WO2023021082A1

WO2023021082A1 - Composition

Info

Publication number: WO2023021082A1
Application number: PCT/EP2022/072947
Authority: WO
Inventors: Simona BIRTIC; Michael LAGUERRE; Lise Laurencon; Ana Paula DO ESPIRITO SANTO
Original assignee: Givaudan Sa
Priority date: 2021-08-19
Filing date: 2022-08-17
Publication date: 2023-02-23
Also published as: GB202111898D0

Abstract

The present invention relates to preservative and/or antimicrobial compositions comprising clove leaves and/or buds extracts.

Description

COMPOSITION

Field of the Invention

The present invention relates to preservative and/or antimicrobial compositions. of the invention

Microbial spoilage of food and beverages is a concern in the food industry today. Food and beverage matrices have varying degrees of sensitivity to microbiological spoilage depending on intrinsic factors of the matrix, such as pH, nutrient content (e.g., juice, vitamin, or micronutrient content; e.g. sauces fats, protein content, sugar level), carbonation level, Brix, dry matter level, water quality (e.g., alkalinity and/or hardness), and preservatives. Spoilage events occur when microorganisms are able to overcome the foods beverage's intrinsic factors and grow. The microorganisms' ability to-overcome these hurdles can be influenced by, among other things, initial contamination level, temperature and package integrity of the beverage against carbonation loss, i.e., in the case of carbonated soft drinks.

Microbiological spoilage can result from one or more yeasts, bacteria, and/or mold microorganisms. For example, yeasts and bacteria are capable of spoiling emulsified and non-emulsified sauces such as mayonnaise, ketchup, vinaigrette, range, carbonated and non-carbonated beverages such as fruit drinks, teas, coffees, enhanced waters, etc. The ability of yeasts and certain bacteria to grow anaerobically enables their growth in carbonated beverages, while molds are restricted to aerobic metabolism, and therefore do not grow. See Stratford, M. et al., Fruit Juices, Fruit Drinks, and Soft Drinks, In The Microbiological Safety & Quality of Food (eds. B. M. Lund, T. C. Baird-Parker, and G. W. Gould, Aspen Publishers 2000). Typically, spoilage by yeasts manifests itself as fermentation with gas and ethanol production, as well as sedimentation, off-flavors and odors, and loss of cloud or emulsion stability. Bacteria tend to produce off-flavors and odors with associated sedimentation. On the other hand, molds may survive but generally are not capable of growth in low oxygen environments and thus, do not spoil carbonated soft drinks except when carbonation is diminished. Mold spoilage of non-carbonated beverages, however, can occur and may be evident after mold mycelial growth, by floating globules, clumps or surface pellicles.

Although yeasts such as Saccharomyces, Zygosaccharomyces, Candida, and Dekkera spp. are often responsible for spoilage incidents in common beverages, acidophilic bacteria such as Lactobacillus, Leuconostoc, Gluconobacter, and Zymomonas spp. and molds like Penicillium and Aspergillus spp. can also spoil cold-filled beverages. Spores of acidophilic, thermophilic bacteria such as Alicycloba- cillus spp. and heat resistant mold spores of Byssochlamys and Neosartorya spp. can survive pasteurization and may spoil non-carbonated hot-filled products such as sport drinks and teas. Packaged waters are susceptible to growth by molds as well.

Protection against microbiological spoilage of beverages can be achieved using chemical preservatives and/or processing techniques such as hot filling, tunnel pasteurization, ultra-high temperature treatment (UHT) or pasteurization followed by aseptic packaging, and/or pasteurization followed by chilling the beverage. Generally, beverages with a pH<4.6 can be chemically preserved, heat processed, and filled into packages such that the product is not re-contaminated. For example, process techniques such as cold filling with chemical preservatives or pasteurization followed by coldfilling may be used to preserve this type of beverage. In a similar manner, this same beverage may be processed using non-preserved techniques such as hot filling, tunnel pasteurization, pasteurization followed by aseptic filling or even requiring the beverage to be chilled, i.e., under refrigeration following the pasteurization step. Beverages having a pH^4.6 must be processed such that spores are destroyed using ultra-high temperatures followed by aseptic filling into packages or retorting sealed packages of product.

However, use of such methods impact the texture, colour and taste of food or beverages. The need is to avoid harsh treatments and keep food or beverage preserved from microbial spoilage. This may be achieved by using preservation ingredients.

Current preservation systems for acidic, shelf-stable carbonated and non-carbonated soft drinks or emulsified and non-emulsified sauces rely on chemically synthesised preservatives (e.g., benzoic and/or sorbic acid). Benzoic and sorbic acids (and salts thereof) effectively inhibit yeast, bacteria, and molds with some exceptions. Benzoic and sorbic acids in food and beverages exist in equilibrium between their dissociated and undissociated forms which is dependent upon the dissociation constant of the acid (pKa) and the beverage pH. The pKa for benzoic acid is 4.19 and the pKa of sorbic acid is 4.76. A food or beverage pH below the pKa of the particular acid pushes the equilibrium towards the undissociated form. The undissociated form is more efficacious against microorganisms, and therefore, weak acid preservatives may be most effective in the low pH range.

However, currently the industry wishes to move away from the use of chemically produced antimicrobials, such as benzoic or sorbic acids.

Moreover, weak acid preservation systems have limitations. Genetic adaptation and subsequent resistance by microorganisms may be one of the biggest concerns. (Piper, P. et al., Weak Acid Adaptation: The Stress Response that Confers Yeasts with Resistance to Organic Acid Food Preservatives, 147 Microbiol. 2635-2642 (2001)). Certain yeasts, such as Z. bailii, Z. bisporus, C. krusei, and S. cerevisiae, have specific genes that enable them to resist the weak acid preservatives and grow, despite their presence and regardless of the co-presence of EDTA or SHMP. Some bacteria, such as Gluconobacter spp., are also thought to be preservative resistant. The levels of weak acids necessary to overcome this resistance have been shown to be far beyond regulatory limits on use levels. Spoilage of preserved sauces, teas, juice-containing beverages, and carbonated beverages is commonly due to preservative-resistant yeasts.

Weak acids are also known to impart a throat or mouth burn when used at high levels. Although there are certain shelf-stable foods and beverages where this may be acceptable, often this sensory perception is considered negative. In addition, non-government organizations and also some international government agencies have raised concerns regarding the use of weak acid preservatives in beverages and foods.

In addition, the other process techniques for low acid foods and beverages (i.e. with a pH^4.6) have limitations. Such low acid food and beverages should be thermally treated to sufficiently destroy spores of Clostridium botulinum and Bacillus cereus. Examples of such processes include UHT and retort. Even after such processing, product should be handled in a way to prevent post-pro- cessing contamination. Research, however, suggests that there may still be various strains of spore forming microorganisms that can survive these different processing techniques. To that end, these processing techniques may not eliminate the potential for spoilage. Moreover, such techniques deplete the taste, the texture and the colour of food, disabling it from its initial freshness and appeal. Natural preservatives having the capability of preserving food and beverages, or cosmetics may be desirable for consumers. Preservatives that could be labeled as natural could also eliminate hot-fill requirements for unpreserved shelf-stable preservative free food, beverages and cosmetics. Thus, it would be desirable to provide a natural preservative and/or a preservative system that inhibits growth of microorganisms to solve at least one of the above-mentioned limitations in the art. of the Invention

In a first aspect, the invention provides Antimicrobial composition comprising an extract obtained or obtainable from Syzygium genus (such as Syzygium aromaticum), such as from the leaves and /or buds.

In a second aspect, the invention relates to antimicrobial composition comprising

(i) at least 0.3 % wt/wt of flavonoids (such as Quercetin, Kaempferol and/or Gossypetin and their derivatives), such as at least 0.5 % , 1%, 2%, 3%, 4%, 5%, 8%, 10%, 12%, 14%, or at least 15 % wt/wt of flavonoids, and/or (ii) at least 0.5% wt/wt of phenolic acids (such as gallic acid, tannic acid and/or capsacin and their derivatives), such as at least 1%, 2%, 3%, 4%, 5%, 8%, 10%, 12%, 14%, 15%, 20%, 25%, 30% or at least 40% wt/wt of phenolic acids and/or at least 0.5 % wt/wt of organic acids (such as quinic acid, malic acid, citric acid and/or tartaric acid and their derivatives), such as at least 1% wt/wt, 2%, 3%, 5%, 8%, 10%, 15%, 20% or at least 30% wt/wt of organic acids

In another aspect, the invention provides an antimicrobial composition comprising gallic acid, quinic acid, malic acid and/or eugenol and/or their derivatives.

In another aspect, the invention provides the use of said extracts and compositions for inhibiting the growth of microorganisms and/or for killing microorganisms in a product (such as a food or beverage product for humans or animals, a nutritional supplement, a nutraceutical formulation, a fragrance or flavouring, a pharmaceutical or veterinary formulation, an oenological or cosmetic formulation).

In another aspect, the invention provides a product (such as a food or beverage product for humans or animals, a nutritional supplement, nutraceutical formulation, a dietary, a sports supplement, a fragrance or flavouring, a pharmaceutical or veterinary formulation, an oenological or cosmetic formulation) comprising an extract as defied herein and/or an antimicrobial composition as define herein.

The details, examples and preferences provided in relation to any one or more of the stated aspects of the present invention will be further described herein and apply equally to all aspects of the present invention. Any combination of the embodiments, examples and preferences described herein below in all possible variations thereof is encompassed by the present invention unless otherwise indicated herein, or otherwise clearly contradicted by context.

Detailed Description of the Invention

The present invention is related to an antimicrobial composition comprising an extract obtained or obtainable from a plant from the Syzygium genus (such as as Syzygium aromaticum).

As will be appreciated by the person skilled in the art, as used herein the term "obtainable from" means that the extract may be obtained from a plant or may be isolated from the plant, or may be obtained from an alternative source, for example by chemical synthesis or enzymatic production. Whereas the term "obtained" as used herein, means that the extract is directly derived from the plant source.

The Syzygium is a genus of flowering plants that belongs to the myrtle family, Myrtaceae. Several species of Syzygium bear fruits that are edible for humans (https://en.wikipedia.org/wiki/Syzygium). Cloves are the aromatic flower buds of the tree Syzygium aromaticum. They are native to the Maluku Islands in Indonesia and are commonly used as a spice.

The extract of the invention may be obtained or be obtainable from any part of the plant such as the leaves, stems, the fruits, the buds (cloves or bud cloves) and mixtures thereof.

In the present application the inventors have surprisingly discovered that the leaves of Syzygium aromaticum have very high antimicrobial properties.

Thus, in a preferred embodiment of the present invention, the extract of the invention is obtained or obtainable from the clove tree Syzygium aromaticum (S. aromaticum) leaves.

In the present invention by "leaves" is meant leaves alone and also leaves mixed with stems.

In another embodiment of the present invention, the extract of the invention is obtained or obtainable from the clove tree Syzygium aromaticum (S. aromaticum) buds (or clove buds).

When the extract of the invention is obtained from the plant material (such as from the leaves and/or buds) different solvents may be used for extraction such as water, an alcohol, an organic solvent, supercritical carbon dioxide extraction or mixtures thereof.

The Syzygium genus extract (such as Syzygium aromaticum leaves extract and/ or bud extract) may be obtained by a process comprising the following steps:

(i) contacting the Syzygium genus plant material (such as leaves and/or buds) (such as Syzygium aromaticum leaves and/or buds) with a solvent; and

(ii) removing the solvent.

Optionally, the leaves may be ground before being contacted with a solvent.

Solvents that may be used in step (i) include alcohols (such as methanol and ethanol), water (including water that has been acidified), organic solvents and mixtures thereof. For example, the organic solvent may be selected from the group consisting of acetic acid, acetone, acetonitrile, benzene, 2-butanone, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane, 1,2-dichloroethane, diethylene glycol, diethyl ether, diglyme (diethylene glycol, dimethyl ether), 1,2- dimethoxy- ethane (glyme, DME), dimethyl- formamide (DMF), dimethyl sulfoxide (DMSO), 1,4-di- oxane, ethyl acetate, ethylene glycol, glycerin, heptane, hexamethylphosphoramide (HMPA), hexamethylphosphorous, triamide (HMPT), hexane, methyl t-butyl, ether (MTBE), methylene chloride, N-methyl-2-pyrrolidinone (NMP), nitromethane, pentane, petroleum ether (ligroine), pyridine, tetrahydrofuran (THF), toluene, triethyl amine, o-xylene, m-xylene and p-xylene. In a preferred embodiment, the organic solvent is acetone.

The alcohol solvent may consist of only alcohol (e.g. 100% alcohol), for example 100% ethanol, or may be a mixture of an alcohol and water (i.e. a hydro-alcoholic solvent), for example, a mix of ethanol and water (hydro-ethanolic solvent), for example, from about 1% to about 99% alcohol (e.g. ethanol, methanol) in water, for example the ratio of water to alcohol is from 10/90% v/v to 90/10% v/v or 30/70% v/v to 70/30% v/v, such as 50/50% v/v or 70/30 v/v. In one preferred embodiment, the ethanol proportion in the water/ethanol blend is a maximum of 50% by weight. In a preferred embodiment the ethanol/water proportion is 30:70 v/v.

Step (i) may be performed at a temperature in a range of from about 20 °C to about 200 °C. For example, the temperature for of step (i) may be in a range of from about 40 °C to about 100 °C, such as 50 °C, 55 °C, 60 °C, 65 °C or 70 °C. Alternatively, step (i) may be performed at the temperature at which the solvent will reflux.

Typically, the ratio of the Syzygium genus (such as Syzygium aromaticum) plant material (such as leaves and/or buds, to solvent mixture used in the extraction process varies from about 1:1 to about 1:20 on a gram to millilitre basis, such as from about 1:3 to about 1:12 For example, a 10 M ratio may be used.

The incubation period (i.e. the period during which the plant material is in contact with the solvent) is typically from about 2 hours to about 24 hours. Any undissolved plant material may be removed from the solvent, for example, by filtration, and re-dissolved in the solvent. The incubation period may then be repeated.

After the plant materials and solvent have been incubated in process step (i), the solvent is separated from any un-dissolved plant material (step ii) and the extracted composition is concentrated (i.e. the solvent is removed) until the extraction composition has a solid component. For example, until all the solvent has been removed and only solid extract remains. Typically, in the process of the invention, at least 30%, such as at least 60%, at least 70%, 80%, 90%, 95% or at least 100% of the solvent and/or liquid is removed.

All of these extractions can be followed by one or multiple purification steps (such as chromatographic process). All of these extractions can be processed with active coal or any decolourization aid mixed to the raw material.

The extract of the invention may be also extracted using other techniques such as Supercritical CO2 extract, subcritical water extract, etc. Thus the extract of the invention may be an aqueous extract, alcohol extract, hidro-alcoholic extract, organic extract (such as an acetone extract), Supercritical CO2 extract, subcritical water extract or mixtures thereof.

The term "aqueous extract" as used herein, refers to the extract obtained from the plant material (such as leaves and/or buds) of a plant of the Syzygium genus (such as Syzygium aromaticum leaves and/or buds) when the extraction from the plant material has been performed using water as the only solvent.

The term "alcohol extract" as used herein, refers to the extract obtained from the plant material (such as leaves and/or buds) of a plant of the Syzygium genus (such as Syzygium aromaticum leaves and/or buds) when the extraction from the plant material has been performed using an alcohol as the solvent.

The term "organic extract" as used herein, refers to the extract obtained from the plant material (such as leaves and/or buds) of a plant of the Syzygium genus (such as Syzygium aromaticum leaves and/or buds) when the extraction from the plant material has been performed using an organic solvent that is not an alcohol, such as acetone

The inventors of the present invention have surprisingly found that the Syzygium aromaticum leaves extracts are substantially higher in phenolic acids than the classical clove bud extracts and provide an antimicrobial effect.

For example, Syzygium aromaticum leaves extracts or "clove leaves extracts" have elevated proportions of gallic acid or quinic acid while the eugenol concentrations are relatively low. There is therefore also provided a Syzygium genus leaves extract (such as Syzygium aromaticum leaves extract) as hereinabove defined, comprising (or consisting essentially/consisting of) at least 3% wt/wt of one or more phenolic compounds, such as at least about 4% wt/wt, at least about 5% wt/wt, at least about 6% wt/wt, at least about 7% wt/wt, at least about 8% wt/wt, at least about 9% wt/wt, at least about 10% wt/wt, at least about 15% wt/wt, at least about 20% wt/wt, at least about 30% wt/wt, at least about 40% wt/wt, at least about 50% wt/wt, at least about 60% wt/wt of one or more phenolic compounds.

Phenolic compounds are defined as compounds that possess an aromatic ring with at least one hydroxyl group, and their structure can vary from simple molecule to complex polymer with high molecular weight. The most adopted classification implies the subdivision of phenolics in two main groups: flavonoids and non-flavonoid polyphenols, and this classification have been commonly used in the literature. Phenolic acids are non-flavonoid polyphenolic compounds present in foodstuffs and are characterized by a carboxyl group linked to benzene ring. Examples of phenolic acids include, without limitation: gallic acid, tannic acid, capsacin, their derivatives and mixtures thereof.

Examples of flavonoids include, without limitation: Quercetin, Kaempferol, Gossypetin and their derivatives (such as Quercetin glucuronide, Kaempferol -3-glucuronide , Gossypetin 7-O-Rhamno- pyranoside).

Other compounds present in the extracts of the invention include organic acids. Examples of organic acid include, without limitation: quinic acid, malic acid, citric acid, tartaric acid, their derivatives and mixtures thereof.

The concentrations (expressed in % wt/wt) of the phenolic compounds, flavonoids, organic acids, eugenol and other molecules refereed herein, are measured using standard techniques such as HPLC. Thus, for avoiding repetition, when referring to a specific % wt/wt of a molecule is to be understand in the present application and in the whole text as the % wt/wt as measured by HPLC (i.e 3% wt/wt of one or more flavonoids, has to be read as "3% wt/wt of one or more flavonoids measured by HPLC")

There is therefore also provided a Syzygium genus leaves extract (such as Syzygium aromaticum leaves extract) as hereinabove defined, comprising (or consisting essentially/consisting of ) at least 3% wt/wt of one or more flavonoids (such as Quercetin, Kaempferol and/or Gossypetin and derivatives), such as at least about 4% wt/wt, at least about 5% wt/wt, at least about 6% wt/wt, at least about 7% wt/wt, at least about 8% wt/wt, at least about 9% wt/wt, at least about 10% wt/wt, at least about 15% wt/wt, at least about 20% wt/wt, at least about 30% wt/wt, at least about 40% wt/wt, at least about 50% wt/wt, at least about 60% wt/wt of one or more flavonoids.

There is also provided a Syzygium genus leaves extract (such as Syzygium aromaticum leaves extract) as hereinabove defined, comprising (or consisting essentially/consisting of) at least 3% wt/wt of one or more phenolic acids (such as gallic acid, tannic acid and/or capsacin their derivatives and mixtures thereof), such as at least about 4% wt/wt, at least about 5% wt/wt, at least about 6% wt/wt, at least about 7% wt/wt, at least about 8% wt/wt, at least about 9% wt/wt, at least about 10% wt/wt, at least about 15% wt/wt, at least about 20% wt/wt, at least about 30% wt/wt, at least about 40% wt/wt, at least about 50% wt/wt, at least about 60% wt/wt of one or more phenolic acids.

There is also provided a Syzygium genus leaves extract (such as Syzygium aromaticum leaves extract) as hereinabove defined, comprising (or consisting essentially/consisting of) at least 3% wt/wt of one or more organic acids (such as quinic acid, malic acid, citric acid and/or tartaric acid and their derivatives), such as at least about 4% wt/wt, at least about 5% wt/wt, at least about 6% wt/wt, at least about 7% wt/wt, at least about 8% wt/wt, at least about 9% wt/wt, at least about 10% wt/wt, at least about 15% wt/wt, at least about 20% wt/wt, at least about 30% wt/wt, at least about 40% wt/wt, at least about 50% wt/wt, at least about 60% wt/wt of one or more organic acids.

There is also provided a Syzygium genus leaves extract (such as Syzygium aromaticum leaves extract) comprising (or consisting essentially/consisting of):

(i) at least 0.3 % wt/wt of one or more flavonoids (such as Quercetin, Kaempferol and/or Gossypetin and their derivatives), such as at least 0.5 % wt/wt , 1% wt/wt, 2% wt/wt, 3% wt/wt, 4% wt/wt, 5% wt/wt, 8% wt/wt, 10% wt/wt, 12% wt/wt, 14% wt/wt, or at least 15 % wt/wt of flavonoids, and/or

(ii) at least 0.5% wt/wt of one or more phenolic acids (such as gallic acid, tannic acid and/or capsacin and their derivatives), such as at least 1% wt/wt, 2% wt/wt, 3% wt/wt, 4% wt/wt, 5% wt/wt, 8% wt/wt, 10% wt/wt, 12% wt/wt, 14% wt/wt, 15% wt/wt, 20% wt/wt, 25% wt/wt, 30% wt/wt or at least 40% wt/wt of phenolic acids and/or

(iii) at least 0.5 % wt/wt of one or more organic acids (such as quinic acid, malic acid, citric acid and/or tartaric acid and their derivatives), such as at least 1%, 2% wt/wt, 3% wt/wt, 5% wt/wt, 8% wt/wt, 10% wt/wt, 15% wt/wt, 20% wt/wt or at least 30% wt/wt of organic acids. There is also provided a Syzygium genus leaves extract (such as Syzygium aromaticum leaves extract) as hereinabove defined, comprising (or consisting essentially/consisting of):

(i) from 0.3 % wt/wt to 15% wt/wt of one or more flavonoids (such as Quercetin, Kaempferol and/or Gossypetin and their derivatives), such as from 0.5 % to 10% wt/wt, such as 0.6% to 6% wt/wt of flavonoids, and/or

(ii) from 0.5% to 30 % wt/wt of one or more phenolic acids (such as gallic acid, tannic acid and/or capsacin and their derivatives), such as from 2% to 15%, such as from 4% to 10% wt/wt such as from 2% to 7% wt/wt of phenolic acids and/or

(iii) from of 0.5 % to 30% wt/wt of one or more organic acids (such as quinic acid, malic acid, citric acid and/or tartaric acid and their derivatives), such as from 1% to 10%, such as from 3% to 15%, such as 2% to 8%, such as from 3 % to 6% wt/wt of organic acids.

Also, there is provided a Syzygium genus leaves extract (such as Syzygium aromaticum leaves extract) as hereinabove defined, comprising (or consisting essentially/consisting of) at least 0.4% wt/wt, such as at least 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 2.2%, 2.4%, 2.6%, 2.8%, 3%, 3.5%, 4%, 5%, 6%, 7%, 8%, 8%, 10% or at least 15% wt/wt of gallic acid and/or its derivatives.

In another embodiment the Syzygium genus leaves extract (such as Syzygium aromaticum leaves extract) of the invention additionally comprises one or more of: quinic acid, malic acid, citric acid, Kaempferol -3-glucuronide, quercetin glucuronide or Gossypetin 7-O-Rhamnopyranoside and their derivatives.

In one embodiment the Syzygium genus leaves extract (such as Syzygium aromaticum leaves extract) comprises (or consist essentially/consist of):

- at least 0.4% wt/wt, such as at least 0.5%, 0.6%, 0.7%, 0.8%, 0.8%, 0.9%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 2.2%, 2.4%, 2.6%, 2.8%, 3%, 3.5%, or at least 4% wt/wt of gallic acid and/or derivatives, and/or

- at least 0.001% wt/wt, such as at least 0.5%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 2.2%, 2.4%, 2.6%, 2.8%, 3%, 3.5%, 4%, 4.5%, 5% or at least 6% wt/wt of quinic acid and/or derivatives.

In one embodiment, the Syzygium genus leaves extract (such as Syzygium aromaticum leaves extract) comprises (or consist essentially/consist of) less than 50 % wt/wt eugenol, such as less than 35 % wt/wt eugenol, such as less than 20% wt/wt, less than 15% wt/wt, less than 10% wt/wt, less than 5% wt/wt, less than 4% wt/wt, less than 3% wt/wt, less than 2% wt/wt, less than 1% wt/wt or less than 0.5% wt/wt eugenol and/or eugenol derivatives.

In one embodiment, the Syzygium genus leaves extract (such as Syzygium aromaticum leaves ex- tract) comprises (or consist essentially/consist of) less than 35 % wt/wt caryophyllene, such as less than 20% wt/wt, less than 15% wt/wt, less than 10% wt/wt, less than 5% wt/wt, less than 4% wt/wt, less than 3% wt/wt, less than 2% wt/wt, less than 1% wt/wt or less than 0.5% wt/wt caryophyllene.

In one embodiment, the extract of the invention further comprises one or more of the following molecules.

In another embodiment, the Syzygium genus leaves extract (such as Syzygium aromaticum leaves extract) of the invention further comprises one or more of the following compounds.

(i) at least 0.5% wt/wt of gallic acid, such as 1% wt/wt, 2% wt/wt, 3% wt/wt, 5% wt/wt, at least 6% wt/wt, at least 10% of gallic acid and/or its derivatives, and/or

(ii) at least 1% wt/wt, 2% wt/wt, 2.5% wt/wt, 3% wt/wt, 3.5% wt/wt, 4% wt/wt, 4.5% wt/wt, 5% wt/wt, 6% wt/wt, at least 8% wt/wt of quinic acid and/or its derivatives, and/or

(iii) less than 35% wt/wt of eugenol and/or its derivatives, such as less than 15% wt/wt of eugenol, less than 10% wt/wt, less than 5 % wt/wt, less than 2% wt/wt , less than 1% wt/wt, such as less than 0.5% wt/wt of eugenol and/or its derivatives.

In one embodiment the Syzygium genus leaves extract (such as Syzygium aromaticum leaves extract) is a water extract that comprises (or consist essentially/consist of):

(i) from 0.5% to 10% wt/wt of gallic acid and/or its derivatives, such as 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10 % wt/wt of gallic acid and/or its derivatives; and /or

(ii) from 1.5% wt/wt to 10% wt/wt of quinic acid and/or its derivatives, such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% wt/wt of qunic acid and/or its derivatives and /or

(iii) less than 3% wt/wt of eugenol and/or its derivatives such as less than 2%, 1%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1% wt/wt of eugenol and/or its derivatives.

In one embodiment the Syzygium genus leaves extract (such as Syzygium aromaticum leaves extract) is an acetone extract that comprises (or consist essentially/consist of):

(i) from 0.01% to 5% wt/wt of gallic acid and/or its derivatives, such as 0.05% wt/wt, 0.08%, 1%, 2%, 3%, 4%, or 5% wt/wt of gallic acid and/or its derivatives; and /or (ii)from 0.01% wt/wt to 5% wt/wt of quinic acid and/or its derivatives, such as 0.05% wt/wt, 0.08%, 1%, 2%, 3%, 4%, or 5% wt/wt of quinic acid and/or its derivatives and /or at least than 3% wt/wt of eugenol and/or its derivatives such as at least 5% wt/wt, 8%, 10%, 15%, 20%, 30%, 35% or 40%% wt/wt of eugenol and/or its derivatives.

In one embodiment, the Syzygium genus leaves extract (such as Syzygium aromaticum leaves extract) is a water extract. In an embodiment, the extraction is done at ambient temperature or at least at 40°C, such as at 50°C.

In one embodiment, the Syzygium genus leaves extract (such as Syzygium aromaticum leaves extract) is an ethanolic or hydroethanolic extract (such as 30:70 v/v ethanol/water). In an embodiment, the extraction is done at ambient temperature or at least at 40°C, such as at 50°C.

In a further embodiment, the extract is obtained or obtainable from the buds of Syzygium genus (such as as Syzygium aromaticum) and is defined herein as "bud extract" or "clove bud extract" of the invention.

The methods of extraction and the solvents that may be used can be used are the same as described previously.

In a preferred embodiment, the clove bud extract of the invention is extracted using water as solvent (clove bud water extract).

Thus in further aspect the invention relates to an antimicrobial composition of the invention comprising a bud extract obtained or obtainable from the buds of Syzygium genus (such as as Syzygium aromaticum clove buds). In a preferred embodiment, the solvent for extraction is water.

There is therefore also provided a Syzygium genus bud extract (such as Syzygium aromaticum bud extract) as hereinabove defined, comprising (or consisting essentially/consisting of) at least 3% wt/wt of one or more phenolic compounds, such as at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60% wt/wt of one or more phenolic compounds.

In one embodiment, the Syzygium genus bud extract (such as Syzygium aromaticum bud extract) comprises (or consisting essentially/consisting of) at least 3% wt/wt of one or more polyphenols, such as at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60% wt/wt of one or more of polyphenols. In one embodiment, the Syzygium genus bud extract (such as Syzygium aromaticum bud extract) comprises (or consisting essentially/consisting of) at least 18% of one or more polyphenols.

Antimicrobial com

First antimicrobial composition of the invention.

The present invention is related to an antimicrobial composition ("first antimicrobial composition of the invention") comprising an extract obtained or obtainable from Syzygium genus (such as Syzygium aromaticum) leaves and/or buds as described previously. All embodiments referring to the extract of the invention can be applied in an analogous manner to the first composition of the invention.

For example, in one embodiment there is also provided an antimicrobial composition as hereinabove defined comprising a Syzygium genus leaves and/or bud extract (such as Syzygium aromaticum leaves and /or bud extract), wherein the extract comprises comprising (or consisting essentially/consisting of):

(i) at least 0.3 % wt/wt of one or more flavonoids (such as Quercetin, Kaempferol and/or Gossypetin and their derivatives), such as at least 0.5 % wt/wt, 1%, 2%, 3%, 4%, 5%, 8%, 10%, 12%, 14%, or at least 15 % wt/wt of flavonoids, and/or

(ii) at least 0.5% wt/wt of one or more phenolic acids (such as gallic acid, tannic acid and/or capsacin and their derivatives), such as at least 1% wt/wt, 2%, 3%, 4%, 5%, 8%, 10%, 12%, 14%, 15%, 20%, 25%, 30% or at least 40% wt/wt of phenolic acids and/or

(iii) at least 0.5 % wt/wt of one or more organic acids (such as quinic acid, malic acid, citric acid and/or tartaric acid and their derivatives), such as at least 1% wt/wt, 2%, 3%, 5%, 8%, 10%, 15%, 20% or at least 30% wt/wt of organic acids.

Also, the invention provides an antimicrobial composition (such as a food antimicrobial composition, a nutraceutical antimicrobial composition or a cosmetic antimicrobial composition) comprising a Syzygium genus bud extract (such as Syzygium aromaticum bud extract), wherein the extract comprises comprising (or consisting essentially/consisting of) at least 3% wt/wt of one or more polyphenols, such as at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60% wt/wt of one or more of polyphenols.

In a preferred embodiment the bud extract is extracted with water as solvent.

In an embodiment, the antimicrobial composition comprises an extract obtained or obtainable from Syzygium aromaticum leaves. In a preferred embodiment, the solvent used for extraction is water, an alcohol (such as ethanol), an organic solvent (such as acetone), supercritical carbon dioxide extraction or mixtures thereof.

In certain embodiments, the antimicrobial composition comprises an extract obtained or obtainable from Syzygium aromaticum leaves that comprises

(i) at least 0.3 % wt/wt of one or more flavonoids (such as Quercetin, Kaempferol and/or Gossypetin, their derivatives and mixtures thereof), such as at least 0.5 % wt/wt, 1% wt/wt, 2% wt/wt, 3% wt/wt, 4% wt/wt, 5% wt/wt, 8% wt/wt, 10% wt/wt, 12% wt/wt, 14% wt/wt, or at least 15 % wt/wt of flavonoids, and/or

(ii) at least 0.5% wt/wt of one or more phenolic acids (such as gallic acid, tannic acid and/or capsacin, their derivatives and mixtures thereof), such as at least 1% wt/wt, 2% wt/wt, 3% wt/wt, 4% wt/wt, 5% wt/wt, 8% wt/wt, 10% wt/wt, 12% wt/wt, 14% wt/wt, 15% wt/wt, 20% wt/wt, 25% wt/wt, 30% wt/wt or at least 40% wt/wt of phenolic acids and/or

(iii) at least 0.5 % wt/wt of one or more organic acids (such as quinic acid, malic acid, citric acid and/or tartaric acid, their derivatives and mixtures thereof), such as at least 1% wt/wt, 2% wt/wt, 3% wt/wt, 5% wt/wt, 8% wt/wt, 10% wt/wt, 15% wt/wt, 20% wt/wt or at least 30% wt/wt of organic acids.

Second composition of the invention

Also, the invention provides an antimicrobial composition (second composition of the invention) comprising (or consisting essentially/consisting of):

(i) at least 0.3 % wt/wt of one or more flavonoids (such as Quercetin, Kaempferol and/or Gossypetin and their derivatives), such as at least 0.5 % , 1%, 2%, 3%, 4%, 5%, 8%, 10%, 12%, 14%, or at least 15 % wt/wt of flavonoids, and/or

(ii) at least 0.5% wt/wt of one or more phenolic acids (such as gallic acid, tannic acid and/or capsacin and their derivatives), such as at least 1% wt/wt, 2%, 3%, 4%, 5%, 8%, 10%, 12%, 14%, 15%, 20%, 25%, 30% or at least 40% wt/wt of phenolic acids and/or (iii) at least 0.5 % wt/wt of one or moreorganic acids (such as quinic acid, malic acid, citric acid and/or tartaric acid and their derivatives), such as at least 1% wt/wt, 2%, 3%, 5%, 8%, 10%, 15%, 20% or at least 30% wt/wt of organic acids, which may be referred to hereinafter as the "second antimicrobial composition of the invention".

In one embodiment, the second antimicrobial composition comprises (or consist essentially/consist of):

(i) from 0.3 % wt/wt to 15% wt/wt of one or more flavonoids (such as Quercetin,

Kaempferol and/or Gossypetin and their derivatives), such as from 0.5 wt/wt % to 10% wt/wt, such as 0.6% wt/wt to 6% wt/wt of one or more flavonoids, and/or

(ii) from 0.5% wt/wt to 30 % wt/wt of one or more phenolic acids (such as gallic acid, tannic acid and/or capsacin and their derivatives), such as from 2% wt/wt to 15% wt/wt, such as from 4% wt/wt to 10% wt/wt such as from 2% wt/wt to 7% wt/wt of one or more phenolic acids and/or

(iii) from of 0.5 % wt/wt to 30% wt/wt of one or more organic acids (such as quinic acid, malic acid, citric acid and/or tartaric acid and their derivatives), such as from 1% wt/wt to 10% wt/wt, such as from 3% wt/wt to 15% wt/wt, such as 2% wt/wt to 8% wt/wt, such as from 3 % wt/wt to 6% wt/wt of one or more organic acids.

In one embodiment, the second antimicrobial composition of the invention comprises (or consist essentially/consist of): less than 50% wt/wt of eugenol and/or its derivatives, such as less than 30% wt/wt, such as less than 15% wt/wt of eugenol, less than 10% wt/wt, less than 5 % wt/wt, less than 2% wt/wt , less than 1% wt/wt, such as less than 0.5% wt/wt of eugenol and/or its derivatives. In another embodiment, the second antimicrobial composition of the invention is essentially free of eugenol (and derivatives).

In one embodiment of the second antimicrobial composition of the invention, the composition comprises gallic acid, quinic acid and/or malic acid and/or their derivatives.

In one embodiment the second antimicrobial composition of the invention comprises (or consist essentially/consist of):

(i) at least 0.5% wt/wt of gallic acid and/or its derivatives, such as at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10 % wt/wt of gallic acid and/or its derivatives; and /or

(ii) at least 1.5 wt/wt of quinic acid and/or its derivatives, such as at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or at least 10% wt/wt of quinic acid and/or its derivatives and /or (iii) less than 50% wt/wt of eugenol and/or its derivatives such as less than 35%, 30%, 20%, 10%, 5%, 3%, 2% or 0.1% wt/wt of eugenol and/or its derivatives.

The flavonoids (such as Quercetin, Kaempferol, Gossypetin, their derivatives and mixtures thereof), phenolic acids (such as gallic acid) and the organic acids (such as malic acid, citric acid) present in the second composition of the invention may be of natural or synthetic origin. In a preferred embodiment, the different compounds are of natural origin such as obtained Syzygium genus leaves (such as Syzygium aromaticum leaves).

In one embodiment, the second antimicrobial composition of the invention comprises gallic acid, and/or quinic acid (and/or its derivatives) purified from Syzygium genus leaves (such as Syzygium aromaticum leaves). For example, the second composition of the invention may comprise from 0.5 v% to about 50% of gallic acid and/or its derivatives, such as from 1% to 10% gallic acid and/or gallic acid derivatives. For example, the enriched extract may comprise from 0.5 % to about 50% of Quinic acid and/or quinic acid derivatives, such as from 1% to 10% Quinic acid and/or quinic acid derivatives.

The first and the second compositions of the invention, may be a food antimicrobial composition, a nutraceutical antimicrobial composition or a cosmetic antimicrobial composition.

The inventors have shown in the examples of the present application that the antimicrobial compositions of the invention prevent the growth of Spoilage microorganisms.

In one embodiment, the antimicrobial compositions of the invention (i.e. the first or second composition of the invention) has antimicrobial activity and/or preservative effect. In one preferred embodiment, the antimicrobial composition comprises Syzygium genus leaves extract and/or buds extracts (such as Syzygium aromaticum leaves and/or buds extract) as described previously. In a preferred embodiment, the leave extract is an acetone extract and/or and hydroalcoholic extract and the bud extract is a water extract and has antimicrobial and/or preservative activity.

As used herein, the term "antimicrobial" or "antimicrobial effect" may refer to a substance (such as an extract of the invention or first or second composition of the invention) that kills microorganisms or stops (inhibits or prevents) their growth. The microorganism may be bacteria, fungi, yeast or a mixture thereof. Methods for assessing the activity of a compound as antimicrobial, such as the methods described in the examples of the present application, are well known by a person skilled in the art. As used herein, the term "preservative" may refer to substance or a chemical (such as a first or second composition of the invention) that is added to products such as food products, beverages, pharmaceutical drugs, paints, biological samples, cosmetics, and many other products to prevent decomposition by microbial growth or by undesirable chemical changes.

In an embodiment, the preservative and/or antimicrobial effect is against spoilage microorganism (such as food and/or beverage spoilage microorganism).

As used herein, the term "Spoilage" refers to the consequence of the microorganism (bacteria yeast and/or mold) growth in the product such as a food or beverage product for humans or animals, a nutritional supplement, nutraceutical formulation, a dietary, a sports supplement, a fragrance or flavouring, a pharmaceutical or veterinary formulation, an oenological or cosmetic formulation. The product components (such as food, beverage or cosmetic formulation components) are utilized as growth substrates by the microorganism (such as bacteria, yeast and /or mold) and are transformed into a vast array of metabolic end products. In this way, the chemical, physical, and sensory properties of the product (such as food, beverage, cosmetic formulation etc) food are changed. Microorganism spoilage becomes evident to the consumer in many ways, depending on the product: signs of food spoilage may include an appearance different from the food in its fresh form, such as a change in color, a change in texture, an unpleasant odour, or an undesirable taste. The item may become softer than normal. If mold occurs, it is often visible externally on the item.

As used herein, the term "food and beverage spoilage microorganism" (such as food and beverage spoilage bacteria, food and beverage spoilage yeast and food and beverage spoilage molds) refers to a microorganism that growths in said food or beverage product and /or can be isolated from said spoiled product and changes the chemical, physical, and/or sensory properties of the food or beverage such a change in colour, a change in texture, an unpleasant odour and / or an undesirable taste. Spoilage microorganism can also produce toxins and other substances that are dangerous to the consumers.

The first antimicrobial composition (such as a food antimicrobial composition) may comprise from about 1% wt/wt to about 100% wt/wt of the extract of the invention as defined previously (Syzyg- ium genus leaves and/or bud extract, such as Syzygium aromaticum leaves and/or bud extract), such as from about 20% to about 80% or from about 40% to about 60% wt/wt of the extract of the invention by weight of the composition. Optionally, the first or second antimicrobial composition of the invention may comprise, consist or consist essential of the compounds or extract combination(s) defined above and a carrier (such as a food, pharmaceutical of cosmetic acceptable carriers). Further it may comprise other antioxidants or antimicrobials, etc.

Third synergistic composition of the invention

The inventors of the present application have shown that the compositions of the invention when is it mixed with a Lamiaceae extract has improved antimicrobial activity if compared with the activity of both components when used alone: this is synergy (see for instance example 6 and 7).

Thus, the first or second antimicrobial composition of the invention may comprise additionally a Lamiaceae extract (third composition of the invention)

Thus, the invention is related to a composition comprising an extract obtained or obtainable from Syzygium aromaticum leaves and/or buds and an extract obtained or obtainable from Lamiaceae (third composition of the invention)

As used herein, the term "Lamiaceae extract" may refer to an extract from a plant of the Lamiaceae family (Lamiaceae material), preferably rosemary, sage, oregano, thyme, mints, and the following genera: Salvia (such as Salvia Apiana and Salvia officinalis), Rosmarinus (such as Rosmarinus officinalis), Lepechinia, Oreganum, Thymus, Hyssopus and mixtures thereof (i.e rosemary and sage, rosemary and Thymus etc).

The extracts may be done as described herein for the clove leave and/or bud extracts.

In a preferred embodiment, the solvent used to extract the Lamiaceae material (such as rosemary and/or salvia leaves) is acetone and/or ethanol/water. Once the plant material and solvent have been incubated, the solvent is separated from residual Lamiaceae material by any suitable separation technique known in the art (like for example filtration).

Further filtration steps can be used. The solvent may be partially or totally removed by any method known in the art such as centrifugation, Rota vap, and any device allowing solvent evaporation or a liquid-liquid way of replacing the solvent. Further filtration steps can be used. For example, in a preferred embodiment, aqueous sodium carbonate (NaHCCh) may be added to dissolve carnosic acid and other organic acids, while base insoluble substances are precipitated out. The solution may be filtered to separate from solid, and the filtrate can be further concentrated under reduced pressure. In a further step, after finishing concentration is achieved, phosphoric acid (H3PO4) may added and the acid insoluble substances (including carnosic acid, carnosol, and carnosic derivatives) are precipitated from the concentrated solution. Additionally, the result may be fileted and the solid precipitate may be subsequently separated from liquid and rinsed with water to remove impurities.

In one embodiment, the antimicrobial composition of the invention comprises an extract obtained or obtainable from the leaves and/or buds of Syzygium genus (such as an extract from the leaves and/or buds of Syzygium aromaticum) and an extract obtained or obtainable from a Lamiaceae (such as leaves and/or stems of rosemary and/or salvia).

In certain embodiments, the antimicrobial composition of the invention comprises an extract obtained or obtainable from the leaves of Syzygium genus (such as an extract from the leaves of Syzygium aromaticum) using acetone and/or ethanol/water as solvent and an extract obtained or obtainable from a Lamiaceae (such as leaves and/or stems of rosemary and/or salvia) using acetone and/or ethanol/water as solvent.

In certain embodiments, the antimicrobial composition of the invention comprises an extract obtained or obtainable from the buds of Syzygium genus (such as an extract from the buds of Syzygium aromaticum) and an extract obtained or obtainable from a Lamiaceae (such as leaves and/or stems of rosemary and/or salvia).

In certain embodiments, the antimicrobial composition of the invention comprises an extract obtained or obtainable from the buds of Syzygium genus (such as an extract from the buds of Syzygium aromaticum) using water as solvent and an extract obtained or obtainable from a Lamiaceae (such as leaves and/or stems of rosemary and/or salvia) using acetone and/or ethanol/water as solvent.

In certain embodiments, the Lamiaceae extract is rich in phenolic diterpenes.

In certain embodiments, the Lamiaceae extract is a Rosemary extract. In a preferred embodiment, the rosemary extract is enriched in phenolic diterpenes.

In certain embodiments, the Lamiaceae extract is a Salvia extract (such as Salvia Apiana and Salvia officinalis). In a preferred embodiment, the Salvia extract is enriched in phenolic diterpenes. In a further embodiment, the Lamiaceae extract is blend of a rosemary extract and a Salvia extract (such as Salvia Apiana and Salvia officinalis).

As used herein, the term "Phenolic diterpenes" may refer to carnosic acid, carnosol, methylcar- nosate, and other phenolic diterpene derivatives (suhc as rosmanol, isorosmanol, 1 1 , 12 -di-O- methylisorosmanol, 12-O-methylcarnosic acid, rosmanol-9-ethyl ether, circi-maritin, Methylated monooxidized product of carnosic acid, genkwanin, epirosmanol, epiisorosmanol, carnosic acid derivative, epirosmanol ethyl ether, cryptotanshinone) and mixtures thereof.

In a preferred embodiment, the Lamiaceae extract (such as a rosemary and/or salvia extract) of the composition of the invention comprises carnosic acid and/or carnosol.

In certain embodiments, the Lamiaceae extract (such as a rosemary and/or salvia extract) may comprise (or consist essentially/consist of): a) from about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% to about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% or 50% by weight of the final composition(wt/wt) of carnosic acid, such as from about 20% to about 80% wt/wt, preferably such as from about 30% to about 50 % wt/wt such as about 40% wt/wt; and/ or b) from about 1% wt/wt, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% to about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% or 50% wt/wt of carnosol by weight of the final composition (wt/wt), such as from about 1% to 20% wt/wt such as from about 2 % to about 10% wt/wt, preferably such as from about 3 % to about 5% wt/wt, such as about 4% wt/wt.

In certain embodiments Lamiaceae extract (such as a rosemary and/or salvia extract) comprises (or consist essentially/consist of) carnosic acid and/or carnosol and the ratio between carnosic acid and carnosol is from 40:1 to 5:1, such as 30:1, 20:1, 10:1 or 5:1.

In a particular embodiment, the Lamiacea extract (such as a rosemary and/or salvia extract) is an extract wherein a majority of the volatile oil components have been removed.

As used herein, the term "volatile oil components" may refer to compounds like essential oils such as: (-)-borneol, (-)-bornyl acetate, (-)-camphor, 1,8-Cineole (eucalyptol) and ver-benone.

In one embodiment the ration between the total % of phenolic diterpenes (such as carno-sic acid and carnosol) / Total % of volatiles oil components (such as (-)-borneol, (-)-bornyl acetate, (-)-cam- phor, 1,8-Cineole (eucalyptol) and verbenone) is not less than 15. In a particular embodiment the antimicrobial composition of the invention further comprises one or more of: carnosic acid, carnosol, methylcarnosate, rosmanol, isorosmanol, 1 1 , 12 -di-O- methyl- isorosmanol, 12-O-methylcarnosic acid, rosmanol-9-ethyl ether, circimaritin, Methylated monooxidized product of carnosic acid, genkwanin, epirosmanol, epiisorosmanol, carnosic acid derivative, epirosmanol ethyl ether, cryptotanshinone.

In certain embodiments the ration between the of phenolic acids (such as gallic acid, tannic acid and/or capsacin), eugenol and the phenolic diterpen(s) (such as carnosic acid) may be 1:30:40 to 1:40:50 such as 1:32:42 or about 1:20:30 or about 5:10:20.

In certain embodiments, the ration between the eugenol and the phenolic diterpene (such as carnosic acid) is from about 1:5 to 5:1, such as 1:1.3, or 1:1.5, or 1:2, or 1:2.5, or 1:3 or 1.3.5, or 1:4 or 1:4.5 or 4.5:1, or 4:1, or 3.5:1, or 3:1, or 2.5:1, or 2:1, or 1.5:1 or 1.3:1, or 1:1

In certain embodiments, the ration between eugenol, gallic acid and carnosic acid may be of at least 5/0.128/6.6.

In certain embodiments, the ratio between eugenol and gallic acid is from about 7:0.128 to about 5:1.

In certain embodiments, the ratio between the one or more polyphenols (for example from a water clove bud extract as previously defined) and the Phenolic dierpens (such as carnosic acid) for example obtained from a Lamiaceae extract as previously defined, may be from about 0.45:1.13 to to about 0.165:0.28, such as 0.23:0.56.

In certain embodiments, the eugenol and/or the gallic acid is provided by the first or second antimicrobial composition of the invention and carnosic acid is provided by a Lamiaceae extract (such as rosemary or salvia extract) as described herein.

In certain embodiments, the antimicrobial effect is against Saccharomyces cerevisiae, Lactobacillus plantarum, SchizoSaccharomyces pombe, Saccharomyces cerevisae, Bysocchlamys nivea, Aspergillus niger, Dekkera anomala and mixtures thereof.

The invention is also related to an antimicrobial composition comprising (or consisting essen- tially/consisting of) at least one phenolic diterpene and eugenol (and derivatives). In a preferred embodiment the phenolic diterpene is carnosic acid. In one embodiment, the ration between the eugenol and the phenolic diterpene (such as carnosic acid) is from about 1:5 to 5:1, such as 1:1.5, 1:2, 1:3, 1:4 or 4:1, 3:1, 2:1, 1.5:1.

The compositions of the invention may be formulated using carriers.

Suitable carriers include inert solid diluents or fillers, sterile aqueous solutions and various organic solvents. Examples of solid carriers are lactose, terra alba, sucrose, cyclodextrin, maltodextrin, dextrin, talc, gelatine, agar, pectin, acacia, magnesium stearate, magnesium hydroxide; stearic acid, arable gum, modified starch and lower alkyl ethers of cellulose, saccharose, silicon dioxide. Examples of liquid carriers are syrup, vegetables oils, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water. Moreover, the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.

The skilled person will understand that the antimicrobial compositions of the invention may typically be provided in solid form, but may be provided in liquid form. By solid form, it is included that the composition may be provided as powder, an amorphous solid, or as a crystalline or part-crystal- line solid. The dried extract may be mixed with a liquid carrier before use.

Antimicrobial and preservative uses

As demonstrated in the examples, the clove leaves extracts of the invention can inhibit the growth of a high spectrum of different spoiler bacteria. Also surprisingly the clove extracts have a very good anti-yeast and anti-mold activity. As it can be seen on table 8, 9 and 10, the clove leaves acetone extract has a strong inhibition effect on different yeast (Zygosaccharomyces bailii, Saccharomyces cerevisiae, Saccharomyces pombe) even at low concentrations.

As demonstrated on table 11, 12 and 13, the leaves acetone extract has a strong inhibition effect on different molds (Dekkera anomala, Aspergillus niger, Bysocchlamys nivea) even at very low concentrations

Moreover, a synergistic inhibitory effect on different bacteria, yeast and mold microorganisms was proved for the combination of the clove leaves extracts with Lamiaceae extracts. As it can be seen from the tables 15, 16, 21, 22 and 23 the clove leaves extract (acetone or ethanol/water) and the rosemary extract or Salvia extracts have a synergistic antimicrobial effect on different microorganisms.

Also a synergistic inhibitory effect of the combination of clove buds water extract with rosemary extract was observed (see tables 17, 18, 19). The extracts of the invention and the antimicrobial compositions of the invention may be utilised in any application in which the antibacterial effect is required.

An extract or a composition of the invention may stabilise any product where there is a risk of microorganism's growth. Thus product in the present invention includes, without limitations food or beverage products for humans or animals, nutritional supplements, nutraceutical formulations, a fragrance or flavoring, pharmaceutical or veterinary formulations, oenological or cosmetic formulations etc.

Therefore, present invention is related to the use of an extract of the invention, a first antimicrobial composition, a second antimicrobial composition and/or a third composition of the invention as antimicrobial and/or as a food preservative.

The present invention is also related to a method for inhibiting the growth of microorganisms and/or for killing microorganisms in a product (such as a food or beverage product for humans or animals, a nutritional supplement, a nutraceutical formulation, a fragrance or flavouring, a pharmaceutical or veterinary formulation, an oenological or cosmetic formulation) wherein the method comprises the step of contacting the product with an extract according to the invention or an antimicrobial composition according to the invention. In this context, "contacting" includes incorporating an extract of the invention or a composition of the invention into the product (such as a foodstuff).

Thus the present invention also relates to the use of an extract according to the invention or an antimicrobial composition as defined herein for inhibiting the growth of microorganisms and/or for killing microorganisms in product (such as a food or beverage product for humans or animals, a nutritional supplement, a nutraceutical formulation, a fragrance or flavouring, a pharmaceutical or veterinary formulation, an oenological or cosmetic formulation).

The present invention also provides a process for processing or producing a food or beverage product for humans or animals, a nutritional supplement, a nutraceutical formulation, a fragrance or flavouring, a pharmaceutical or veterinary formulation, an oenological or cosmetic formulation comprising applying or incorporating into said product (a food or beverage product for humans or animals, a nutritional supplement, a nutraceutical formulation, a fragrance or flavouring, a pharma- ceutical or veterinary formulation, an oenological or cosmetic formulation) an extract of the invention or any of the antimicrobial composition as defined herein for having a food preservative and/or antimicrobial effect.

When used as an antimicrobial and/or preservative agent for the products (such as food or beverage product for humans or animals, a nutritional supplement, a nutraceutical formulation, a fragrance or flavouring, a pharmaceutical or veterinary formulation, an oenological or cosmetic formulation), an effective amount of the extract of the invention, the first, the second and/or the third antimicrobial composition of the invention may be added at any appropriate step in the production process of said product (food or beverage product, etc.) to achieve the desired effect (i.e antimicrobial and/or preservative).

The different components of the first, second or third compositions of the invention may be used together as a unique formulation (i.e used simultaneously) or may be, separately or sequentially added to the product that is need to be protected against bacterial, yeast and/or mold spoilage.

Thus, the invention is related to a method for inhibiting the growth of microorganisms and/or for killing microorganisms in a product wherein the method comprises the step of simultaneously, separately or sequentially adding a first or a second composition as defined herein and a Lamiaceae extract to a product.

The invention is related to a method for inhibiting the growth of microorganisms and/or for killing microorganisms in a product wherein the method comprises the step of simultaneously, separately or sequentially adding a composition as defined herein (first or second composition of the ivnetion), carnosic acid and/or carnosol to a product.

The invention is related to a method for processing or preparing a food or beverage product for humans or animals, a nutritional supplement, a nutraceutical formulation, a fragrance or flavouring, a pharma-ceutical or veterinary formulation, an oenological or cosmetic formulation comprising the step of simultaneously, separately or sequentially adding a composition as defined herein and a Lamiaceae extract to a product for having a food preservative and/or antimicrobial effect.

The invention is related to a method for processing or preparing a food or beverage product for humans or animals, a nutritional supplement, a nutraceutical formulation, a fragrance or flavouring, a pharma-ceutical or veterinary formulation, an oenological or cosmetic formulation comprising the step of simultaneously, separately or sequentially adding a composition as defined herein, carnosic acid and/or carnosol to a product for having a food preservative and/or antimicrobial effect.

According to the present invention, the antimicrobial properties of the extract of the invention, the first, second or third compositions of the invention are harnessed as a preservative in compositions such as beverages and food, cosmetic formulation etc. by using an effective amount of the first compositions or the second compositions of the invention, wherein the preservative achieves microbial stability of at least one microorganism chosen from mold, yeast and bacteria in said product (beverage, food, cosmetic formulation etc.).

In certain embodiments of the uses or methods described herein, the preservative and/or antimicrobial effect is against a food and/or beverage spoilage microorganism.

In certain embodiments of the uses or methods described herein, the preservative and/or antimicrobial effect is against bacteria, more precisely food and beverage spoilage bacterial. The antibacterial effect may be against acid-lactic bacteria, thermo-acidophilic bacteria, xerophilic bacteria and/or acidophilic bacteria.

As used herein, the term "Acid lactic bacteria" refers to an order of gram-positive, low-GC, acid- tolerant, generally nonsporulating, non-respiring, either rod-shaped (bacilli) or spherical (cocci) bacteria that share common metabolic and physiological characteristics. These bacteria produce lactic acid as the major metabolic end product of carbohydrate fermentation. Examples of acid lactic bacteria are the Lactobacillus genus (such as Lactobacillis plantarum), Leuconostoc genus or Lactococcus genus.

As used herein, the term "Thermo-acidophilic" refers to an extremophilic microorganism that is both thermophilic and acidophilic; i.e., it can grow under conditions of high temperature and low pH. Examples of Thermo-acidophilic bacteria are bacteria from the Al icyclobacillus genus such as Al icyclobacil lus acidoterrestris and Alicyclobacill us acidocaldarius. This type of bacteria are able to grow in acidic conditions, while the spores are able to survive typical pasteurization procedures.

As used herein, the term "Xerophilic bacteria" refers to bacteria that are tolerant to high osmotic pressure.

As used herein, the term "Acidophilic bacteria" refers to bacteria that thrive under acidic and high acidic conditions. Examples are Al icyclobacill us genus (such as Al icyclobacill us acidoterrestris or Al- icyclobacil I us acidocaldarius ), Gluconobacter genus and Zymomonas genus.

The anti-bacteria effect (bacterial growth inhibited, decreases and/or limited) may be against bacteria that belong to the genus and/or species mentioned before or combination thereof. In certain embodiments of the uses or methods described herein, the preservative and/or antimicrobial effect is against yeast, more precisely food and beverage spoilage yeast. Typically, spoilage by yeasts manifests itself as fermentation with gas and ethanol production, as well as sedimentation, off-flavours and odours, and loss of cloud or emulsion stability. Examples of yeast spoilage include "blown cans" of soft drinks, cloudy re-fermented wine, pink or red slime dripping from refrigerated meat, white yeast colonies on food, and tainted fruit juices.

Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. In one embodiment, the yeast is able to degrade glucose or fructose to ethanol, even in the presence of oxygen (Crabtree effect) (such as Saccharomyces cerevisiae or Dekkera anomala), is tolerant to high osmotic pressure (xerophilic) (such as SchizoSaccharomyces pombe) and /or is tolerate high ethanol concentrations (such as Dekkera anomala).

Common genera (or genus) of yeast include: Candida Spp, Saccharomyces Spp. (such as Saccha- romices cerevisiae and/or Saccharomyces pombe), Dekkera Spp. (such as D. anomala), Brettano- myces spp. and/or Zygosaccharomyces Spp (such as Zygosaccharomyces baillii).

The anti-yeast effect (yeast growth inhibited, decreases and/or limited) may be against yeast that belongs to the genus and/or species mentioned before or any combination thereof.

In certain embodiments of the uses or methods described herein, the preservative and/or antimicrobial effect is against molds. As use herein the term "mold" or "mould" refers to a fungus that grows in the form of multicellular filaments called hyphae. Molds are a large and taxonomically diverse number of fungal species in which the growth of hyphae results in discoloration and a fuzzy appearance, especially on food. Molds are considered to be microbes and do not form a specific taxonomic or phylogenetic grouping, but can be found in the divisions Zygomycota and Ascomycota. Common genera of molds include:

Acremonium, Alternaria, Aspergillus, Byssochlamys, Cladosporium, Fusarium, Mucor, Penicillium, Rhizopus, Stachybotrys, Trichoderma and Trichophyton. More precisely food and beverage spoilage molds such as the molds selected from Byssochlamys genus (such as Byssochlamys nivea), Penicillium genum and Aspergillus genum (such as Aspergillum niger). Aspergillum niger is a conidial fungus with an asexual spore-forming structure that is highly thermotolerant, adaptive UV radiations and able to grow at low aw food products (growth at aw > 0.80) and within a wide range of pH (2 - 8). It is also producer of mycotoxin and therefore a dangerous food and beverage spoiler.

The antimold effect (mold growth inhibited, decreases and/or limited) may be against molds that belong to the genus and/or species mentioned before or any combination thereof. In certain embodiments of the uses or methods described herein, the antimicrobial effect is against Saccharomyces cerevisiae, Lactobacillus plantarum, SchizoSaccharomyces pombe, Saccharomyces cerevisae, Bysocchlamys nivea, Aspergillus niger, Dekkera anomala and mixtures thereof.

The invention also relates to preservative and/or antimicrobial composition (such as an anti-bacterial, anti-yeast or anti-mold) comprising an effective amount of a first composition of the invention and/or an effective amount of a second composition of the invention.

"Effective amount" is the amount necessary in order to achieve a specific effect, in accordance with what one of ordinary skill in the art would be readily able to determine through routine experimentation. For example, with regard to the present disclosure, an effective amount of an extract of the invention to a food product or foodstuff, e.g. meat, fish and poultry (both fresh/unprocessed and processed), a sauce like an emulsified sauce or non-emulsified sauce (such as ketchup) or beverage, to extend the longevity of the food, e.g. red color to the fresh meat, fish and poultry, is an amount which is determined to provide the red color longevity based on known parameters which include, but are not limited to the concentration of the extract of the invention, the volume and/or surface area of the meat, fish and poultry, and the atmospheric environment conditions of the meat, fish and poultry. Similarly, the effective amounts of the flavonoids (such as Quercetin, Kaempferol, Goss- ypetin, their derivatives and mixtures thereof), the phenolic acids (such as gallic acid, tannic acid, capsacin, their derivatives and mixtures thereof) and the organic acids (such as quinic acid, malic acid, citric acid, tartaric acid, their derivatives and mixtures thereof), to extend the longevity of red color to the meat, fish and poultry are determined in a similar way.

The effective amount of an extracts of the invention or the compositions of the invention depends on the nature of the food product or beverage. For example, the extracts of the invention may be present in the beverage product in an amount ranging from about 50 ppm to about 20,000 ppm such as from about 250 ppm to about 5000 ppm in high-nutrient (e.g., juice, vitamin, nitrogen, etc.) beverages, or for example from about 100 ppm to about 1000 ppm in low nutrient beverages (e.g., beverages lacking vitamin(s), low levels of nitrogen, etc., and about < 3% juice), and further for example, from about 250 ppm to about 1000 ppm such as 250 ppm to 750 ppm in low acid beverages.

In certain embodiments of the uses or methods described herein, the extract of the invention and/or the compositions of the invention are present in the product in an amount from about 0.001% to about 10% by weight of the Product (such as a foodstuff), such as from about 0.025% to about 5%, or from about 0.05% to about 2.5% by weight of the Product (such as a foodstuff). For example, the extract or the compositions of the invention may be present in the Product (such as a foodstuff) in an amount from about 25ppm to about 20000pmm, such as from about 250 ppm to about 2000 ppm, such as 500 ppm or 1000 ppm.

In certain embodiments of the uses or methods described herein, the extract of the invention and/or the compositions of the invention are present in the product in an amount from at least 0.001% wt/wt by weight of the Product (such as a foodstuff), such as at least about 0.025%, at least 0.05%, at least 0.01%, at least 0.1%, at least 1%, at least 2.5 %, at least about 5% by weight of the Product (such as a foodstuff). For example, the extract or the compositions of the invention may be present in the Product (such as a foodstuff) in an amount from at least about 25ppm, at least 250 ppm, at least 500ppm, at least lOOOppm, at least 20000pmm wt/wt.

In certain embodiments of the uses or methods described herein, the extract of the invention and/or the compositions of the invention (the first, second antimicrobial and/or third composition of the invention) is incorporated into the product so that the concentration of flavonoids (such as Quercetin, Kaempferol, Gossypetin, their derivatives and mixtures thereof) in the final product is from about 12ppm, 15ppm, 20 ppm, 30, 40, 50, 60, 70, 80, 90, 100 ppm, to about 6%, 5%, 4%, 4%, 3%, 2%, 1% (lOOOOppm), 9000ppm, 8000ppm, 7000ppm, 6000ppm, 5000ppm, 4000ppm, 3000ppm, 2000ppm, lOOOppm, 800ppm, 600ppm, 500ppm, 400ppm, 300ppm to 200ppm wt/wt, such as from lOOppm to 2000ppm; and /or the concentration of phenolic acids (such as gallic acid, tannic acid, capsacin, their derivatives and mixtures thereof) in the final product is from about 12ppm, 15ppm, 20 ppm, 30, 40, 50, 60, 70, 80, 90, 100 ppm, to about 6%, 5%, 4%, 4%, 3%, 2%, 1% (lOOOOppm), 9000ppm, 8000ppm, 7000ppm, 6000ppm, 5000ppm, 4000ppm, 3000ppm, 2000ppm, lOOOppm, 800ppm, 600ppm, 500ppm, 400ppm, 300ppm to 200ppm wt/wt, such as from 50ppm to lOOOppm; and /or the concentration of organic acids (such as quinic acid, malic acid, citric acid, tartaric acid, their derivatives and mixtures thereof) in the final product is from about 12ppm, 15ppm, 20 ppm, 30, 40, 50, 60, 70, 80, 90, 100 ppm, to about 6%, 5%, 4%, 4%, 3%, 2%, 1% (lOOOOppm), 9000ppm, 8000ppm, 7000ppm, 6000ppm, 5000ppm, 4000ppm, 3000ppm, 2000ppm, lOOOppm, 800ppm, 600ppm, 500ppm, 400ppm, 300ppm to 200ppm wt/wt, such as from 50ppm to lOOOppm.

In certain embodiments of the uses or methods described herein, the extract of the invention and/or the compositions of the invention (the first, second antimicrobial and/or third composition of the invention) is incorporated into the product so that the concentration of flavonoids (such as Quercetin, Kaempferol, Gossypetin, their derivatives and mixtures thereof) in the final product is from at least about 12ppm, 15ppm, 20 ppm, 30, 40, 50, 60, 70, 80, 90, 100 ppm, 500ppm, lOOOppm, 2000ppm, such as at least lOOOOppm wt/wt, such as at least 1%, 2%, 3%, 4%, 5% or at least 6% wt/wt; and /or the concentration of phenolic acids (such as gallic acid, tannic acid, capsacin, their derivatives and mixtures thereof) in the final product is from about from at least about 12ppm, 15ppm, 20 ppm, 30, 40, 50, 60, 70, 80, 90, 100 ppm, 500ppm, lOOOppm, 2000ppm, such as at least lOOOOppm wt/wt, such as at least 1%, 2%, 3%, 4%, 5% or at least 6% wt/wt, such as from 50ppm to lOOOppm; and /or the concentration of organic acids (such as quinic acid, malic acid, citric acid, tartaric acid, their derivatives and mixtures thereof) in the final product is from about from at least about 12ppm, 15ppm, 20 ppm, 30, 40, 50, 60, 70, 80, 90, 100 ppm, 500ppm, lOOOppm, 2000ppm, such as at least lOOOOppm wt/wt, such as at least 1%, 2%, 3%, 4%, 5% or at least 6% wt/wt, such as from 50ppm to lOOOppm.

In certain embodiments of the uses or methods described herein, the extract of the invention and/or the compositions of the invention (the first, second antimicrobial and/or third composition of the invention) is incorporated into the product so that the concentration of eugenol (and derivatives) in the final product is from about 12ppm, 15ppm, 20 ppm, 30, 40, 50, 60, 70, 80, 90, 100 ppm, to about 6%, 5%, 4%, 3%, 2%, 1% (lOOOOppm), 9000ppm, 8000ppm, 7000ppm, 6000ppm, 5000ppm, 4000ppm, 3000ppm, 2000ppm, lOOOppm, 800ppm, 600ppm, 500ppm, 400ppm, 300ppm to 200ppm wt/wt, such as from 50ppm to lOOOppm wt/wt.

In certain embodiments of the uses or methods described herein, the extract of the invention and/or the compositions of the invention (the first, second antimicrobial and/or third composition of the invention) is incorporated into the product so that the concentration of eugenol (and derivatives) in the final product at least about 12ppm, 15ppm, 20 ppm, 30, 40, 50, 60, 70, 80, 90, 100 ppm, 500ppm, lOOOppm, 2000ppm, such as at least lOOOOppm wt/wt, such as at least 1%, 2%, 3%, 4%, 5% or at least 6% wt/wt, such as at least 50ppm or at least lOOOppm wt/wt.

In certain embodiments of the uses or methods described herein, If the composition of the invention (the first, second antimicrobial and/or third composition of the invention) also comprises a Lamiaceae extract (such as a rosemary and/or salvia extract), said antimicrobial composition of the invention is incorporated into the product so that the concentration of phenolic diterpene(s) (such as carnosic acid) in the final product is from about 12ppm, 15ppm, 20 ppm, 30, 40, 50, 60, 70, 80, 90, 100 ppm, to about 6%, 5%, 4%, 3%, 2%, 1% (lOOOOppm), 9000ppm, 8000ppm, 7000ppm, 6000ppm, 5000ppm, 4000ppm, 3000ppm, 2OOOppm, lOOOppm, 800ppm, 600ppm, 500ppm, 400ppm, 300ppm to 200ppm wt/wt, such as from 50ppm to lOOOppm wt/wt.

In certain embodiments of the uses or methods described herein, If the composition of the invention (the first, second antimicrobial and/or third composition of the invention) also comprises a Lamiaceae extract (such as a rosemary and/or salvia extract), said antimicrobial composition of the invention is incorporated into the product so that the concentration of phenolic diterpene(s) (such as carnosic acid) in the final product is of at least about 12ppm, 15ppm, 20 ppm, 30, 40, 50, 60, 70, 80, 90, 100 ppm, 500ppm, lOOOppm, 2000ppm, such as at least lOOOOppm wt/wt, such as at least 1%, 2%, 3%, 4%, 5% or at least 6% wt/wt, such as from at least 50ppm or at least lOOOppm wt/wt.

In certain embodiments of the uses or methods described herein, the extract of the invention and/or the compositions of the invention may be applied or incorporated into said product in the form a dry powder or a liquid form, to the product (such as a foodstuff).

In certain embodiments of the uses or methods, there is an improvement of the colour life and/or an inhibition, decrease or limitation of the microbial growth (such as bacterial, yeast and/or molds growth) in said product (such as a food or beverage). The antibacterial effect may be against acid- lactic bacteria, thermo-acidophilic bacteria (such as Lactobacillus genus (such as Lactobacillis plantarum), Leuconostoc genus and/or Lactococcus genus), Xerophilic bacteria (such as Alicycloba- cillus genus like Al icycl obaci 11 us acidoterrestris or Al icycl obaci 11 us acidocal-darius) and/or acidophilic bacteria (such as Alicyclobacillus genus (such as Alicyclobacillus acidoterrestris or Alicyclobacillus acidocaldarius ), Gluconobacter genus and/or Zymomonas genus), or combinations thereof.

In certain embodiments of the uses or methods, the preservative and/or antimicrobial effect is against yeast, more precisely food and beverage spoilage yeast. In one embodiment, the yeast is selected from the genus Candida Spp, Saccharomyces Spp. (such as Saccharomices cerevisiae and/or Saccharomyces pombe), Dekkera Spp. (such as D. anomala), Brettanomyces spp. and/or Zy- gosaccharomyces Spp (such as Zygosaccharomyces baillii). The anti-yeast effect may be against one or more yeast, such as the yeast that belongs to the genus and/or species mentioned before or combinations thereof.

In certain embodiments of the uses or methods, the preservative and/or antimicrobial effect is against molds such as Acremonium, Alternaria, Aspergillus, Byssochlamys, Cladosporium, Fusarium, Mucor, Penicillium, Rhizopus, Stachybotrys, Trichoderma and Trichophyton. More precisely food and beverage spoilage molds such as the molds selected from Byssochlamys genus (such as Bysso- chlamys nivea), Penicillium genum and Aspergillus genum such as Aspergillum niger. The anti-mold effect may be against one or more molds, such as the molds that belong to the genus and/or species mentioned before or combinations thereof.

The antimicrobial effect may be against one or more of the bacteria, yeast and molds described before.

In certain embodiments of the uses or methods, there is prevention on the change of colour of the product, a prevention on the change in texture, a prevention of an unpleasant odour and / or a prevention of un-desirable taste.

Testing of the antimicrobial properties may be determined by art-recognised methods. These include the ones described in the examples.

In a preferred embodiment of the uses and methods described herein, the Syzygium genus leaves are Syzygium aromaticum leaves.

Foodstuff and other products

The present invention further provides a product (such as food or beverage product for humans or animals, a nutritional supplement, a nutraceutical formulation, a fragrance or flavouring, a pharmaceutical or veterinary formulation, an oenological or cosmetic formulation) comprising a Syzygium genus leaves and/or buds extract (or extracts of the invention) or a stabilising and/or antimicrobial composition of the invention (first, second antimicrobial and/or third composition of the invention). In a preferred embodiment, the Syzygium species is Syzygium aromaticum.

The extracts or antimicrobial compositions of the invention are suitable for use in a wide range of foodstuffs and other products.

As used herein, "food" or "foodstuff" refers at least to an edible food product, such as, a solid, semisolid or liquid (such as beverage) food stuff product known in the art. Mention may be made of food products and beverage such as, but not limited to, emulsified or non-emulsified sauces and any other edible composition that can be spoiled as a result of microorganism contamination.

As used herein, the term "beverage" or "beverage composition" refers to a liquid drink that is appropriate for human or animal consumption. Mention may be made, of beverages, but not limited to, for example, energy drinks, flavored water, fruit smoothies, sport drinks, fruit juices and juices concentrates (e.g., juice drinks and full strength fruit juice), carbonated sodas/juices, shakes, protein drinks (e.g., dairy, soy, rice or other), meal replacements, drinkable dairy yogurts, drinkable soy yogurts, teas, coffees, cola drinks, fortified waters, low acid beverages as defined in 21 C.F.R §113, acidified beverages as defined in 21 C.F.R. §114, syrups, cordials, dilutables such as squashes, health drinks, functional beverages (e.g., nutraceuticals), nectars, tonics, horchata (i.e., vegetable and/or rice components made into a beverage), frozen carbonated beverages and frozen uncarbonated beverages.

In a preferred embodiment, the food product is selected from beverages such as carbonated and non-carbonated beverages (such as fruit drinks such as juices, juices concentrates, teas, coffees, enhanced waters etc.); emulsified or non-emulsified sauces (such as mayonnaise, ketchup, etc) and meat, meat analogues and cultured meat.

Food encompasses the following general food categories, as defined by the Food and Drug Ad-min- istration (FDA): baked goods and baking mixes, including all ready-to-eat and ready-to-bake products, flours, and mixes requiring preparation before serving; beverages, alcoholic, including malt beverages, wines, distilled liquors, and cocktail mix; beverages and beverage bases, non-alcoholic, including only special or spiced teas, soft drinks, coffee substitutes, and fruit and vege-table flavored gelatin drinks; breakfast cereals, including ready-to-eat and instant and regular hot cereals; cheeses, including curd and whey cheeses, cream, natural, grating, processed, spread, dip, and miscellaneous cheeses; chewing gum, including all forms; coffee and tea, including regular, decaffeinated, and instant types; condiments and relishes, including plain seasoning sauces and spreads, olives, pickles, and relishes, but not spices or herbs; confections and frostings, including candy and flavored frosting, marshmallows, baking chocolate, and brown, lump, rock, maple, powdered, and raw sugars; dairy product analogs, including nondairy milk, frozen or liquid creamers, coffee whiteners, toppings, and other nondairy products; egg products, including liquid, frozen, or dried eggs, and egg dishes made therefrom, i.e., egg roll, egg foo young, egg salad, and frozen multicourse egg meals, but not fresh eggs; fats and oils, including margarine, dressings for salads, butter, salad oils, shortenings and cooking oils; fish products, including all prepared main dishes, salads, appetizers, frozen multicourse meals, and spreads containing fish, shellfish, and other aquatic animals, but not fresh fish; fresh eggs, including cooked eggs and egg dishes made only from fresh shell eggs; fresh fish, including only fresh and frozen fish, shellfish, and other aquatic animals; fresh fruits and fruit juices, including only raw fruits, citrus, melons, and berries, and home-prepared "ades" and punches made therefrom; fresh meats, including only fresh or home-frozen beef or veal, pork, lamb or mutton and home-prepared fresh meat-containing dishes, salads, appetizers, or sandwich spreads made therefrom; fresh poultry, including only fresh or home-frozen poultry and game birds and home-prepared fresh poultry-containing dishes, salads, appetizers, or sandwich spreads made therefrom; fresh vegetables, tomatoes, and potatoes, including only fresh and home-prepared vegetables; frozen dairy desserts and mixes, including ice cream, ice milks, sherbets, and other frozen dairy desserts and specialties; fruit and water ices, including all frozen fruit and water ices; gelatins, puddings, and fillings, including flavored gelatin desserts, puddings, custards, parfaits, pie fillings, and gelatin base salads; grain products and pastas, including macaroni and noodle products, rice dishes, and frozen multicourse meals, without meat or vegetables; gravies and sauces, including all meat sauces and gravies, and tomato, milk, buttery, and specialty sauces; hard candy and cough drops, including all hard type candies; herbs, seeds, spices, seasonings, blends, extracts, and flavorings, including all natural and artificial spices, blends, and flavors; jams and jellies, home-prepared, including only home-prepared jams, jellies, fruit butters, preserves, and sweet spreads; jams and jellies, commercial, including only commercially processed jams, jellies, fruit butters, preserves, and sweet spreads; meat products, including all meats and meat containing dishes, salads, appetizers, frozen multicourse meat meals, and sandwich ingredients prepared by commercial processing or using commercially processed meats with home preparation; milk, whole and skim, including only whole, lowfat, and skim fluid milks; milk products, including flavored milks and milk drinks, dry milks, toppings, snack dips, spreads, weight control milk beverages, and other milk origin products; nuts and nut products, including whole or shelled tree nuts, peanuts, coconut, and nut and peanut spreads; plant protein products, including the National Academy of Sciences/National Research Council "reconstituted vegetable protein" category, and meat, poultry, and fish substitutes, analogues, and extender products made from plant proteins; poultry products, including all poultry and poultry-containing dishes, salads, appetizers, frozen multicourse poultry meals, and sandwich ingredients prepared by commercial processing or using commercially processed poultry with home preparation; processed fruits and fruit juices, including all commercially processed fruits, citrus, berries, and mixtures; salads, juices and juice punches, concentrates, dilution, "ades", and drink substitutes made therefrom; processed vegetables and vegetable juices, including all commercially processed vegetables, vegetable dishes, frozen multicourse vegetable meals, and vegetable juices and blends; snack foods, including chips, pretzels, and other novelty snacks; soft candy, including candy bars, chocolates, fudge, mints, and other chewy or nougat candies; soups, home-prepared, including meat, fish, poultry, vegetable, and combination home-prepared soups; soups and soup mixes, including commercially prepared meat, fish, poultry, vegetable, and combination soups and soup mixes; sugar, white, granulated, including only white granulated sugar; sugar substitutes, including granulated, liquid, and tablet sugar substitutes; and sweet sauces, toppings, and syrups, including chocolate, berry, fruit, corn syrup, and maple sweet sauces and toppings.

In a preferred embodiment, the animal or human food and beverage products are selected from meat products (such as raw meat, cooked meat, raw poultry products, cooked poultry products, raw seafood products, cooked seafood products, meat analogues, ready to eat meals), cooking sauces (such as pasta sauces and ketchups, table sauces), pasteurised and unpasteurised soups, salad dressings and other oil-in-water emulsions e.g. mayonnaise, hollandaise, burger sauce or bearnaise water-in-oil emulsions, dairy products, bakery products, confectionary products, fruit products and foods with fat based or water containing filings. Preferably, the foodstuff comprises an oil-in-water- emulsion or an oil-in-water emulsion.

The foodstuff typically contains the extract of the invention, the first, second and/or third antimicrobial composition of the invention in an amount sufficient to stabilise the product (such as a foodstuff), for example, by reducing, inhibiting or preventing the growth of an undesired or spoiler microorganism or killing said microorganism. Typically, the extract or stabilising composition is present in the foodstuff in an amount from about 0.001% to about 10% by weight of the foodstuff. Such as from about 0.025% to about 5%, or from about 0.05% to about 2.5%. For example, the extract or the compositions of the invention may be present in the foodstuff in an amount from about 25ppm to about 20000pmm, such as from about 250 ppm to about 2000 ppm, such as 500 ppm or 1000 ppm.

When the extract of the invention, the first and/or the second antimicrobial composition of the invention is incorporated into a foodstuff, such as egg and oil based sauces, e.g. mayonnaise, hollandaise or bearnaise, as well as providing a stabilising effect by reducing, inhibiting or preventing the amount of oxidation over a given period relative to the amount of oxidation that would have occurred in the absence of the stabilising composition, the extract or the compositions of the invention should not adversely affect the colour of the foodstuff in which it has been incorporated.

Therefore, it is an object of the invention to provide a antimicrobial composition that achieves the desired stabilising effect and additionally, can be incorporated into a foodstuff, such as egg and oil based sauces, e.g. mayonnaise, hollandaise or bearnaise, or non-emulsified sauces such as ketchup, with no identifiable or a minimal change in the colour or taste of the foodstuff, such that the change would be unacceptable to the consumer. In one embodiment, the extract of the invention, the first second antimicrobial and/or the third composition of the invention is incorporated into the product so that the concentration of flavonoids (such as Quercetin, Kaempferol, Gossypetin, their derivatives and mixtures thereof) in the final product is from about 12ppm, 15ppm, 20 ppm, 30, 40, 50, 60, 70, 80, 90, 100 ppm, to about 6%, 5%, 4%, 4%, 3%, 2%, 1% (lOOOOppm), 9000ppm, 8000ppm, 7000ppm, 6000ppm, 5000ppm, 4000ppm, 3000ppm, 2000ppm, lOOOppm, 800ppm, 600ppm, 500ppm, 400ppm, 300ppm to 200ppm wt/wt, such as from lOOppm to 2000ppm; and /or the concentration of phenolic acids (such as gallic acid, tannic acid, capsacin, their derivatives and mixtures thereof) in the final product is from about 12ppm, 15ppm, 20 ppm, 30, 40, 50, 60, 70, 80, 90, 100 ppm, to about 6%, 5%, 4%, 4%, 3%, 2%, 1% (lOOOOppm), 9000ppm, 8000ppm, 7000ppm, 6000ppm, 5000ppm, 4000ppm, 3000ppm, 2000ppm, lOOOppm, 800ppm, 600ppm, 500ppm, 400ppm, 300ppm to 200ppm wt/wt, such as from 50ppm to lOOOppm; and /or the concentration of organic acids (such as quinic acid, malic acid, citric acid, tartaric acid, their derivatives and mixtures thereof) in the final product is from about 12ppm, 15ppm, 20 ppm, 30, 40, 50, 60, 70, 80, 90, 100 ppm, to about 6%, 5%, 4%, 4%, 3%, 2%, 1% (lOOOOppm), 9000ppm, 8000ppm, 7000ppm, 6000ppm, 5000ppm, 4000ppm, 3000ppm, 2000ppm, lOOOppm, 800ppm, 600ppm, 500ppm, 400ppm, 300ppm to 200ppm wt/wt, such as from 50ppm to lOOOppm.

In one embodiment, the extract of the invention, the first second antimicrobial and/or the third composition of the invention is incorporated into the product so that the concentration of flavonoids (such as Quercetin, Kaempferol, Gossypetin, their derivatives and mixtures thereof) in the final product is at least about 12ppm, 15ppm, 20 ppm, 30, 40, 50, 60, 70, 80, 90, 100 ppm, 500ppm, lOOOppm, 2000ppm, such as at least lOOOOppm wt/wt, such as at least 1%, 2%, 3%, 4%, 5% or at least 6% wt/wt, such as at least lOOppm or at least 2000ppm; and /or the concentration of phenolic acids (such as gallic acid, tannic acid, capsacin, their derivatives and mixtures thereof) in the final product is of at least about 12ppm, 15ppm, 20 ppm, 30, 40, 50, 60, 70, 80, 90, 100 ppm, 500ppm, lOOOppm, 2000ppm, such as at least lOOOOppm wt/wt, such as at least 1%, 2%, 3%, 4%, 5% or at least 6% wt/wt, such as at least 50ppm or at least lOOOppm; and /or the concentration of organic acids (such as quinic acid, malic acid, citric acid, tartaric acid, their derivatives and mixtures thereof) in the final product is of at least about 12ppm, 15ppm, 20 ppm, 30, 40, 50, 60, 70, 80, 90, 100 ppm, 500ppm, lOOOppm, 2000ppm, such as at least lOOOOppm wt/wt, such as at least 1%, 2%, 3%, 4%, 5% or at least 6% wt/wt, such as at least 50ppm or at least lOOOppm.

In one embodiment If the antimicrobial composition of the invention that is present in a product (such a food product) also comprises a Lamiaceae extract, said antimicrobial composition of the invention is incorporated into the product so that the concentration of phenolic diterpene(s) (such as carnosic acid) in the final product is from about 12ppm, 15ppm, 20 ppm, 30, 40, 50, 60, 70, 80, 90, 100 ppm, to about 6%, 5%, 4%, 3%, 2%, 1% (lOOOOppm), 9000ppm, 8000ppm, 7000ppm, 6000ppm, 5000ppm, 4000ppm, 3000ppm, 2000ppm, lOOOppm, 800ppm, 600ppm, 500ppm, 400ppm, 300ppm to 200ppm wt/wt, such as from 50ppm to lOOOppm.

For the avoidance of doubt, preferences, options, particular features and the like indicated for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all other preferences, options particular features and the like as indicated for the same or other aspects, features and parameters of the invention.

When we use the term "comprising" or "comprises" we mean that the extract or composition being described must contain the listed ingredient(s) but may optionally contain additional ingredients. When we use the term "consisting essentially of" or "consists essentially of" we mean that the extract or composition being described must contain the listed ingredient(s) and may also contain small (for example up to 5% by weight, or up to 1 % or 0.1 % by weight) of other ingredients provided that any additional ingredients do not affect the essential properties of the extract or composition. When we use the term "consisting of" or "consists of" we mean that the extract or composition being described must contain the listed ingredient(s) only.

These terms can be applied in an analogous manner to processes, methods and uses.

Unless otherwise stated herein, the weight percentages listed are based on the total weight of the extract, for example the total weight of the dry extract.

The term "about" as used herein, e.g. when referring to a measurable value (such as an amount of weight of a particular component in the composition or reaction mixture), refers to variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or particularly, ±0.1%, of the specified amount.

As used herein, references to a material being "substantially free" of another material may refer to the material consisting of less than 1% by weight (e.g. less than 0.1%, such as less than 0.01% or less than 0.001%, by weight) of that other material. The terms "a" and "an" are defined as one or more unless expressly stated otherwise or constrained by other language herein. An element or feature proceeded by "a" or "an" may be interpreted as one of the recited element or feature, or more than one of the element or feature.

All amounts, unless

otherwise are amount in weight percent (% wt/wt).

It should be understood that when an amount in weight percent (% wt/wt) is described in the present disclosure, it is intended that any and every amount within the range, including the end points, is to be considered as having been expressly disclosed. For example, the disclosure of "a range of from about 1 to about 10" is to be read as indicating each and every possible number along the continuum between about 1 and about 10. It is to be understood that the inventors appreciate and understand that any and all data points within the range are to be considered to have been specified, and that the inventors have possession of the entire range and all points within the range.

When a concentration is expressed as "ppm", the concentration is parts per million by weight based on the total weight of the consumable. It should be understood that when a range of values is described in the present disclosure, it is intended that any and every value within the range, including the end points, is to be considered as having been disclosed. For example, "a range of from 1 ppm to 1000 ppm" of a component of the composition is to be read as indicating each and every possible number along the continuum between 1 and 1000. It is to be understood that the inventors appreciate and understand that any and all values within the range are to be considered to have been specified, and that the inventors have possession of the entire range and all the values within the range.

Provided below in Table 1 are non-limiting examples of various types of microorganism such as molds, yeasts and bacteria, that commonly contaminate a beverage or food. Aspergillus spp. and Penicillium spp. represent common mold genera that can readily grow in non-carbonated beverages such as juice drinks and enhanced waters if not controlled by preservation or heating. Byssochlamys spp. are examples of heat resistant molds that survive pasteurization and grow in, for example, unpreserved isotonic sport drinks and teas. The yeasts, Zygosaccharomyces spp., Saccharomyces spp. can be problematic to acidic shelf stable beverages in part due to their potential resistance to sorbic and benzoic acid. Dekkera spp. are a genus of yeasts that are uniquely tolerant to high levels of carbonation, and thus can grow in and spoil carbonated beverages. Lactobacillus spp. are acidophilic bacteria that can spoil non- carbonated beverages. The genus Al icyclobacillus spp. is a spore-forming bacterium that can survive pasteurization and grow at elevated temperatures of beverages such as isotonic sport drinks and juices. Bacillus spp. and Clostridium spp. are sporeforming bacteria that may survive mild pasteurization treatments and spoil low- acid food and beverage products.

5

Table 1.

Example 1. Preparation of clove leaves extracts

Reflux 0 Dried clove leaves (Syzygium aromaticum) were ground into powder of approx. 3-4mm size. 450g samples of these were refluxed with stirring for 2 hours with 10 times W/V i.e. 4500ml of the solvents shown below. This reflux was repeated with the same volume of solvent, the extracts were then filtered and dried.

• Solvent A - 100% distilled water

• Solvent B - 70% distilled water/30% ethanol

• Solvent C - 70% ethanol/30% distilled water

• Solvent D - 100% acetone

Percentages of ethanol/water are by volume.

Brown flowable powders were obtained with solvents A-C, with mass yields between about 20%0 and about 34%. 1.1. Dynamic extraction at ambient temperature

Dried clove leaves were ground into powder of approx. 3-4mm size. 100g sample of these were extracted at ambient temperature with stirring for 2 hours with 10 times W/V i.e. 1000ml of water. This cold extraction was repeated with the same volume of water, the extract was then filtered and dried.

1.2. Dynamic extraction at 50°C

Dried clove leaves were ground into powder of approx. 3-4mm size. 100g sample of these were extracted at 50°C with stirring for 2 hours with 10 times W/V i.e. 1000ml of water. This extraction was repeated with the same volume of water, the extract was then filtered and dried.

1.3. Percolation at 50°C

Dried clove leaves were ground into powder of approx. 3-4mm size. 100g sample of these were extracted at 50°C by percolation for 2 hours with 10 times W/V i.e. 1000ml of water. This extraction was repeated with the same volume of water, the extract was then filtered and dried.

All of these extractions can be processed with active coal mixed to the raw material.

1.4. Clove extract composition

Eugenol contents were quantified by GC-FID to try to explain this difference and summarized in the Table 2.

Table 2. Eugenol content of the four extracts obtained with solvents A-D

Table 3. Quantification in HPLC-UV

Example 2. Preparation of clove bud extracts

The clove Bud was grinding before extraction. It was extracted with boiling water (100°C) with 2 covers of 2 hours each under mechanical stirring. Once the extraction was completed, the water was filtered to remove the plant residues. The resulting water was concentrated under reduced pressure to dryness. The dry extract contains about 10 - 15% polyphenol.

Example 3. Preparation of rosemary extracts

The detailed procedure to prepare the composition of Rosemary extract was described in the US Patent No. 5,859,293 (PCT W096/34534), which is incorporated herein by reference in its entirely. The procedure is briefly summarized as follows: The Rosemary leaf was extracted with acetone at room temperature. After the extraction was completed, the acetone extract was filtered to separate the solution from Rosemary leaf and concentrated under reduced pressure to make concentrated native extract. At this time, the concentrated extract can be dried directly in a vacuum oven under mild heat to make a powdered extract, which is a composition comprising about 15%-30% carnosic acid and I %-3% carnosol. Or to the concentrated native extract, aqueous sodium carbonate (NaHCCh) was added to dissolve carnosic acid and other organic acids, while base insoluble substances were precipitated out. The solution was filtered to separate from solid, and the filtrate was further concentrated under reduced pressure. Once finishing concentration is achieved, phosphoric acid (H3PO4) was added and the acid insoluble substances (including carnosic acid, carnosol, and carnosic derivatives) were precipitated from the concentrated solution. Through filtering, the precipitated solid was subsequently separated from liquid and rinsed with water to remove impurities. Last, the solid was dried in a vacuum oven and then milled into powder which is a composition containing about 40-85% carnosic acid, 2- 10% carnosol, and 2- 10% 12-O-methylcarnosic acid.

Example 4. Preparation of Salvia extracts.

Salvia officinalis or Salvia apiana leaves were extracted with acetone at reflux (56,4°C) for 2 hours under mechanical stirring. After the extraction was completed, the acetone extract was filtered to remove the plant residues. The resulting acetonic extract was concentrated under reduced pressure to dryness. The dry extract contains about 10-20% carnosic acid and I %-2% carnosol. Example 5. Antimicrobial effect measurements

Products were screened for their inhibitory activity against different spoilage microorganisms. This activity was assessed in vitro and quantified through qPCR. 5.1 Microorganisms

A number of spoilage microorganisms were selected to conduct an in vitro screening (Table #). During this experiment, the microorganisms were cultured in the presence of different concentrations of the ingredients and growth inhibition was measured. The microorganisms were obtained from TNO's database or they were ordered. Each strain was cultured in specific conditions of medium and temperature prior to the screening.

Table 4. Details on the spoilage microorganisms and on their growing conditions. These were optimized during the preparation phase and applied during the screening phase.

Incubations

During the experiment, the microorganisms were cultured in the presence of different concentrations of the test ingredients and their growth inhibition was measured by qPCR. The screening was performed in 96-deep well plates. Each ingredient was tested at 6 concentrations: 1%, 0,5%, 0,25%>, 0,125%, 0.061% and 0.031% or lower concentrations. Some ingredients were not easily dissolved despite the use of DMSO. For these ingredients, the concentration range was slightly adjusted to ensure dissolution of the ingredients even at the highest concentration tested. Tables describing Examples contain information on the exact concentration range tested for each clove extract.

Table 5. Composition of extracts

5.2. Clove leaves extract antimicrobial effect on Bacteria.

5.2.1 Clove leaves acetone extract antimicrobial effect on Alicyclobacillus acidoterrestris.

As it can be seen on table 6, the clove acetone extract has a strong inhibition effect on Alicyclobacillus acidoterrestris even at low concentrations.

Table 6. Clove leaves acetone extract antimicrobial effect on Alicyclobacillus acidoterrestris

5.2.2. Clove leaves ethanol/water (30:70) extract antimicrobial effect on Alicyclobacillus acidoterrestris.

As demonstrated on table 7 , the clove leaves ethanol/water also has a strong inhibition effect on Alicyclobacillus acidoterrestris even at low concentrations. Table 7. Clove leaves ethanol/water (30:70) extract antimicrobial effect on Alicyclobacillus aci- doter restr is

5.3. Clove leaves extracts antimicrobial effect on Yeast 5.3.1. Clove leaves acetone extract antimicrobial effect on Zygosaccharomyces bailii.

As it can be seen on table 8, 9 and 10, the clove leaves acetone extract has a strong inhibition effect on different yeast (Zygosaccharomyces bailii, Saccharomyces cerevisiae, Saccharomyces pombe) even at low concentrations.

Table 8. Clove leaves acetone extract antimicrobial effect on Zygosaccharomyces bailii.

5.3.2. Clove leaves acetone extract antimicrobial effect on Saccharomyces cerevisiae. Table 9. Clove leaves acetone extract antimicrobial effect on Saccharomyces cerevisiae

2.3.3. Clove leaves acetone extract antimicrobial effect on Saccharomyces pombe.

Table 10. Clove leaves acetone extract antimicrobial effect on Saccharomyces pombe

5 5.4. Clove leaves acetone extract antimicrobial effect on Molds

As demonstrated on table 11, 12 and 13, the leaves acetone extract has a strong inhibition effect on different molds (Dekkera anomala, Aspergillus niger, Bysocchlamys nivea) even at very low concentrations. 0

5.4.1. Clove leaves acetone extract antimicrobial effect on Dekkera anomala:

Table 11. Clove acetone extract antimicrobial effect on Dekkera anomala:

5.4.2. Clove leaves acetone extract antimicrobial effect on Aspergillus niger

Table 12. Clove leaves acetone extract antimicrobial effect on Aspergillus niger:

5.4.3. Clove leaves acetone extract antimicrobial effect on Bysocchlamys nivea.

Table 13. Clove leaves acetone extract antimicrobial effect on Bysocchlamys nivea:

Example 6. Clove leaves acetone extract in combination with Rosemary extract has a synergistic effect on Saccharomyces cerevisiae.

As it can be seen from the table 15, the clove leaves extract (acetone) and the rosemary extract have a very low antimicrobial effect (6% or 0.5 % of inhibition) on Saccharomyces cerevisiae while the combination of both shows antimicrobial effect on Saccharomyces cerevisiae. This is a synergistic effect.

Table 14. Concentration of the main active components in the rosemary and clove extracts

Table 15. Synergistic effect on Saccharomyces cerevisiae

Table 16. Synergistic effect of clove leaves extract and rosemary extract on Lactobacillus planta- rum

Example 6. Clove bud extracts.

The following results were obtained using clove bud extracts obtained by water extraction and formulated and dried using modified starch as a carrier. Thus obtained extract comprised 18% polyphenols. Polyphenol measurements were run using Folin-Ciocalteu method. Table 17. Synergistic effect of clove buds water extract and rosemary extract against Dekkera

Table 18. Synergistic effect of clove buds water extract and rosemary extract against SchizoSac- charomyces pombe

Tabel 19. Synergistic effect of Clove buds water extract and rosemary extract against Saccharomyces cerevisae.

Example 7. Salvia acetone extract. Salvia officinalis or Salvia apiana extracts comprise phenolic diterpenes, notably carnosic acid from 10-20%.

Table 20. Salvia acetone extract inhibition of Saccharomyces cerevisiae

Table 21. Synergistic effect of Clove leaves acetone extract and salvia acetone extract.

Table 22. Synergistic effect of Clove leaves EtOH/water extract and salvia acetone extract.

Table 23. Synergistic effect of Clove leaves acetone extract extract and salvia acetone extract.

Claims

1. Antimicrobial composition comprising an extract obtained or obtainable from Syzygium aro- maticum leaves.

2. Antimicrobial composition according to claim 1, wherein the solvent used for extraction is water, an alcohol (such as ethanol), an organic solvent (such as acetone), supercritical carbon dioxide extraction or mixtures thereof.

3. Antimicrobial composition according to claim 1 or 2, wherein the extract obtained or obtainable from Syzygium aromaticum comprises

(iv) at least 0.3 % wt/wt of one or more flavonoids (such as Quercetin, Kaempferol and/or Gossypetin, their derivatives and mixtures thereof), such as at least 0.5 % wt/wt, 1% wt/wt, 2% wt/wt, 3% wt/wt, 4% wt/wt, 5% wt/wt, 8% wt/wt, 10% wt/wt, 12% wt/wt, 14% wt/wt, or at least 15 % wt/wt of flavonoids, and/or

(v) at least 0.5% wt/wt of one or more phenolic acids (such as gallic acid, tannic acid and/or capsacin, their derivatives and mixtures thereof), such as at least 1% wt/wt, 2% wt/wt, 3% wt/wt, 4% wt/wt, 5% wt/wt, 8% wt/wt, 10% wt/wt, 12% wt/wt, 14% wt/wt, 15% wt/wt, 20% wt/wt, 25% wt/wt, 30% wt/wt or at least 40% wt/wt of phenolic acids and/or

(vi) at least 0.5 % wt/wt of one or more organic acids (such as quinic acid, malic acid, citric acid and/or tartaric acid, their derivatives and mixtures thereof), such as at least 1% wt/wt, 2% wt/wt, 3% wt/wt, 5% wt/wt, 8% wt/wt, 10% wt/wt, 15% wt/wt, 20% wt/wt or at least 30% wt/wt of organic acids.

4. Antimicrobial composition according to any of the preceding claims, wherein the extract obtained or obtainable from Syzygium aromaticum comprises less than 60 % wt/wt eugenol and/or eugenol derivatives, such as less than 40% wt/wt, less than 35% wt/wt, less than 20% wt/wt, less than 15% wt/wt, less than 10% wt/wt, less than 5% wt/wt, less than 4% wt/wt, less than 3% wt/wt, less than 2% wt/wt, less than 1% wt/wt or less than 0.5% wt/wt eugenol and/or eugenol derivatives.

5. Antimicrobial composition according to any of the preceding claims, wherein the extract obtained or obtainable from Syzygium aromaticum comprises

(i) at least 0.5% wt/wt of gallic acid, such as 1% wt/wt, 2% wt/wt, 3% wt/wt, 5% wt/wt, at least 6% wt/wt, at least 10% of gallic acid and/or its derivatives, and/or (ii) at least 1% wt/wt, 2% wt/wt, 2.5% wt/wt, 3% wt/wt, 3.5% wt/wt, 4% wt/wt, 4.5% wt/wt, 5% wt/wt, 6% wt/wt, at least 8% wt/wt of quinic acid and/or its derivatives and/or

(iii) less than 40 % wt/wt eugenol and/or eugenol derivatives, such as less than 35% wt/wt, such as less than 20% wt/wt, less than 15% wt/wt, less than 10% wt/wt, less than 5% wt/wt, less than 4% wt/wt, less than 3% wt/wt, less than 2% wt/wt, less than 1% wt/wt or less than 0.5% wt/wt eugenol and/or eugenol derivatives. Antimicrobial composition according to any of claims 1 to 5 further comprising a Lamiaceae extract. Antimicrobial composition according to any of claims 1 to 5 further comprising one or more of: carnosic acid, carnosol, methylcarnosate, rosmanol, isorosmanol, 11 , 12 -di-O- methyli- sorosmanol, 12-O-methylcarnosic acid, rosmanol-9-ethyl ether, circimaritin, Methylated monooxidized product of carnosic acid, genkwanin, epirosmanol, epiisorosmanol, carnosic acid derivative, epirosmanol ethyl ether, cryptotanshinone. Antimicrobial composition according to any of the preceding claims, wherein the antimicrobial effect is against one or more of the microorganism selected from one or more of: a) yeast of the following genus: Candida, Saccharomyces (such as Saccharomices cerevisiae, Saccharomyces pombe), Dekkera (such as D. anomala), Brettanomyces, Zygosaccharomyces (such as Zygosaccharomyces baillii or SchizoSaccharomyces pombe) or combinations thereof, and/or b) molds selected from Byssochlamys genus (such as Byssochlamys nivea), Penicillium genus genus, Dekkera (such as D. anomala), Aspergillus genu (such as Aspergillum niger). Antimicrobial composition comprising an extract obtained or obtainable from Syzygium aro- maticum, wherein the extract is a water extract from the buds and comprises at least 3% wt/wt of one or more polyphenols, such as at least about 4% wt/wt, at least about 5% wt/wt, at least about 6% wt/wt, at least about 7% wt/wt, at least about 8% wt/wt, at least about 9% wt/wt, at least about 10% wt/wt, at least about 15% wt/wt, at least about 20% wt/wt, at least about 30% wt/wt, at least about 40% wt/wt, at least about 50% wt/wt, at least about 60% wt/wt of one or more of polyphenols.

10. Antimicrobial composition comprising

(i) at least 0.3 % wt/wt of flavonoids (such as Quercetin, Kaempferol and/or Gossypetin, and their derivatives), such as at least 0.5 % wt/wt, 1% wt/wt, 2% wt/wt, 3% wt/wt, 4% wt/wt, 5% wt/wt, 8% wt/wt, 10% wt/wt, 12% wt/wt, 14% wt/wt, or at least 15 % wt/wt of flavonoids, and/or

(ii) at least 0.5% wt/wt of phenolic acids (such as gallic acid, tannic acid and/or capsacin and their derivatives), such as at least 1% wt/wt, 2% wt/wt, 3% wt/wt, 4% wt/wt, 5% wt/wt, 8% wt/wt, 10% wt/wt, 12% wt/wt, 14% wt/wt, 15% wt/wt, 20% wt/wt, 25% wt/wt, 30% wt/wt or at least 40% wt/wt of phenolic acids and/or

(iii) at least 0.5 % wt/wt of organic acids (such as quinic acid, malic acid, citric acid and/or tartaric acid and their derivatives), such as at least 1% wt/wt, 2% wt/wt, 3% wt/wt, 5% wt/wt, 8% wt/wt, 10% wt/wt, 15% wt/wt, 20% wt/wt or at least 30% wt/wt of organic acids.

11. Antimicrobial composition according to claims 9 or 10, comprising less than 40 % wt/wt eugenol and/or eugenol derivatives, such as less than 35% wt/wt, such as less than 20% wt/wt, less than 15% wt/wt, less than 10% wt/wt, less than 5% wt/wt, less than 4% wt/wt, less than 3% wt/wt, less than 2% wt/wt, less than 1% wt/wt or less than 0.5% wt/wt eugenol and/or eugenol derivatives.

12. Antimicrobial composition according to any of claims 9 to 11 further comprising a Lamiaceae extract.

13. Antimicrobial composition according to any of claims 9 to 11 further comprising one or more of: carnosic acid, carnosol, methylcarnosate, rosmanol, isorosmanol, 1 1 , 12 -di-O- methyli- sorosmanol, 12-O-methylcarnosic acid, rosmanol-9-ethyl ether, circimaritin, Methylated monooxidized product of carnosic acid, genkwanin, epirosmanol, epiisorosmanol, carnosic acid derivative, epirosmanol ethyl ether, cryptotanshinone.

14. Method for inhibiting the growth of microorganisms and/or for killing microorganisms in a product wherein the method comprises the step of contacting the product with an antimicrobial composition according to claims 1 to 7, wherein the solvent used for extraction is water.

15. Method according to claim 14, wherein the microorganism is selected from one or more of: a) yeast of the following genus: Candida, Saccharomyces (such as Saccharomices cere- visiae, Saccharomyces pombe), Dekkera (such as D. anomala), Brettanomyces, Zygosac- charomyces (such as Zygosaccharomyces baillii or SchizoSaccharomyces pombe) and/or b) molds selected from Byssochlamys genus (such as Byssochlamys nivea), Penicillium genus, Dekkera (such as D. anomala), Aspergillus genus (such as Aspergillum niger).

16. The use of an antimicrobial composition according to claims 1 to 13 for inhibiting the growth of microorganisms and/or for killing microorganisms in a product.

17. Method for inhibiting the growth of microorganisms and/or for killing microorganisms in a product wherein the method comprises the step of contacting the product with an antimicrobial composition according to claims 1 to 13.

18. Method for inhibiting the growth of microorganisms and/or for killing microorganisms in a product wherein the method comprises the step of simultaneously, separately or sequentially adding a composition as defined in any of claims 1 to 5 and a Lamiaceae extract to a product.

19. Method for inhibiting the growth of microorganisms and/or for killing microorganisms in a product wherein the method comprises the step of simultaneously, separately or sequentially adding a composition as defined in any of claims 1 to 5, carnosic acid and/or carnosol to a product.

20. Method for inhibiting the growth of microorganisms and/or for killing microorganisms in a product wherein the method comprises the step of simultaneously, separately or sequentially adding a composition as defined in any of claims 9 to 11 and a Lamiaceae extract to a product.

21. Method for inhibiting the growth of microorganisms and/or for killing microorganisms in a product wherein the method comprises the step of simultaneously, separately or sequentially adding a composition as defined in any of claims 9 to 11, carnosic acid and/or carnosol to a product. Method for processing or preparing a food or beverage product for humans or animals, a nutritional supplement, a nutraceutical formulation, a fragrance or flavouring, a pharmaceutical or veterinary formulation, an oenological or cosmetic formulation comprising applying to or incorporating into said product, an antimicrobial composition according to claims 1 to 13 for having a food preservative and/or antimicrobial effect. Method for processing or preparing a food or beverage product for humans or animals, a nutritional supplement, a nutraceutical formulation, a fragrance or flavouring, a pharmaceutical or veterinary formulation, an oenological or cosmetic formulation comprising the step of simultaneously, separately or sequentially adding a composition as defined in any of claims 9 to 11 and a Lamiaceae extract to a product for having a food preservative and/or antimicrobial effect. Method for processing or preparing a food or beverage product for humans or animals, a nutritional supplement, a nutraceutical formulation, a fragrance or flavouring, a pharmaceutical or veterinary formulation, an oenological or cosmetic formulation comprising the step of simultaneously, separately or sequentially adding a composition as defined in any of claims 9 to 11, carnosic acid and/or carnosol to a product for having a food preservative and/or antimicrobial effect. Use or method according to any of claims 16 to 24, wherein the microorganism is selected from one or more of: a) acid lactic bacteria such as Lactobacillus genus (such as Lactobacillis plantarum), Leu- conostoc genus or Lactococcus genus; thermo-acidophilic bacteria such as Alicyclo- bacillus genus (such as Alicyclobacillus acidoterrestris and Alicyclobacillus acidocal- darius); Xerophilic bacteria and/or acidophilic bacteria such as Gluconobacter and Zymomonas genus, b) from yeast of the following genus: Candida, Saccharomyces (such as Saccharomices cerevisiae, Saccharomyces pombe), Dekkera (such as D. anomala), Brettanomyces, Zygosaccharomyces (such as Zygosaccharomyces baillii or SchizoSaccharomyces pombe) or combinations thereof, and/or c) molds selected from Byssochlamys genus (such as Byssochlamys nivea), Penicillium genus genus, Dekkera (such as D. anomala), Aspergillus genu (such as Aspergillum niger) or combinations thereof.

26. Use or method according to any of claims 16 to 25, wherein the product is selected from a food or beverage product for humans or animals, a nutritional supplement, a nutraceutical formulation, a fragrance or flavouring, a pharmaceutical or veterinary formulation, an oeno¬

5 logical or cosmetic formulation.

27. A food or beverage product for humans or animals, a nutritional supplement, nutraceutical formulation, a dietary, a sports supplement, a fragrance or flavouring, a pharmaceutical or veterinary formulation, an oenological or cosmetic formulation comprising a composition ac¬0 cording to any of claims 1 to 13.

28. Composition comprising an extract obtained or obtainable from Syzygium aromaticum leaves and/or buds and an extract obtained or obtainable from Lamiaceae.

29. Method for inhibiting the growth of microorganisms and/or for killing microorganisms in a product wherein the method comprises the step of simultaneously, separately or sequentially adding an extract obtained or obtainable from Syzygium aromaticum leaves and/or buds and an extract obtained or obtainable from Lamiaceae to a product. 0