WO2024256584A1

WO2024256584A1 - Compositions and uses thereof

Info

Publication number: WO2024256584A1
Application number: PCT/EP2024/066452
Authority: WO
Inventors: Ana Paula DO ESPIRITO SANTO; Amelie ROUGER
Original assignee: Givaudan SA
Current assignee: Givaudan SA
Priority date: 2023-06-16
Filing date: 2024-06-13
Publication date: 2024-12-19
Anticipated expiration: 2025-12-16
Also published as: CN121335628A; KR20260019650A; EP4727373A1

Abstract

The present invention relates to antimicrobial compositions that comprise amino acids and organic acids and to their uses.

Description

COMPOSITIONS AND USES THEREOF

TECHNICAL FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

The need to improve food preservation methods is enormous. It has been estimated that about one quarter of the word's food supply is lost as a result of microbial spoilage, and food-borne microbial infections represent a constant and serious threat to human health.

Several bacterial species that may contaminate and grow in foodstuffs and crops are pathogenic or produce toxins and cause a range of food-poisoning diseases. Despite substantial improvement in the technology and hygiene, food products may be exposed to spoilage and pathogenic bacteria in the food-handling environment and the number of food poisoning is still increasing in most of the countries.

Food preservation techniques, e.g. heat processing, freezing, ultrasound, irradiation, and modified atmosphere packaging, significantly reduce microbial load but of particular concern is the evidence that processed foods are being contaminated with microorganisms following processing and prior to packaging. Of rinsing concern in the food industry is microbial spoilage of various foods such as dairy and meat products, dressings, spreads, margarines and seafood.

There has been extensive research conducted in the field of food safety to develop effective anti-microbial compositions, which function as anti-fungal and anti-bacterial compositions.

SUMMARY OF THE INVENTION

The inventors have discovered that compositions with certain combinations of amino acids and organic acids leads to a synergistic effect in the antimicrobial activity of the compositions.

Therefore, in a first aspect, the present invention relates to an antimicrobial composition, hereinafter the antimicrobial composition of the invention, comprising:

(i) A first component which is amino acid selected from the group consisting of L-lysine hydrochloride and L-Alanine hydrochloride, or a combination thereof, and (ii) A second component which is an organic acid selected from the group comprising cinnamic acid, tartaric acid, succinic acid, malic acid or a combination thereof, or a composition comprising organic acids.

In a second aspect, the present invention relates to a food composition that comprises the antimicrobial composition of the invention.

In another aspect, the present invention relates to the use of the antimicrobial composition according of the invention for obtaining a shelf-stable food composition, for decreasing the content of microorganisms in a food composition, for increasing the shelf life of a food composition or for preservation of fresh or cooked food or food compositions.

In another aspect, the present invention relates to a method for obtaining a shelf-stable food composition or for increasing the shelf life of a food composition comprising contacting the foodstuff with the antimicrobial composition of the invention.

In another aspect, the present invention relates to a method for reducing the content of microorganisms in a food composition, wherein the method comprising contacting the food product with the antimicrobial composition of the invention.

DETAILED DESCRIPTION OF THE INVENTION

As previously mentioned, the present invention discloses compositions comprising amino acids and organic acids with antimicrobial effect that present a synergistic antimicrobial effect in comparison with the antimicrobial activity of the individual components.

Antimicrobial composition of the invention.

As such, a first aspect of the present invention relates to an antimicrobial composition, hereinafter the antimicrobial composition of the invention, comprising:

(i) A first component which is amino acid selected from the group consisting of L-lysine hydrochloride and L-Alanine hydrochloride, or a combination thereof, and

(ii) A second component which is an organic acid selected from the group comprising cinnamic acid, tartaric acid, succinic acid, or malic acid or a combination thereof or a composition comprising organic acids.

The term “antimicrobial” as used herein refers to any compound capable of inhibiting the growth or propagation of microorganisms, and/or killing microorganisms; antimicrobial compounds include bactericides, bacteriostats, bacteriostatics, bacteriostatins, fungicides, fungistats, algaecides and algistats, depending on the dose level applied, system conditions and the level of microbial control desired.

As used herein, the term "synergistic" or "synergy" refers to a result that is more than merely additive.

Several synergistic examples are provided in the present document.

For example:

Compound A at dosage a% --> partial bacteria growth observed

Compound B at dosage b% --> growth bacteria observed

Compound C at dosage c% --> partial bacteria growth observed

Blend of compound A at dosage a% + compound B at dosage b% + compound C at c% --> no bacteria growth observed.

Or for example:

Compound A at dosage a% --> mould growth observed

Compound B at dosage b% --> mould growth observed

Blend of compound A at dosage a% + compound B at dosage b% --> no mould growth observed.

Also, the synergistic effect may be an effect on a broader spectrum of microorganisms, for example single compounds may be only active for certain types of microorganism while the combination of those actives results in a broader protection (broader antimicrobial effect on a larger number of microorganisms)

The first component of the antimicrobial composition of the invention is an amino acid selected from the group consisting of L-lysine hydrochloride and L-alanine hydrochloride or a combination thereof.

In one embodiment, the first component of the antimicrobial composition of the invention is L-lysine hydrochloride.

In one embodiment, the first component of the antimicrobial composition of the invention is L-Alanine hydrochloride. In one embodiment, the first component of the antimicrobial composition of the invention is L-lysine hydrochloride and L-Alanine hydrochloride.

Lysine (symbol Lys or K) is a a-amino acid that is a precursor to many proteins. It contains a a-amino group (which is in the protonated -NH⁺³ form under biological conditions), a a-carboxylic acid group (which is in the deprotonated -COO- form under biological conditions), and a side chain lysyl ((CH2)4NH2), classifying it as a basic, charged (at physiological pH), aliphatic amino acid. The "L-amino acid" form is the form recognised as natural by the body.

Alanine (symbol Ala or A), or a-alanine, is an a-amino acid that is used in the biosynthesis of proteins. It contains an amine group and a carboxylic acid group, both attached to the central carbon atom which also carries a methyl group side chain. Consequently, its IIIPAC systematic name is 2-aminopropanoic acid, and it is classified as a nonpolar, aliphatic a-amino acid. The L-isomer of alanine (left-handed) is the one that is incorporated into proteins. The right-handed form, D-alanine, occurs in polypeptides in some bacterial cell walls and in some peptide antibiotics, and occurs in the tissues of many crustaceans and molluscs as an osmolyte. In the antimicrobial composition of the invention, the first component is the L-alanine form.

The term “hydrochloride” refers to an acid salt resulting, or regarded as resulting, from the reaction of hydrochloric acid with an organic base (e.g. an amine). In the antimicrobial composition of the invention, the L-lysine or the L-alanine are in their hydrochloride form, i.e. lysine HCI (CAS: 10098-89-2) or alanine HCI (CAS number: 6003-05-0). In the present invention, the terms L-lysine hydrochloride and L-lysine-HCI and the terms L- alanine hydrochloride and L-alanine-HCI are used interchangeably.

The second component of the antimicrobial composition of the invention is an organic acid selected from the group comprising cinnamic acid, tartaric acid, succinic acid and malic acid or a combination thereof or a composition comprising organic acids.

The term “organic acids” as used herein refers to an organic compound with acidic properties. The most common organic acids are the carboxylic acids, whose acidity is associated with their carboxyl group -COOH. Sulfonic acids, containing the group - SO2OH, are relatively stronger acids. Alcohols, with -OH, can act as acids but they are usually very weak. The relative stability of the conjugate base of the acid determines its acidity. Other groups can [also] confer acidity, usually weakly: the thiol group -SH, the enol group, and the phenol group. In biological systems, organic compounds containing these groups are generally referred to as organic acids. Examples of organic acids, without limitation are, lactic acid, acetic acid, formic acid, citric acid, oxalic acid, uric acid, malic acid and tartaric acid.

Cinnamic acid is an organic compound with the formula C6H5-CH=CH-COOH. It is a white crystalline compound that is slightly soluble in water, and freely soluble in many organic solvents. Classified as an unsaturated carboxylic acid, it occurs naturally in a number of plants. It exists as both a cis and a trans isomer. In a particular embodiment, the cinnamic acid is a cis isomer. In another particular embodiment, the cinnamic acid is a trans isomer. Cinnamic acid is obtained from oil of cinnamon, or from balsams such as storax. It is also found in shea butter. Cinnamic acid is defined in the entry of the database CAS Registry with the number 621-82-9 as well as structurally by formula (i)

Formula (i)

In a particular embodiment, the cinnamic acid has a natural origin as is obtained from cinnamon or balsams. In another particular embodiment, the cinnamic acid has a synthetic origin.

Tartaric acid is a white, crystalline organic acid that occurs naturally in many fruits, most notably in grapes, but also in bananas, tamarinds, and citrus. Its salt, potassium bitartrate, commonly known as cream of tartar, develops naturally in the process of fermentation. It is commonly mixed with sodium bicarbonate and is sold as baking powder used as a leavening agent in food preparation. The acid itself is added to foods as an antioxidant E334 and to impart its distinctive sour taste. Naturally occurring tartaric acid is a useful raw material in organic chemical synthesis. Tartaric acid, an alpha- hydroxy-carboxylic acid, is diprotic and aldaric in acid characteristics, and is a dihydroxyl derivative of succinic acid. Tartaric acid is defined in the entry of the database CAS Registry with the number 87-69-4 as well as structurally by formula (ii).

Formula (ii)

In a particular embodiment, the tartaric acid has a natural origin as is obtained from bananas, tamarinds or citrus. In another particular embodiment, the tartaric acid has a synthetic origin.

In a particular embodiment, the tartaric acid is present in the antimicrobial composition from about 0.1% to about 80%, based on the total weight of the antimicrobial composition. In other embodiments the tartaric acid is present in the antimicrobial composition from about 0.1% to about 3%, from about 2% to about 50% or even from about 5% to about 30% or from about 20% to about 40%%, based on the total weight of the antimicrobial composition.

Succinic acid is a dicarboxylic acid with the chemical formula (CH2)2(CC>2H)2. In living organisms, succinic acid takes the form of an anion, succinate, which has multiple biological roles as a metabolic intermediate being converted into fumarate by the enzyme succinate dehydrogenase in complex 2 of the electron transport chain which is involved in making ATP, and as a signaling molecule reflecting the cellular metabolic state. Succinic acid is defined in the entry of the database CAS Registry with the number 110- 15-6 as well as structurally by formula (iii).

Formula (iii)

In a particular embodiment, the succinic acid has a natural origin. In another particular embodiment, the succinic acid has a synthetic origin. Malic acid is an organic compound with the molecular formula C4H6O5. It is a dicarboxylic acid that is made by all living organisms, contributes to the sour taste of fruits, and is used as a food additive. Malic acid has two stereoisomeric forms (L- and D- enantiomers), though only the L-isomer exists naturally. The salts and esters of malic acid are known as malates. Malic acid is the main acid in many fruits, including apricots, blackberries, blueberries, cherries, grapes, mirabelles, peaches, pears, plums, and quince and is present in lower concentrations in other fruits, such as citrus. The malate anion is an intermediate in the citric acid cycle. Malic acid is defined in the entry of the database CAS Registry with the number 6915-15-7 as well as structurally by formula (iv).

Formula (iv)

In a particular embodiment, the malic acid has a natural origin as is obtained from apricots, blackberries, blueberries, cherries, grapes, mirabelles, peaches, pears, plums, quince or citrus. In another particular embodiment, the malic acid has a synthetic origin.

In another particular embodiment, the second component of the antimicrobial composition of the invention is an organic acid selected from the group comprising cinnamic acid, tartaric acid, succinic acid and malic acid.

In another particular embodiment, the second component of the antimicrobial composition of the invention is a combination or organic acids selected from the group consisting of: a combination of cinnamic acid and tartaric acid, a combination of cinnamic acid and succinic acid, a combination of cinnamic acid and malic acid, a combination of tartaric acid and succinic acid, a combination of tartaric acid and malic acid, a combination of succinic acid and malic acid, a combination of cinnamic acid tartaric acid and succinic acid, a combination of cinnamic acid, tartaric acid and malic acid, a combination of tartaric acid, succinic acid and malic acid, a combination of cinnamic acid, succinic acid and malic acid, a combination of cinnamic acid, tartaric acid and malic acid, and a combination of cinnamic acid, tartaric acid, succinic acid and malic acid.

In another particular embodiment, the antimicrobial composition of the invention comprises tartaric acid, succinic acid and cinnamic acid. In a particular embodiment, the tartaric acid, succinic acid and cinnamic acid are present at a ratio of about 1 :0.1 :0.2 to about 1 :1 :2.

In another embodiment, the tartaric acid and the cinnamic acid are present at a ratio of about 10:1 to about 10:1 , preferably from about 5:1 to about 1:5, even more preferably from about 3:1 to about 1 :3.

In another particular embodiment, the antimicrobial composition of the invention comprising a composition that comprises organic acids as the second component comprises one or more of quinic acid, malic acid, citric acid and shikimic acid.

Quinic acid is a cyclitol, a cyclic polyol, and a cyclohexanecarboxylic acid. It is a colorless solid that can be extracted from plant sources. Quinic acid is implicated in the perceived acidity of coffee. The compound is obtained from cinchona bark, coffee beans, and the bark of Eucalyptus globulus. Quinic acid is defined in the entry of the database CAS Registry with the number 77-95-2 as well as structurally by formula (v).

Formula (v)

In a particular embodiment, the quinic acid has a natural origin as is obtained from plants, such as cinchona bark, coffee beans, and the bark of Eucalyptus globulus. In another particular embodiment, the quinic acid has a synthetic origin.

Citric acid is an organic compound with the chemical formula HOC(CO2H)(CH2CO2H)2 It is a colorless weak organic acid. It occurs naturally in citrus fruits. In biochemistry, it is an intermediate in the citric acid cycle, which occurs in the metabolism of all aerobic organisms. Citric acid occurs in a variety of fruits and vegetables, most notably citrus fruits. Lemons and limes have particularly high concentrations of the acid; it can constitute as much as 8% of the dry weight of these fruits. Citric acid is defined in the entry of the database CAS Registry with the number 77-92-9 as well as structurally by formula (vi).

Formula (vi)

In a particular embodiment, the citric acid has a natural origin as is obtained from citrus fruits. In another particular embodiment, the citric acid has a synthetic origin.

Shikimic acid more commonly known as its anionic form shikimate, is a cyclohexene, a cyclitol and a cyclohexanecarboxylic acid. It is an important biochemical metabolite in plants and microorganisms. Its name comes from the Japanese flower shikimi. Shikimic acid is defined in the entry of the database CAS Registry with the number 138-59-0 as well as structurally by formula (vii).

Formula (vii)

In a particular embodiment, the shikimic acid has a natural origin as is obtained from plants. In another particular embodiment, the shikimic acid has a synthetic origin. In other embodiments, other acids such as formic acid, propionic acid, ferulic acid, levulinic acid and/or chlorogenic acid may also be present in the antimicrobial composition in a suitable amount either individually or in combination with the other organic acids described herein. The other acids such as formic acid, ferulic acid, levulinic acid and/or chlorogenic acid may be of natural and/or synthetic origin.

In another particular embodiment, the antimicrobial composition of the invention comprising a composition that comprises organic acids as the second component is a cranberry extract.

Cranberries are a group of evergreen dwarf shrubs or trailing vines in the subgenus Oxycoccus of the genus Vaccinium.

The term “cranberry extract” refers to the extract obtained from the cranberry, including the whole fruit, and/or skin of the cranberry fruit, and/or the pulp and/or the residues of the fruit after juicing of the fruit. The fruit or the parts may be fresh, frozen or dried. Juice that is recovered from any processing of the fruit any also be processed to recover cranberry extract.

The person skilled in the art will be aware of known methods for providing an extract of cranberry, since these are well known in the art. In a preferred embodiment of the antimicrobial composition of the invention, the extract of cranberry is obtained from a cranberry juice concentrate that is diluted in a solution ethanol/water.

In another particular embodiment, the cranberry extract is substantially free of polyphenols. The person skilled in the art will be aware of known methods for providing an extract of cranberry substantially free of polyphenols. In a preferred embodiment, the extract of cranberry that is obtained from a cranberry juice concentrate and is diluted in a solution ethanol/water, is also percolated through a chromatographic column to separate the organic acids fraction from the polyphenolic fraction.

Polyphenols are compounds with aromatic ring and several hydroxyl groups and often have functional groups beyond hydroxyl groups. Polyphenolic compounds are usually referred to as a diverse group of naturally occurring compounds containing multiple phenolic functionalities. Polyphenols include flavonoids, phenolic acid, tannic acid, stilbenes, lignans and ellagitannin. Flavonoids include flavones, flavonols, flavanols, flavanones, isoflavones, proanthocyanidins, and anthocyanins. Phenolic acid include caffeic acid. Lignans are polyphenols derived from phenylalanine found in Flax seed and other cereals. Thus, in a particular embodiment, the cranberry extract is substantially free of flavonoids, phenolic acid, tannic acid, stilbenes, lignans and ellagitannin.

The term “substantially free” as used herein means that the cranberry extract does not comprise more than 0.01 g/L of polyphenols. In a more particular embodiment the cranberry extract does not comprise more than 0,75 g/L, more than 0,50 g/L, more than 0,25 g/L, more than 0,01 g/L of polyphenols.

In another particular embodiment, the cranberry extract comprises polyphenols and is rich in organic acids. In a particular embodiment, the concentration of polyphenols in the cranberry extract is of between 0% and 50%.

The term “rich in organic acids” as used herein means that the cranberry extract comprises at least 30% weight per volume (w/w) of organic acids. In a more particular embodiment, the cranberry extract comprises more than 35% (w/v) of organic acids, more in particular, the cranberry extract comprises more than 37% (w/v) of organic acids.

In another particular embodiment, the composition of the invention comprising organic acids comprises quinic acid, malic acid, citric acid and shikimic acid and are present at a ratio of about 78: 67,5 :46,5: 1 by weight.

In another particular embodiment, the antimicrobial composition of the invention comprises:

(i) L-lysine as the first component and cinnamic acid as the second component,

(ii) L-lysine hydrochloride as the first component and a combination of cinnamic acid and tartaric acid as the second component,

(iii) L-lysine hydrochloride as the first component and succinic acid as the second component,

(iv) L-lysine hydrochloride as the first component and tartaric acid as the second component,

(v) L-lysine hydrochloride as the first component and malic acid as the second component,

(vi) L- alanine as the first component and cinnamic acid as the second component,

(vii) L- alanine hydrochloride as the first component and a combination of cinnamic acid and tartaric acid as the second component,

(viii) L- alanine hydrochloride as the first component and succinic acid as the second component, (ix) L- alanine hydrochloride as the first component and tartaric acid as the second component,

(x) L- alanine hydrochloride as the first component and malic acid as the second component,

(xi) L-alanine hydrochloride as the first component and succinic acid as the second component, or

(xii) L-alanine hydrochloride as the first component and a cranberry extract as the second component.

In a particular embodiment, the antimicrobial composition of the invention comprises L- lysine hydrochloride as the first component and cinnamic acid as the second component and are present at a ratio of about 1 : 13 to 1 : 17, preferably 1 :15.

In another particular embodiment, the antimicrobial composition of the invention comprises L-lysine hydrochloride as the first component and a combination of cinnamic acid and tartaric acid as the second component and are present at a ratio of about 1 : 2: 5.

In another particular embodiment, the antimicrobial composition of the invention comprises L-lysine hydrochloride as the first component and succinic acid as the second component and are present at a ratio of about 1 : 1 ,5 to 1:2,5.

In another particular embodiment, the antimicrobial composition of the invention comprises L-lysine hydrochloride as the first component and tartaric acid as the second component and are present at a ratio of about 1 : 1 ,5 to 1: 2,5

In another particular embodiment, the antimicrobial composition of the invention comprises L-alanine hydrochloride as the first component and malic acid as the second component and are present at a ratio of about 1 :18 to 1 :22, preferably 1 : 20.

In another particular embodiment, the antimicrobial composition of the invention comprises L-alanine hydrochloride as the first component and succinic acid as the second component and are present at a ratio of about 1:18 to 1 :22, preferably 1 : 20.

In another particular embodiment, the antimicrobial composition of the invention comprises L-alanine hydrochloride as the first component and cranberry extract as the second component and are present at a ratio of about 2:1 , 1 :1 or 1 : 1 ,5. Food composition of the invention

In a second aspect, the present invention relates to a food composition, hereinafter the food composition of the invention that comprises the antimicrobial composition of the invention, wherein the food composition has an acidic pH.

The antimicrobial composition of the invention may be present in the food composition at least about 0.05%, at least about 0.1%, preferably at least about 0.5%, preferably at least about 1.0% or even 2%, 3%, 4% or 5%, based on the total weight of the food composition. In another embodiment, the antimicrobial composition of the invention may be present in the food composition from about 0.3% to about 5%, preferably from about 0.5% to about 3.5%, based on the total weight of the food composition.

The term “food composition” or “foodstuff” as used herein refers to a composition which is suitable for human or animal consumption. The food composition includes, without limitation, eggs, egg-based products, including but not limited to mayonnaise, salad dressings, sauces, ice creams, egg powder, modified egg yolk and products made therefrom; baked goods, including breads, cakes, sweet dough products, laminated doughs, liquid batters, muffins, doughnuts, biscuits, crackers and cookies; confectionery, including chocolate, candies, caramels, halawa, gums, including sugar free and sugar sweetened gums, bubble gum, soft bubble gum, chewing gum and puddings; frozen products including sorbets, preferably frozen dairy products, including ice cream and ice milk; dairy products, including cheese, butter, milk, coffee cream, whipped cream, custard cream, milk drinks and yoghurts; mousses, whipped vegetable creams, meat products, including processed meat products; meat analogues, plant based products, plant based non-dairy products, edible oils and fats, aerated and non-aerated whipped products, oil-in- water emulsions, water-in-oil emulsions, margarine, shortening and spreads including low fat and very low fat spreads; dressings, mayonnaise, dips, cream based sauces, cream based soups, beverages, syrups, juice-based compounds, spice emulsions, sauces, beverages, patisseries, oils, bakery, filling and edible ink.

The term “beverage” as used herein refers to any potable or edible liquid.

In a preferred embodiment, the food composition of the invention is a syrup. A syrup is a condiment that is a thick, viscous liquid consisting primarily of a solution of sugar in water, containing a large amount of dissolved sugars but showing little tendency to deposit crystals. Its consistency is similar to that of molasses. The viscosity arises from the hydrogen bonds between the dissolved sugar, which has many hydroxyl (OH) groups, and the water. In a more preferred embodiment, the food composition of the invention is a fruit syrup. A fruit syrup is a concentrated fruit juice used as sweetener. In a particular embodiment, the fruit syrup is selected from the group consisting of: banana syrup, berries syrup, orange syrup, grape syrup, grenadine syrup, passion fruit syrup, kiwi syrup, cranberry syrup and peach syrup.

In a preferred embodiment, the food composition of the invention is a grenadine syrup.

In certain embodiments, the food composition of the invention has an acidic pH, that means that the pH of the food composition of the invention is lower than 5.

The term acidic food product refers to those products that have a pH lower than 5, such as beverages, concentrates and syrups for beverages, fruit preparations, vegetable preparations, sauces, dressings, canned pet food. In a particular embodiment, the pH of the food composition of the invention is between 1 and 5, preferably between 3 and 4,5.

In certain embodiments, the food composition of the invention has a neutral or basic pH. The term neutral or basic food product refers to those products that have a pH higher than 5, such as meat, meat analogues, non-fermented dairy, dairy alternatives, bakery, soups, vegetable soups, etc. In a particular embodiment, the pH is between 5 and 6.5.

The food composition of the invention comprises the antimicrobial composition of the invention, i.e. comprises the first component and the second component of the antimicrobial composition of the invention.

In a particular embodiment, the food composition of the invention comprises the first component of the antimicrobial composition of the invention at a concentration of between 0,001 % and 0,5 % weight per weight (w/w) and the second component of the antimicrobial composition of the invention at a concentration between 0,001 % and 0,8 % (w/w).

In a more particular embodiment, the food composition of the invention comprises the first component of the antimicrobial composition of the invention at a concentration of between 0,01% and 0,4 % (w/w). In another more particular embodiment, the food composition of the invention comprises the first component of the antimicrobial composition of the invention at a concentration of between 0,05 % and 0,4 % (w/w). In another more particular embodiment, the food composition of the invention comprises the first component of the antimicrobial composition of the invention at a concentration of between 0,05 % and 0,3 % (w/w). In another more particular embodiment, the food composition of the invention comprises the second component of the antimicrobial composition of the invention at a concentration between 0,001 % and 0,7 % (w/w). In another more particular embodiment, the food composition of the invention comprises the second component of the antimicrobial composition at a concentration between 0,01 % and 0,6 % (w/w). In another more particular embodiment, the food composition of the invention comprises the second component of the antimicrobial composition at a concentration between 0,01 % and 0,5 % (w/w). In another more particular embodiment, the food composition of the invention comprises the second component of the antimicrobial composition at a concentration between 0,01 % and 0,4% (w/w). In another more particular embodiment, the food composition of the invention comprises the second component of the antimicrobial composition at a concentration between 0,01 % and 0,3 % (w/w).

In a particular embodiment, the food composition of the invention comprises the antimicrobial composition of the invention which comprises L-lysine hydrochloride as the first component and cinnamic acid as the second component and are present at a ratio of about 1 : 13 to 1 : 17, preferably 1 :15.

In another particular embodiment, the food composition of the invention comprises the antimicrobial composition of the invention which comprises L-lysine hydrochloride as the first component and a combination of cinnamic acid and tartaric acid as the second component and are present at a ratio of about 1 : 2: 5.

In another particular embodiment, the food composition of the invention comprises the antimicrobial composition of the invention which comprises L-lysine hydrochloride as the first component and succinic acid as the second component and are present at a ratio of about 1 : 1,5 to 1 :2,5.

In another particular embodiment, the food composition of the invention comprises the antimicrobial composition of the invention which comprises L-lysine hydrochloride as the first component and tartaric acid as the second component and are present at a ratio of about 1 : 1,5 to 1 : 2,5

In another particular embodiment, the food composition of the invention comprises the antimicrobial composition of the invention which comprises L-alanine hydrochloride as the first component and malic acid as the second component and are present at a ratio of about 1 :18 to 1:22, preferably 1 : 20. In another particular embodiment, the food composition of the invention comprises the antimicrobial composition of the invention which comprises L-alanine hydrochloride as the first component and succinic acid as the second component and are present at a ratio of about 1 :18 to 1 :22, preferably 1 : 20.

In another particular embodiment, the food composition of the invention comprises the antimicrobial composition of the invention which comprises L-alanine hydrochloride as the first component and cranberry extract as the second component and are present at a ratio of about 2:1 , 1 :1 or 1 :1 ,5.

In another particular embodiment of the food composition of the invention, the first component of the antimicrobial composition of the invention is L-lysine hydrochloride and the second component of the antimicrobial composition of the invention is cinnamic acid, and the L-lysine hydrochloride from the antimicrobial composition is present at a concentration between 0,05 % and 0,2 % (w/w), and the cinnamic acid from the antimicrobial composition is present at a concentration between 0,005% and 0,05 % (w/w). In a more particular embodiment, the L-lysine hydrochloride from the antimicrobial composition is present a concentration of 0, 15 %(w/w) and the cinnamic acid from the antimicrobial composition is present at a concentration of 0,01 % (w/w),

In another particular embodiment of the food composition of the invention, the first component of the antimicrobial composition of the invention is L-lysine hydrochloride and the second component antimicrobial is a composition is a combination of cinnamic acid and tartaric acid and wherein the L-lysine hydrochloride from the antimicrobial composition is present at a concentration between 0,05% and 0,2% (w/w) and the cinnamic acid and the tartaric acid from the antimicrobial composition are present at concentrations between 0,01 % and 0,15% and 0,01 % and 0,1% (w/w) respectively. In a more particular embodiment, the L-lysine hydrochloride from the antimicrobial composition is present at a concentration of 0,1 % (w/w) and the cinnamic acid and the tartaric acid from the antimicrobial composition are present at concentrations of 0,05% and 0,02% respectively (w/w).

In another particular embodiment of the food composition of the invention, the first component of the antimicrobial composition is L-lysine hydrochloride and the second component antimicrobial composition is succinic acid and wherein the L-lysine hydrochloride from the antimicrobial composition is present at a concentration between 0,1% and 0,3% (w/w) and the succinic acid from the antimicrobial composition is present at a concentration between 0,05 % and 0,2 % (w/w). In a more particular embodiment, the L-lysine hydrochloride from the antimicrobial composition is present at a concentration of 0,15% (w/w) and the succinic acid from the antimicrobial composition is present at 0,1% (w/w).

In another particular embodiment of the food composition of the invention, the first component of the antimicrobial composition is L-lysine hydrochloride and the second component antimicrobial composition is tartaric acid and wherein the L-lysine hydrochloride from the antimicrobial composition is present at a concentration between 0,1 % and 0,3% (w/w) and the tartaric acid from the antimicrobial composition is present at 0,05 % and 0,2% (w/w). In a more particular embodiment, the L-lysine hydrochloride from the antimicrobial composition is present at a concentration of 0,15% (w/w) and the tartaric acid from the antimicrobial composition is present at 0,1% (w/w).

In another particular embodiment of the food composition of the invention, the first component of the antimicrobial composition is L-lysine hydrochloride and the second component antimicrobial composition is malic acid and wherein the L-lysine hydrochloride from the antimicrobial composition is present at a concentration of 0,1 % and 0,3% (w/w) and the malic acid from the antimicrobial composition is present at 0,05 % and 0,2 % (w/w). In a more particular embodiment, the L-lysine hydrochloride from the antimicrobial composition is present at a concentration of 0,15% (w/w) and the malic acid from the antimicrobial composition is present at 0,1 % (w/w).

In another particular embodiment of the food composition of the invention, the first component of the antimicrobial composition is L-alanine hydrochloride and the second component antimicrobial composition is succinic acid and wherein the L-alanine hydrochloride from the antimicrobial composition is present at a concentration of 0,05 % and 0.3% (w/w) and the succinic acid from the antimicrobial composition is present at 0,005 % and 0.1% (w/w). In a more particular embodiment, the L-alanine hydrochloride from the antimicrobial composition is present at a concentration of 0,2% (w/w) and the succinic acid from the antimicrobial composition is present at 0,01% (w/w).

In another particular embodiment of the food composition of the invention, the first component of the antimicrobial composition is L-alanine hydrochloride and the second component of the antimicrobial composition is a composition enriched in organic acids wherein the L-alanine hydrochloride from the antimicrobial composition is present at a concentration of 0,1 % and 0,3% (w/w) and the composition enriched in organic acids is present at 0, 1 % and 0,3% (w/w). In a more particular embodiment, the L-alanine hydrochloride from the antimicrobial composition is present at a concentration of 0,2% (w/w) and the composition enriched in organic acids is present at 0,2% (w/w).

In another particular embodiment of the food composition of the invention, the first component of the antimicrobial composition is L-alanine hydrochloride and the second component of the antimicrobial composition is a composition enriched in organic acids wherein the L-alanine hydrochloride from the antimicrobial composition is present at a concentration of 0,1% and 0,3% (w/w) and the composition enriched in organic acids is present at 0,1% and 0,3% (w/w). In a more particular embodiment, the L-alanine hydrochloride from the antimicrobial composition is present at a concentration of 0,3% (w/w) and the composition enriched in organic acids is present at 0,2% (w/w).

In another particular embodiment of the food composition of the invention, the first component of the antimicrobial composition is L-alanine hydrochloride and the second component of the antimicrobial composition is a composition enriched in organic acids wherein the L-alanine hydrochloride from the antimicrobial composition is present at a concentration of 0,005% and 0,1% (w/w) and the composition enriched in organic acids is present at 0,05% and 0,2% (w/w). In a more particular embodiment, the L-alanine hydrochloride from the antimicrobial composition is present at a concentration of 0,05% (w/w) and the composition enriched in organic acids is present at 0,1% (w/w).

In another particular embodiment of the food composition of the invention, the first component of the antimicrobial composition is L-alanine hydrochloride and the second component of the antimicrobial composition is a composition enriched in organic acids wherein the L-alanine hydrochloride from the antimicrobial composition is present at a concentration of 0,05 % and 0,2% (w/w) and the composition enriched in organic acids is present at 0,1 % and 0,3% (w/w). In a more particular embodiment, the L-alanine hydrochloride from the antimicrobial composition is present at a concentration of 0,1 % (w/w) and the composition enriched in organic acids is present at 0,2% (w/w).

In a particular embodiment of the food composition of the invention, the tartaric acid and the cinnamic acid and are present at a ratio of from about 1 :10 to about 10:1 , such as from about 1 :5 to 5.1 such as about 1 :2.

In another particular embodiment, the antimicrobial composition of the invention comprises tartaric acid, succinic acid and cinnamic acid. In a particular embodiment, the tartaric acid, succinic acid and cinnamic acid are present at a ratio of about 1 :0.1 :0.2 to about 1 :1 :2. The expert will know how to adjust the quantity of the first component and the second component of the antimicrobial composition of the invention in the food composition of the invention. For example, the concentrations of the first component and the second component may vary depending on the food composition.

In a preferred embodiment, for the manufacture of beverages, concentrates, emulsions and syrups for beverages, sauces, dressings, fruit preparations, vegetable preparations, fermented dairy products, fermented meat or canned pet food, the food composition of the invention comprises L-lysine HCI as the first component of the antimicrobial composition of the invention and a combination of cinnamic acid and tartaric acid as the second component of the antimicrobial composition of the invention. In a more particular embodiment, the food composition of the invention comprises a concentration of L- Lysine HCI varying from 0,01 % to 0,5%, a concentration of cinnamic acid varying from 0,0005% to 1% and a concentration of tartaric acid varying from 0,001% to 1 %.

In a more preferred embodiment, for the manufacture of a syrup, the composition of the invention comprises L-lysine HCI at a concentration of 0,1%, tartaric acid at a concentration of 0,05% and cinnamic acid at a concentration between 0,02% and 0,04%.

In another particular embodiment, the food composition of the invention, in addition to the antimicrobial composition of the invention, also comprise other additives, like herbs; spices; flavouring agents; colouring agents; emulsifiers (like fatty acid monoglycerides) or antioxidants.

In another aspect, the present invention relates to a nutraceutical or dietary supplement comprising the antimicrobial composition of the invention and a food or dietary acceptable adjuvant.

All previous embodiments and definitions described in relation with the previous aspects are equally valid for the current aspect and its embodiments.

The term “nutraceutical” as used herein refers to substances useful in both nutritional and pharmaceutical fields of application. Thus, nutraceutical compositions can be used as supplements to food and beverages and as pharmaceutical formulations for enteral or parenteral application which may be solid formulations, such as capsules or tablets, or liquid formulations, such as solutions or suspensions.

The expression “food or dietary acceptable adjuvant” as used herein refers to any food material that can be used together with the main foodstuff material in order to enhance, preserve, and improve the characteristics of the foodstuff main material. Adjuvants can also serve as fillers, diluents, viscosity regulators, etc.

Uses of the invention

In another aspect, the present invention relates to the use of the antimicrobial composition of the invention for obtaining a shelf-stable food composition composition, for decreasing the content of microorganisms in a foodstuff, for increasing the shelf life of food composition, for preservation of fresh or cooked food or food composition.

The term “food composition” or "foodstuff¹ as used herein is intended to mean a substance which is suitable for human or animal consumption. In addition, the term foodstuff as used herein also is meant to include food materials which are used in the preparation of a foodstuff. As used herein the tern foodstuff includes, without limitation, eggs, egg-based products, including but not limited to mayonnaise, salad dressings, sauces, ice creams, egg powder, modified egg yolk and products made therefrom; baked goods, including breads, cakes, sweet dough products, laminated doughs, liquid batters, muffins, doughnuts, biscuits, crackers and cookies; confectionery, including chocolate, candies, caramels, halawa, gums, including sugar free and sugar sweetened gums, bubble gum, soft bubble gum, chewing gum and puddings; frozen products including sorbets, preferably frozen dairy products, including ice cream and ice milk; dairy products, including cheese, butter, milk, coffee cream, whipped cream, custard cream, milk drinks and yoghurts; mousses, whipped vegetable creams, meat products, including processed meat products; edible oils and fats, aerated and non-aerated whipped products, oil-in- water emulsions, water-in-oil emulsions, margarine, shortening and spreads including low fat and very low fat spreads; dressings, mayonnaise, dips, cream based sauces, cream based soups, beverages, syrups, spice emulsions, sauces, beverages, patisseries, oils, bakery, filling and edible ink.

In a preferred embodiment, the foodstuff is a product selected from a group consisting of: eggs, mayonnaise, salad dressings, sauces, ice creams, egg powder, modified egg yolk, breads, cakes, sweet dough products, laminated doughs, liquid batters, muffins, doughnuts, biscuits, crackers, cookies, chocolate, candies, caramels, halawa, gums, bubble gum, soft bubble gum, chewing gum, puddings, sorbets, ice cream, ice milk, cheese, butter, milk, coffee cream, whipped cream, custard cream, milk drinks, yoghurts, mousses, whipped vegetable creams, meat, processed meat products, oils, edible oils, fats, oil-in-water emulsions, water-in-oil emulsions, margarine, mayonnaise, dips, cream based sauces, cream based soups, beverages, syrups, spice emulsions, sauces, patisseries, dairy products, oils, bakery, filling and edible ink.

In a more preferred embodiment, the foodstuff is a syrup. In a more preferred embodiment, the foodstuff is a fruit syrup. In a particular embodiment, the fruit syrup is selected from the group consisting of: banana syrup, berries syrup, orange syrup, grape syrup, grenadine syrup, passion fruit syrup, kiwi syrup, cranberry syrup and peach syrup. In a preferred embodiment, the food composition of the invention is a grenadine syrup.

The term “microorganism” refers to any non-cellular or unicellular (including colonial) organism. Microorganisms include all prokaryotes. Microorganisms include bacterial spores, mycobacteria, protozoa, non-enveloped viruses, fungal spores, vegetative fungi, yeast, vegetative bacteria (including cyanobacteria), enveloped viruses, and other virus (e.g. virinos, viroids, phages), and algae (including lichens). The term is used interchangeably herein with "microbe." In certain embodiments, the microorganisms may be spoilage microorganism (food spoilage microorganisms).

The term “spoilage microorganism” as used herein refers to any organism or agent that can produce that food can become unsuitable for human consumption due to the outgrowth of said microorganisms. These spoilage microorganisms are associated with impairment of food quality attributes, such as taste, texture, color, and nutritional value. The food spoilage can be related to quality parameters or safety issues. The majority of spoilage microorganisms present no particular food safety hazard while others can be pathogenic and/or produce toxins.

In a particular embodiment, the spoilage microorganism is a filamentous fungi, a yeast or a bacteria.

The term “pathogen” as used herein refers to any organism or agent that can produce disease. A pathogen may also be referred to as an infectious agent, or simply a germ. Foodborne pathogens, or pathogens that can be present in a foodstuff, are organisms or agents that contaminate food during production and processing, but also during storage and transport before consuming. During their growth these microorganisms can secrete different components, including toxins, into the extracellular environment. Other harmful substances can be also liberated and can contaminate food after disintegration of food pathogens. Some bacterial and fungal toxins can be resistant to inactivation, and can survive harsh treatment during food processing. In a particular embodiment, these agents are bacteria and fungi, some viruses, prions and protozoa. In a more particular embodiment, the pathogen is a filamentous fungi, a yeast or a bacteria.

The term “preservation” refers to processes that make food more resistant to microorganism growth and slow the oxidation of fats. This slows down the decomposition and rancidification process.

As an expert may know the content of the antimicrobial composition of the invention for the manufacture of the foodstuffs will be dependent on the type of foodstuff as well as the intended use of such product. Thus in another embodiment, the invention relates to a food composition comprising an effective amount of an antimicrobial 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 a an antimicrobial composition of the invention to be applied to a food product or foodstuff, e.g. meat, fish and poultry (both fresh/unprocessed and processed) or beverage (such as syrups) etc, to obtain a shelf-stable food composition preparation and/or for increasing the shelf life of a food composition, is an amount which is determined to reduce the content of microorganisms, and include known parameters which include, but are not limited to, the concentration of the components of the antimicrobial composition of the invention, the volume and/or surface area of the food composition (such as meat, fish and poultry, beverages etc) and the atmospheric environment conditions of said food composition.

The effective amount of the antimicrobial composition of the invention may depend on the nature of the food product or beverage. For example, In certain embodiments, the product (such as food or beverage) obtained by the above mentioned method(s) comprising a food product (such as food or beverage) and an antimicrobial composition of the invention has from about 12ppm, 15ppm, 20 ppm, 30, 40, 50, 60, 70, 80, 90, 100 ppm, to about 6%, 5%, 4%, 4%, 3%, 2%, 1 % (or 10000ppm), 9000ppm, 8000ppm, 7000ppm, 6000ppm, 5000ppm, 4000ppm, 3000ppm, 2000ppm, 1000ppm, 800ppm, 600ppm, 500ppm, 400ppm, 300ppm to 200ppm w/w, such as from 50ppm to 1000ppm of L-lysine hydrochloride in the final product.

For example, In certain embodiments, the product (such as food or beverage) obtained by the above mentioned method(s) comprising a food product (such as food or beverage) and an antimicrobial composition of the invention has from about 12ppm, 15ppm, 20 ppm, 30, 40, 50, 60, 70, 80, 90, 100 ppm, to about 6%, 5%, 4%, 4%, 3%, 2%, 1 % (or 10000ppm), 9000ppm, 8000ppm, 7000ppm, 6000ppm, 5000ppm, 4000ppm, 3000ppm, 2000ppm, 1000ppm, 800ppm, 600ppm, 500ppm, 400ppm, 300ppm to 200ppm w/w, such as from 50ppm to 1000ppm of cinnamic acid in the final product.

For example, In certain embodiments, the product (such as food or beverage) obtained by the above mentioned method(s) comprising a food product (such as food or beverage) and an antimicrobial composition of the invention has from about 12ppm, 15ppm, 20 ppm, 30, 40, 50, 60, 70, 80, 90, 100 ppm, to about 6%, 5%, 4%, 4%, 3%, 2%, 1 % (or 10000ppm), 9000ppm, 8000ppm, 7000ppm, 6000ppm, 5000ppm, 4000ppm, 3000ppm, 2000ppm, 1000ppm, 800ppm, 600ppm, 500ppm, 400ppm, 300ppm to 200ppm w/w, such as from 50ppm to 1000ppm of tartaric acid in the final product. In certain embodiments, the product (such as food or beverage) obtained by the above mentioned method(s) comprising a food product (such as food or beverage) and an antimicrobial composition of the invention has less than 400 ppm of tartaric acid. In other embodiments, the tartaric acid may be present from about 50 ppm to about 400 ppm, from about 100 ppm to about 400 ppm, from about 100 ppm to about 300 ppm.

For example, In certain embodiments, the product (such as food or beverage) obtained by the above mentioned method(s) comprising a food product (such as food or beverage) and an antimicrobial composition of the invention has from about 12ppm, 15ppm, 20 ppm, 30, 40, 50, 60, 70, 80, 90, 100 ppm, to about 6%, 5%, 4%, 4%, 3%, 2%, 1 % (or 10000ppm), 9000ppm, 8000ppm, 7000ppm, 6000ppm, 5000ppm, 4000ppm, 3000ppm, 2000ppm, 1000ppm, 800ppm, 600ppm, 500ppm, 400ppm, 300ppm to 200ppm w/w, such as from 50ppm to 1000ppm of succinic acid in the final product.

For example, In certain embodiments, the product (such as food or beverage) obtained by the above mentioned method(s) comprising a food product (such as food or beverage) and an antimicrobial composition of the invention has from about 12ppm, 15ppm, 20 ppm, 30, 40, 50, 60, 70, 80, 90, 100 ppm, to about 6%, 5%, 4%, 4%, 3%, 2%, 1 % (or 10000ppm), 9000ppm, 8000ppm, 7000ppm, 6000ppm, 5000ppm, 4000ppm, 3000ppm, 2000ppm, 1000ppm, 800ppm, 600ppm, 500ppm, 400ppm, 300ppm to 200ppm w/w, such as from 50ppm to 1000ppm of L-alanine hydrochloride in the final product.

For example, In certain embodiments, the product (such as food or beverage) obtained by the above mentioned method(s) comprising a food product (such as food or beverage) and an antimicrobial composition of the invention has from about 12ppm, 15ppm, 20 ppm, 30, 40, 50, 60, 70, 80, 90, 100 ppm, to about 6%, 5%, 4%, 4%, 3%, 2%, 1 % (or 10000ppm), 9000ppm, 8000ppm, 7000ppm, 6000ppm, 5000ppm, 4000ppm, 3000ppm, 2000ppm, 1000ppm, 800ppm, 600ppm, 500ppm, 400ppm, 300ppm to 200ppm w/w, such as from 50ppm to 1000ppm of cranberry extract in the final product.

Methods for improving shelf-life of food products of the invention

In a particular embodiment, the foodstuff is a syrup. In a more particular embodiment, the foodstuff is a fruit syrup. In a still more particular embodiment, the foodstuff is a grenadine syrup.

In another particular embodiment, the fruit syrup has an acidic pH.

In another aspect, the present invention relates to a method for reducing the content of microorganisms in a food composition, wherein the method comprising contacting the food composition with the antimicrobial composition of the invention.

The term “microorganism” already defined before, refers to any non-cellular or unicellular (including colonial) organism. Microorganisms include all prokaryotes. Microorganisms include bacterial spores, mycobacteria, protozoa, non-enveloped viruses, fungal spores, vegetative fungi, yeast, vegetative bacteria (including cyanobacteria), enveloped viruses, and other virus (e.g. virinos, viroids, phages), and algae (including lichens). The term is used interchangeably herein with "microbe."

The term “reducing the content of microorganisms” refers to a reduction of at least a 0.5 Log CFU of microorganisms.

In a particular embodiment, the microorganisms are filamentous fungi or a yeast.

In certain embodiments, the antimicrobial composition of the invention is effective against yeast.

In certain preferred embodiments, the antimicrobial composition of the invention is effective against a mould or a filamentous fungus. In certain preferred embodiments, the antimicrobial composition of the invention is effective against bacteria.

In certain embodiments, the antimicrobial composition of the invention is effective against yeast, mould, filamentous fungus and/or bacteria.

A fungus is any member of the group of eukaryotic organisms that includes microorganisms such as yeasts and molds, as well as the more familiar mushrooms. In a particular embodiment, the filamentous fungi is a species from a genus selected from the group consisting of the Penicillium genus, the Aspergillus genus, the Byssochlamys genus, the Rhizopus genus, the Thalaromyces genus, the Cladosporium genus, the Cephalosporium genus, the Emericellopsis genus, the Pleurotus genus, the Tolepocladium genus, the Trichoderma genus, the Fusarium genus, the Claviceps genus, the Aureobasidium genus and the Giberella genus.

In a still more particular embodiment, the filamentous fungi from the Penicillium genus is P. digitalis, P. glabrum, P. crysogenum, P. corylophylum or P. albicans or wherein the filamentous fungi from the Aspergillus genus is A. oryzae, A. flavus, A. glabrum or A. fumigatus.

Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. In a particular embodiment, the yeast is a species from a genus selected from the group Schizosaccharomyces, Candida, Hanseniaspora, Nadsonia, Saccharomycodes, Wickerhamia, Lipomyces, Ambrosiozyma, Pachysolen, Arthroascus, Pachytichospora, Citeromyces, Pichia, Clavispora, Saccharomyces, Cyniclomyces, Saccharomycopsis, Kluyveromyces, Debaryomyces, Schwanniomyces, Dekkera, Sporopachydermia, Guilliermondella, Stephanoascus, Hansenula, Torulaspora, Issatchenkia, Wickerhamiella, Yarrowia, Lodderomyces, Wingea and Zygosaccharomyces genus.

The antimicrobial effect of the antimicrobial composition of the invention may be against one or more of the fungi or a yeast genus mentioned herein.

In certain embodiments, the antimicrobial composition of the invention is effective against (i.e inhibits) one or more of the following microorganisms: Zygosaccharomyces rouxii, Zygosaccharomyces bailii, Aspergillus brasiliensis, Aspergillus niger, Penicillium roqueforti, Candida tropicalis, Pichia farinose.

In certain embodiments, the antimicrobial composition of the invention is effective against all of the following microorganisms: Zygosaccharomyces rouxii, Zygosaccharomyces bailii, Aspergillus brasiliensis, Aspergillus niger, Penicillium roqueforti, Candida tropicalis, Pichia farinose, Candida albicans, Saccharomyces cerevisiae.

In certain preferred embodiments, the antimicrobial composition of the invention is effective against acetic acid bacteria.

Thus, in certain embodiments, the present invention relates to a method for obtaining a shelf-stable food composition or for increasing the shelf life of a food composition, and/or for reducing the content of acetic acid bacteria of a food composition comprising contacting the food composition with the antimicrobial composition of the invention.

In a particular embodiment of the methods of the invention, the antimicrobial composition is contacted with the food product under conditions leading to the food composition of the invention.

KITS

The present invention also relates to a kit comprising the different elements of an antimicrobial composition of the invention described herein. In certain embodiments, the kit comprises the elements already mixed and ready to use (for example as a blend) and optionally instructions of how to use said ingredients.

In certain embodiments, the kit comprises the different elements separately and optionally instructions of how to use said ingredients. For example, the kit may comprise

A first component selected from one or more of L-lysine hydrochloride and L-alanine hydrochloride, and the further component of the kit of the invention is one or more of cinnamic acid, tartaric acid, succinic acid, malic acid and cranberry extract.

For example, the kit may comprise a combination or organic acids selected from the group consisting of: a combination of cinnamic acid and tartaric acid, a combination of cinnamic acid and succinic acid, a combination of cinnamic acid and malic acid, a combination of tartaric acid and succinic acid, a combination of tartaric acid and malic acid, a combination of succinic acid and malic acid, a combination of cinnamic acid tartaric acid and succinic acid, a combination of cinnamic acid, tartaric acid and malic acid, a combination of tartaric acid, succinic acid and malic acid, a combination of cinnamic acid, succinic acid and malic acid, a combination of cinnamic acid, tartaric acid and malic acid, and a combination of cinnamic acid, tartaric acid, succinic acid and malic acid.

(i) L-lysine as the first component and cinnamic acid as the second component,

(vi) L-alanine hydrochloride as the first component and succinic acid as the second component, or

(vii) L-alanine hydrochloride as the first component and a cranberry extract as the second component. and optionally instructions of how to use said ingredients.

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

The invention will be described by way of the following examples which are to be considered as merely illustrative and not limitative of the scope of the invention.

EXAMPLES

Materials

Yeast Extract Glucose Chloramphenicol broth medium (YGC) [composition in g/liter: Yeast extract 5.0; D(+)glucose 20.0; chloramphenicol 0.1], pH adjusted to 3,2 with orthophosphoric acid 1M.

Yeast Extract Glucose Chloramphenicol agar medium (YGC) [composition in g/liter: Yeast extract 5.0; D(+)glucose 20.0; chloramphenicol 0.1 ; agar-agar 14.9],

Grenadine syrup model [composition in g/liter : white cristallin sugar 400; water 205; mix of fruits puree (raspberry, blackcurrant, elderberry) 210; citric acid 5], pH 3.2, 57 °Brix.

Buffer solution [KH2PO4 3 g; Na2HPO4 6 g; Tween 80 0.10 ml; distilled H20 to 1 liter].

Organic acids (cinnamic, tartaric, malic, succinic) were used in their powder form, 100 % pure.

Cranberry extract used in the tests contains 38.6 % of organic acids [composition in g / 100ml: quinic acid 15,6; malic acid 13,5; citric acid 9,3; shikimic acid 0,2], For obtaining this extract, the cranberry juice concentrate was diluted in a solution ethanol/water and percolated through a chromatographic column to separate the organic acids fraction from the polyphenolic fraction. Then, the organic acids fraction was concentrated under vacuum and spray-dried to obtain the final powder.

Amino acids alone (alanine, cysteine, glycine and lysine) or in their hydrochloride form (alanine HCI, cysteine HCI, glycine HCI and lysine HCI) were used in their powder form, 100% pure. Hydrochloric acid [HCI, 37%; Merck] was diluted in sterile distiled water according to the desired molarity.

Ortho-phosphoric acid (H3PO4, 85%; Meek) was used for pH correction.

Sodium chloride (NaCI, Merk) solution 0.9% in deionized sterile water was used for dilutions of culture of microorganisms.

The microorganisms used in the in vitro and callenge tests are from DSMZ collection (Deutsche Sammlung von Mikroorganismen und Zellkulturen) or from private collection of spoilage microorganisms.

Methods

Challenge tests against moulds

Spores of moulds were harvested from 14-day-old cultures incubated at 30 °C on Yeast Extract Glucose Chloramphenicol Agar medium (YGC) by flooding the agar plates with buffer solution and harvesting the spores using sterile rake shaped spreaders. Spore suspensions were filtered through a column of glass whool to remove hyphal fragments and then kept at 6°C until use. Counting was done using a Neubauer chamber. Viability assays were done using YGC agar medium.

The application matrices were inoculated in the surface with 2 - 3 Log of spores I ml and incubated at 25°C for at least, 7 days.

In vitro tests were performed in YGC medium, pH adjusted to 3,5 with ortho-phosphoric acid. Each microplate well received 200pl of different conditions. The YGC medium without preservative or test compounds was used as negative control. Tests were done in duplicate.

Fruit syrups, mainly grenadine syrup were used as application matrix.

Tests in syrups were performed in sterile tubes containing 5 ml of the application matrix, in duplicate.

The efficacy or not of the test compounds was determined through visual detection of the germination of the spores: (+) means no activity against mould evidenciated by mycelium growth, ( - ) means inhibitory activity against mould evidenciated by absence of mycelium. Challenge tests against yeasts

Yeasts strains were incubated, separately, at 30 °C in Yeast Extract Glucose Chloramphenicol broth medium (YGC). After the culture period, counting were done on YGC agar medium. Dilutions with NaCI 0.9% were made to obtain an inoculation rate of 2 - 3 Log CFU I ml of the application matrices. The test tubes were capted and incubated at 25°C for at least, 7 days.

In vitro tests were performed in duplicate, as for moulds, but incubated at 25°C in a microplate reader under orbicular agitation (150rpm) every 5 minutes and absorbance recording at 600nm until the stationary phase was reached. Stationary phase can vary from 16 to 72h. Results are average of the difference between the absorbance at T final and absorbance at TO [Abs Tf - Abs TO], So, greater the difference between these two measurements, greater the growth of the yeast in the given medium.

As for moulds, challenge tests were carried in fruit syrups - mainly grenadine syrup - in duplicate.

Viability platings were performed on YGC agar at different type points for, at least, 7 days. Considering the inoculation rate, results are given as the difference of the concentration of yeasts at the end of the test and the concentration of yeasts at the day of inoculation [Log CFU/ml at day final - Log CFU/ml at day 0], If this difference is higher than 1 Log CFU/ml, it means yeast growth and if it is lower than 1 Log CFU/ml, it means inhibition of the yeast growth.

Results

L-Lysine HCI and cinnamic acid

Test against yeast Zygosaccharomyces rouxii DSM7525 in application (in syrup).

Table 1. Test against yeast Zygosaccharomyces rouxii DSM7525 (day 28)

When lysine HCI or cinnamic acid are applied alone, at 0,15 and 0,01 % respectively, in a grenadine syrup, they do not inhibit the growth of Zygosaccharomyces rouxii DSM7525. Surprisingly, when lysine HCI and cinnamic acid are applied in combination, at the same above dosages, the growth of Zygosaccharomyces rouxii DSM7525 is inhibited.

At day 0, the syrup was inoculated with 3,7 Log CFU/ml with the yeast Zygosaccharomyces rouxii DSM7525. Considering this inoculation rate, a difference [Log CFU/ml at day 28 - Log CFU/ml at day 0] higher than 1 Log CFU/ml, means yeast growth and if it is lower than 1 Log CFU/ml, it means inhibition of the yeast growth.

L-Lysine HCI, cinnamic acid and tartaric acid

Table 2. Test against mould Aspergillus brasiliensis in syrup (day 28)

When lysine HCI, cinnamic acid or tartaric acid are applied alone, at 0,1 , 0,02 and 0,05% respectively, in a grenadine syrup, they do not inhibit the growth of Aspergillus brasiliensis. But, when lysine HCI, tartaric acid and cinnamic acid are applied in combination, at the same above dosages, the growth of Aspergillus brasiliensis is inhibited.

Table 3. Test against mould Aspergillus niger in syrup (day 28)

When lysine HCI, cinnamic acid or tartaric acid are applied alone, at 0.1 , 0.02 and 0.05% respectively, in a grenadine syrup, they do not inhibit the growth of Aspergillus niger. But, when lysine HCI, tartaric acid and cinnamic acid are applied in combination, at the same above dosages, the growth of Aspergillus niger is inhibited.

L-Lysine HCI and succinic acid

Table 4. Test against yeast in vitro

When lysine HCI or succinic acid are applied alone, at 0.15 and 0.01% respectively, in a grenadine syrup, they do not inhibit the growth of Zygosaccharomyces rouxii DSM7525.

Surprisingly, when lysine HCI and succinic acid are applied in combination, at the same above dosages, the growth of Zygosaccharomyces rouxii DSM7525 is inhibited.

L-Lysine HCI and tartaric acid

Table 5. Test against yeast in vitro

When lysine HCI or tartaric acid are applied alone, at 0.15 and 0.01% respectively, in a grenadine syrup, they do not inhibit the growth of Zygosaccharomyces rouxii DSM7525. Surprisingly, when lysine HCI and tartaric acid are applied in combination, at the same above dosages, the growth of Zygosaccharomyces rouxii DSM7525 is inhibited.

L-alanine HCI and malic acid

Table 6. Tests against yeast in vitro

When alanine HCI or malic acid are applied alone, at 0.2 and 0.1 % respectively, in culture medium, they do not inhibit the growth of Zygosaccharomyces bailii. Surprisingly, when alanine HCI and malic acid are applied in combination, at the same above dosages, the growth of Zygosaccharomyces bailii is inhibited.

L-alanine HCI and succinic acid

Table 7. Tests against yeast in vitro

When alanine HCI or succinic acid are applied alone, at 0.2 and 0.1 % respectively, in culture medium, they do not inhibit the growth of Zygosaccharomyces bailii. Surprisingly, when alanine HCI and succinic acid are applied in combination, at the same above dosages, the growth of Zygosaccharomyces bailii is inhibited.

L-alanine HCI and cranberry extract

Table 8. Test against mould Aspergillus brasiliensis in syrup

When alanine HCI or cranberry extract are applied alone, at 0.1 and 0.2% respectively, in a grenadine syrup, they do not inhibit the growth of Aspergillus brasiliensis. Surprisingly, when alanine HCI and cranberry extract are applied in combination, at the same above dosages, the growth of Aspergillus brasiliensis is inhibited. Table 9. Test against mould Aspergillus niger in syrup

When alanine HCI or cranberry extract are applied alone, at 0.1 and 0.2% respectively, in a grenadine syrup, they do not inhibit the growth of Aspergillus niger. Surprisingly, when alanine HCI and cranberry extract are applied in combination, at the same above dosages, the growth of Aspergillus niger is inhibited. Table 10. Test against mould Penicillium roqueforti in syrup

When alanine HCI or cranberry extract are applied alone, at 0.1 and 0.2% respectively, in a grenadine syrup, they do not inhibit the growth of Penicillium roqueforti. Surprisingly, when alanine HCI and cranberry extract are applied in combination, at the same above dosages, the growth of Penicillium roqueforti is inhibited.

Test against yeasts in vitro

Table 11. Test against yeast Candida tropicalis in vitro

When alanine HCI or cranberry extract are applied alone, at 0.05 and 0.1% respectively, in culture medium, they do not inhibit the growth of Candida tropicalis. Surprisingly, when alanine HCI and succinic acid are applied in combination, at the same above dosages, the growth of Candida tropicalis is inhibited.

Table 12. Test against yeast Zygosaccharomyces bailii DSM70834 in vitro

When alanine HCI or cranberry extract are applied alone, at 0.05 and 0.1 % respectively, in culture medium, they do not inhibit the growth of Pool of Zygosaccharomyces bailii strains DSM70834 and DSM70492. Surprisingly, when alanine HCI and succinic acid are applied in combination, at the same above dosages, the growth of Pool of

Zygosaccharomyces bailii is inhibited.

Table 13. Test against yeast Pichia farinosa 2065 in vitro

When alanine HCI or cranberry extract are applied alone, at 0.05 and 0.1 % respectively, in culture medium, they do not inhibit the growth of Pichia farinosa. Surprisingly, when alanine HCI and succinic acid are applied in combination, at the same above dosages, the growth of Pichia farinosa is inhibited.

Test against pool of yeast Zyc/osaccharomyces bailii strains DSMZ 70834 and DSMZ 70492 in applicatipon (syrup) Table 14. Test against pool of yeast Zygosaccharomyces bailii strains

When alanine HCI or cranberry extract are applied alone, at 0.1 and 0.2% respectively, in a grenadine syrup, they do not inhibit the growth of Pool of Zygosaccharomyces bailii strains DSMZ 70834 and DSMZ 70492. Surprisingly, when alanine HCI and cranberry extract are applied in combination, at the same above dosages, the growth of those yeast strains is inhibited.

Claims

1. An antimicrobial composition comprising:

(i) A first component which is amino acid selected from the group consisting of L-lysine hydrochloride and L-Alanine hydrochloride, or a combination thereof; and

(ii) A second component which is an organic acid selected from the group comprising cinnamic acid, tartaric acid, succinic acid, malic acid or a combination thereof or a composition comprising organic acids.

2. The antimicrobial composition of claim 1 wherein the composition comprising organic acids comprises one or more of quinic acid, malic acid, citric acid, formic acid, propionic acid, ferulic acid, levulinic acid, chlorogenic acid and shikimic acid or combinations thereof.

3. The antimicrobial composition of claim 2 wherein the composition comprising organic acids is a cranberry extract, optionally, wherein the cranberry extract is substantially free of polyphenols.

4. The antimicrobial composition according to claims 2 to 3 wherein the composition comprising organic acids comprises quinic acid, malic acid, citric acid and shikimic acid and are present at a ratio of about 78:67,5:46,5:1 by weight.

5. The antimicrobial composition of any of claims 1 to 4 wherein

(i) the first component is L-lysine hydrochloride and the second component is cinnamic acid,

(ii) the first component is L-lysine hydrochloride and the second component is a combination of cinnamic acid and tartaric acid.

(iii) the first component is L-lysine hydrochloride and the second component is succinic acid,

(iv) the first component is L-lysine hydrochloride and the second component is tartaric acid,

(v) the first component is L-lysine hydrochloride and the second component is malic acid,

(vi) the first component is L- alanine hydrochloride and the second component is cinnamic acid, (vii) the first component is L- alanine hydrochloride and the second component is a combination of cinnamic acid and tartaric acid.

(viii) the first component is L- alanine hydrochloride and the second component is succinic acid,

(ix) the first component is L- alanine hydrochloride and the second component is tartaric acid,

(x) the first component is L- alanine hydrochloride and the second component is malic acid,

(xi) the first component is L-alanine hydrochloride and the second component is succinic acid, or

(xii) the first component is L-alanine hydrochloride and the second component is a cranberry extract.

6. A food composition that comprises the antimicrobial composition according to any of claims 1 to 5, wherein the food composition has an acidic pH.

7. The food composition according to claim 6 wherein the first component of the antimicrobial composition is present at a concentration of between 0,001 and 0,5 % (w/w) and wherein the second component of the antimicrobial composition is present at a concentration of between 0,001 and 0,8 % (w/w).

8. The food composition according to claim 6 wherein the

(i) the first component of the antimicrobial composition is L-lysine hydrochloride and the second component of the antimicrobial composition is cinnamic acid and wherein the L-lysine hydrochloride from the antimicrobial composition is present at a concentration of 0,15% (w/w) and the cinnamic acid from the antimicrobial composition is present at 0,01 % (w/w),

(ii) the first component of the antimicrobial composition is L-lysine hydrochloride and the second component antimicrobial is a composition is a combination of cinnamic acid and tartaric acid and wherein the L-lysine hydrochloride from the antimicrobial composition is present at a concentration of 0, 1 % (w/w) and the cinnamic acid and the tartaric acid from the antimicrobial composition are present at concentrations of 0,05% and 0,02% respectively (w/w), (iii) the first component of the antimicrobial composition is L-lysine hydrochloride and the second component antimicrobial composition is succinic acid and wherein the L-lysine hydrochloride from the antimicrobial composition is present at a concentration of 0,15% (w/w) and the succinic acid from the antimicrobial composition is present at 0,1 % (w/w),

(iv) the first component of the antimicrobial composition is L-lysine hydrochloride and the second component antimicrobial composition is tartaric acid and wherein the L-lysine hydrochloride from the antimicrobial composition is present at a concentration of 0,15% (w/w) and the tartaric acid from the antimicrobial composition is present at 0,1 % (w/w),

(v) the first component of the antimicrobial composition is L-lysine hydrochloride and the second component antimicrobial composition is malic acid and wherein the L-lysine hydrochloride from the antimicrobial composition is present at a concentration of 0,15% (w/w) and the malic acid from the antimicrobial composition is present at 0,1% (w/w),

(vi) the first component of the antimicrobial composition is L-alanine hydrochloride and the second component antimicrobial composition is succinic acid and wherein the L-alanine hydrochloride from the antimicrobial composition is present at a concentration of 0,2% (w/w) and the succinic acid from the antimicrobial composition is present at 0,01% (w/w),

(vii) the first component of the antimicrobial composition is L-alanine hydrochloride and the second component of the antimicrobial composition is a composition enriched in organic acids wherein the L-alanine hydrochloride from the antimicrobial composition is present at a concentration of 0,2% (w/w) and the composition enriched in organic acids is present at 0,2% (w/w),

(viii) the first component of the antimicrobial composition is L-alanine hydrochloride and the second component of the antimicrobial composition is a composition enriched in organic acids wherein the L-alanine hydrochloride from the antimicrobial composition is present at a concentration of 0,3% (w/w) and the composition enriched in organic acids is present at 0,2% (w/w),

(ix) the first component of the antimicrobial composition is L-alanine hydrochloride and the second component of the antimicrobial composition is a composition enriched in organic acids wherein the L-alanine hydrochloride from the antimicrobial composition is present at a concentration of 0,05% (w/w) and the composition enriched in organic acids is present at 0,1% (w/w),

(x) the first component of the antimicrobial composition is L-alanine hydrochloride and the second component of the antimicrobial composition is a composition enriched in organic acids wherein the L-alanine hydrochloride from the antimicrobial composition is present at a concentration of 0, 1% (w/w) and the composition enriched in organic acids is present at 0,2% (w/w),

9. Use of the antimicrobial composition according to any one of claims from 1 to 5 for obtaining a shelf-stable food composition, for decreasing the content of microorganisms in a food composition, for increasing the shelf life of a food composition or for preservation of fresh or cooked food or food composition.

10. A method for obtaining a shelf-stable food preparation or for increasing the shelf life of a food composition comprising contacting the foodstuff with the antimicrobial composition according to any of claims 1 to 5.

11. The food composition according to claims 6 to 8 or method according to claim 10 wherein the food composition is a syrup.

12. A method for reducing the content of microorganisms in a food composition, wherein the method comprising contacting the food composition with the antimicrobial composition according to any of claims 1 to 5.

13. The use according to claim 9 or the method according to claim 12 wherein the microorganisms is a filamentous fungi or a yeast.

14. The use or the method according to claim 13 wherein the filamentous fungi is a species from a genus selected from the group consisting of the Penicillium genus, the Aspergillus genus, the Rhizopus genus, the Thalaromyces genus, the Cladosporium genus, the Cephalosporium genus, the Emericellopsis genus, the Pleurotus genus, the Tolepocladium genus, the Trichoderma genus, the Fusarium genus, the Claviceps genus, the Aureobasidium genus and the Giberella genus and I or wherein the yeast is a species from a genus selected from the group Schizosaccharomyces, Candida, Hanseniaspora, Nadsonia, Saccharomycodes, Wickerhamia, Lipomyces, Ambrosiozyma, Pachysolen, Arthroascus, Pachytichospora, Citeromyces, Pichia, Clavispora, Saccharomyces, Cyniclomyces, Saccharomycopsis, Kluyveromyces, Debaryomyces, Schwanniomyces, Dekkera, Sporopachydermia, Guilliermondella, Stephanoascus, Hansenula, Torulaspora, Issatchenkia, Wickerhamiella, Yarrowia, Lodderomyces, Wingea and Zygosaccharomyces genus.

15. The use or the method according to claim 14 wherein the filamentous fungi from the Penicillium genus is P. digitalis, P. glabrum, P. crysogenum, P. corylophylum or P. albicans or wherein the filamentous fungi from the Aspergillus genus is A. oryzae, A. flavus, A. glabrum or A. fumigatus.

16. The food composition according to claim 6, wherein the:

(i) the first component of the antimicrobial composition is L-alanine hydrochloride and the second component of the antimicrobial composition is cinnamic acid and wherein the L-alanine hydrochloride from the antimicrobial composition is present at a concentration of 0.15% (w/w) and the cinnamic acid from the antimicrobial composition is present at 0.01 % (w/w); or

(ii) the first component of the antimicrobial composition is L-alanine hydrochloride and the second component antimicrobial is a composition is a combination of cinnamic acid and tartaric acid and wherein the L- alanine hydrochloride from the antimicrobial composition is present at a concentration of 0.1 % (w/w) and the cinnamic acid and the tartaric acid from the antimicrobial composition are present at concentrations of 0.05% and 0.02% respectively (w/w); or

(iii) the first component of the antimicrobial composition is L-alanine hydrochloride and the second component antimicrobial composition is succinic acid and wherein the L-alanine hydrochloride from the antimicrobial composition is present at a concentration of 0.15% (w/w) and the succinic acid from the antimicrobial composition is present at 0.1 % (w/w); or

(iv) the first component of the antimicrobial composition is L-alanine hydrochloride and the second component antimicrobial composition is tartaric acid and wherein the L- alanine hydrochloride from the antimicrobial composition is present at a concentration of 0.15% (w/w) and the tartaric acid from the antimicrobial composition is present at 0.1 % (w/w); or (v) the first component of the antimicrobial composition is L-alanine hydrochloride and the second component antimicrobial composition is malic acid and wherein the L-alanine hydrochloride from the antimicrobial composition is present at a concentration of 0.15% (w/w) and the malic acid from the antimicrobial composition is present at 0.1% (w/w).