NZ523687A

NZ523687A - Antimicrobial agent

Info

Publication number: NZ523687A
Application number: NZ523687A
Authority: NZ
Inventors: Dieter Elsser; Andrew John Morgan; Linda Valerie Thomas; Shukun Yu
Original assignee: Danisco
Priority date: 2000-09-27
Filing date: 2001-09-27
Publication date: 2005-03-24
Also published as: WO2002026060A1; GB2381456A; GB2381456B; JP2004509908A; EP1322189A1; CA2423139A1; CN1466422A; NZ523686A; WO2002026061A1; GB0302473D0; JP2004509634A; AU2001290135A1; CN1466423A; EP1322188A1; AU2001290133A1; GB2381196A; CA2423134A1; GB0302415D0

Abstract

An antimicrobial composition comprising a cyclic compound having Formula I, wherein R1 and R2 are independently selected from -OH, =O, and -OC(O)R', wherein R' is a hydrocarbyl group; wherein R3 is selected from -OH, =O, a substitutent comprising an -OH group and -OC(O)R', wherein R' is a H or a hydrocarbyl group, wherein R4 and R5 are each independently selected from a hydrocarbyl group, H, OH, =O, and -OC(O)R', wherein R' is a H or a hydrocarbyl group or wherein R4 and R5 represent a bond with an adjacent atom on the ring of the cyclic compound; and wherein said compound comprises at least one ester group. Also disclosed a process for preventing and/or inhibiting the growth of, and/or killing, micro-organisms in a material, and the use of a cyclic compound having Formula I.

Description

523687 WO 02/26061 PCT/GBO1/04330 1 ANTIMICROBIAL AGENT The present invention relates to antimicrobial agents. More specifically, the invention relates to the antimicrobial activity of a series of anhydrofructose derivatives.

Food degradation from various sources is recognized in the literature and individual chemicals are known which will inhibit one aspect or another of degradation derived from a single source. Degradation, and the loss of colour or flavour of freshly cut plant parts are known to be caused by oxidation, enzymes, microbes, and metal ions. For example, 10 acidulants are known to prevent microbial degradation by maintaining a relatively low pH environment but their effectiveness is only temporary.

Listeria monocytogenes is one example of an organism which can contaminate certain foodstuffs and which exhibits resistance to many physical and chemical treatments. 15 Listeria monocytogenes is a gram-positive bacillus that causes serious infection, mainly in immunocompromised patients and newborn infants. Meningitis and bacteremia are the most frequent manifestations of listeriosis.

Bacillus cereus is another common cause of food poisoning. Two distinct clinical 20 syndromes have been identified, the first having a short incubation period of about 4 hours, the second having an incubation period of about 17 hours. B. cereus food poisoning is initiated when the spore forms survive cooking and the contaminated food is allowed to reach temperatures that permit germination of the spore and elaboration of an enterotoxin.

Salmonella, of which there are over two thousand different strains, is a further cause of food poisoning in humans. Salmonella is a genus of rod-shaped Gram-negative Enterobacteriaceae flat inhabit the intestine and cause infections such as gastroenteritis and typhoid. If invasive, they can cause enteric fevers (for example, typhoid caused by 30 Salmonella typhi, or paratyphoid fever caused by Salmonella paratyphi). Other strains of Salmonella are associated with food poisoning (usually Salmonella Typhimurium, Salmonella panama or Salmonella Enteritidis, the latter notorious for the contamination PCT/GBO1/04330 2 of poultry) and occasionally septicaemia in non-intestinal tissues.

It is well known in the art that Salmonella cannot propagate at pH values below 4.5. As a consequence, mildly acid products such as fine food and non-fermented meat products are 5 especially susceptible to attack by Salmonella.

For meat products, nitrite is often used as a preservative. However, the addition of nitrite is restricted for toxological reasons (due to its acute toxicity, together with the dangers associated with nitrosamine formation). As a result, Salmonella is only inhibited at 10 concentrations of nitrite beyond 1,000 ppm, which are far beyond legal limits.

Instead, it has been shown that combinations of nitrite and sorbic acid can increase the effectiveness against Salmonella [Inhibition of Salmonella by Sodium Nitrite and Potassium Sorbate in Frankfurters, Journal of Food Science, 47, 1982, p. 1615 ff]. 15 Inhibition has been observed at concentrations beyond 50 ppm of nitrite combined with 2600 ppm sorbic acid.

Other agents such as bacteriocins (Nisin) are unable to inhibit Salmonella in food, whereas benzoic acid is unsuitable because the inhibitory effect can only be observed in 20 acid products. The inhibitory effect of phytogenic ingredients (or "natural substances") such as oil extracts from different spices, has also been tested, but again the concentrations required for achieving the inhibitory effect on Salmonella were too high and the sensorical influence on the food was too strong.

Thus, to date, the use of chemical substances has been severely limited because on the one hand they have to be safe from a toxicological view point, but on the other hand they must not influence the product sensorically.

The present invention seeks to alleviate the problems associated with prior art chemical 30 substances and to provide new antimicrobial compositions based on anhydrofructose derivatives. In particular, the invention seeks to provide antimicrobial agents that are suitable for use in foodstuffs/feed.

In a first aspect, the invention provides an antimicrobial composition comprising a cyclic compound having Formula I, wherein R1 and R2 are independently selected from -OH, =0, and -0C(0)R', wherein R' is a hydrocarbyl group; wherein R3 is selected from -OH, =0, a substituent comprising an -OH group and -0C(0)R', wherein R' is a H or a hydrocarbyl group; wherein R4 and R5 are each independently selected from a hydrocarbyl group, H, OH, =0, and -0C(0)R', wherein R' is 10 a H or a hydrocarbyl group or wherein R4 and R5 represent a bond with an adjacent atom on the ring of the cyclic compound; and wherein said compound comprises at least one ester group.

A second aspect of the invention provides a process for preventing and/or inhibiting the 15 growth of, and/or killing, microorganisms in a material, the process comprising the step of contacting fhe material with a cyclic compound having Formula I, wherein R1 and R2 are independently selected from -OH, =0, and -0C(0)R', wherein R' is a 20 hydrocarbyl group; wherein R3 is selected from -OH, =0, a substituent comprising an -OH group and -0C(0)R\ wherein R' is a H or a hydrocarbyl group; wherein R4 and R5 are each independently selected from a hydrocarbyl group, H, OH, =0, and -00(0)R', wherein R' is aHora hydrocarbyl group or wherein R4 and R5 represent a bond with an adjacent atom on the ring of the cyclic compound; and wherein said compound comprises at least one ester 25 group. intellectual property"" ofrce of nz 1 0 AUG 2004 received PCT/GBO1/04330 4 In a third aspect, the invention relates to the use of a compound having Formula I, R1 .. "R5 I wherein R1 and R2 are independently selected from -OH, =0, and -0C(0)R's wherein R' is a 5 hydrocarbyl group; wherein R3 is selected from -OH, =0, a substituent comprising an -OH group and -0C(0)R', wherein R' is a H or a hydrocarbyl group; wherein R4 and R5 are each independently selected from a hydrocarbyl group, H, OH, =0, and -0C(0)R', wherein R' is a H or a hydrocarbyl group or wherein R4 and R5 represent a bond with an adjacent atom on the ring of the cyclic compound; and wherein said compound comprises at least one ester 10 group; for preventing and/or inhibiting the growth of, and/or killing, microorganisms in a material. . . ...

It will be appreciated that by the term "ester group" it is meant a group of the formula X-C(0)0-Y wherein X and Y are hydrocarbyl groups.

Preferably, the material is a foodstuff or feed. Thus, in a preferred aspect, the present invention relates to antimicrobial substances that are suitable for use in foodstuffs and/or feed to inhibit food poisoning and spoiling bacteria contained therein.

In another preferred embodiment, the material is a home product, a body care product or a cosmetic product, for example, a body lotion.

By way of definition, the term "antimicrobial" refers to a substance that kills or prevents or inhibits the growth or reproduction of microorganisms. Antimicrobials are generally classified according to the type of microorganism they are effective against. For example, antibacterial substances are effective against bacteria, antifungal substances are effective against fungi, including yeast, and antiviral substances are effective against viruses. Certain antimicrobials can be used internally, for example antibiotic medications, whereas other antimicrobials are for external use only, such as antiseptics.

PCT/GBO1/04330 As used herein, the term "hydrocarbyl group" means a group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, hydroxy, carboxyl, epoxy, acrylic, hydrocarbon, N-acyl, or cyclic group etc. In addition to the possibility of the 5 substituents being a cyclic group, a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and 10 include, for instance, sulphur, nitrogen and oxygen.

In a more preferred aspect, the cyclic compound of the invention is a compound having Formula II II wherein R1, R2, R3, R4, and R5 are as defined hereinabove.

Preferably, the cyclic compound is a compound having Formula HI o m wherein R1, R2, R3, R4, and R5 are as defined hereinabove.

In one preferred embodiment, said cyclic compound is of Formula IV, 6 F f IV 1 ^ wherein R and R are independently selected from -OH, =0, and -0C(0)R', wherein R' is a 5 hydrocarbyl group; wherein R3 is selected from -OH, =0, a substituent comprising an -OH group and -0C(0)R', wherein R' is a H or a hydrocarbyl group; wherein R4 and R5 are each independently selected from a hydrocarbyl group, H, OH, =0, and -0C(0)R', wherein R' is a H or a hydrocarbyl group or wherein R4 and R5 represent a bond with an adjacent atom on the ring of the cyclic compound; wherein R6 and R7 are each independently selected from a 10 hydrocarbyl group, H, OH, =0, and -0C(0)R\ wherein R' is a H or a hydrocarbyl group or wherein R4 and R5 represent a bond with an adjacent atom on the ring of the cyclic compound; and wherein said compound comprises at least one ester group.

More preferably, said cyclic compound is of formula V, wherein R1, R2, R3, R4 R5, R6 and R7 are as defined hereinabove.

Preferably, R1 is selected from -OH, =0, and -0C(0)R', wherein R' is a hydrocarbyl group.

Preferably, R2 is selected from -OH, =0, and -0C(0)R', wherein R' is a hydrocarbyl group.

V Preferably, R3 is selected from a substituent comprising an -OH group and -0C(O)R', wherein R' is a H or a hydrocarbyl group.

PCT/GBO1/04330 7 Even more preferably, R3 is -0C(0)R', wherein R' is a H or a hydrocarbyl group.

Even more preferably, R3 is -0C(0)R', wherein R' is a hydrocarbyl group.

In one preferred embodiment, R3 is -0C(0)R', wherein R' is R" group.

Preferably, R' and/or R" is a branched or unbranched, substituted or unsubstituted alkyl group.

More preferably, R' and/or R" is (CH2)PCH3, wherein p is from 1 to 24.

Even more preferably, R' and/or R'' is a Cg alkyl group.

In an another preferred embodiment, R' and/or R" is a Cn alkyl group.

In an another preferred embodiment, R' and/or R" is a Ci6 or a Cig alkyl group.

In one preferred embodiment of the invention, R3 is of the formula -(CH2)n-OC(0)-(CH2)pCH3, wherein n and p are each independently from 1 to 24.

More preferably, R3 is of the formula -(CH2)n-OC(0)-(CH2)7CH3, wherein n is from 1 to 24, preferably from 1 to 20, preferably from 1 to 10, preferably from 1 to 5, or preferably 1, 2, or 3.

In an alternative preferred embodiment, R3 is of the formula -(CH2)n-OC(0)-(CH2)nCH3, 25 wherein n is from 1 to 24, preferably from 1 to 20, preferably from 1 to 10, preferably from 1 to 5, or preferably 1,2, or 3.

In one preferred embodiment, R4 is selected from a hydrocarbyl group, H, OH, =0, and -0C(O)R', wherein R' is a H or a hydrocarbyl group.

In a particularly preferred embodiment, R4 is selected from a hydrocarbyl group, H, OH, and =0. 8 In one preferred embodiment, R5 is selected from a hydrocarbyl group, H, OH, =0, and -0C(0)R', wherein R' is a H or a hydrocarbyl group.

In a particularly preferred embodiment, R5 is selected from a hydrocarbyl group, H, OH, and =0.

In one especially preferred embodiment, the compound is esterified anhydrofructose wherein at least one OH group of anhydrofructose is esterified to form a -0C(0)R"' group, wherein R'" is a hydrocarbyl group.

Preferably, R"' is a branched or unbranched, substituted or unsubstituted alkyl group.

Even more preferably, R'" is (CH2)PCH3, wherein p is from 1 to 24, More preferably still, R"' is a Cg alkyl group.

In an alternative preferred embodiment, R'" is a C12 alkyl group.

In another preferred embodiment, R"' is a Ci6 or a Cig alkyl group 25 In one preferred embodiment of the invention, the cyclic compound is of the formula: In one preferred embodiment, R4 and R5 represent a bond with an adjacent atom on the ring of the cyclic compound. 0 p= 1-24 In one preferred embodiment of the invention, cyclic compound is of the formula: p = 1-24 More preferably, the cyclic compound is selected from lie following: o o OH OH More preferably, the cyclic compound is selected from the following: Q o J7 OH Preferably, the compound of the invention is a derivative of Ascopyrone P, Ascopyrone M, Ascopyrone T, Ascopyrone Ti, Ascopyrone T2, Ascopyrone T3, and mixtures thereof.

Even more preferably, the compound of the invention is selected from esterfied Ascopyrone P, esterfied Ascopyrone M, esterfied Ascopyrone T, esterfied Ascopyrone Ti, esterfied 15 Ascopyrone T2, esterfied Ascopyrone T3, and mixtures thereof.

The structures of Ascopyrone P, Ascopyrone M, Ascopyrone T, Ascopyrone Ti, Ascopyrone T2 and Ascopyrone T3 are shown below. ur>n-L h0ch2 Hu Ascopyrone M Ascopyrone P Ascopyrone T HOCH2 HOCH2 HOCH2 un un ■un u oh Ascopyrone Tj Ascopyrone T2 Ascopyrone T3 Ascopyrone is a known compound. In 1978 and 1981, a group of American scientists prepared Ascopyrone P by pyrolysis of amylopectin, amylose and cellulose at the Wood 5 Chemistry laboratory in Montana, with the intention of using Ascopyrone P as a starting material for organic synthesis [Shafizadeh, F., Furneaux R.H., Stevenson, T.T., and Cochran, T.G., 1 ^-Anhydro^-deoxy-D-g/ycero-hex-1 -en-3-ulose and other pyrolysis products of cellulose, Carbohydr. Res. 67(1978): 433-447; Stevenson, T.T., Stenkmap, R.E., Jensen, L.H., Cochran, T.T., Shafizadeh, F., and Furneaux R.H., The crystal 10 structure of 1,5-anhydro-4-deoxy-D-g/ycero-hex-l -en-3-ulose, Carbohydr. Res. 90(1981): 319-325]. They characterized Ascopyrone P by, for example, *H and 13C NMR, and IR spectroscopy techniques. A 3-dimensional structure of Ascopyrone P was provided. The yield of Ascopyrone P obtained by pyrolysis was under 3% and complicated separation methods had to be used.

The natural occurrence of Ascopyrone P in some species of very scarcely studied fungi collected from the Alps has been taught [M.-A. Baute, G. Deffieux, J. Vercauteren, R. Baute, and Badoc A., Enzymatic activity degrading 1,4-a-glucans to Ascopyrones P and T in Pezizales ad Tuberales, Phytochernistry, 33 (1993): 41-45]. The occurrence of 20 Ascopyrone P in fungi immediately prompted the hypothesis that Ascopyrone P would act as an antibiotic. However, Ascopyrone P did not function satisfactorily as an antibiotic in the disclosed tests. 11 Ascopyrone P and Ascopyrone T can be produced enzymatically from 1,5-anhydro-D-fructose using cell-free extract prepared from the fungi of the order Pezizales, such as Plicaria leiocarpa mdAnthracobia melaloma, and the order of Tuberales, such as, Tuber melanosporum. Ascopyrone T] is the dihydrate form of Ascopyrone T, whereas Ascopyrone T2 and T3 are the tautomeric monohydrate forms of Ascopyrone T.

Ascopyrone M can be produced from 1,5-anhydro-D-fructose by EDTA-sensitive dehydratases isolated from the fungi Morels, such as Morchella vulgaris, Gyromitres, pezizes, such as Peziza echinospora.

Ascopyrone M, P and T can. also be produced chemically by treating 1,5-anhydro-D-fructose with alkali under mild conditions [Studies on the degradation of some pentoses and of 1,5-anhydro-D-fructose, the product of the starch-degrading enzyme a-l,4-glucan lyase; Thesis, Ahmad, T., The Swedish University of Agricultural Sciences, Sweden, 15 1995].

When the compound of the present invention is prepared by chemical means, it may be prepared in accordance with one of the following methods: (1) Ascopyrone P may be produced by treating 1,5-anhydro-D-fructose with non-aqueous acid at elevated temperature, for example at 70 °C. (2) Ascopyrones (for example, Ascopyrone P, T and M) may be produced from 1,5-anhydro-D-fructose by alkaline treatment according to Ahmad, T., 1995.

The structures of all ascopyrones produced were confirmed by NMR techniques.

Preferably, the compound of the present invention is prepared by enzymatic means as disclosed in M.-A. Baute et al, [Phytochernistry, 33 (1993): 41-45). For example 30 ascopyrones (such as, Ascopyrone P, T and M) may be produced from 1,5-anhydro-D-fructose using enzymatic methods as disclosed in M.-A. Baute et al.

PCT/GBO1/04330 12 In a particularly preferred embodiment, the compound is selected from the following: OH € OH °hjt 0 OH, HO OH oh^r O OH CHjOH -o OH OH' OH OH OAc AcO u OH p= 1.2....24 or an esterified derivative thereof.

In a preferred embodiment, the cyclic compound having formula I has an antimicrobial effect against gram positive bacteria and yeasts.

Preferably, the cyclic compound having formula I has an antimicrobial effect against a microorganism selected from Listeria, Salmonella, Bacillus, Saccharomyces, Pseudomonas, Clostridium, Lactobacillus, Brochothrix, Micrococcus, Yersinia, Enterobacter and Zygosaccharomyces, Staphylococcus, Escherichia.

Even more preferably, the cyclic compound having formula I has an antimicrobial effect against a microorganism selected from Listeria monocytogenes, E. coli, Staphylococcus PCT/GBO1/04330 13 aureus, Listeria innocua, Salmonella Typhimurium, Salmonella sp., Bacillus cereus, Bacillus subtilis, Saccharomyces cerevisiae, Saccharomyces cerevisiae var. paradoxus, Saccharomyces carlsbergensis, Pseudomonas fluorescens, Clostridium sporogenes, Lactobacillus sake, Brochothrix thermosphacta, Micrococcus luteus, Yersinia enterocolitica, 5 Enterobacter aerogenes and Zygosaccharomyces bailii.

Even more preferably, the cyclic compound having formula I has an antimicrobial effect against a micro-organism selected from Listeria monocytogenes, E. coli, Bacillus cereus, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Pseudomonas fluorescens, 10 Clostridium sporogenes, Lactobacillus sake, Brochothrix thermosphacta and Micrococcus luteus.

In a highly preferred aspect a derivative of the compound of formula I is a compound of the formula "Y° This compound (3,6-di-Oacetyl-l ,5-anhydro-4-deoxy-D-g/>'cero-hex-3-enopyranose-2-ulose) may be prepared in accordance with the teaching of Andersen et al. (1998), Structure of 1,5-anhydro-D-fructose: X-ray analysis of crystalline acetylated dimeric forms, J. Carbohydr. Chem. 17:1027-1035.

The aspect of the present invention wherein the derivative of the compound of formula I is an ester is particularly preferred because the compound may be lipophilic and/or may 25 have both hydrophobic and hydrophilic properties. When the compound has both hydrophobic and hydrophilic properties the compound readily resides at a water/oil interface of an emulsion.

The residence of the compound at a water/oil interface of an emulsion may allow it to act 30 as an emulsifier. Thus the present invention may further provide compounds having a 14 dual functional effect. The compounds may act both as an antimicrobial and as an emulsifier.

Many of the compounds of the present invention can be derived from 1,5-anhydrofructose. 5 1,5-Anhydrofructose is monoketo sugar found in bacteria, red algae, fungi and mammals. In red algae and fungi 1,5-anhydrofructose is produced by the action of a-l,4-glucan lyase [EC 4.2.2.13] from floridean starch and glycogen, respectively.

When the compound of the present invention is prepared from 1,5-anhydro-D-fructose, 10 preferably the 1,5-anhydro-D-fructose is prepared in accordance with GB-A-2296717. In other words, preferably the 1,5-anhydro-D-fructose is prepared by a method comprising treating an a-l,4-glucan with the enzyme a-l,4-glucan lyase characterised in that enzyme is used in substantially pure form.

Preferably, the cyclic compound of the invention comprises a five or a six membered ring.

The compounds of the present invention comprise at least one ester group. Thus, as used herein the term "ester" includes mono-, di-, tri- and poly-esters.

In a preferred aspect the compound of formula I is a diester wherein the R1 substituent is an -OH group and wherein the ester linkages are formed from the -OH group of the R4 substituent and from the -OH group of the R3 substituent.

As mentioned above, in a particularly preferred embodiment of the invention, the 25 compound is 6-0-acyl-l,5-anhydro-D-fructose, as represented below.

The preparation of 6-0-acyl- 1,5-anhydro-D-fructose may be addressed by a chemical approach or by an enzymatic approach, in accordance with the methods detailed in WO The chemical approach may comprise the following reaction to synthesise C12 esters of 5 anhydrofructose: The reaction is carried out with lauroyl chloride and pyridine. The acylation sites were assigned through derivatisation of NH2OR followed by separation and NMR of the 10 products. The products were found to be 50% 6-O-acyl- 1,5-anhydro-D-fructose 11% 3-O-acyl- 1,5-anhydro-D-fructose A similar method may be used to prepare other ester derivatives of anhydrofructose.

The enzymatic approach to prepare 6-O-acyl-l ,5-anhydro-D-fructose may comprise the use of lipases and proteases. In aqueous solution lipases and proteases cleave ester linkages. Lipases are sugar specific and proteases fatty acid specific. However, Synthesis 1990, 112-115 discloses that lipases and proteases in non-aqueous solution offer a 20 reversal of activity, and form ester bonds. Thus lipases and proteases in non-aqueous solution may be used in the preparation of a compound in accordance with the present invention.

In accordance with J. Chem. Soc. Per kin Trans. I, 1995, 2203-2222 lipases were 25 screened to identify suitable lipases for the preparation of compounds in accordance with the present invention. Screening with pyridine identified Candida antarctica, Pseudomonas cepacia, Pseudomonas fluorescens, and hog pancreas. Screening with tBuOH:pyridine 2:1 identified Candida antarctica, Candida cylindracea, 00/56745.

PCT/GBO1/04330 16 Pseudomonas cepacia, Pseudomonas fluorescens, hog pancreas.

Thus preferably the compound in accordance with the present invention is prepared with a lipase obtained from Candida antarctica, Pseudomonas cepacia, Pseudomonas 5 fluorescens, hog pancreas, or Candida cylindracea.

Preferably the compovmd in accordance with the present invention is prepared with lipase from Candida antarctica. Candida antarctica may be obtained from Novo Nodisk A/S, Denmark under the name Novozym 435.

The enzymatic approach was demonstrated by the enzymatic acylation of 1,5-anhydro-D-fructose with lauric acid to form 6-0-acyl-1,5-anhydro-D-fructose.

Laurie acid Solvent 3 A molecular sieve.

Temperature Reaction time Conversion (mol/mol) (w/w) (OC) (h) 1 tert-BuOH - 40 24 21 % 1 tert-BuOH 1 (powd.) 40 24 56% 1 tert-BuOH 1 (powd.) 40 72 62 % 1 acetone 1 (powd.) 24 55% 1 tert-BuOH 45 24 56% 1 tert-BuOH 45 24 61% 1 tert-BuOH 45 24 66% 3 tert-BuOH 45 24 73% 3 tert-BuOH (powd.) 45 24 78% 3 tert-BuOH (powd.) 45 48 quantitative 3 acetone 72 quantitative The chemical approach may comprise the quantitative conversion with lauric, palmitic and stearic acid of 1,5-anhydro-D-fructose to 6-O-acyl-1,5-anhydro-D-fructose as follows: The reaction forms a composition comprising monomer ketone/dimer type 1/dimer type 2 -1:3:1. The mixture may be purified by chromatography on silica to give approximately 17 70% yield.

The cyclic compound of the invention may be used alone, or in combination with other components, for example, one or more preservatives, one or more chelators (such as 5 EDTA sodium salt, polyphosphate or citrate) and/or one or more antioxidants (such as ascorbate, isoascorbate, ascorbate palmitate, BHA or BHT).

By way of definition, in the broadest sense, the tenn "preservative" is intended to encompass all substances which inhibit the development of, or kill, micro-oganisms. In a narrower 10 sense, it is generally understood that preservatives are used in concentrations of 0.5 % or less. Food additives which are allowed to be used as preservatives are listed in the Regulation No. 95/2/EG of the European Parliament and Council of 20 February 1995, relating to food additives other than colouring agents and sweeteners.

Typical food preservatives permitted in the EU which are suitable for use in combination with the compounds of the invention include sorbic acid, benzoic acid, PHB ester (p-hydroxybenzoate), and sulphur dioxide. The mode of action of these preservatives, together with their range of effects are listed below.

Sorbic Acid fE200 to 2031: Mode of action: inhibits different enzymes in the cells of the microorganisms.

Range of effects: mainly against yeasts and moulds as well as catalase-positive bacteria. Catalase-negative bacteria as well as lactic acid bacteria and Clostridia are not inhibited. Effective concentration: 500 - 3000 ppm.

Permitted maximum quantities in food: up to 2000 ppm in potato dough, processed cheese, packed bread, fine bakery products, emulsified sauces etc.

Benzoic Acid (E210 to 213): Mode of action: inhibits exchange of oxygen through the cellular membrane and affects 30 the enzymatic structure.

Range of effects: for acid products only, up to approx. pH 4.5; inhibits yeasts and moulds, restricted inhibition of bacteria (no, or only very little, inhibition of lactic acid 18 bacteria and Clostridia).

Permitted maximum quantities in food: 500 ppm in aspic, fruit preparations, marmalades etc.

PHB Ester (p-hydroxybenzoate) (E214 to 219) Mode of action: damages the bacterial membrane because of the surface activity, poisonous to protoplasm because of protein denaturation.

Range of effects: mainly inhibits yeasts and fungi, but also Gram-positive bacteria in a pH range between 3.0 and 8.0.

Effective concentration: sensorical influence at concentrations beyond approx. 0.08 %.

Sulphur Dioxide (E220 to 224; E 226 to 227) Mode of action: depends on pH to a great extent, in practice it is only effective at acidic pH values (< 4,0). Very complex mechanisms.

Range of effects: mainly antibacterial, above all against Gram-negative, aerobic bacteria. Effective concentrations: 250 - 500 ppm for inhibition of aerobic, Gram-negative bacteria, 800 - 2000 ppm against Gram-positive bacteria, yeasts, and moulds.

Permitted maximum quantity in food products: max. 2000 ppm in dry fruits, grape juice concentrate for home production of wine, in some cases only max. quantities of 20 - 30 20 ppm are permitted.

For more specific applications, the compounds of the present invention may also be used in combination with the following preservatives: biphenyl, diphenyl, orthophenylphenol, thiabendazol, nisin, natamycin, hexamethylentetramine, dimethyldicarbonate, boric acid, 25 sodiumtetraborate, nitrite, propionic acid and propionate, and lysozyme. The mode of action of these preservatives, together with their range of effects and specific uses are listed below.

Biphenvl. Diphenvl (E 2301 30 Range of effects: Inhibition of moulds.

Substance for treatment of fruits: surface treatment of citrus fruits.

Permitted maximum quantity: 70 ppm PCT/GBO1/04330 19 Orthophenylphenol (E 231 / E 232) As with E230, limited to treatment of fruits as a surface treatment for citrus fruits.

Thiabendazol (E 233) Surface treatment of citrus fruits and bananas.

Nisin (E 234) Mode of action: Disturbance of membrane functions.

Range of effects: Gram-positive bacteria, no influence on Gram-negative bacteria. 10 Permitted maximum quantity in food products (EU): 3ppm in semolina pudding and similar products, 12.5 ppm (= 12.5 IU/g) in ripened cheese and processed cheese, 10 ppm in clotted cream, 10 ppm in mascarpone.

Natamvcin (Pimaricin) (E235) Mode of action: specifically attacks cell membrane, where - in general - an interaction with sterines occurs which increases the permeability of the membrane.

Range of effects: Moulds and yeasts, not effective against bacteria. Usual dosage rates are below approx. 50 mg / 1. Maximum level is 1 mg/dm2 on the surface, with a maximum penetration of 5 mm.

Applications: surface treatment of hard, semi-hard and semi-soft cheese and of dried, cured sausages.

Hexamethvlentetramine (E 239) Hexamethylentetramine is formed by adding ammonia to formaldehyde in an aqueous 25 solution. The microbicidal effect is due to the formaldehyde.

Permitted only for Provolone cheese (25 ppm residual quantity).

Dimethvldicarbonate (E 242) Permitted only for non-alcoholic drinks, non-alcoholic wine, and liquid concentrate.

Boric Acid. Sodiumtetraborate (E284 / E 285) Permitted only for caviar.

Nitrite (E 249 and E 250) Permitted in the form of nitrite curing salt for treatment of meat products ("red products"). For cured and dried meat products which are not heat treated and for other cured meat products an addition of 150 ppm has been fixed as a guideline. These concentrations do 5 not show a preservative effect. They are mainly added for their technological properties (formation of colour, taste) as well as for their antioxidant effects.

Propionic Acid and Propionate CE 280. E 281. E 282. and E 283") Mode of action: similar to sorbic acid, pH < 4.5 is optimal.

Accumulation in the cell leads to inhibition of enzymes.

Range of inhibition: moulds are inhibited at an pH of 5.5 by concentrations of 125 to 12500 ppm, for inhibition of bacteria higher concentrations are necessary (> 16000 ppm). Application: Sliced and packaged bread.

Permitted maximum quantity: 3000 ppm.

Lvsozvme (E 1105) Permitted only for ripened cheese.

Permitted maximum quantity: quantum satis.

Studies by the applicant of the inhibitive effects of the present compounds have been tested in a medium (Elliker broth) with an almost neutral pH (pH 6.8) and have been shown to be effective against both Gram-positive and Gram-negative bacteria. As many of the preservatives described above show an inhibitory effect mainly at low pH, the use of the compounds of the present invention clearly broadens the potential range of 25 applications.

In principle, the use of substances for chemical preservation depends on the following factors: (a) Toxicological harmlessness the effects of the substance when applied acutely, subchronically, and for a long term period. 21 • Testing of acute toxicity (LD50), cinetics and metabolism, pharmacological effects, genotoxicity, etc. (b) Technological / food chemical aspects: • Solubility in water: as growth takes place in the aqueous phase, a preservative has to be water-soluble • Reaction with food ingredients, problem of off-flavours (sensory acceptance) • Interferences with food ingredients (e.g. destruction of vitamin B1 by sulphuric acid) The antimicrobial effectiveness of chemical substances in food and feed products is thus determined by a range of different factors. Among others, the composition of the population of micro-organisms, the composition of the food product (ingredients, pH, water activity, content of salt, etc.), the packaging, time-temperature-conditions, etc. are 15 key factors that influence the inhibitory activities of the antimicrobial agent.

The invention will now be described only by way of example, and with reference to the accompanying figures, wherein: Figure 1 shows a photograph of well diffusion tests on M. luteus (top plate), B. cereus (middle two plates), and CI. Sporogenes (bottom two plates) treated with the following: Upper right segment: 3 % C8 anhydrofructose ester; Middle right segment: 0.3 % C$ anhydrofructose ester; Lower right segment: 3 % C12 anhydrofructose ester; Lower left segment: 0.3 % C12 anhydrofructose ester; Middle left segment: equivalent methanol control at 25 % methanol; Upper left segment: equivalent methanol control at 2.5 % methanol.

Figure 2 shows a photograph of a well diffusion test on M. luteus treated with the 30 following: Segment 1: 3 % C8 anhydrofructose ester; Segment 2: 0.3 % Cs anhydrofructose ester; 22 Segment 3: 3 % Cn anhydrofructose ester; Segment 4: 0.3 % C12 anhydrofructose ester; Segment 5: equivalent methanol control at 25 % methanol; Segment 6: equivalent methanol control at 2.5 % methanol.

EXAMPLES CHEMICAL SYNTHESIS The compounds of the invention were prepared, characterised and purified in accordance with the general methods disclosed in WO 00/56745.

MATERIALS AND METHODS TEST STRAINS All microorganisms were taken from storage at -80 °C. Most organisms were tested as vegetative cell suspensions from overnight broth culture. Bacillus and Clostridium species were tested as endospore suspensions prepared earlier and stored at 4 °C.

For broth cultures and Bioscreen testing most bacteria were grown in Brain Heart Infusion (BHI, Oxoid, pH 7.4). Lactobacillus sake A10 was grown in de Man, Rogosa, Shaipe medium (MRS, Oxoid). Yeasts were grown in Sabouraud Liquid medium (SLM, Oxoid). Most bacteria were cultured at 30 °C. Lactic acid bacteria were grown on solid medium in enriched CO2 atmosphere. Clostridium species were grown in Reinforced 25 Clostridial Medium (RCM) at 37 °C anaerobically. Brochothrix thermosphacta and yeasts were grown at 25 °C.

Bioscreen testing An automated Microbiology Reader Bioscreen C was used to measure growth curves of 30 the strains in the presence and absence of test samples. The Bioscreen C measures the development of turbidity (i.e. growth) kinetically by vertical photometry in 200 wells of a honeycomb microtitre plate, simultaneously. The system consists of a Bioscreen C 23 analyser, which is an incubator and measurement unit, integrated with a PC, software (BioLink v 5.30), printer and a 'Honeycomb 2' cuvette multiwell plate. Growth curve data can be analysed within the BioLink software or exported to programs such as Excel.

Protocol To a 14 mg sample was added 50 jul of 100% methanol. 66.7 |il of IMS was then added (industrial methylated spirit, 96% ethanol) to make a 12% (w/v) solution.

For the test, this solution was then diluted 1 in 4 in sterile distilled water. This was 10 necessary because the level of alcohol in the sample would otherwise be inhibitory to the test micro-organism. This made a final solution of 3% (w/v).

The test sample could not be filter sterilised because too much would have been lost, and only ca. 470 pi was available. The sample had been handled aseptically and it was hoped 15 that it was sterile. For the same reason the pH of the sample was not measured.

The sample was then tested at 0.3% concentration in the Bioscreen. However it was immediately realised that this may be problematic because the AF-ester 1 test sample was milky-white and turbid. Unfortunately, when this was added to the Bioscreen wells, the 20 initial turbidity was too high for any microbial growth to be discerned. Therefore to ascertain if any inhibition had occurred, viable counts were taken of the inoculum, and then after 24 h incubation in the Bioscreen at 30 °C, by sampling directly from the Bioscreen plate. Inhibition could then be assessed by comparison with the final numbers achieved in the control wells that contained 2.5% alcohol.

Results of Bioscreen BS021100 AF ester 1 = Cs ester of anhydrofructose (structure shown in claim 32 - LHS). 24 Table 1 Test strain Initial count (cfu/ml) Count after 24 h at 30 °C (cfu/ml) 2.5% alcohol control 0.3% AF-ester 1 B. cereus 204 lxlO3 1.3x10' 2.4x10* L. monocytogenes S23 1.2 x I03 1.1 x 10y <10* Lb. sake A10 A H—» O S: 1.9x10* A i—» 0K E. coli SI5 .3 x 102 1.1x10" 3.7 xlO7 Ps. fluorescens 3756 3.6x10* 1.1 x 10y 2.3 x 10' S. cerevisiae 9763 3.6 xW 2.0x10° 2x 101 S. carlsbergensis 6418 .2 x 10* 9.7 xlO4 1 x 10A Conclusions The results in Table 1 show that AF-ester 1 was inhibitory towards all the microorganisms tested. The order of inhibitory activity was as follows: Gram positives > yeasts > Gram negatives. The sample was particularly effective against L. monocytogenes, but was also very effective against Bacillus. There was evidence of cidal activity towards L. monocytogenes, and possibly the yeasts.

Anhydrofructose ester 1: Cidal test A preliminary cidal experiment was undertaken with the sample that had earlier been tested in Bioscreen with viable count confirmation. This had shown good activity. For the 15 cidal experiment the chosen test organism was L. monocytogenes S23, because this had shown the greatest sensitivity in the growth inhibition testing.

Protocol Aliquot 3 x 890 ml 10 mM HEPES buffer, pH 7. To the control test was added 100 ml 20 water, to the other control test was added 100 ml equivalent alcohol control and to the test sample was added 100 ml AF ester 1. To all tests were added 10 ml of an overnight culture. The samples were left at ambient temperature for 2 L A viable count was carried out. Note: the AF ester 1 precipitated out during the test.

PCT/GBO1/04330 Results Tests Viable count (cfu/ml) Control/water 3.1 x 10® Control/alcohol 2.0 x 107 Test/AF ester 1 2.4 xlO7 From the results it was concluded that AF ester 1 does not have any cidal activity.

Testing of new samples: Anhydrofructose ester C8 (AFC8) = Cg ester of anhydrofructose (structure shown in claim 32 - LHS) Anhydrofructose ester C12 (AFC12) = C12 ester of anhydrofructose (structure shown in claim 32 -RHS) Glucose ester C8 (GC8) - control 15 Glucose ester C12 (GC12) - control AF esters were dissolved in water by either heating at 70 °C for 10 - 15 min, or 100 °C for 5-10 minutes. Both methods were unsuccessful, and the esters were eventually tested as 0.5 % (w/v) solutions in 50:50 methanol/water that had been heated. AFC8 did not 20 dissolve, but the others were better.

Results: No zones observed for equivalent methanol controls.

Table 2 Test strain Well diffusion zone (mm) tested against 0.5% (wt/vol) extracts AFC8 AFC12 Glucose C8 Glucose C12 B. cereus 204 0 6.82 0 0 CI. sporogenes Campden 3.90 11.30 0 +/- (3.50) L. monocytogenes S23 0 0 0 0 Lb. sake A10 0 0 0 0 Br. thermosphacta CRA7883 0 7.50 0 0 Micrococcus luteus 0 8.95 0 0 E. coli S15 0 0 0 0 PCT/ GBO1/04330 26 Ps. fluorescens 327 0 0 0 0 S. cerevisiae ATCC 9763 0 +/- (10.00) 0 0 S. carlsbergensis CRA6413 0 0 0 0 Results of Bioscreen run BS191200 Table 3 Strain (Control: average OD without methanol) Level tested % (wt/vol) Final - min OD for 18 or 2* h growth at 30 °C AFC8 AFC1 2 GC8 GC12 Control: Equivalent Methanol level B. cereus 204 (0.80) 0.05 0 0 0.242 0 0.194 0.025* 0.168 0 0.785 0.561 0.619 B. cereus Campden (0.72) 0.05 0 0 0.882 0.437 0.75 0.025* 0.8 0 0.777 0.641 0.707 L. monocytogenes S23 (0.66) 0.05 0 0 0.242 0.19 0.27 0.025 0.027 0 0.514 0.379 0.537 Lb. sake A10 (0.90) 0.05 0 0 0.245 0.392 0.123 0.025 0.535 0.376 0.81 0.785 0.477 E. coli S15 (0.95) 0.05 0.573 0.478 0.654 0.745 0.716 0.025 0.753 0.703 0.818 0.875 0.851 E. coli CRA109 (0.86) 0.05 0 0 0.285 0.149 0.187 0.025 0.697 0.738 0.789 0.759 0.84 Ps. fluorescens 3756 (1.2) 0.05 0 0 0 0 0 0.025 0.068 0.192 0.82 0.953 0.973 Ps. fluorescens 327 (0.37) 0.05 0 0 0 0 0 0.025 0.12 0.178 0.21 0.354 0.219 *AF CI 2 showed total inhibition of Bc204 and Be Campden at a minimum level tested of 0.0125%.

Controls for this run were based on the final - OD for 18 or 24 h growth at 30 °C for growth in equivalent methanol levels. Inhibition by the AF esters was judged by whether 10 the number was lower than the number derived for the methanol control.

CONCLUSIONS • The well diffusion results showed that 0.5% AFC8 and AFC12 both had anti-clostridial activity, and AFC12 had activity against Bacillus, Brochothrix, 15 Micrococcus and perhaps yeasts, but not L. monocytogenes, or gram negatives (GN). 27 • Bioscreen results were from tests with 0.05% samples.

• Bioscreen confirmed the order of activity was as follows: AFC12 > AFC8. Bioscreen also confirmed activity against Bacillus, but activity was also observed against L. monocytogenes, and Lb sake, as well as some activity against gram negatives (GN).

Description of Bioscreen analysis: BS040101 0.3% AF ester was made up in 2.5% methanol. Serial dilutions were made. The following concentrations were tested: 0.3, 0.15, 0.075, 0.038 and 0%. APP was made up in water. The samples were analysed after 24 h at 30 °C (Table 4).

Table 4 Strain AF ester C8 AF ester C12 Be 204 Total inhibition at 0.15% Inhibition to 0.075% Total inhibition at 0.038% Lm S23 Total inhibition at 0.15% Inhibition to 0.038% Total inhibition at 0.038% Lbs A10 Total inhibition at 0.3% Inhibition to 0.15% Total inhibition at 0.15% Inhibition to 0.038% Ec S15 No inhibition at 0.3% No inhibition at 0.3% Psf3756 Inhibition to 0.3% Inhibition to 0.3% Viable counts from BS040101 Inhibition was judged by whether the final count in the presence of either AF ester was lower than the final count in 2.5 % methanol (control). The results are shown in Table 5.

Table 5 Final viable count after 24 l (cfu/ml) Strain 0.3% AFE C8 0.3% AFE C12 2.5% methanol control Be 204 1 x 10* 1.5 x 10J 1x10s Lm S23 2.5 x 10" 1.5 x10s 2.9 x 10y Lbs A10 <103 <10s 3.7 xlO7 Ec S15 3.5 x 10' 1.7 x 10y 2.3 x 10y Psf3756 1.7x10* 2.7 x 108 3.2 x 10y See 9763 9.4 x 104 7.4 x 10* 4.5 x 106 Sea 6413 3.3 x 104 nd 1.4 xlO4 28 Well diffusion testing The results for M. luteus, B. cereus 204, B. cereus Campden, CI. sporogenes 1.221, and CI. sporogenes Campden are illustrated in Figures 1 and 2 and Table 6. None of the 5 methanol control tests gave any diffusion zones. Code for the wells: 1 = 3% AFEC8, 2 = 0.3% AFEC8, 3 = 3% AFEC12, 4 = 0.3% AFEC12, 5 = equivalent methanol control at 25% methanol, 6 = equivalent methanol control at 2.5% methanol.

Table 6 Test strain Well diffusion zone (mm) tested against 0.3% and 3% (wt/vol) extracts AFEC8 AFEC8 AFEC12 AFEC12 3% 0.3% 3% 0.3% B. cereus 204 2.29 0 7.75 1.27 B cereus Campden <0.5 0 3.23 <0.5 CI sporogenes 1.221 6.00 0 16.83 .06 CI. sporogenes Campden 6.15 .05 14.15 6.15 L, monocytogenes S23 1.62 0 2.26 +/- L. monocytogenes 272 2.36 0 +/- 8.82 0 Lb. sake A10 +/- 0 +/- (2.8) +/- Br. thermosphacta CRA7883 0.98 0 7.55 +/- Micrococcus luteus 3.95 0 9.42 2.33 E. coli SI5 0 0 0 0 Ps. fluorescens 327 0 0 0 0 Ps fluorescens 3756 0 0 0 0 S. cerevisiae ATCC 9763 E E + E S. carlsbergensis CRA6413 E E + E CODE (for yeasts): E = enhanced growth; + = zone of inhibition observed.

Further testing of APP Well diffusion zones at 3% vs CI. sporogenes Campden (4.72) and Br. thermosphacta 7883 (2.96) PCT/GBO1/04330 29 Viable counts of BS040101 Table 7 Strain Viable count in 0.3% APP Be 204 7.0 x 101 LmS23 .4 x 107 Lbs A10 1.9 x 10B Ec S15 6.2 xlO8 Psf3756 A I—4 o See 9763 3.1 xlO5 Sea 1.1 x 10s Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant are, or related fields, are thus intended to fall within the scope of the following claims.

Claims

30 CLAIMS

1. An antimicrobial composition comprising a cyclic compound having Formula I, R3 5 R1' R4 R2^R5 I wherein R1 and R2 are independently selected from -OH, =0, and -0C(0)R\ wherein R' is a hydrocarbyl group wherein R3 is selected from -OH, =0, a substituent comprising an -OH group and -0C(0)R', 10 wherein R' is a H or a hydrocarbyl group; wherein R4 and R5 are each independently selected from a hydrocarbyl group, H, OH, =0, and -0C(0)R', wherein R' is a H or a hydrocarbyl group or wherein R4 and R5 represent a bond with an adjacent atom on the ring of the cyclic compound; and wherein said compound comprises at least one ester group. 15

2. A composition according to claim 1 wherein said material is a foodstuff or feed.

3. A composition according to claim 1 or 2 wherein the cyclic compound is a compound 20 having Formula II n ,1 t>2 r>3 t>4 j r>5 . wherein R , R , R, R , and R are as defined in claim 1. 25

4. A composition according to any one of the preceding claims wherein the cyclic compound is a compound having Formula III intellectual property office of n.z t0 AUG 2004 RECEIVED 31 r1 r4 r2 in wherein R1, R2, R3, R4, and R5 areas defined in claim 1. 5

5. A composition according to any one of the preceding claims wherein said cyclic compound is of Formula IV, IV wherein R1 and R2 are independently selected from -OH, =0, and -0C(0)R\ wherein R' is a 10 hydrocarbyl group wherein R3 is selected from -OH, =0, a substituent comprising an -OH group and -0C(0)R\ wherein R' is a H or a hydrocarbyl group; wherein R4 and R5 are each independently selected from a hydrocarbyl group, H, OH, =0, and -0C(0)R', wherein R' is a H or a hydrocarbyl group or wherein R4 and R5 represent a 15 bond with an adjacent atom on the ring of the cyclic compound; wherein R6 and R7 are each independently selected from a hydrocarbyl group, H, OH, =0, and -0C(0)R', wherein R' is a H or a hydrocarbyl group or wherein R4 and R5 represent a bond with an adjacent atom on the ring of the cyclic compound; and wherein said compound comprises at least one ester group. 20

6. A composition according to any one of the preceding claims wherein said cyclic compound is of formula V, intellectual property OFFICE OF N.Z 2 0 APR 2001* RECEIVED 32 r3 v wherein R1, R2, R3, R4, R5, R6 and R7 are as defined in claim 5. 5

7. A composition according to any one of the preceding claims wherein R1 is selected from -OH, =0, and -0C(0)R\ wherein R' is a hydrocarbyl group

8. A composition according to any one of the preceding claims wherein R2 is selected from -OH, =0, and -0C(0)R', wherein R' is a hydrocarbyl group 10

9. A composition according to any one of the preceding claims wherein R3 is selected from a substituent comprising an -OH group and -0C(0)R', wherein R' is a H or a hydrocarbyl group; 15

10. A composition according to any one of the preceding claims wherein R3 is -0C(0)R', wherein R' is a H or a hydrocarbyl group;

11. A composition according to any one of the preceding claims wherein R3 is -0G(0)R', wherein R' is a hydrocarbyl group; 20

12. A composition according to any one of the preceding claims wherein R3 is -0C(0)R\ wherein R' is R" group;

13. A composition according to any one of the preceding claims wherein R' and/or R" is a 25 branched or unbranched, substituted or unsubstituted alkyl group.

14. A composition according to any one of the preceding claims wherein R' and/or R" is (CH2)PCH3, wherein p is from 1 to 24. intellectual property office of n.2 2 0 APR 2004 received 33

15. A composition according to any one of the preceding claims wherein R' and/or R" is a Cg alkyl group. 5

16. A composition according to any one of the preceding claims wherein R' and/or R" is a C12 alkyl group

17. A composition according to any one of the preceding claims wherein R3 is of the formula -(CH2)n-0C(0)-(CH2)pCH3, wherein n and p are each independently from 1 to

18. A composition according to any one of the preceding claims wherein R3 is of the formula -(CH2)n-0C(0)-(CH2)7CH3, wherein n and p are each independently from 1 to 24.

19. A composition according to any one of the preceding claims wherein R3 is of the formula -(CH2)n-0C(0)-(CH2)nCH3, wherein n and p are each independently from 1 to 24. 20

20. A composition according to any one of the preceding claims wherein R4 is selected from a hydrocarbyl group, H, OH, =0, and -0C(0)R', wherein R' is a H or a hydrocarbyl group.

21. A composition according to any one of the preceding claims wherein R4 is selected from a hydrocarbyl group, H, OH, and =0. 25

22. A composition according to any one of the preceding claims wherein R5 is selected from a hydrocarbyl group, H, OH, =0, and -0C(0)R', wherein R' is a H or a hydrocarbyl group.

23. A composition according to any one of the preceding claims wherein R5 is selected from 30 a hydrocarbyl group, H, OH, and =0.

24. A composition according to any one of the preceding claims wherein R4 and R5 represent _ i intellectual propehu 1 office of n.z I 2 0 APR 2004 I RECEIVED 34 a bond with an adjacent atom on the ring of the cyclic compound;

25. A composition according to any one of the preceding claims wherein the compound is esterified anhydrofructose wherein at least one OH group of anhydrofructose is esterified to 5 form a -0C(0)R''' group, wherein R'" is a hydrocarbyl group.

26. A composition according to claim 25 wherein R'" is a branched or unbranched, substituted or unsubstituted alkyl group. 10

27. A composition according to claim 25 wherein R'" is (CH2)PCH3, wherein p is from 1 to 24.

28. A composition according to claim 25 wherein R"' is a Cg alkyl group. 15

29. A composition according to claim 25 wherein R'" is a C12 alkyl group

30. A composition according to any one of the preceding claims wherein the cyclic compound is of the formula: 0 p -1-24 20

31. A composition according to any one of the preceding claims wherein the cyclic compound is of the formula: o intellectual property office of n.z 2 0 APR 2004 received 35

32. A composition according to any one of the preceding claims wherein said cyclic compound is selected from the following:

33. A composition according to any one of the preceding claims wherein said cyclic compound is selected from the following: o o 10

34. A composition according to any one of the preceding claims wherein the compound is a derivative of Ascopyrone P, Ascopyrone M, Ascopyrone T, Ascopyrone Ti, Ascopyrone Ascopyrone T3, and mixtures thereof.

35. A composition according to any one of the preceding claims wherein the compound is 15 selected from esterified Ascopyrone P, esterified Ascopyrone M, esterified Ascopyrone T, esterified Ascopyrone Ti, esterified Ascopyrone T2, esterified Ascopyrone T3, and mixtures thereof.

36. A composition according to any one of the preceding claims wherein the compound is 20 selected from the following: f intellectual property ofrce of n.z 2 0 APR 2004 RECEIVED 36 CH,OH intellectual property ofrce of n.z 2 0 APR 2004 received CHjOH OH OH OH AcO p = 1,2....24 or an esterified derivative thereof.

37. A composition according to any one of the preceding claims wherein the cyclic compound having formula I has an antimicrobial effect against a microorganism selected from Listeria, Salmonella, Bacillus, Saccharomyces, Pseudomonas, Clostridium, Lactobacillus, Brochothrix, Micrococcus, Yersinia, Enterobacter and Zygosaccharomyces, Staphylococcus, and Escherichia. 10

38. A composition according to any one of the preceding claims wherein the cyclic compound having formula I has an antimicrobial effect against a microorganism selected from Listeria monocytogenes, E. coli, Staphylococcus aureus, Listeria innocua, Salmonella Typhimurium, Salmonella sp., Bacillus cereus, Bacillus subtilis, Saccharomyces cerevisiae, 15 Saccharomyces cerevisiae var. paradoxus, Saccharomyces carlsbergensis, Pseudomonas fluorescens, Clostridium sporogenes, Lactobacillus sake, Brochothrix thermosphacta, 37 Micrococcus luteus, Yersinia enterocolitica, Enterobacter aerogenes and Zygosaccharomyces bailii.

39. A composition according to any one of the preceding claims wherein the cyclic compound having formula I has an antimicrobial effect against a micro-organism selected from Listeria monocytogenes, E. coli, Bacillus cereus, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Pseudomonas fluorescens, Clostridium sporogenes, Lactobacillus sake, Brochothrix thermosphacta and Micrococcus luteus.

40. A composition according to any one of the preceding claims wherein said compound of formula I is used in combination with one or more of an antioxidant, a preservative and/or a chelator.

41. A process for preventing and/or inhibiting the growth of, and/or killing, microorganisms in a material, the process comprising the step of contacting the material with a cyclic compound having Formula I, wherein R1 and R2 are independently selected from -OH, =0, and -0C(0)R\ wherein R' is a hydrocarbyl group wherein R3 is selected from -OH, =0, a substituent comprising an -OH group and -0C(0)R', wherein R' is a H or a hydrocarbyl group; wherein R4 and R5 are each independently selected from a hydrocarbyl group, H, OH, =0, A e and -0C(0)R', wherein R' is a H or a hydrocarbyl group or wherein R and R represent a bond with an adjacent atom on the ring of the cyclic compound; and wherein said compound comprises at least one ester group.

42. A process according to claim 41 wherein said material is a foodstuff or feed. I intellectual property oppice of n.z 2 0 APR 2004 RECEIVED 38

43. A process according to claim 41 or 42 wherein the cyclic compound is a compound having Formula II n wherein R1, R2, R3, R4, and R5 are as defined in claim 41.

44. 44 A process according to any one of claims 41 to 43 wherein the cyclic compound is a compound having Formula HI in wherein R1, R2, R3, R4, and R5 are as defined in claim 41.

45. 45 A process according to any one of claims 41 to 44 wherein said cyclic compound is of Formula IV, wherein R1 and R2 are independently selected from -OH, =0, and -0C(0)R', wherein R' is a hydrocarbyl group wherein R3 is selected from -OH, =0, a substituent comprising an -OH group and -0C(0)R\ wherein R' is a H or a hydrocarbyl group; wherein R4 and R5 are each independently selected from a hydrocarbyl group, H, OH, =0, and -0C(0)R', wherein R' is a H or a hydrocarbyl group or wherein R4 and R5 represent a IV intellectual property ofrce of. n1 2 0 APR 2004 RECEIVED 39 bond with an adjacent atom on the ring of the cyclic compound; wherein R6 and R7 are each independently selected from a hydrocarbyl group, H, OH, =0, and -0C(0)R', wherein R' is a H or a hydrocarbyl group or wherein R4 and R5 represent a bond with an adjacent atom on the ring of the cyclic compound; 5 and wherein said compound comprises at least one ester group.

46. A process according to any one of claims 41 to 45 wherein said cyclic compound is of formula V, r3 -O -R Rf R4 r2 10 V wherein R1, R2, R3, R4, R5, R6 and R7 are as defined in claim 45.

47. 47 A process according to any one of claims 41 to 46 wherein R1 is selected from -OH, =0, and -0C(0)R', wherein R' is a hydrocarbyl group 15

48. A process according to any one of claims 41 to 47 wherein R2 is selected from -OH, =0, and-0C(0)R', wherein R'is a hydrocarbyl group

49. A process according to any one of claims 41 to 48 wherein R3 is selected from a 20 substituent comprising an -OH group and -0C(0)R', wherein R' is a H or a hydrocarbyl group;

50. A process according to any one of claims 41 to 49 wherein R3 is -0C(0)R\ wherein R' is a H or a hydrocarbyl group; 25

51. A process according to any one of claims 41 to 50 wherein R3 is -0C(0)R\ wherein R' is a hydrocarbyl group; , mtellectual property ! ofrce of n.z 2 0 APR 2004 RECEIVED 40

52. A process according to any one of claims 41 to 51 wherein R is -0C(0)R\ wherein R' is R" group;

53. A process according to any one of claims 41 to 52 wherein R' and/or R" is a branched or 5 unbranched, substituted or unsubstituted alkyl group.

54. A process according to any one of claims 41 to 53 wherein R' and/or R" is (CH2)pCH3, wherein p is from 1 to 24. 10

55. A process according to any one of claims 41 to 54 wherein R' and/or R" is a Cg alkyl group.

56. A process according to any one of claims 41 to 55 wherein R' and/or R" is a C12 alkyl group 15

57. A process according to any one of claims 41 to 56 wherein R3 is of the formula -(CH2)n-0C(0)-(CH2)PCH3, wherein n and p are each independently from 1 to 24.

58. A process according to any one of claims 41 to 57 wherein R3 is of the formula -(CH2)n-20 0C(0)-(CH2)7CH3, wherein n and p are each independently from 1 to 24. •j

59. A process according to any one of claims 41 to 58 wherein R is of the formula -(CHhV 0C(0)-(CH2)iiCH3, wherein n and p are each independently from 1 to 24. 25

60. A process according to any one of claims 41 to 59 wherein R4 is selected from a hydrocarbyl group, H, OH, =0, and -0C(0)R', wherein R' is a H or a hydrocarbyl group.

61. A process according to any one of claims 41 to 60 wherein R4 is selected from a hydrocarbyl group, H, OH, and =0. 30

62. A process according to any one of claims 41 to 61 wherein R5 is selected from a hydrocarbyl group, H, OH, =0, and -0C(0)R\ wherein R' is a H or a hy 2 0 APR 2004 received 41

63. A process according to any one of claims 41 to 62 wherein R5 is selected from a hydrocarbyl group, H, OH, and =0. 5

64. A process according to any one of claims 41 to 63 wherein R4 and R5 represent a bond with an adjacent atom on the ring of the cyclic compound;

65. A process according to any one of claims 41 to 64 wherein the compound is esterified anhydrofructose wherein at least one OH group of anhydrofructose is esterified to form a - 10 0C(0)R''' group, wherein R"' is a hydrocarbyl group.

66. A process according to claim 65 wherein R"' is a branched or unbranched, substituted or unsubstituted alkyl group. 15

67. A process according to claim 65 wherein R'" is (CH2)pCH3, wherein p is from 1 to 24.

68. A process according to claim 65 wherein R'" is a Cg alkyl group.

69. A process according to claim 65 wherein R'" is a C12 alkyl group 20

70. A process according to any one of claims 41 to 69 wherein the cyclic compound is of the formula: o p = 1-24 25

71. A process according to any one of claims 41 to 70 wherein the cyclic compound is of the formula: intellectual property office of n.z 2 0 APR 2004 received 42

72. A process according to any one of claims 41 to 71 wherein said cyclic compound is selected from the following:

73. A process according to any one of claims 41 to 72 wherein said cyclic compound is selected from the following: 10

74. A process according to any one of claims 41 to 73 wherein the compound is a derivative of Ascopyrone P, Ascopyrone M, Ascopyrone T, Ascopyrone Ti, Ascopyrone T2, Ascopyrone T3, and mixtures thereof. 15

75. A process according to any one of claims 41 to 74 wherein the compound is selected from esterified Ascopyrone P, esterified Ascopyrone M, esterified Ascopyrone T, esterified Ascopyrone T1, esterified Ascopyrone T2, esterified Ascopyrone T3, and mixtures thereof. 20

76. A process according to any one of claims 41 to 75 wherein the compound is selected from the following: intellectual property office of n2 2 0 APR 2004 RECEIVED 43 intellectual propehiy oppicf of n.z 20 APR 2004 received CHJOH CH-OH AcO p = 1,2....24 or an esterified derivative thereof. ■5

77. A process according to any one of claims 41 to 76 wherein the cyclic compound having formula I has an antimicrobial effect against a microorganism selected from Listeria, Salmonella, Bacillus, Saccharomyces, Pseudomonas, Clostridium, Lactobacillus, Brochothrix, Micrococcus, Yersinia, Enterobacter and Zygosaccharomyces, Staphylococcus, 10 and Escherichia.

78. A process according to any one of claims 41 to 77 wherein the cyclic compound having formula I has an antimicrobial effect against a microorganism selected from Listeria monocytogenes, E. coli, Staphylococcus aureus, Listeria innocua, Salmonella Typhimurium, 15 Salmonella sp., Bacillus cereus, Bacillus subtilis, Saccharomyces cerevisiae, Saccharomyces cerevisiae var. paradoxus, Saccharomyces carlsbergensis, Pseudomonas fluorescens, OFFICE OF N.Z 2 0 APR 2004 received 44 Clostridium sporogenes, Lactobacillus sake, Brochothrix thermosphacta, Micrococcus luteus, Yersinia enterocolitica, Enterobacter aerogenes and Zygosaccharomyces bailii.

79. A process according to any one of claims 41 to 78 wherein the cyclic compound having 5 formula I has an antimicrobial effect against a micro-organism selected from Listeria monocytogenes, E. coli, Bacillus cereus, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Pseudomonas fluorescens, Clostridium sporogenes, Lactobacillus sake, Brochothrix thermosphacta and Micrococcus luteus. 10

80. A process according to any one of claims 41 to 79 wherein said compound of formula I is used in combination with one or more of an antioxidant, a preservative and/or a chelator.

81. Use of a compound having Formula I, wherein R1 and R2 are independently selected from -OH, =0, and -0C(0)R', wherein R' is a hydrocarbyl group wherein R3 is selected from -OH, =0, a substituent comprising an -OH group and -0C(0)R', 20 wherein R'is a H or a hydrocarbyl group; wherein R4 and R5 are each independently selected from a hydrocarbyl group, H, OH, =0, and -0C(0)R\ wherein R' is a H or a hydrocarbyl group or wherein R4 and R5 represent a bond with an adjacent atom on the ring of the cyclic compound; and wherein said compound comprises at least one ester group; 25 for preventing and/or inhibiting the growth of, and/or killing, microorganisms in a material.

82. Use according to claim 81 wherein said material is a foodstuff or feed. 15 I

83. Use according to claim 81 or 82 wherein the cyclic compound is a compound having 45 Formula II :\ intellectual property office of n.z 2 0 APR 2004 received r1 r4 r2 h wherein R1, R2, R3, R4, and R3 are as defined in claim 81.

84. Use according to any one of claims 81 to 83 wherein the cyclic compound is a compound having Formula III r1 r4 r2 m 10 wherein R1, R2, R3, R4, and R3 are as defined in claim 81.

85. Use according to any one of claims 81 to 84 wherein said cyclic compound is of Formula IV, r r 15 IV wherein R and R are independently selected from -OH, =0, and -0C(0)R', wherein R' is a hydrocarbyl group wherein R3 is selected from -OH, =0, a substituent comprising an -OH group and -0C(0)R', wherein R'is a H or a hydrocarbyl group; 20 wherein R4 and R5 are each independently selected from a hydrocarbyl group, H, OH, =0, and -0C(0)R\ wherein R' is a H or a hydrocarbyl group or wherein R4 and R5 represent a bond with an adjacent atom on the ring of the cyclic compound; intellectual property office of n.z 2 0 APR 2004 46 received wherein R6 and R7 are each independently selected from a hydrocarbyl group, H, OH, =0, and -0C(0)R', wherein R' is a H or a hydrocarbyl group or wherein R4 and R5 represent a bond with an adjacent atom on the ring of the cyclic compound; and wherein said compound comprises at least one ester group. 5

86. Use according to any one of claims 81 to 85 wherein said cyclic compound is of formula V, r3 -O V 10 wherein R1, R2, R3, R4, R5, R6 and R7 are as defined in claim 85.

87. Use according to any one of claims 81 to 86 wherein R1 is selected from -OH, =0, and -0C(0)R', wherein R' is a hydrocarbyl group 15

88. Use according to any one of claims 81 to 87 wherein R2 is selected from -OH, =0, and -0C(0)R\ wherein R' is a hydrocarbyl group

89. Use according to any one of claims 81 to 88 wherein R3 is selected from a substituent comprising an -OH group and -0C(0)R', wherein R' is a H or a hydrocarbyl group; 20

90. Use according to any one of claims 81 to 89 wherein R3 is -0C(0)R', wherein R' is a H or a hydrocarbyl group;

91. Use according to any one of claims 81 to 90 wherein R3 is -0C(0)R\ wherein R' is a 25 hydrocarbyl group;

92. Use according to any one of claims 81 to 91 wherein R3 is -0C(0)R', wherein R' is R" group; intellectual property office of n.z 20 APR 2004 received 47

93. Use according to any one of claims 81 to 92 wherein R' and/or R" is a branched or unbranched, substituted or unsubstituted alkyl group. 5

94. Use according to any one of claims 81 to 93 wherein R' and/or R" is (CH2)PCH3, wherein p is from 1 to 24.

95. Use according to any one of claims 81 to 94 wherein R' and/or R" is a Cg alkyl group. 10

96. Use according to any one of claims 81 to 95 wherein R' and/or R" is a C12 alkyl group

97. Use according to any one of claims 81 to 96 wherein R3 is of the formula -(CH2)n-0C(0)-(CH2)PCH3, wherein n and p are each independently from 1 to 24. 15

98. Use according to any one of claims 81 to 97 wherein R3 is of the formula -(CH2),,-0C(0)-(CH2)7CH3, wherein n and p are each independently from 1 to 24.

99. Use according to any one of claims 81 to 98 wherein R3 is of the formula -(CH2)n-0C(0)-(CH2)hCH3, wherein n and p are each independently from 1 to 24. 20

100. Use according to any one of claims 81 to 99 wherein R4 is selected from a hydrocarbyl group, H, OH, =0, and -0C(0)R', wherein R' is a H or a hydrocarbyl group.

101. Use according to any one of claims 81 to 100 wherein R4 is selected from a hydrocarbyl 25 group, H, OH, and =0.

102. Use according to any one of claims 81 to 101 wherein R5 is selected from a hydrocarbyl group, H, OH, =0, and -0C(0)R', wherein R' is a H or a hydrocarbyl group. 30

103. Use according to any one of claims 81 to 102 wherein R5 is selected from a hydrocarbyl group, H, OH, and =0. 48

104. Use according to any one of claims 81 to 103 wherein R4 and R5 represent a bond with an adjacent atom on the ring of the cyclic compound;

105. Use according to any one of claims 81 to 104 wherein the compound is esterified 5 anhydrofructose wherein at least one OH group of anhydrofructose is esterified to form a - 0C(0)R''' group, wherein R"' is a hydrocarbyl group.

106. Use according to claim 105 wherein R'" is a branched or unbranched, substituted or unsubstituted alkyl group.

107. Use according to claim 105 wherein R'" is (CH2)PCH3, wherein p is from 1 to 24.

108. Use according to claim 105 wherein R"' is a Cg alkyl group. 15

109. Use according to claim 105 wherein R'" is a C12 alkyl group

110. Use according to any one of claims 81 to 109 wherein the cyclic compound is of the formula: o 20 p = 1-24

111. Use according to any one of claims 81 to 110 wherein the cyclic compound is of the formula: o -H-t OH OH p = 1-24 intellectual property office of n.z 20 APR 2004 received 49

112. Use according to any one of claims 81 to 111 wherein said cyclic compound is selected from the following: 0 o 11 OH

113. Use according to any one of claims 81 to 112 wherein said cyclic compound is selected from the following: o o OH O c£> 10

114. Use according to any one of claims 81 to 113 wherein the compound is a derivative of Ascopyrone P, Ascopyrone M, Ascopyrone T, Ascopyrone Ti, Ascopyrone T2, Ascopyrone T3, and mixtures thereof.

115. Use according to any one of claims 81 to 114 wherein the compound is selected from 15 esterified Ascopyrone P, esterified Ascopyrone M, esterified Ascopyrone T, esterified Ascopyrone T1, esterified Ascopyrone T2, esterified Ascopyrone T3, and mixtures thereof.

116. Use according to any one of claims 81 to 115 wherein the compound is selected from the following: 20 INTELL|CTUAl property OFRCF OF ISI.z 2 0 APR 2004 received 50 20 APR 2004 received CH,OH CH2OH OH OH OH OAc AcO p = 1,2....24 or an esterified derivative thereof. 10 15

117. Use according to any one of claims 81 to 116 wherein the cyclic compound having formula I has an antimicrobial effect against a microorganism selected from Listeria, Salmonella, Bacillus, Saccharomyces, Pseudomonas, Clostridium, Lactobacillus, Brochothrix, Micrococcus, Yersinia, Enterobacter and Zygosaccharomyces, Staphylococcus, and Escherichia.

118. Use according to any one of claims 81 to 117 wherein the cyclic compound having formula I has an antimicrobial effect against a microorganism selected from Listeria monocytogenes, E. coli, Staphylococcus aureus, Listeria innocua, Salmonella Typhimurium, Salmonella sp., Bacillus cereus, Bacillus subtilis, Saccharomyces cerevisiae, Saccharomyces cerevisiae var. paradoxus, Saccharomyces carlsbergensis, Pseudomonas fluorescens, Clostridium sporogenes, Lactobacillus sake, Brochothrix thermosphacta, Micrococcus 51 luteus, Yersinia enterocolitica, Enterobacter aerogenes and Zygosaccharomyces bailii.

119. Use according to any one of claims 81 to 118 wherein the cyclic compound having formula I has an antimicrobial effect against a micro-organism selected from Listeria 5 monocytogenes, E. coli, Bacillus cereus, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Pseudomonas fluorescens, Clostridium sporogenes, Lactobacillus sake, Brochothrix thermosphacta and Micrococcus luteus.

120. Use according to any one of claims 81 to 119 wherein said compound of formula I is 10 used in combination with one or more of an antioxidant, a preservative and/or a chelator.

121. An antimicrobial composition as claimed in claim 1 substantially as herein described with reference to any Example thereof.

122. A process as claimed in claim 41 substantially as herein described with reference to any Example thereof.

123. A use as claimed in claim 81 substantially as herein described with reference to any Example thereof.