However, the invention may have application outside this field.

BACKGROUND ART There are a number of preparations and compositions used in the preservation of foods or in pharmaceuticals, as anti-microbial compositions. Such preparations and compositions may be derived from other living organisms (such as existing natural plant remedies, and antibiotics derived from the genus Penicillium for example), or may be synthetically produced.

The function of such preparations and compositions is to combat both the microbes themselves, and the detrimental consequences of their microbial activity. In the food industry, such consequences include the short shelf-life of many foods because of food spoilage, and food poisoning in humans and animals eating foodstuffs containing pathogenic microbes, and so forth.

Food poisoning occurs as a result of the effects of microbial toxins on a person or animal consuming food containing pathogenic bacteria. Food spoilage occurs as a result of the effects of microbial damage to food. Unfortunately, microbial toxins are not necessarily destroyed by heat to which food may be exposed during cooking, reheating and so forth.

A number of bactericidal or food preservative compositions have been developed for application to the surface of a foodstuff to kill or inhibit growth of pathogenic bacteria.

Some such compositions have been derived from bacteria themselves, or are a synthetic equivalent, such as those disclosed in New Zealand Patent Specification Nos. 232512,244737 and 236730.

Yet other compositions include anti-oxidant materials extracted from vegetable material including herbs (such as rosemary or sage as disclosed in New Zealand Patent Specification Nos. 270064,226188, and 191609), or extracts from the leaves of a range of other plant genera (such as disclosed in New Zealand Patent Specification Nos. No. 239403,216525,210182,212305, and 257479).

Yet further compositions or preparations rely on other chemical compounds (as disclosed in New Zealand Patent Specification Nos. 177554,206260, and 236730).

Sodium metabisuphite is one such chemical used as a preservative. However, whilst useful, it is not effective against all microbes.

The problem typically involved in the preservation of foods, or the treatment of animals with anti-microbial pharmaceuticals, is that such preparations and compositions are required to be safe for consumption, whilst at the same time being effective anti-microbial compositions. In addition, cost is always a factor in the manufacture of such compositions.

However, emphasis is being increasingly placed on ensuring the negative effects of microbes in food are significantly reduced, if not eliminated. Some reasons for the changing emphasis include: a) different manufacturing practices;

b) legislative changes; and c) altered consumer expectations-specifically arising out of food poisoning incidences and/or related to food hygiene in general.

One or all of these changes impact on what foodstuffs contain, how they are processed, and so forth.

For example, in relation to meats, such as bacon, these changes have resulted in the fact that bacon as purchased ex-supermarket (at least in New Zealand) now has: a) a much higher water content (Aw values often > 95); b) a higher carbohydrate/sucrose content; c) a much lower residual nitrite level (the upper legal limit currently being 125ppm); d) a faster manufacture time (2 days is typical); and e) lower salt levels (of approximately 2%).

The current manufacturing practice of preparing a high free-water, low-salt (1.5%- 2.0% is usual), minimally heat-treated, liquid-smoke prepared product, along with an associated long-period (14 day) supermarket holding (at temperatures in the range 2- 10°C), results in a bacon which is a suitable substrate for not only halophilic and haloduric organisms, but most spoilage bacteria as well.

The minimum Aw for most spoilage bacteria is generally considered as 0.91.

However, the growth of Acinetobacter, Enterobacter aerogenes, Bacillus subtilis, Clostridium botulinum, Escherichia coli, and Pseudomonas, which have Aw values in the range together with Staphylococcus aureus (min. Aw 0.86) would not be inhibited at current bacon Aw levels. The water environment is also suitable

for encouraging and sustaining the population growth of yeasts and moulds.

Residual nitrite in bacon typically inhibits the growth of Clostridium species, but only at levels in excess of 200 ppm. The currently permitted maximum legal level of 125ppm residual nitrite is at the lower level of what is generally considered inhibitory. Other factors may contribute to Clostridium inhibition. Such factors include pH, salt level, oxygen availability, heat treatment, and Eh. However, these conditions in bacon permit Clostridium growth.

Further, the range of additives now permitted in bacon, because of legislative changes, includes carbohydrates, mono-and di-saccharides, and gums, which are not only supportive of microbial growth, but are attended by their own resident microbial populations.

The results of a study on organisms which may be found in retail bacons at any present time, have indicated that: a) The organisms potentially capable of growing in bacon have been detected growing there as contaminants; and b) once a bacterial type is growing in bacon, its growth continues to be supported.

Accordingly, a need has been identified to produce an anti-microbial composition that would be permitted to be added to bacon (as well as other foodstuffs), whilst at the same time complying with New Zealand (together with the now-associated Australian and ANZFA legislation), and Codex Alimentarius, USFDA Regulations, and other such legislative regulations related to foodstuffs.

It would be a further advantage if such a composition also had applications beyond the food industry, such as being capable of use in the pharmaceutical industry.

It would also be an advantage if such compositions were capable of substantially extending the shelf-life of foodstuffs.

Further, when using a composition or preparation as a food preservative or associated with pharmaceuticals, it would be an advantage if the composition was effective in killing a wide range of bacteria, fungi, and viruses, along with some protozoans of particular concern in the food and beverage industries. It would also be a further advantage if only a small quantity of the composition was required to achieve this effect.

In addition, particularly in the food and pharmaceutical industries, it is important that any composition or preparation does not impart undesirable flavour to the foodstuff being treated, or to pharmaceuticals required to be administered orally.

Further, where such compositions are to operate as an effective food preservative, such compositions need to be capable of being completely dispersed throughout the foodstuffs. This is particularly relevant in the curing of meat products where there is a need to distribute any preservative throughout the whole meat, including the fat barriers. In most situations however, this is enhanced by the use of a carrier substance.

It would also be a further advantage if the composition could be used with a range of foodstuffs (such as dog rolls, poultry feed, sauces, brines (for meats such as bacon, ham, corned beef and so forth), seafood (both processed and live), meat mixes, meat pies, casings for sausages, and in the treatment of preserved vegetables, and so forth.

A further advantage would include the ability to apply the composition via a range of different methods, such as being added and mixed into the foodstuffs directly, being sprayed onto the foodstuffs, having the foodstuffs immersed in a solution containing the preservative, and also in some cases enabling specific foodstuffs (such as

molluscs) to be treated in vivo with the composition.

Most existing compositions typically display some, but not necessarily all of these above mentioned advantageous features.

It is therefore an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice.

Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only.

DISCLOSURE OF INVENTION According to one aspect of the present invention there is provided an anti-microbial composition isolated from at least one member of an active spice group as herein defined.

The term"active spice group"shall mean and include, unless otherwise specified, members of the following defined group: a) Plant matter derived from the family of plants Myristicae-and in particular M. fragrans.

However, use of this term is not to be seen as limiting the scope of this specification.

According to another aspect of the present invention, there is provided an anti- microbial composition substantially as described above wherein the preferred members selected from the active spice group include nutmeg and mace.

According to another aspect of the present invention, there is provided an anti- microbial composition substantially as described above wherein said composition is isolated by a method wherein aromatic components are separated from non-aromatic components.

In this specification, the term"aromatic"shall mean and include components aromatic to the olfactory senses. However, use of this term is not to be seen as limiting.

According to another aspect of the present invention, there is provided an anti- microbial composition substantially as described above wherein the composition is substantially odourless and/or neutral tasting.

According to another aspect of the present invention there is provided a method of preparing an anti-microbial composition substantially as described above wherein aromatic components are separated from non-aromatic components.

According to another aspect of the present invention there is provided a method of preparing an anti-microbial preparation using an anti-microbial composition substantially as described above and including an additional step of: a) combining said composition with a carrier suitable for the intended use of the composition and/or preparation.

In the present invention carriers may include liquid carriers and solid carriers in various forms (including polysaccharides, gums, salts, other sugars, sodium lactate, and so forth).

According to another aspect of the present invention there is provided a method of preserving foodstuffs comprising the use of an anti-microbial composition and/or preparation substantially as described above.

According to another aspect of the present invention there is provided a method of preserving foodstuffs using an anti-microbial composition and/or preparation substantially as described above including the optional step of adding either or both an additional spice group and a herb to the composition or the preparation.

The term"additional spice group"shall mean and include members of the following defined groups: a) Plant matter derived from plants of the family Piperaceae-in particular P. nigrum. b) Plant matter derived from Eugenia caryophyllata and/or Carvophvllis aromatica. c) Plant matter derived from Zingiberaceae-in particular Z. officinale. d) Plant matter derived from Capsicum frutescens.

According to another aspect of the present invention there is provided a method of using an anti-microbial composition and/or preparation substantially as described above for microbial control.

According to another aspect of the present invention there is provided a method of using an anti-microbial composition and/or preparation substantially as described above for at least one of bacterial, fungal, viral and protozoan control.

The term microbe shall, for the purposes of this specification, mean and include bacteria, viruses, microscopic fungi and protozoans. However, use of this term should not be seen as limiting this specification.

A number of spices have been tested for their anti-microbial properties by the applicant. These include, nutmeg, mace, cloves, ginger, pepper and capsicum.

Extracts from all of these display anti-microbial activity. However, nutmeg is the preferred spice as the anti-microbial composition derived from it has been found to be most effective at killing a range of microbes commonly associated with foodstuffs (the results of the tests are provided in Table 1).

However, in other embodiments of the present invention, combinations of spice oils derived from the range of spices indicated above may also be used. Table 2 illustrates the results of trials demonstrating the synergistic effect of various combinations of spice oils and the effects of nutmeg alone, on a range of microbes commonly associated with food. While the combinations tested preferably included a concentration of the nutmeg extract at least equal to the concentration of the additional spice oil, the concentrations of the spice oils in the combinations may vary.

The seed kernel and arillus of nutmeg both possess a strong aromatic odour and taste, due to the presence of volatile and fixed oils. Processing nutmegs is known to yield from 8 to 15% of volatile oil. Twenty-five percent to 30% or more of expressed oil is also present in nutmeg. This is commonly known as'oil of mace'.

The volatile oils which nutmeg contains have in the past been used as a remedial composition, and have considerable stimulating properties. The spice taken in small quantities is also known historically to, or is purported to, assist digestion, dispel flatulency, strengthen the viscera, and stop dysentery.

The (volatile) oil has typically only been used in medicine for flavouring or covering the taste of other medicines, and is prepared by dissolving the volatile oil of nutmeg in strong alcohol to achieve that purpose. The volatile and fixed oils have also been used in chronic rheumatism, and in the preparation of lotions for the hair. The chief use, however, of nutmeg and mace is as condiments.

In preferred embodiments of the present invention oleoresin nutmeg is used to obtain an anti-microbial composition for both preserving foods and for use as a pharmaceutical product. Oleoresin nutmeg is preferred for its ease of use and availability. Further, it is easier to isolate and concentrate the anti-microbial composition from the oleoresin nutmeg, thereby increasing the efficiency of the

process. However, the ground spice or the oils may also be used, and also provide effective anti-microbial isolates. In addition, other plant matter derived from nutmeg may be used.

In tests conducted to determine the effectiveness of the potential anti-microbial compositions in nutmeg, the oil/oleoresin preparations were found to be the most effective. Results of tests relating to same are provided in table 3.

In preferred embodiments, the quantity of the anti-microbial composition used as a foodstuff preservative is generally determined by the final weight of the foodstuff being treated. Typically, this will be between. 006%-. 008% of the final weight of the foodstuff. However, concentrations outside this range may also be used in some applications. For example, lower levels have been found to be useful as bacteriostatic compositions, while levels higher than 0.05% have been effective in cleaning applications.

Other factors will also affect how much anti-microbial composition is applied to the foodstuff. For example, the type of foodstuff being preserved or treated will dictate the concentration of anti-microbial composition used. Where the composition is applied to a curing mixture or brine for curing meats, the seepage of fluids from the meat may dictate that a greater concentration of the composition be applied (probably in the range of. 008% of the final weight of the cured meat). However, the final amount of composition used is such that it preferably does not impart any flavour to the cured meat.

Bacon is one example of a popular, commonly consumed cured meat. However, a range of pathogenic bacteria can be associated with this food type. Table 4 indicates a number of types of Salmonellae occurring in samples of bacon, and the likely origin of the bacteria. The effectiveness of nutmeg (particularly oleoresin nutmeg) as an anti-microbial composition is illustrated with particular reference to the treatment

of bacon, in Tables 5,6,7,8,9 and 10. The effectiveness as an anti-microbial composition is illustrated with particular reference to chicken and fish in Tables 17 and 18.

The results show that nutmeg is an effective anti-microbial composition with a broad spectrum of activity against both Gram positive and Gram negative organisms.

The oleoresin preparation was preferably used as it is readily available, easily transported, and contains higher concentrations of the antimicrobial compounds.

However, raw plant material such as the green wood, bark, sap and leaves of the nutmeg tree (Myristica fragrans) are to be examined as potential sources of anti- microbial compositions.

The results obtained from pre-processed materials such as oleoresin nutmeg and nutmeg oils are also generally applicable to other pre-processed materials, such as ground nutmeg and other preparations thereof. Extraction of the ground spice using appropriate solvents (such as Bloor's or Folch solvents) yielded the same anti- microbial composition.

The effectiveness of oleoresin nutmeg as an antibacterial composition was <BR> <BR> determined and measured by in vivo LIDO comparisons with pertinent antibiotics (such as penicillin G against gram +vc; Tetracycline T against gram-vc; Bacitracin against Micrococci; and Cephalosporin against Clostridia). The results indicate an effectiveness within the range 47-52% of that of the comparable antibiotic.

Where the composition is applied to live shellfish, such as mussels, the quantity is preferably reduced, as lower concentrations have been found to be effective. This in part is also dependent on the way the composition is applied. For example, where the composition is applied directly to the shellfish, lower amounts of the composition may be used, as compared with the amount of the composition if added to the water

medium in which the shellfish are living. Where the composition is added to the animal's environment (rather than applied directly to the animal), the composition typically concentrates in the animal over time, due to the animal cycling the water through its system (particularly when feeding).

Table 11 illustrates the results of tests conducted to determine the effect of nutmeg oil on molluscan microflora using mussels as the test animal. However, the effects of the anti-microbial treatment has been demonstrated to extend to a range of other molluscan species.

The antimicobial composition extracted from nutmeg is preferably used to combat a range of bacteria and fungi, along with some viruses and protozoans. The organisms against which the anti-microbial composition has been demonstrated to be effective are listed in Tables 12 and 13. However, other organisms may also be susceptible to the composition.

It is an advantage that the composition has demonstrated its effectiveness in killing a wide range of bacteria, fungi, and viruses, along with some protozoans. Accordingly, its versatility may reduce the need to rely on a range of different substances as food preservatives or associated with anti-microbial pharmaceuticals. It is a further advantage when only a small quantity of the composition is required to achieve this effect, thereby making it a substantially cost effective product.

In preferred embodiments of the present invention, the anti-microbial composition may be applied to a foodstuff (or be administered) in a number of ways.

Its incorporation into a brine into which a foodstuff is immersed, has already been stated with reference to the curing of meats (Tables 7 and 8). The ability to apply the composition directly into live animals, or the cycling of the composition in the animal's environment has also been discussed (refer also to Table 11).

In addition, the other most commonly preferred methods of application include the addition of the composition into a foodstuff mixture, and the spray application onto the foodstuff (Table 14 details the effectiveness of nutmeg oil in spray trials).

However, other application methods may be employed as required, and depending on the foodstuff to be treated.

Where the anti-microbial composition is in a dry form and is to be used in brines/cures, the composition is preferably used in conjunction with a carrier substance. The carrier substance improves the dispersion of the composition through the brine/cure, and enables the composition to remain in suspension. When the composition is in an emulsion form, there is less of a need to use a carrier, as the composition may be mixed into or agitated with the brine to obtain the required dispersion.

Typically, the carrier preferably includes at least one of a gum, a sugar, and a salt.

The sugar may be used to improve the effectiveness of the brine (it is known that cures/brines work better with carbohydrates present in the cure). Alternately, sugar may be used if there is already a high salt content, and using salt as the carrier would make the cured foodstuff too salty for consumers.

While the use of gums, sugars and salt as carriers is a known practice, the composition may be used with any other suitable carrier, or with a combination of carriers, as required to achieve the desired application of the anti-microbial composition on to or into the food being treated. In addition the type of carrier used will be dependent on the foodstuff being treated.

The ability to completely disperse the composition/preparation throughout the foodstuffs being treated contributes to the composition's effectiveness as a food preservative. This is particularly relevant in the curing of meat products where there is a need to distribute any preservative throughout the whole meat, including the fat

barriers. The ability to use the composition with a range of carrier substances, is also an advantage.

Nutmeg oil is typically steam distilled from dried kernels of ripe seeds of nutmeg.

The oil is known to consist of 60-80% d-Camphene, about 8% d-pinne; dipentene, d-borneol, l-terpineol, about 6% geraniol and safrol, and about 4% myristicin, varying according to the source (geographic origin) of the nutmeg.

The anti-microbial components of nutmeg oil (prepared as earlier described, or obtained as an oil extract), are preferably isolated and concentrated by standard filtration techniques, using a standard peristaltic pump (although any pumping means, including gravity separation, may be employed) in conjunction with an eluting solvent. In preferred embodiments the eluting solvent includes a solution of water: methanol: pyridine in proportions of 3: 3: 1 (v: v: v). Another preferred solvent includes water/salt mixtures. However, any suitable eluting solution may be used (some of which have been referenced earlier).

On the otherhand, the anti-microbial compositions of nutmeg oleoresin (purchased in the oleoresin form, or extracted using a solution of water : ethanol in proportions of 24: 1 (v: v)) are preferably obtained through the fractional distillation of oleoresin at a constant temperature of 39° C (maintained via a water bath or other suitable means) and at a pressure of lOkPa.

While the above methods are preferred for preparing, isolating and/or concentrating the anti-microbial compositions from raw plant material, and subsequently from nutmeg oil and nutmeg oleoresin, any suitable processes for the extraction of the anti-microbial compositions may be employed.

Other suitable processes for the extraction of the anti-microbial compositions also include chromatographic separations, (including high performance liquid

chromatography, HPLC; thin layer chromatography, TLC; and gas liquid chromatography, GLC) have also been successful.

BEST MODES FOR CARRYING OUT THE INVENTION Further aspects of the present invention will become apparent from the following description which is given by way of example only.

EXAMPLE 1 Nutmeg oil is preferably separated into its anti-microbial components by standard filtration techniques. Preferably the components are isolated via gel filtration, using a Sephadex G-15 column, in conjunction with an eluting solvent.

In preferred embodiments the eluting solvent includes a solution of water: methanol: pyridine in proportions of 3: 3: 1, (v: v: v). However, any suitable eluting solution may be used.

Samples (0.5 mL) of nutmeg oil are preferably injected onto the filtration column and separated by pumping at 50mL/hr using a standard peristaltic pump (although any suitable pumping means, including gravity separation, may be employed) The components of the anti-microbial composition present in 3mL aliquots of the gel filtration fractions can be detected as emerging peaks by absorbance at 180,200,220 nm, and identified by IR (solvent and mull) absorbance spectra. The identified nutmeg oil components are provided in Table 15.

The results of synergistic and complementary antibacterial activities assayed using plate cultures and by mixing 0.5mL amounts of the fractions (obtained from the gel filtration) in all possible permutations are presented in Tables 16 and 17.

EXAMPLE 2 Nutmeg oleoresin is preferably extracted using a solution of water: ethanol in proportions of 24: 1 (v: v).

It was found to be unrealistic to fractionally distil nutmeg oleoresin by the usual procedures, but a partial concentration of the antibacterial components was achieved by distilling oleoresin at a constant temperature of 39° C (water bath) and pressure of 1 OkPa.

The distillate was found to possess only the major aroma components, and had no antibacterial activity. On the otherhand, the concentrated residue was 18% higher than the original oleoresin in antibacterial activity.

The lack of any unpleasant odour or taste is an advantage particularly in the food and pharmaceutical industries, where it is preferable that any composition or preparation be a neutral falvour so as not to impart undesirable qualities to the foodstuff being treated, or to pharmaceuticals required to be administered orally.

The antibacterial activities of the residue and extract were determined (as for the gel filtration-separated components), against E. coli, P. aeruginosa, and S. aureus. The results are provided in Table 17.

Also included in Table 17 are the results from more extensive antibacterial profiles on test bacteria using: a) the water-extract alone; b) the water-extract residue; c) isolated gel filtration fractions which screened positive for antibacterial effect;

d) the pooled fractions of (c); and e) pooled gel filtration fractions in permutations of three.

EXAMPLE 3 Results of tests with the anti-microbial composition suggest one advantage will be its capability to extend the shelf-life of foodstuffs beyond that currently available.

It is also an advantage that the composition is capable of being used with a range of foodstuffs. Some examples include dog rolls, poultry feed, sauces, brines (for meats such as bacon, ham, corned beef and so forth), seafood (both processed and live), raw chicken, raw fish, meat mixes, meat pies, casings for sausages, and in the treatment of preserved vegetables, and so forth. Use of the composition therefore obviates the need to source and use different anti-microbial compositions as previously required for different food types.

There are a number of recipes available for curing meats, or as brines for vegetables and so forth. While some recipes may incorporate nutmeg as a flavouring, the use of nutmeg extracts in concentrations which are effective as anti-microbial compositions for preserving food, while at the same time being both flavourless and odourless is not known.

Typically different foods have different properties. Therefore, it is preferable to accommodate such differences when applying the anti-microbial composition (s), to ensure effective penetration of the anti-microbial composition, and therefore ensure the food is preserved as required.

Accordingly, different food types require different carriers to ensure optimum penetration of the anti-microbial composition.

The following preferred combinations are provided by way of example only, and it should be appreciated that other combinations may also be used. a) Bacon, Ham Manufactured and Processed Hams Oleoresin nutmeg Carrageenan gum (0.0066% w: w finished product) Locust gum (0.0033% w: w finished product) Dextrose b) Corned Beef Oleoresin nutmeg Carrageenan gum (below 0.008% w: w finished product) Dextrose c) Sausages and Hamburger Mixes, Meat Pies Oleoresin nutmeg Guar gum (0.01% w: w finished product) d) Sausage Casings Oleoresin nutmeg Phosphates Guar gum (below 0.008% w: w finished product) e) Fish, including Molluscs Oleoresin nutmeg Xanthan gum (0.008% w: w finished product)

f) Vegetables Oleoresin nutmeg Xanthan gum g) Mincemeat Labelled as spiced beef/mutton/pork/lamb as appropriate.

Oleoresin nutmeg Guar gum (less than 0.008% w: w finished product) All of the above examples may be applied to different carriers-for example, sugars, salts, phosphates and so forth.

The versatility of the composition is further illustrated by the ability to apply the composition to foodstuffs via a range of different methods. Such methods include, adding and mixing the composition and/or preparation into the foodstuffs directly, spraying it onto the foodstuffs, having the foodstuffs immersed in a solution containing the composition or preparation, and also in some cases enabling specific foodstuffs (such as molluscs) to be treated in vivo with the composition.

EXAMPLE 4 An advantage of the anti-microbial composition is that it also has applications beyond the food industry, such as being capable of use in the pharmaceutical industry.

Anti-microbial efficacies were determined in vitro by disk/plate assay and broth culture growth. While this technique comprises only the first stage in pharmaceutical assessment procedures, the results are clinically of significance with the following microbes particularly:

Bacteria Bacteroides fragilis: Nosocomial infections; mouth ulcers/diseases.

Bacillus cereus: Food poisoning; resident in cereals (used in some cures).

Brucella abortus: Brucellosis. Relevant to butchery.

Bacillus anthracis: Anthrax (tested against non- pathogenic strain). Relevant to butchery.

Leptospira interrogane: Letospirosis. Relevant to butchery.

Haemophilus influenzae: Bacterial meningitis. Most common (6 weeks-2 years).

Neisseria meningitidis: Effective against this.

Borditello pertussis: Whooping cough (Pertussis).

Corynebacterium: Includes C. diphtheriae (diphtheria).

Streptococcus: Includes S. pneumonie (Pneumococcal pneumonia).

Listeria: Includes L. monocytoWgenes (Listeriosis).

Legionella: Legionnaires disease.

Clostridia

Salmonella (includes S tvphimurium) Escherichia (includes E. coli) The most serious (and most Staphylococcus (includes S. aureus) common) organisms Shigella sonnai responsible for food Enterobacter poisoning Proteus Klebsiella S. typhi: Typhoid fever Shigella sonnai: Shigellosis Campylobacter jejeuni: Campylobacter infection.

Citrobacter: Nosocomial infection.

Neisseria gonerrhoeae: Gonerrhoeae.

Yersinia enterocolitica: Similar to Y. pestis (plague).

Vibrio: Cholera, gastroenteritis.

Viruses Only a cursory evaluation was carried out. The following virus types were destroyed. Clinical significance is indicated.

Virus Type Clinical Significance Icosatedral nucleocapsid (s. s. RNA) Poliomyelitis (all 3 serotypes)

Icosatedral nucleocapsid (s. s. RNA) HAV (Hepatits A virus) Icosatedral nucleocapsid (s. s. RNA) Type E Hepatitis (HEV) Helical symmetry Mumps Myovirus Family Myoviridae and resident Coliphage T3 in bacteria as bacteriophage.

Particularly E. coli, and of interest in pathogenicity of E. coli.

[Also tested, and found to be destroyed, were S. aureus that were methicillin resistant. These are generally termed Methicillin Resistant S. aureus (MRSA) and are a significant clinical problem.] Protozoans: Amoeboid: Giardia lamblia (Giordiosis).

Paramecia: Intestinal.

Fungi. Yeasts and Moulds Tinea: Ringworm.

Athlete's Foot (Tinea pedis).

Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope thereof as defined in the appended claims.

TABLE OFPOTENTIALOLLANTI-BACTERIALSSCREENING BACTERIAOIL Percent kill at weightby NutmegPepperCapetcumGingerMace 6950403122Acinetobacter74 6253402610Acromonas81 575037225Alcaligenes88 896147392710Bacillussubtilis Bacilius604740271089 636140295Chromobacterium91 646040295Crtrobacter79 5310039305Clostridium94 546750245Corynebacterium81 838047240Enterobacter82 868450265Escherichia87 626042284Flavobacterium88 695744967Lactobacillus89 715241905Leuconostoc90 7050472410Microbacterium91 644146205Micrococcus90 7240452710Pediococcus90 804360250Proteus92 954270205Pscudomonas93 724144215Salmonella94 70464405Serratia90 91844350Staphylococcus87 949130105Steptococcus89 818830245Vibrio82 Antibacterialactivityofsalectedspicaells,measuredfortwenty-f ourhours,Table1: TABLE 2: SYNERGISTIC EFFECTS OF SPICE OILS Spices Combination Percent of Viable Organisms Surviving E.coliS.aureusCl.perSal.ent[%massconc.] Nutmeg: Pepper 22 25 5 10 0.002,0.002 Nutmeg: Pepper 10 11 2 5 0.003,0.003 Nutmeg: Cloves 24 29 6 11 0.002,0.002 Nutmeg: Mace 26 20 3 12 0.002,0.002 Nutmeg: Capsicum 10 6 4 10 0.002,0.002 Nutmeg, 0.002 26 37 6 12 Nutmeg, 003 10 15 2 6 Nutmeg, 0. 004 4 11 0. 5 2 Table 2 : The effectsofvariousconbinationsantibacterial of spice effectsThe of nutmeg alone, at three concentration levels, shown,asacomparisonarealso asbefore.Assayprocedure TABLESCREENINGOFNUTMEGOLLS: BACTERIA OIL SOURCE Percent kill at 0.004% by weight A C 6970Acinetobacter62 8080Acromonas74 8285Alcaligines70 658888Bacillussubtilis Bacillus 888868 Bacterotdes fragilis NT NT 83 NTNT74Brucellsabortus 8990Chromobacterium78 7778Citrobacter69 Clostridium889192 aium 68 80 80 7980terobacter64 Es 88 87 8686Flavobacterium84 NTNT79Haemophilusinfluenzae LActobacillus st 87 88 NT NT 83 8890Leuconostoc84 'uia 53 80 81 tctobactewm S6 86 89 crococcus 62 88 Neisseria ganocown NT NT 81 Pedioooccus 69 84 88 Proteus 79 90 91 Pseudomonas 78 92 92 9193Salmonella84 8889Serratia77 NT NT 84 8887Staphylococcus70 8888Streptococcus70 Vibrio 64 81 82 NTNT86Yersiniaenterocolitica :Effectivencesofthreenutmegoils,(A,LionelHitchenEssentialOil ,Table3 EastIndian;B,LionelHitochenSOrNutmegHX2046;NutmegOil, C,andCo,OleoresinNutmeg).MessuredagainstselectedHall bactaia. NT- not tested.

TABLE 4 : INSURVEYSAMPLESIDENTIFIED OriginaimonellaFrequency Type % Total AnimalFeed.anatum10 S.choleraesius 80 Animal S.enteritidis 60 Animal S.hirschfddii 10 Animal S. paratyphi A 5 Human S. paratyphi Human5 S.Human10 S.Animla?Human?10 @ Table 4 : The likely origins of the samonellae ssp most commonly found insamples.Thefrequencycolumnindicatesthesurvey inn=20samplespercentageoccurence TABLE 6: MICROBES IDENTIFIED IN BACONS <BR> <BR> <BR> <BR> <BR> <BR> BACON<BR> Source Description Bacterial types: CFUs<BR> <BR> <BR> <BR> ClostridieStreptococcaeE.coliTPCSalmonellae <BR> /gm /10g/50g /10g /10g<BR> <BR> 800.00040Pr1,000201pieces <BR> <BR> <BR> 100,0001shoulder 1,00012Pr <BR> <BR> <BR> <BR> <BR> <BR> <BR> 100,0001middle 30 Pr 30<BR> 2 middle 110 ND ND 40 ND<BR> <BR> 3 shoulder 640,000 5 Pr 620 12<BR> <BR> 5,30010ND450ND4shoulder <BR> <BR> @@ 460 ND<BR> <BR> <BR> <BR> <BR> <BR> <BR> 4 shoulder 5,300 10 ND 450 ND<BR> <BR> 4 middle 5,100 10 ND 400 @@<BR> <BR> <BR> <BR> 5 shoulder 42,000 20 Pr 700 5<BR> 5 middle 47,000 20 Pr 5<BR> <BR> <BR> <BR> 6 shoulder 600,000 10 Pr 810 10<BR> <BR> <BR> <BR> 6 middle 700,000 5 Pr 840 10<BR> 7 shoulder 4,500 10 Pr 550 10<BR> <BR> <BR> <BR> 7 middle 4,500 10 Pr 500 10<BR> @ 650 5<BR> 8 shoulder 57,000 NDND"Os<BR> 8 middle 52,000 ND ND 650 5 :Bacterialtypesandnumbersidentifiedinbaconspreparedatvarious marufacturingplantslocatedthroughoutNewZealandandTable6 aucldandSupermarketspurclasedin Colonyformingunit;TPC,Totalplatecount;Pr,Present;ND,notdetec tedattheindicatedlevel.AbbreviauonsCFU, TABLE 6:6: GROWTH OF MICROBES IN BACONS BACON MICRO-ORGANISMS Percent C. F. U.

Aw Salt E. coli Cl. botulinum S. aureus. St. faecalis S. typhimuriuffi 98 2.1 2.105 1.104 1.10' 1.104 5.104 97 2.0 2.1052.105 4.10' 1.104 1.104 3. 104 96 2.4 1.1051.105 4.10' 6.10' 4.10' 1.104 91 2.7 4.104 3.10' 5.10' 3.10' 4.104 90 2.8 4.10'4.10' 2.10' 4.10'4.10' 2.10' 3.104 88 2.9 6.101 1.10' 3.10'3.10' 2.10' 2. 10' Table 6: Growth of selected organisms in typical bacon types available ex Supermarket.

Samples of each bacon were inoculated with 1 ml of a single culture containing 100 CFU, prior to incubation as described in the text.

Abbreviations: CFU, Colony forming unit. Aw, available water Salt, sodium chloride, E. coli, Escherichia coli: Cl botulin Clostridium botulinum: S.aureus, Staphylococcus aureus; St. faecalis Streptococcus faecalis: S. typhimurion. Salmonella tryphimurium.

TABLE 7: MICROBES IN NUTMEG-CURED BACON Organism BACTERIAL NUMBERS (CFU/gm) Raw Meat Nutmeg Cure Control Cure -A B C A B C Clostridium 500 0 0 25 36 40 Coliforms 580 106 0 0 410 400 500 Enterobacter 620 127 15 0 550 660 800 Klebsiella 135 55 10 0 120 140 150 Lactobacillus 740 180 19 0 590 600 800 Salmonella 70 10 0 0 70 85 95 Staphylococcus. 210 40 0 0 160 170 210 Streptococcus 640 137 20 0 200 210 230 Table 7: Bacterial populations in the carcass, (Raw Meat) and two sides of a single cured pig. One side of the pig was treated with a cure including nutmeg (Nutmeg Cure); the other side treated with an identical cure, except with no nutmeg (Control Cure). Nutmeg was added to the former cure to give a final level of 0.002% by weight in the finished bacon.

The columns A, B, C, refer to the samples taken at the following times during manufacture: A immediately post-pumping, 4 hours after commencing manufacture; B. immediately post-smoking, 48 hours after manufacture commence; C. after holding for a further 24 hours, that is, 72 hours after manufacture commenced.

TABLE 8: MICROBES IN NUTMEG-CURED BACON - CONTINUED Elapsed time-0 hrs 22 hrs 68 hrs 4 days7 days10 days|15 days Manufacture Stage-Raw meat Pump-OfN Sliced Sale 4°C 4°C 4°C BACTERIA 3, 000 1,300 510 ND ND ND ND A2, (2, 900) (3,400) (3,900) (4,500) (6,000) (7,100) BaciDi/LactobaåDi 30 000 8,000 6,000 2,000 750 ND ND (30,000) (31,000) (33,000) (50,000) (100,000) (200,000) Clostridia 57 21 ND ND ND ND ND 57 (48) Enterobacter 2, 000 300 ND ND ND ND ND Enterobacter 2,000 300 ND ND ND ND ND (2, 500) (5, 000) (5, 500) (7, 000) (9, 000) (11, 000) Escherichia 1, 000 ND ND ND ND ND ND _ (2, 600) (3,500) (4,100) (4,000) (4,000) (3,700) Pseudomonas 10, 000 5,400 860 50 ND ND ND (11,000) (13, 000) (15,000) (17,000) (2Q000) 26, 000 ? Salmonellae (110) 100 ND ND ND ND ND ND Total Plate Court 58,000 16, 000 9,000 3, 000 1,000 200 ND (49,000) (53,000) (63,000) (84,000) (161,000) (257, 000) Table8: Bacterial populadons in the carcass (Raw Meat) and two sides of a sinble cured pig during manufacture. One side of the pig was treated with a cure containing nutmeg (initial nutmeg level 0.0055% ww, reducing through drip loss to 0.00275% w w in the final bacon.) The other side of the pig was treated with a cure omitting nutmeg but otherwise identical. Data in bold type refers to bacterial populations in the nutmeg-cured side. The bracketed standard-type figures beneath refer to standard-cure populations (both as manufacture details with total elapsed times were; pump, 4hrs; left overnight, 22hrs; smoke, 26hrs; freezer, 52hrs; slice, pack, 68hrs; then hold for sale. ND = Not detected. TABUS 9 : INHIBITORY EFFECT OF OLEORESIN NUTMEG [Nutmeg] ViableOrganismsSurvivingof % E. Coli S. aurcus Cl. per. Sal. ent. St. faecalis 100100100100100 0.001 56 50 30 43 42 0.002 26 37 6 12 2 0. 003 10 15 2 6 1 0. 004 4 11 1 2 0 5010.0052 0.006 0 2 0 0 0 0.007 0 0 0 0 0 0.008 0 0 0 0 0 0000.0090 0000.0100 Table 9: The inhibitory effect of Oleoresin nutmeg on the growth of some clinicalsignificance.Thenutmegconcentrationismicro-organisms of shown as a percentage of the mass of the growth numberofThe CFUs 24hrincubationwascomparedwiththeCFUsafter detected on the uninhibited niedium in order to determine the percentage of organisms surviving. Death rate curves are shown elsewhere. From these data the M.L.C (Minimum Inhibitory Concentration) and hence the use rate for oleoresin nutmeg was set at 0.005% (by weight) of the finished product.

TABLE 10: Ru-T RATES AT VARIOUS NUTMEG LEVELS @ N/N, Viable Organieme Time (hours) Table 10: Fraction (NIN.) of initial number (N@) of organisms remaining after treatment with: 0. 01% ("-") ; 0. 004% (o-o); 0. 003% (x-x) ; and 0.002% (@@@) Thecurve(@@@)wasuntreated.theinitialpopulation(N@)wasnutmego il. <BR> <P> 10'organisms/mL.

TABLE 11: EFFECT OF NUTMEG OIL ON MOLLUSCAN MICROFLORA Organism BACTERIAL NUMBERS (CFU's/gm) ImmersionCycling1Cycling2RawSpray 0.004%0.004%0.004%-0.004% Bacillus 65S 45 20 10 C 5000lostridium19 Enterobacter 60 10 0 0 0 0050Escherichia110 avobacterium 820 48 20 10 0 3010100ctobacillus700 501500icrococcus740 Proteus 460 10 3 15 0 Pseudomonas 520 29 6 5 0 2718200Serratia540 Streptococcus 550 20 0 10 0 Table 11 : The microblal population (CFU's/gm tissue) of mussels, ex Auckland before(Raw)andafterthreeselectednutmegtreatments;FishShop, Spraying,Spraying,Immersion, and thecyclingtreatments,nutmegFor oil at final concentration 0.004% was theliveholdingthrough tank for, cycling 1,12 hours; cycling 2,40 hours.

The percentage use rates were calculated as: Spray and Immersion 0.004%w:wofmolluscmass.- 0.004%w:woftheamountofcirculatingwater.Cycling- TABLE 12: ORGANISMS AGAINST WHICH NUTMEG EXTRACTS WERE FOUND TO BE EFFECTIVE BACTERIA Acinetobacter Acromonas Alcaligenes Bacillus subtilis Bacillus cereus Bacteroidesfragilis Brucella abortus Chromobacterium Citrobacter Clostridium Corynebacterium Enterobacter Escherichia Haemophilus influenzae Lactpbaciltus Leptospirainterrogans Leuconostoe Usteria Microbacterium Microcoocus Neisseriagonorrhocac Pediococcus Proteus Pseudomonas Salmonella Serratia Shigella sonnei Staphylococcus Streptococcus Vibrio Yersiniaenterocolitica MOULDS, YEASTS & FUNGI Aspergillus Cephalosporium Cladosporium Mucor Rhizopus Mycoderma Saccharomyces Ascinycetes (Candida) <BR> <BR> <BR> <BR> <BR> <BR> <BR> PROTOZOANS<BR> <BR> <BR> <BR> Giardia Iamblia Trichomonas AMUSES Adenovirus ColiphageT8 Aeromonas myovirus TABLE 18: THE ANTI-MYCOTIC EFFECTS OF NUTMEG SpeciesINHIBITION 50% Reduction Death Time: Days Tune: Days Moulds: 8Alternaria5 4Aspergillus2 Botrytis ineffective ineffective Cephalosporium 3 5 Cladosporium 4 6 Fusarium 5 7 Mucor 3 4 4pus3 Yeasts: Candida 3 4 7ycoderma3 Rhodotorula 5- Saccharomyces 2 4 6orulopsis5 Table 13: Times (approximate) to achieve 50% and 100% kills of selected yeasts and moulds, by nutmegoilinculturegrowth.(w:W) TABLE 14: SPRAY EFFECTIVENESS OF NUTMEG OIL Numbers(CFU/10cm2)[NUTMEG]Micro-organism g/cm2 E. Coli S. aureus Cl. botulinum S. enteritidis No spray 75 75 90 81 Water only 75 75 86 80 0.001 12 32 13 15 0.002 10 5 2 11 0.003 153 0.004 1 2 0 3 0.005 011 0.010 000 Table 14: The effectiveness of nutmeg oil in spray application. Four sets of agar surfaces were each inoculated with aliquots of growth culture of one of the above organisms. Each set consisted of right separate surfaces, each of 200cm2. The resulting thirty-two surfaces were each sprayed as shown above, four being untreated, and four used as controls, being sprayed with water only. These results for surface spraying are similar to the inhibition trials described in Table 9 TABLE 16: IDENTIFICATION OF NUTMEG OIL COMPON Description of Nutmeg Oil Extract Aqueous Gel Oleoresin IUPAC NAME Present Fracs 4 Confirmed NH@-Gly-ala-pro-cys-gly-cys-his-glu- glu-COOH Present Fracn 5 Confirmed NH,-Gly-his-cys-pro-cys-gly-gly-COOH Present Confirmed2-NH2-4-0-L-D-glucopyranosyl-L-D-6 3-me-glucopyranosyl-D-mannopyranose. Present Fracn 10 Confirmed 5- (phenylimino)-2-pentanone Present Fracn 10 Tentative 5-(4-NH2-phenyl@mino)-2-pentanone Present Fracn 12 Confirmed 4,6-dihydroxy purine Present Fracn 24 Tentative Methyl guanine Present Fracn 2S Tentative Short chain fatty aåd Present Fracn 26 Confirmed 3-OH-pinane Present Fracn 26 Tentative Aldehyde Table 16: Identification of the chemicals contributing to the antibacterial activity of nutmeg oil and extracts thereof The extracts were prepared by the following methods: Aqueous; extract of oleoresin as described in text : Gel; oleoresin components isolated by gel filtration. See text, table 16, for details of fractionation method and the fractions obtained thereby.

Oleoresin; the residue from a commercial oleoresin distilled at lOkPa, 39°C.

TABLE 16: GEL FILTRATION OF NUTMEG COMPONENTS Sample Elution Volume Antibacterial Activity Solubility No. mL A B C Water Ethanol 010 ---@13 2 l6 ---319 ---422 HiHiHiSolSol525 6 28 Hi Hi tu Sol Sol 7 31 Med Med Med Sol Sol 8 34 Low Low Low Sol Sol 9 37--- 10 40 ---1143 --MedSol?1246 ---1349 1452 HiHiHiSolIns1555 HiHiHiSolIns1658 ---1761 18 64 - - - -1967 -2070 21 73- 22 76- ---2379 HiHiHiSolSol2482 HiHiHiSolSol2585 Med--SolSol2688 27 91 Med--Sol Sol ---2894 ---2997 ---30100 Table 16: Antibacterial activities of gel filtration fractions against: E. coli (A); P. aeruginosa, (B); S. aureus (C): Hi = high ; Med = medium; Low = low ; - = nil efficacy. Solubilities of such components: Sol = soluble; Ins = insoluble;? = slightly soluble. TABLEPROFILESOFSELECTEDANTI-BACTERIAL NUTMEGEXTRAITS BACTERIA EXTRACT SOURCE ; PERCENT KILL 1 2 3 4 5 E. coli 87% 10% 62% (24) 89% 90% (10) P. aeruginosa 92% 12% 74% (16) 93% 96% (10) S. sureus 87% 9% 70% (S) 89% 92% (4) Adnetobacter 71% 11% 60% (6) 74% 78% (10) Alcaligcnes80%13%59% (8) 82% 86% (4) Bacillus 87% 12% 61% (7) 89% 91% (4) 6%72%all76%85%(4)Brucella74% Citrobacter 78% 5% 77% (24) 79% 80% (4) 3%91%(15)92%94%(10)Clostridium91% 4%82%(24)83%83%Corynebacterium81% 4%81%all84%84%Enterobacter83% 2%85%(27)88%88%Flavobacterium87% 3%88%all90%90%Lactobacillus89% 2%85%(7)86%86%Leptospira83% 1%89%(8)89%89%Micrococcus88% 2%87%all90%90%Pediococcus89% Proteus 92% 3% 91% (24) 94% 94% Salmonella95%5%93%all96%96% 7%89%(12)90%90%Serratia90% 6%86%(26)90%90%Streptococcus88% 5%82%(35)87%87%Vibrio81% 5%81%all88%92%(4)Yersinia86% Table 17 : Antibacterial activities of 5 nutmeg extracts against selected bacteria.

The figures denote the percentage kill after twenty four hours; incubation at 30° C. The sources of the extracts (1-5) are described in the text. In column 3 the parenthetical figure denotes the individual filtration sample giving the greatest antimicrobial activity which is displayed alongside at left. Under extract source 5 are recorded the percentage kills by combinations of three gel filtration fractions. Only two fractions (4) and (10) (shown in parentheses) were additionally active to the fractions described in pooled fraction 4. Percentages in column 5 without adjacent parenthetical description are thus derived from fractions within those assayed in pooled group 4.

TABLE 18: EFFECTIVENESS ON CHICKEN SAMPLE 1 Uncooked frozen chicken, ex Supermarket TESTED FOR: Common pathogens, standard bacterial plate count, yeasts and moulds, Clostridium ssp.

SAMPLE 2 Treated uncooked frozen chicken, treated by immersion for 20 minutes Bacteria Sample 1 Sample 2 Cl7U per g. CFU Klebsiella ssp 400 ND/lOOg Klebsiella pneumoniae 200 ND/lOOg Enterobacter ND/lOOg Escherichia incl. E. coli 3,500 ND/100g E. coli >3,000 Salmonella 500 ND/lOOg Predominent: S >350 ND/lOOg typhimurium Shigella Campylobacter 350 ND/lOOg Staphylococcus 1,000 ND/lOg Predominent: S aureus >550 ND/10 Streptococcus 1550 Predominent: S 400 ND/10 pneumonie Clostridia 100 ND Identified: C perfringens. Absent C. botulinum Yeasts & Moulds 100 Standard Plate Count: 12,500 <500/g Predominent ssp Lactobacilli TABLE 19: EFFECTIVENESS ON FISH <BR> <BR> <BR> <BR> <BR> SAMPLE1<BR> <BR> One sample of fish, raw, gutted, fresh, chilled (4°C) ex Supermarket.

SAMPLE 2 One sample fish, as above, but treated by immersion for 20 minutes. Bacteria Sample 1 Sample 2 CFU per g. CFU per g >5,500120E.coli Salmonella ssp300Absent/lOOg 800Absent/100gKlebsiellaSSD 1,100Absen/100gCitrobacterssp S.ureus >6, 000 Absent/lOg Streoticiccus ssp 2, 000 >50/g Clostridiassp 50 Absent/lOOg Standard Plate Count >20, 000 200