitivity of P. roqeforti to sorbate. The spore suspension was prepared as described in example 1. In all cases the amount of colony forming unitis / ml in this experiment was 104. The experiment was further executed as described in example 1. The MIC for sorbate of P. roqueforti after 96 hours of incubation was 1250 ppm, both proadifen and tannic acid did not have any inhibiting effect at a concentration of 300 μM. It was demonstrated that P. roqueforti has a MIC of 800 ppm when 100 μM of proadifen was added. In case of adding 200 μM of proadifen the MIC was even reduced to 200 ppm. In case 300 μM of proadifen was applied no growth at all was observed during 72 hours. It was further demonstrated that adding 200 μM of tannic acid reduced the MIC value of P. roqueforti towards sorbate from 1250 ppm to 800 ppm. These results confirm the results obtained in example 1 , Table 3.
In an additional experiment, the effect of proadifen and resveratrol, a cytochrome p450 inhibitor isolated from grapes, was examined in the same apple juice (pH adjusted to 5.0) as the one used in example 2, the same way as described in example 2. The MIC for sorbate of P. roqueforti in this food matrix after 96 hours of incubation was 1400 ppm, while both proadifen and resveratrol showed no inhibiting effect at a concentration up to 1500 μM. It was demonstrated that adding 300 μM of proadifen or resveratrol reduced the MIC value of P. roqueforti towards sorbate from 1400 ppm to 900 ppm.
These results clearly demonstrate that the sensitivity of P. roqueforti towards sorbate was increased when the cytochrome p450 inhibitor proadifen, tannic acid or resveratrol was added to an anti-microbial composition comprising sorbate. Example 4: Increase of sensitivity of A. niger towards sorbate when the cytochrome p450 inhibitors proadifen or tannic acid were applied In this experiment, the cytochrome p450 inhibitors proadifen and tannic acid were tested in PDB medium to examine if this would increase the sensitivity of A. niger to sorbate. The experiment was executed as described in example 3. The MIC for sorbate of A niger after 96 hours of hcubation was 5000 ppm, proadifen did not have any inhibiting effect at a concentration of 800 μM; tannic acid did not have any inhibiting effect at a concentration of 1500 μM. It was demonstrated that A niger has a MIC of 2200 ppm when 200 μM of proadifen was added. In case of adding 300 μM of proadifen the MIC was further reduced to 1400 ppm.
It was demonstrated that adding 200 μM of tannic acid reduced the MIC value of A. niger towards sorbate from 5000 ppm to 4000 ppm.
In an additional experiment, the effect of proadifen and tannic acid were examined in the same apple juice as the one used in example 2 (pH adjusted to 5.0) the same way as described in example 2.
The MIC for sorbate of A niger in this food matrix after 96 hours of incubation was 1400 ppm, while both proadifen and tannic acid showed no inhibiting effect at a concentration up to 1500 μM.
It was demonstrated that adding 300 μM of proadifen reduced the MIC value of A niger towards sorbate to 900 ppm, while adding 300 μM of tannic acid reduced the MIC value to
1200 ppm.
These results clearly demonstrate that the sensitivity of A niger towards sorbate was increased when the cytochrome p450 inhibitor proadifen or tannic acid was added to an anti-microbial composition comprising sorbate. Example 5: Increase of sensitivity of S. cerevisiae towards natamycin when the cytochrome p450 inhibitor tannic acid was applied
In this experiment, the cytochrome p450 inhibitor tannic acid was tested in PDB medium to examine if this would increase the sensitivity of S. cerevisiae to natamycin. The experiment was executed as described in example 2. The MIC for natamycin of S. cerevisiae after 72 hours of incubation was 1.25 ppm, tannic acid did not have any inhibiting effect at a concentration of 400 μM. It was demonstrated that S. cerevisiae has a MIC of 0.1-0.4 ppm when 100 μM of tannic acid was added. In case of adding 300 μM of tannic acid no growth was observed during 72 hours.
These results clearly demonstrate that the sensitivity of S. cerevisiae towards natamycin was increased when the cytochrome p450 inhibitor tannic acid was added to an antimicrobial composition comprising natamycin.
Example 6: Increase of sensitivity of P. roqueforti towards natamycin when the cytochrome p450 inhibitors tannic acid and proadifen were applied
In this experiment, the cytochrome p450 inhibitors tannic acid and proadifen were tested in PDB medium to examine if this would increase the sensitivity of P. roqueforti to natamycin. The experiment was executed as described in example 4. The MIC for natamycin of P. roqueforti after 72 hours of incubation was 3.0 ppm. We concluded that tannic acid did not have any inhibiting effect at a concentration of 800 μM. The same holds true for proadifen when used at 400 μM. It was demonstrated that P. roqueforti has a MIC of 2.0 ppm when 100 μM of tannic acid was added. In case of adding 200 μM of proadifen the MIC was reduced to 1.25 ppm; at a concentration of 300 μM of proadifen no growth was observed during 48 hours.
These results clearly demonstrate that the sensitivity of P. roqueforti towards natamycin was increased when the cytochrome p450 inhibitor tannic acid or proadifen was added to an anti-microbial composition comprising natamycin. Example 7: Increase of sensitivity of A. niger towards natamycin when the cytochrome p450 inhibitors tannic acid or resveratrol were applied In this experiment the cytochrome p450 inhibitors tannic acid and resveratrol were tested in PDB medium to examine if this would increase the sensitivity of A niger to natamycin. The experiment was executed as described in example 4. The MIC for natamycin of Aniger after 72 hours of incubation was 2.5 ppm, tannic acid did not have any inhibiting effect at a concentration of 800 μM, resveratrol at 1500 μM. It was demonstrated that A.niger has a MIC of 2.0 ppm when 300 μM of tannic acid was added. In case of adding 300 μM of resveratrol the MIC was reduced to 1.75 ppm.
These results clearly demonstrate that the sensitivity of A. n/ger towards natamycin was increased when the cytochrome p450 inhibitor tannic acid or resveratrol was added to an anti-microbial composition comprising natamycin.
Example 8: Increase of sensitivity of Bacillus subtilis towards nisin when the cytochrome p450 inhibitors proadifen or reveratrol were applied In this experiment, the cytochrome p450 inhibitors proadifen and resveratrol were tested to examine if this would increase the sensitivity of Bacillus subtilis to the antibacterial agent nisin. For this experiment a fresh spore suspension of Bacillus subtilis ATCC32234 was prepared in Iso Sensitest Broth (from Oxoid lot nr. 369978) using well-known methods. A stock suspension containing 5.7 x 104 spores/ml was obtained. The minimal inhibitory concentrations of the cytochrome p450 inhibitors were determined as described in example 1. However, in this experiment the microtiterplates were incubated at 30°C for 48 hours in Iso Sensitest Broth (the pH was 6.5). It was found that the cytochrome p450 inhibitors proadifen and resveratrol at concentrations of respectively >100 μM and >1000 μM could not inhibit the growth of the Bacillus. The minimum inhibitory concentration of nisin was 10 ppm. It was demonstrated that the minimal inhibitory concentration for nisin of B. subtilis after 48 hours of incubation was 7.5 ppm when 10 μM of proadifen was added and 5 ppm when 100 μM of resveratrol was added. These results clearly show that the cytochrome p450 inhibitors, at concentrations were these compounds do not have any inhibiting effect, reduce the minimal inhibitory concentration of Bacillus subtilis for nisin considerably.

Claims

1. An anti-microbial composition comprising at least one anti-microbial agent and at least one inhibitor of a cytochrome p450 protein.
2. An anti-microbial composition according to claim 1, wherein the anti-microbial agent is selected from the group consisting of an anti-bacterial, an anti-microbial agent and mixtures thereof.
3. An anti-microbial composition according to claim 2, wherein the anti-microbial agent is a weak acid preservative and/or natamycine.
4. An anti-microbial composition according to claim 3, wherein the weak acid preservative is sorbic acid, propionic acid, benzoic acid, a phydroxybenzoic acids, lactic acid, citric acid or an alkali metal or alkali earth metal salt thereof, or mixtures thereof.
- i
5. An anti-microbial composition according to any one of claims 1 to 4, wherein the inhibitor of a cytochrome p450 protein is a food grade inhibitor.
6. An anti-microbial composition according to any one of claims 1 to 5, wherein the inhibitor of a cytochrome p450 protein is tannic acid and/or resveratrol.
7. A food product comprising an anti-microbial composition as defined in any one of claims 1 to 6.
8. A food product according to claim 7, which comprises (d) from 0.5 ppm to 25 ppm of natamycine and/or (e) from 0.25 to 50 ppm nisine and (b) from 10 μM to 300 μM of an inhibitor of a cytochrome p450 protein. Use of an anti-microbial composition as defined in any one of claims 1 to 6 for the prevention of microbial growth in a food product.
PCT/EP2005/052785 2004-06-16 2005-06-16 Anti-microbial composition for food products WO2005122800A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP04076776 2004-06-16
EP04076776.6 2004-06-16

Publications (1)

Publication Number Publication Date
WO2005122800A1 true WO2005122800A1 (en) 2005-12-29

Family

ID=34972094

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2005/052785 WO2005122800A1 (en) 2004-06-16 2005-06-16 Anti-microbial composition for food products

Country Status (2)

Country Link
AR (1) AR049516A1 (en)
WO (1) WO2005122800A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112584709A (en) * 2018-08-10 2021-03-30 三得利控股株式会社 Microorganism proliferation inhibitor, method for preventing microorganism contamination, and beverage

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5895681A (en) * 1996-06-20 1999-04-20 Thomas J. Lipton, Division Of Conopco, Inc. Method of preserving tea containing beverages
WO1999053763A1 (en) * 1998-04-17 1999-10-28 Pillarisetti Venkata Subba Rao An environment friendly acaricide formulation
US6060075A (en) * 1997-08-27 2000-05-09 Vittal Mallya Scientific Research Foundation Check mite composition and a process for preparing the same
US6132787A (en) * 1997-04-25 2000-10-17 The Procter & Gamble Company Antimicrobial combinations of a sorbate preservative natamycin and a dialkyl dicarbonate useful in treating beverages and other food products and process of making
US20020039981A1 (en) * 2000-07-18 2002-04-04 Lopes John A. Concentrated sanitizing compositions for cleaning food and food contact surfaces

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5895681A (en) * 1996-06-20 1999-04-20 Thomas J. Lipton, Division Of Conopco, Inc. Method of preserving tea containing beverages
US6132787A (en) * 1997-04-25 2000-10-17 The Procter & Gamble Company Antimicrobial combinations of a sorbate preservative natamycin and a dialkyl dicarbonate useful in treating beverages and other food products and process of making
US6060075A (en) * 1997-08-27 2000-05-09 Vittal Mallya Scientific Research Foundation Check mite composition and a process for preparing the same
WO1999053763A1 (en) * 1998-04-17 1999-10-28 Pillarisetti Venkata Subba Rao An environment friendly acaricide formulation
US20020039981A1 (en) * 2000-07-18 2002-04-04 Lopes John A. Concentrated sanitizing compositions for cleaning food and food contact surfaces

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112584709A (en) * 2018-08-10 2021-03-30 三得利控股株式会社 Microorganism proliferation inhibitor, method for preventing microorganism contamination, and beverage

Also Published As

Publication number Publication date
AR049516A1 (en) 2006-08-09

Similar Documents

Publication Publication Date Title
Cerrutti et al. Inhibitory effects of vanillin on some food spoilage yeasts in laboratory media and fruit purees
Rawat Food Spoilage: Microorganisms and their prevention
Gatto et al. Phenolic extracts from wild edible plants to control postharvest diseases of sweet cherry fruit
Bansal et al. Minimally processed foods: Overview
US5895680A (en) Foodstuff preservation
US5738888A (en) Beverage preservation
AU2005286540B2 (en) Antimicrobial composition
Fitzgerald et al. The potential application of vanillin in preventing yeast spoilage of soft drinks and fruit juices
EP2034856A1 (en) Beverage compositions comprising a preservative system
Nieva et al. Fruit and vegetable smoothies preservation with natural antimicrobials for the assurance of safety and quality
Baraiya et al. Improvement of postharvest quality and storability of jamun fruit (Syzygium cumini L. Var. Paras) by zein coating enriched with antioxidants
US20130012428A1 (en) Liquid antimicrobial compositions
WO2008100406A2 (en) Beverage compositions comprising polylysine and at least one weak acid
Irkin et al. Chitosan Coating of Red Table Grapes and Fresh‐Cut Honey Melons to Inhibit F usarium oxysporum Growth
Marino et al. Chitosan-propolis combination inhibits anthracnose in'Hass' avocados
CA3179786A1 (en) Fermented onion composition
Wang et al. Sodium alginate edible coating augmented with essential oils maintains fruits postharvest physiology during preservation: A review
Bist et al. Role of microorganisms in post-harvest loss of agricultural products: A review
US5895681A (en) Method of preserving tea containing beverages
Tumbarski et al. FOOD INDUSTRY APPLICATIONS OF PROPOLIS: A REVIEW.
WO2005122800A1 (en) Anti-microbial composition for food products
AU2021343866A1 (en) Preservative and antimicrobial compositions
Saranraj et al. Role of organic acids and hydrogen peroxide in fruit juice preservation: A review
WO2007063043A1 (en) Improved anti-microbial composition
de Oliveira et al. Chitosan-based coatings and plant extracts

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

WWW Wipo information: withdrawn in national office

Country of ref document: DE

122 Ep: pct application non-entry in european phase