AU2011213719A1 - Nanoemulsion compositions - Google Patents

Abstract

Abstract The present invention relates to nanoemulsion compositions comprising water, at least one edible oil and a modified starch emulsifier. The nanoemulsion compositions are formed by high energy dispersion and allow enhanced loading of bioactive compounds and combinations of compounds that are difficult to dissolve. Food preservative compositions and functional foods comprising the nanoemulsion composition are also described.

Description

Australian Patents Act 1990 - Regulation 3.2 ORIGINAL COMPLETE SPECIFICATION STANDARD PATENT Invention Title Nanoemulsion compositions The following statement is a full description of this invention, including the best method of performing it known to me/us: P/00/0 I I C I Al - la Field of the Invention The present invention relates to nanoemulsion compositions comprising water, an edible 5 oil and a modified starch emulsifier. The nanoemulsion compositions are formed by high energy dispersion and allow enhanced loading of bioactive compounds and combinations of compounds that are difficult to dissolve. The compositions are able to solubilise hydrophilic, amphiphilic and hydrophobic compounds. Food preservative compositions comprising the nanoemulsion and a functional food composition are also described 10 together with a method of preserving food. Background of the Invention Nanoemulsions have a unique potential to solubilise high amounts of hydrophilic or hydrophobic compounds and therefore increase bioavailability of such compounds. 15 Nanoemulsions are emulsion systems that have three main phases, water, oil and interface. Despite their potential, few food grade nanoemulsions have been developed for commercial use. The lack of development relating to food matrices and in part stability of food grade nanoemulsions is blamed on limited choices for food grade surfactants. low oil solubilisation capacity and stability of soluble compounds (Garti, Spernath, Aserin, & 20 Lutz, 2005). Food matrices are unique and multiphase in nature rendering them more complex. A suitable food grade nanoemulsion delivery system should be multipurpose, easy to use and flexible enough for different functions in food. Furthermore, best results may be obtained 25 with concentrated and uniformly distributed bioactive compounds. Food grade nanoemulsions reported in literature are often complex, involve many different solvent components and are too sensitive to environmental changes and therefore lack stability (Sagalowicz & Leser, 2010). These complex systems often have high concentrations of emulsifiers that must be declared as additives and as such do not get consumer approval 30 (Sagalowicz & Leser, 2010). Furthermore, high concentrations of emulsifier increases the cost of the food and may result in off-flavors (McClements, 2010). An ideal food grade -2 emulsifier must have legislative approval as well as being efficient at lower/food acceptable concentrations. Furthermore, some useful bioactives may only be solubilised in solvents that are 5 unacceptable for human consumption or could advantageously be avoided. For example. propolis, a natural medicine produced by honey bees, is solubilised in ethanol or other organic solvents. There is a need for food grade nanoemulsions that are able to solubilise a range of 10 bioactive ingredients, hydrophilic, amphiphilic and hydrophobic, and are stable, dilutable and consumer acceptable. Summary of the Invention The present invention is predicated in part on the discovery that stable nanoemulsions that 15 can dissolve or hold bioactive molecules in dispersion may be formed with edible oils. such as food grade plant and animal oils, and modified starches with surface active properties that enable them to act as emulsifiers. In a first aspect of the invention, there is provided a nanoemulsion composition comprising 20 water, at least one edible oil and a modified starch emulsifier; wherein the average particle size of the dispersed oil phase in the nanoemulsion is less than 500 ni and with the proviso that any emulsifier, other than the modified starch emulsifier, is present in an amount of less than 3% w/w of the nanoemulsion composition. 25 In some embodiments, the at least one edible oil is a vegetable or nut oil such as canola oil. peanut oil, safflower oil, soybean oil or olive oil. In some embodiments, the modified starch emulsifier is an alkali metal C 5

-C

12 alkenyl succinate modified starch, such as starch sodium-6-octenylsuccinate. In some embodiments, the oil is present in an amount in the range of 5 % to 30 % v/v, especially 5 % to 20 % v/v of the composition. In some 30 embodiments. the modified starch emulsifier is present in an amount in the range of 2.5 % to 10 % w/v, especially 3 % to 7.5 % w/v, most especially about 5 % w/v of the composition.

In some embodiments, the nanoemulsion composition further comprises a bioactive compound, especially a bioactive compound that is difficult to solubilise in ingestible solvents, such as pharmaceutically acceptable carriers or food compositions. 5 In some embodiments, the bioactive compound is a pharmaceutical or nutraceutical compound, for example, propolis. In another aspect of the invention, there is provided a food preservative composition 10 comprising the nanoemulsion of the invention and a bioactive food preserving compound. In some embodiments, the food preserving compound is an antioxidant or antibacterial phytochemical such as gallic acid, tocopherols, ascorbic acid, ascorbic esters, vanillic acid, vanillin, quercetin, catechin, caffeic acid, rutin and curcumin. In some embodiments, the 15 preservative composition is stable to dilution enabling concentrated compositions to be made. In a further aspect of the invention, there is provided a method of preserving food comprising exposing the food to a food preserving composition according to the invention. 20 In some embodiments, the food is a perishable fresh food, such as fish, meat, fruit and vegetables, especially fish. In some embodiments, the exposing is achieved by dipping the food in the preservative composition or by spraying the preservative composition on the food. 25 In another aspect of the invention, there is provided a functional food comprising the nanoemulsion composition of the invention and at least one vitamin, mineral, prebiotic, antioxidant, plant sterol or plant extract. 30 Description of the Invention The singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise.

-4 As used herein, the term "about" refers to a quantity, level, value, dimension, size or amount that varies by as much as 20%, 15%, 10% or 5% to a reference quantity, level, value, dimension, size or amount. 5 Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. 10 As used herein the term "nanoemulsion" refers to a mixture of oil, water and emulsifier in which one of the oil and water forms a dispersed phase and the other forms a carrier phase and the particle size of the dispersed phase ranges from 50 nrn to less than 500 nm. especially 100 to less than 500 nm, 100 to 300 nm or 140 nm to 280 nm. Nanoemulsions 15 are also referred to as mini-emulsions, fine-dispersed emulsions, sub-micron emulsions. unstable microemulsions and translucent emulsions. As used herein, the term "bioactive compound" refers to a compound that has pharmaceutical, nutraceutical or preservative properties. Bioactive compounds are often 20 used in pharmaceutical nutraceutical, cosmetic, functional foods and preservatives and may have properties such as antiseptic, antioxidant, anti-inflammatory, antibiotic or antimicrobial properties. The term phytochemicall" as used herein refers to a plant derived bioactive chemical. In 25 particular, the phytochemicals referred to herein have antioxidant and/or antibacterial activity or are suitable for inclusion in functional foods. In some cases the phytochemicals may be modified synthetically, for example, by esterification of carboxylic acids or hydroxy groups. 30 The term "functional food" refers to a food or beverage having health benefits or disease preventing properties beyond the basic function of supplying nutrients. Functional foods -5 include processed foods which are fortified with health promoting additives such as vitamins, antioxidants, prebiotics and hormones. As used herein, the term "alkenyl" refers to a straight chain or branched hydrocarbon group 5 having 2 to 20 carbon atoms and having one or more double bonds between carbon atoms. Where appropriate, the alkenyl group may have a specified number of carbon atoms. For example, "Cs-C 12 -alkenyl" includes groups having 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms in a linear or branched arrangement and one or more double bonds. Examples of suitable alkenyl groups include, but are not limited to ethenyl, propenyl, isopropenyl, butenyl, 10 butadienyl, pentenyl, pentadienyl, hexenyl, hexadienyl, heptenyl, acetenyl, nonenyl, decenyl, undecenyl and docecenyl. In a first aspect of the invention, there is provided a nanoemulsion composition comprising water, at least one edible oil and a modified starch emulsifier, wherein the average particle 15 size of the dispersed oil phase in the nanoemulsion is less than 500 nm and with the proviso that any emulsifier, other than the modified starch emulsifier, is present in an amount of less than 3% w/w of the composition. In some embodiments, edible oil is an oil or fat from animal or plant origin, fatty acids. 20 lipids and fatty esters or mixtures thereof. The fats and oils may have a broad molecular weight range, however, in some embodiments, the oil or fat has at least some components having a high molecular weight range, for example in the range of 250 to 600 Da. In some embodiments, the oil is a plant oil such as a vegetable oil, nut oil or algal oil. Examples of suitable vegetable oils include rapeseed oil, especially canola oil, olive oil, peanut oil. 25 coconut oil, soybean oil, sunflower oil, safflower oil, corn oil, grape seed oil, linseed oil. jojoba oil, palm oil, avocado oil, cotton seed oil, evening primrose oil, borage oil, sea buck thorn oil, rice bran oil and sesame oil. Suitable nut oils include hazelnut oil. walnut oil. macadamia nut oil and almond oil. Suitable algal oils are those derived from algae. such as those produced for food or biofuel applications. In some embodiments. the oil is an 30 animal oil or fat such as butter, tallow, chicken fat, pork fat., beef fat and fish oils. In particular embodiments, the at least one edible oil is selected from rapeseed oils including canola oil, olive oil, safflower oil, soybean oil, peanut oil, corn oil, nut oils, linseed oil, rice -6 bran oil, and sesame oil, and mixtures thereof, especially canola oil, olive oil, nut oils and sesame oil, and mixtures thereof, more especially canola oil. In some embodiments, the edible oil may be a mixture of edible oils such as a mixture of 5 animal and/or plant oils, for example, those oils listed above. In some embodiments, the vegetable oil component comprises at least one oil and one or more other food grade hydrocarbon components. Suitable food grade hydrocarbon components include food grade essential oils such as limonene, or other citrus peel oils, sweet orange oil, peppermint oil, lemon oil, eucalyptus oil, clove oil, spearmint oil, oregano oil, tea tree oil, 10 camphor oil, cedar oil, rosewood oil, myrtle oil and neem oil. The modified starch emulsifier is a starch ester emulsifier especially a starch ester emulsifier having a degree of substitution from 1.2 up to about 2.2 of one or more aX-(Cs

C

15 alkenyl)(C 3

-C

7 )dicarboxylic acids, especially where the alkenyl group is a straight 15 chain alkenyl group. Particular modified starch emulsifiers include alkali metal C 5

-C

12 alkenyl succinate substituted starch. The dicarboxylic acid may be selected from malonic acid, succinic acid, glutaric acid, adipic acid and pimelic acid, especially malonic acid. succinic acid or glutaric acid, most especially succinic acid. The alkali metal may be lithium, sodium or potassium, especially sodium or potassium, most especially sodium. 20 The alkenyl group may be a straight chain or branched alkenyl group, especially a branched alkenyl group having one to three double bonds. Examples of suitable alkenyl groups include I-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 2,4-pentadienyl, 1-hexenyl. 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 2,4-hexadienyl, 1,3,5-hexatrienyl, I -heptenyl. 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 2,4-heptadienyl. 3.5 25 heptadienyl, 4,6-heptadienyl, 2,4,6-heptatrienyl, 1 -octenyl, 2-octenyl, 3-octenyl, 4-octenyl. 5-octenyl, 6-octenyl, 7-octenyl, 2,4-octadienyl, 3,5-octadienyl, 4,7-octadienyl, 3,5.7 octatrienyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl. 8-nonenyl, 3,5-nonadienyl, 3,6-nonadienyl, 6,8-nonadienyl, 4,6,8-nonatrienyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 30 7,9-decadienyl, 5,7,9-decatrienyl, 1-undecenyl, 2-undecenyl, 3-undecenyl. 4-undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl. 9-undecenyl. 10-undecenyl. 6.8 undecadienyl, 7,9-undecadienyl, 8,10-undecadienyl, 6.8,10-undecatrienyl, I-dodecenyl.

-7 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dondecenyl, 8-dodecenyl, 10-dodecenyl, 11-dodecenyl, 8,10-dodecadienyl, 7,9-dodecadienyl, 9,11 dodecadienyl and 6,8,10-dodecatrienyl, especially 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, I-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 5 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, more especially l-octenyl, 2-octenyl, 3-octenyl, 4-octenyl. 5 octenyl, 6-octenyl, 7-octenyl, most especially 6-octenyl. In a particular embodiment, the modified starch emulsifier is starch sodiurn-6-octenyl succinate, marketed under the tradename HI-CAP" 100 or NARLEX. RTM. PPEI388. 10 In some embodiments, the nanoemulsion does not contain both canola oil and H-l CAP 100. The nanoemulsions of the invention are oil-in-water emulsions. In some embodiments, the 15 edible oil is present, either as a single or a mixture of oils, in the nanoemulsion in an amount in the range of 5% to 30% v/v, especially 5% to 20% v/v, 5% to 15% v/v or 7% to 12% v/v of the composition, especially about 10% v/v of the composition. In some embodiments, when the edible oil component is mixed with another food grade 20 hydrocarbon, the at least one edible oil comprises at least 50 % of the dispersed phase of the nanoemulsion. In some embodiments, the modified starch emulsifier is present in an amount in the range of 2% to 10% w/v of the composition, especially 2.5% to 10% w/v or 3% to 7.5% w/v of" 25 the composition, more especially about 5% w/v of the composition. In some embodiments, the modified starch emulsifier is the only emulsifier present. In other embodiments, the nanoemulsion composition may comprise emulsifiers other than the modified starch emulsifier, in amounts of less than about 3% w/w. For example. other 30 emulsifiers that may be present include, but are not limited to, lecithin, caseinate, B-casein. whey proteins such as -lactalbumin, Tween 20, Tween 40, Tween 80 , sorbitan esters -8 such as sorbitan monostearate or those sold under the trade mark SPAN, and Citreami"' M emulsifiers. In some embodiments, the nanoemulsion composition further comprises a bioactive agent 5 such as a pharmaceutical, nutraceutical or food preservative, especially one that is difficult to solubilise or is sparingly soluble in commonly used pharmaceutical or nutraceutical carriers. In some embodiments, the bioactive compound is propolis or ibuprofen. In one aspect of the invention, the nanoemulsion is used in food technology, particularly in 10 the preparation of functional foods or in the preservation of food, especially perishable food. In a particular aspect, the present invention provides a food preservative composition comprising the nanoemulsion of the invention and at least one food preserving compound. 15 The food preserving compound may be any hydrophilic, amphiphilic or hydrophobic preservative compound or mixture of compounds able to be used in foods. In a particular embodiment, the food preservative compound is an antioxidant and/or antimicrobial phytochemical. Examples of suitable phytochemicals include phenolic compounds 20 including monophenols, flavonoids, phenolic acids, hydroxycinnamic acids, lignans or phytoestrogens, tyrosol esters, stilbenoids and punicalagins, terpenes such as carotenoids. monoterpenes, saponins, phytosterols, tocopherols and triterpenoids, betalains such as betacyanins and betaxanthins, and vitamins such as Vitamin E, ascorbic acid and its esters. essential oils or mixtures of any of these compounds. The phytochemicals may also be in 25 the form of plant extracts, rather than isolated compounds. Suitable phenolic compounds include gallic acid, ellagic acid, salicylic acid, tannic acid, vanillin, vanillic acid and its esters, capsaician, curcumin, quercetin, carvacrol, gingerol. kaempferol, myricertin, rutin, isorhamnetin, hesperidin, naringenin, silybin, eriodictyol, 30 apigenin, tangeritin, luteolin, catechins such as catechin, gallocatechin. epicatechin. epigallocatechin, epigallocatechin gallate, epicatechin-3-gallate, theaflavin, theaflavin-3 gallate, theaflavin-3'-gallate, theaflavin-3,3'-digallate, thearubigins; anthocyanins and -9 anthocyanidins such as pelargonidin, peonidin, cyanidin, delphinidin, malvidin and petunidin; isoflavones such as daidzein, genisteine and glycitein, dihydroflavonols, chalcones, coumestans such as coumestrol; caffeic acid, chlorogenic acid. cinnamic acid, ferulic acid, coumarin; lignans such as natairesinol, secoisolariciresinol, pinoresinol and 5 lariciresinol; tyrisol esters such as tyrosol, hydroxytyrosol, oleocanthal and oleuropein: stilbenoids such as resveratrol, pterostilbene and piceatannol; and punicalagins. Suitable terpenes include carotenes such as ax-carotene; 1-carotene, y-carotene, 6-carotene, lycopene, neurosporene, phytofluene and phytoene; xanthophylls such as canthaxanthin, 10 cryptoxanthin, zeaxanthin, astaxanthin, lutein and rubixanthin; phytosterols such as campesterol, p-sitosterol, y-sitosterol and stigmasterol; tocopherols such as a-tocophenol. triterpenoids such as oleanolic acid, ursolic acid, betulinic acid and moronic acid. Suitable betalains include betacyanins such as betanin, isobetanin, probetanin and 15 neobetanin; and betaxanthins such as indicaxanthin and vulgaxanthin. In a particular embodiment, the bioactive compound is a phytochemical such as gallic acid. tocopherols, ascorbic acid and its esters, vanillic acid, vanillin, P-carotene. carvacrol. quercetin, catechins, caffeic acid, rutin and curcum in, especially gallic acid. 20 In some embodiments, more than one phytochemical is present in the preserving composition. Advantageously, the nanoemulsion of the invention is able to solubilise phytochemicals of widely varying hydrophobicity, for example, a hydrophilic compound and a hydrophobic compound that are not normally mixable. This may allow synergism 25 between combinations of compounds to provide a better preservative result than either compound alone. For example, combinations of vanillic acid and butylated hydroxyl anisole or vanillic acid and quercitin result in organoleptically acceptable levels ol trimethylamine (TMA) in fish for longer periods of time. 30 In some embodiments, the preserving composition is diluted with water before use to provide a concentration suitable for use. Advantageously, the nanoemulsion is stable to dilution.

- 10 The amount of preservative compound in the nanoemulsion may be any amount up to the capacity of the nanoemulsion to adsorb any further preserving compound. In some embodiments, the amount forms a concentrate composition which is subsequently diluted 5 prior to use in preserving food. In other embodiments, the amount of preservative compound is suitable for direct application to food. The amount of preservative compound will depend on the specific compound used, the food to be preserved and the method of exposing the food. Typical amounts applied to food will be in the order of pg to mg per litre of nanoemulsion, for example between 100 pg and 15 g per litre, especially 250 pg to 10 15 g, 1 mg to 15 mg, I mg to 10 g, I mg to 7.5 g, 10 mg to 5 g, 50 mg to 2.5 g, 50 mg to I g, 50 mg to 900 mg, 50 mg to 800 mg, 50 mg to 700 mg, 50 mg to 600 mg, 50 mg to 500 mg, 100 mg to 400 mg, 100 mg to 300 mg, or 100 mg to 250 mg per litre. In some embodiments, the nanoemulsion composition comprising the bioactive compound 15 is a concentrated composition which is diluted prior to use. In some embodiments, the nanoemulsion composition comprises the bioactive compound at the time of preparation of the nanoemulsion. In other embodiments, the bioactive compound is added after formation of the nanoemulsion, for example, immediately before 20 use. In yet another aspect, there is a method of preserving food comprising exposing the food to a food preservative composition as described above. 25 In some embodiments, the food is a perishable food. As used herein, perishable foods are foods that are normally eaten fresh and not subject to preserving techniques such as canning, salting and pickling. Perishable foods require refrigeration to remain fresh and include seafood, such as fish, crustaceans and shellfish, meat, fruit and vegetables. In a particular embodiment, the perishable food is seafood, especially fish. In another 30 particular embodiment, the perishable food is meat. In yet another particular embodiment. the perishable food is a fruit. In a further particular embodiment, the perishable food is a vegetable.

-li The food may be exposed to the preservative composition by any suitable method that provides an effective amount of preservative into the food. Suitable methods include dipping, spraying, painting, glazing, injecting and vacuum infusion, especially dipping and 5 spraying. In some embodiments, where the preservative composition is diluted with water before the food is exposed to it, the composition after dilution may comprise 200 Pg to 5 mg per litre of preservative compound, especially about 200 pg to 2400 ptg per litre. 10 The food is exposed to the preservative composition for a period of time suitable to allow adequate adsorption of the preserving compound into the food or sufficient adsorption on the surface of the food. In some embodiments, such as where spraying or painting is used, the amount used is sufficient but not excessive and after treatment, the food remains in 15 contact with the preserving composition. If an excess of composition is used, the excess unadsorbed composition may be washed off after a period of time sufficient for adequate adsorption of preserving composition. In embodiments that involve dipping, the time required will depend on the food being treated. However, in most cases I to 30 minutes. especially 5 to 20 minutes, more especially about 10 minutes will be sufficient for 20 adsorption of preservative composition. In another embodiment, the preserving composition is applied to perishable fruits or vegetables immediately before or after harvest to prevent spoilage. For example, fruit may be harvested and sprayed with the preservative composition to prevent spoilage caused by 25 microorganisms such as fungi, bacteria or viruses, especially fungi. In yet another aspect of the invention there is provided a functional food comprising the nanoemulsion composition of the invention and at least one vitamin, mineral, prebiotic, antioxidant, plant sterol or plant extract. 30 Vitamins are nutrients essential for normal growth, vitality and well being. Vitamins include Vitamin A (retinol and its precursors such as carotinoids), Vitamin B I (thiamine), - 12 Vitamin B2 (riboflavin), Vitamin B3 (Niacin), Vitamin B6 (pyridoxal phosphate). Vitamin B12 (cobalamine), Vitamin C (ascorbic acid), Vitamin E (a-tocopherol and tocotrienols), Vitamin B9 (folic acid), Vitamin B5 (panthothenic acid) and Vitamin K (phylloquinone). 5 Suitable minerals include trace minerals required for human or animal nutrition, for example Fe++, Fe+++, Mg++ and Ca++. The phytochemical antioxidants mentioned may also be incorporated into the nanoemulsion composition to form an ingredient of a functional food. 10 Suitable prebiotics include fructooligosaccharides such as inulin, galactooligosaccharides and short or long chain oligosaccharides. Suitable sterols include plant sterols such as capesterol, stigrnasterol, brassicasterol, 15 sitosterol and ergosterol. Other suitable sterols include cholesterol and its derivatives. Suitable plant extracts include those that have biological benefits such as antioxidant and/or antibacterial properties, for example, grape seed extract, tea extract and apple 20 extract. The functional good may comprise bioactive compounds including peptides, such as nisin. The nanoemulsion compositions of the invention may be prepared by any method suitable 25 for preparation of a nanoemulsion including low energy methods and high energy methods. In some embodiments, the nanoemulsion is formed by mixing the oil, water and emulsifier and optionally a food preservative or functional food component in a homogenizer or Microfluidizer T. In some embodiments, the nanoemulsion is formed using a two step process, initial mixing to form a coarse emulsion followed by high shear 30 mixing in a microfluidizer thereby forming the nanoemulsion.

- 13 In some embodiments, the bioactive compound is present in the composition when the nanoemulsion is formed. In other embodiments, the bioactive compound is incorporated in the nanoemulsion composition after the nanoemulsion is formed, for example. by simple stirring of the nanoemulsion with the added bioactive compound. 5 In order that the nature of the present invention be clearly understood and put into practical effect, specific embodiments will now be described by way of the following non-limiting examples. 10 Brief Description of Figures Figure 1 is a graphical representation of the migration of gallic acid (ppm) from nanoemulsion formulations when fish muscle is dipped in the nanoemulsion from 0, 10, 20 and 30 minutes. 15 Figure 2 is a graphical representation of the adsorption of gallic acid by fish muscle from nanoemulsion dip solutions as compared to adsorption from water at a gallic acid concentration of 1200 pg/L and at 0, 10, 20 and 30 minutes. Figure 3 is a graphical representation of the influence of hydrophobicity (expressed as log 20 P) on the solubility of various phytochernicals. Figure 4 is a graphical representation showing effects of combination of phytochemicals on TMA levels. NT = untreated, BNE = blank nanoemulsion, VAQ = vanillic acid and quercetin combination, VA = vanillic acid, VAB = vanillic acid and BHA and Q = 25 quercetin. Figure 5 is a graphical representation of the effect of plant extracts on TMA production in fish. NT = untreated, BNE = blank nanoemulsion, TN = tea extract nanoemulsion. GSN = grape seed extract nanoemulsion, AN - apple extract nanoemulsion, TW = tea in water. 30 GSW = grape seed extract in water, AW = apple extract in water.

- 14 Figure 6 is a graphical representation showing the formation of nanoemulsions with different oils. Figure 7 is a graphical representation showing mixing of vanillic acid pre-processing and 5 post-processing of nanoemulsions. Figure 8 is a graphical representation showing the effect of dipping various foods and the residual concentration of vanillic acid in nanoemulsion for 20 and 40 minute intervals. 10 EXAMPLES Example 1: Formation of Nanoemulsions Materials Commercial canola oil was purchased from a local food market, Hi-Cap 100 modified starch was purchased from National Starch and Chemical (Sydney, Australia). 15 Phytochemicals and solvents (methanol, ethyl acetate and hexane) were purchased from Sigma Aldrich. Limonene was purchased from Australian Botanical Products Pty Ltd. Methods i. Formation of coarse nanoemulsion 20 The formation of nanoemulsions involved two stages. Initially coarse emulsions were prepared with an Ultraturrax mixer. HI-CAP 100 was hydrated at 60'C for 3 hours before mixing with other ingredients, such as canola oil, water and phenolic compounds. Proper initial mixing was achieved by Ultraturrax mixer at 19000 rpm for 1 min. 25 ii. Formation of nanoemulsion The coarse emulsions were passed through an air driven Microfluidizer (Model M-1 10 1 Microfluidics, USA). This involved feeding the coarse emulsion through a 200 mL stainless steel reservoir. Microfluidizer splits the liquid into two opposing channels in a ceramic interaction chamber with stream pore size of 75 ptm and the two streams collide 30 head on. The resultant amplification of pressure in the interaction chamber is 232 times the external air flow rate through the device. Different pressures were tested ranging from 35 - 15 MPa to 90 MPa while the number of passes through Microfluidizer (cycles) ranged from I to 7. iii. Particle Size measurement 5 The emulsions were then subjected to particle size measurement using the laser light scattering method of the Malvern Mastersizer@ 2000; the samples were diluted in a ratio of 1:3 with Millipore water before particle size analysis. The emulsion system that displayed visual stability was then subjected to storage where the particle size was measured at 7 day intervals for a total period of 28 days. 10 iv. Total Phenol Assay Total phenol contents were measured by the methods described by Singleton and Rossi (Cited in Theeshan & Aruoma, 2004) using Folin-Ciocalteu reagent (FCR). Briefly, nanoemulsion samples were collected after centrifugation and 20 tL samples were diluted 15 into 1980 pL water or with ethanol (depending upon the solubility of relevant antioxidant). 10 1 .L of the above diluted sample was taken in the plastic cuvettes having 790 pL- of water followed by 50 pL of FCR and after 3 minutes 150 pL of sodium carbonate. The contents were thoroughly shaken and placed in dark for 2 hours before being analysed by spectrophotometer at 765 nm. 20 Varying the oil used (limonene, canola oil), amounts of oil, amounts of emulsifier and amounts of presence of phytochemicals demonstrated effects on nanoemulsion formation as shown in Table 1. 25 Optimization of nanoemulsion formation Table I Particle size of emulsions generated with various formulations processed ith a microfluidizer at 95 MPa* Cycles Particle size (nm) achieved with various formulations A B C U V W X Y Z Q R S 0 3554 2010 3294 2611 3045 2943 2553 2404 2553 4048 3265 195 - 16 1 734 1508 2752 859 1273 1716 1196 1545 2541 1355 214 179 2 802 1522 2773 876 1346 1783 1226 1791 2109 1164 200 173 3 872 1149 2798 852 1418 1902 1221 1854 2445 1116 193 169 4 860 1237 1737 996 1316 1888 1221 1797 1854 1046 187 158 5 812 1224 nm 971 1256 1690 1426 2102 2156 nm 193 154 6 801 1184 nm 969 1455 1666 1315 1862 2389 nm nm 142 *= composition for the letters are shown in the table below, letters nn= particle size not measured due to instability, formulation T did not form a nanoemulsion COMPOSITION(%) A B C Q R 5 T U V W X Y Z

HI-CAP

T

I100 15 10 10 3 5 5 10 5 7 10 5 7 10 Canolaoil 0 0 0 0 10 5 0 0 0 0 0 0 0 Limonene 20 20 10 7 0 5 10 10 0 30 10 25 30 Gallicacid 1 1 1 0 0 0 0 0 25 0 1 1 1 BHA 1 1 1 0 0 0 0 0 0 0 1 1 1 Table I shows that the presence of canola oil resulted in the formation of a nanoemulsion 5 in the first pass through the microfluidizer. In contrast, nanoemulsions were not formed when limonene was used as the only oil.

-17 - :1- 00 en \o r n -1 0q - -t - -n - -4 0C, In - 00 -, u -U 2 ~ cx C',N - c- -n - U, U r e - - N ' - N ) CI 00 CtI t al n\; C (N C C0' 0 In - t InP 00 L f -zC)00 \0 NN'- 0 \J o - - - It C: Nx ' NN 0 In (71 m C\ 00 N 'I rl N -~ 'NN~t~ttt~ 5u U-E rmj (., 2 2 : C: r-4 r4rC. .2 -T 1 r- r~t N - N r, c V- - r-(4 N - ('1 C N o NNNNNNC \ C Pt1t0 00 10 c cv 00 00) -00 o( 0 On 'n In - ,I DC D C N ' NNNNN(N0\0 N E tn U it -It C) Nr - 00 rn N4 U ( mn rn - m e 00 11 C 0.) 0 I~S tn * a) U 00 CIO t~ rn - 0 - It N a, - o n r. C- Ol ~ e00 0 Inr 0 0 Ct 0 t0 UPt In7 -0'00 tn( aC r~~j <D 00 N I Pt In N---o In ~ ~ ~ ~ ~ . r--TCr 0 -r U- tn -In tn \.O rn tn -18 Table 3: Effect of biopolymer concentration on storage stability of nanoemulsions at different number of cycles Cycles Storage time (days) at Storage time (days) at Storage time (days) at 2.5% Hi Cap 5% Hi Cap 7% Hi Cap 1 7 14 21 28 1 7 14 21 28 1 7 14 21 28 1 206 293 307 307 341 220 225 211 209 219 215 217 201 227 202 2 211 256 276 298 299 213 215 213 211 215 215 203 219 210 214 3 199 257 277 270 289 192 206 199 204 202 198 201 178 205 202 4 191 259 266 279 295 191 190 196 191 198 194 196 199 204 200 5 193 255 271 276 285 191 201 209 204 209 194 177 206 203 187 5 Table 4 Effect of the loading of phytochemicals individually and as combination (0.5% w/v) number of cycles and storage time on the particle size (nm) of nanoemulsion R* Cycles Storage Time (Days) and particle sizes (nim) T AP VA GAT GAQ 1 7 1 2 2 1 7 1 2 2 1 7 1 2 2 1 7 1 2 2 l 7 1 2 2 12 I 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 0 8 0 1 1 0 2 0 1 1 1 1 0 5 0 0 1 2 2 2 1 2 1 2 2 7 3 6 4 1 8 0 7 3 6 3 2 6 3 8 8 8 2 8 6 9 3 6 4 0 2 1 1 1 2 2 1 2 2 2 2 1 2 2 1 2 2 2 2 2 2 2 2 2 2 2 9 9 9 0 0 8 0 0 0 1 9 0 0 9 0 1 1 1 2 1 0 2 0 1 1 6 8 7 0 5 7 4 5 9 3 8 0 0 7 7 1 3 2 1 7 1 8 6 1 1 3 I I 1 I I 1 2 1 2 2 I I I 1 2 1 2 1 2 2 1 2 1 2 2 9 9 9 9 9 9 0 9 0 0 9 9 9 9 0 9 1 9 1 2 9 2 9 0 1 7 4 3 4 8 7 1 9 1 5 5 7 8 5 5 6 08 1 0 9 1 1 7 I 4 1 1 1 I 1 1 1 I I 1 1 2 1 2 2 2 2 I 2 2 1 2 8 9 9 9 9 9 9 9 9 9 9 9 9 9 0 9 1 0 1 1 9 1 0 7 0 4 3 4 3 3 3 8 7 8 7 0 5 8 6 1 2 8 8 5 9 9 3 2 9 1 5 I I I 1 I I 1 2 I I I I I I 1 1 2 2 2 2 2 2 1 2 2 9 9 9 8 9 8 9 0 9 9 8 9 9 9 9 9 1 0 2 0 0 0 9 0 0 0 0 1 9 8 8 5 1 8 8 6 1 6 6 9 0 1 7 1 2 1 7 5 6 2 6 I I I I I I I 1 2 1 1 1 1 1 1 2 2 2 2 1 2 1 2 2 8 8 9 9 9 8 9 9 9 2 8 8 9 9 9 9 1 0 I I 8 0 9 0 0 4 1 0 1 3 7 4 3 6 I 8 9 4 4 3 3 4 9 8 8 7 0 9 -1 3 7 I I 1 I 1 1 1 1 1 1 1 1 1 1 I 1 2 2 2 1 2 1 2 2 5 8 8 9 8 8 9 9 9 9 8 8 9 9 9 9 1 I I 8 0 9 0 0 9 6 7 0 9 4 3 9 9 9 5 9 5 9 7 5 0 3 8 6 4 7 1 3 Letters and symbol represent T=a-tocopherol, AP= ascorbyl palmitate, VA= vanillic acid, GAT= gallic acid and tocopherols, GAQ= gallic acid and tocopherols *= formulation 10% canola and 5% HICAP" 100, individual phenolic compounds are loaded at 0.5% w/v level, whereases combination of phenolics is added at 0.5% w/v each. 10 - 19 Table 5 Effect of individual phenolic loading, number of cycles and storage time on the particle size of nanoemulsion R * Cycles Storage Time (Days) and particle sizes (nm) T AP VA Q GA 1 7 1 2 2 1 7 1 2 2 1 7 1 2 2 1 7 1 2 2 1 7 1 2 2 1 2 2 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2? 2 2 1 0 9 1 1 1 5 3 3 3 1 2 2 2 1 I I I I 1 0 9 1 0 0 2 9 4 2 I 4 3 8 7 6 I 8 2 3 7 5 7 4 7 8 5 2 4 6 3 2 1 2 2 2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 1 8 1 0 0 9 0 4 3 3 2 9 I I I 1 0 1 9 I 1 0 0 I I 9 9 5 5 6 1 2 8 0 3 1 9 5 0 5 3 8 1 4 2 0 6 6 6 1 6 3 1 I I 1 I 2 2 2 2 2 11 2 2 2 2 I 1 2 2 2 9 9 9 9 9 0 5 3 2 2 9 1 0 1 0 8 0 0 0 0 8 9 0 0 0 9 5 6 5 9 5 5 6 7 2 4 1 2 0 7 0 9 7 2 6 8 5 3 4 6 4 I I 1 1 I 2 2 2 2 2 1 2 1 2 2 1 2 2 1 2 1 2 2 2 2 8 9 9 9 8 0 4 3 3 3 9 0 9 0 0 7 0 0 9 0 7 0 0 0 0 6 4 2 8 2 1 7 1 9 4 4 9 1 3 3 7 3 3 6 8 2 0 1 7 6 5 I I I I I 2 2 2 2 22 2 2 2 2 1 2 N N N N N 6 9 9 9 9 9 5 4 4 2 8 0 0 0 0 0 0 0 9 0 VI M M M M 2 6 2 4 2 8 1 1 8 9 9 7 3 2 0 1 3 4 5 5 6 1 1 1 1 I 1 2 2 2 2 1 2 2 2 1 2 2 2 2 N N N N N 8 9 9 9 8 9 5 4 4 4 9 0 0 0 0 9 0 0 0 0 M M M M NIM 6 1 2 1 8 7 5 0 0 3 6 2 1 4 3 7 1 4 2 3 7 I 1 I I I I 2 2 2 2 1 2 1 1 2 1 2 2 1 1 N N N N N 8 8 9 9 8 9 6 3 3 2 9 0 9 8 0 9 0 0 9 9 M M M M N 4 6 0 4 7 3 1 6 6 8 4 1 1 5 3 4 2 6 7 4 Letters T= a-tocopherol, AP= ascorbyl palmitate, VA= vanillic acid, Q= quercetin and GA= gallic acid, NM= particle size not measure, formulation 10% canola and 5% HICAP"' 100, phenolic compounds are loaded at 0.25% w/v level 5 As shown in Table 2, limonene alone does not form nanoemulsions, however, when it is combined with canola oil in any combination, such as 25%, 50% or 75% w/w, a nanoemulsion may be formed. 10 Table 3 demonstrates that low emulsifier concentrations reduce stability of' the nanoemulsions of formulation R. Different bioactive compounds can be added to nanoernulsions at either low (0.25% w/v, as shown in Table 5) or at higher concentrations (0.5% w/v, as shown in Table 4), either alone 15 or in combination, without compromising the stability of the nanoemulsion.

- 20 Example 2: Solubility of Phytochemicals Materials Commercial canola oil was purchased from a local food market, Hi-Cap 100 modified starch was purchased from National Starch and Chemical (Sydney, Australia). Phytochemicals and 5 solvents (acetonitrile, methanol, ethyl acetate and hexane) were all purchased from Sigma Aldrich. Formation offood grade emulsions The formation of nanoemulsions involved two stages. Initially coarse emulsions were 10 prepared with an Ultraturrax mixer. Ingredients (10% v/v canola oil, 90% V/V water and 5% W/V hydrated Hi-Cap) were mixed passed through the Ultraturrax mixer at 19000 rpm for 1 min. The coarse emulsions were then passed through an air driven Microfluidizer (Model M I 10 L Microfluidics, USA). The operating pressure was 95 MPa with three passes through the Microfluidizer 15 Measurement of Phytochemical solubility Nanoemulsions were prepared with an excess of test phenolic compound in order to measure the maximum total solubility. Similarly solubility in water and oil was measured by adding an excess of test compound into the solvent in a Falcon tube. These tubes were subjected to 20 rotary shaking overnight followed by centrifugation and filtration. All of the above solutions were subjected to centrifugation at 5000 g. 10 min. to remove solids and were filtered using 0.45 tm nylon filters. For water solubility, samples were drawn directly, whereas for oil solubility testing the phenolics were solvent extracted by washing the oil 3 times with I part oil:5 parts volume ethyl acetate. Ethyl acetate was evaporated under nitrogen flush and the 25 residue was dissolved in 40% acetonitrile solution for HPLC analysis HPLC Analysis Nanoemulsion samples were first washed five times with hexane to remove oil, and then extracted with a 20% acetonitrile solution containing 0.1% formic acid to remove the 30 phytochermical. These diluted samples were then centrifuged with trichloroacetic acid to -21 precipitate and remove proteins. Samples were further centrifuged at 5000 rpm for 10 min and filtered through 0.45 im nylon filters prior to injection into HPLC. HPLC chromatography was accomplished using a Waters 2690 Alliance H-PLC System and a 5 Waters 996 Photodiode Array Detector. The acquisition was controlled using MassLynx V3.5 software (Micromass Ltd., Manchester, UK). The samples were analysed on an XTerraTM MS C18 reverse phase column, 150 x 2.1 mm, 3.5 im (Waters) connected to a 10 mm x 2.1 mm XTerraTM MS C18, 3.5 im guard column (Waters) at a constant temperature (27 0 C). A Waters Alliance 2690 Autosample rack, set at 18 'C, was used with injection volumes of 10 10 pAL. The mobile phase flow rate was 0.25 mL/min. For the HPLC analysis, the mobile phase solvents were: 0.1% formic acid in 2% acetonitrile (A) and 0.1% formic acid in 80% acetonitrile (B). The following mobile phase gradient for phase B was used: isocratic elution 5% for I min, linear gradient elution from 15 5% to 25% in 29 min, to 30% in 5 min, to 55% in 5 min, isocratic elution at 55% for 5 min, then gradient elution to 80% for 5 min, then phase B was reduced to 5% over 5 min, and maintained at 5% for another 5 min. The total acquisition time was 60 min for each sample. UV spectra were recorded from 200 to 400 nm using a diode array detector. Quantification was carried out at 290 nrm for gallic acid and vanillic acid, and at 390 nm for quercetin. 20 Solubilities oJ phytochenicals The results showed that the solubilities of vanillic acid, quercetin, rutin, caffeic acid, vanillin, curcumin, ascorbyl palmitate, quercetin, vanillic acid and BHA were higher in nanoemulsions than their respective solubilities in either water or oil. This suggests that phytochemical 25 loaded nanoemulsions may have important uses in fresh food preservation. Solubility data (Table 6) suggest that such nanoemulsions could act as universal solvent for phytochemicals. This is evident from the increase in solubility for compounds that range from hydrophilic to hydrophobic. The enhanced solubility can have many potential benefits as 30 functional compounds in the diverse area of phytochemical applications in food science.

- 22 Table 6 Solubility ofphytochemicals into water, oil and nanoemulsions Phytochemical Solubility (mg/L) at 25'C Solubility enhancement (%) Oil Water Nanoemulsion Vs. Oil Vs. Water Rutin 134±5 33±4 2043±70 1524 6190 Caffeic acid 805±32 1248±21 2982±23 370 238 Vanillin 3346±15 2386±54 7705±71 230 322 Catechin 306±9 3837±69 3738±58 1221 97 Curcumin 130±2 0 462±21 355 NC BHA 00 22±1 2234± 123 NC 10154 Gallic acid 0 ±0 11770 ±117 7441± 395 NC 63 Vanillic acid 452 ± 9 765± 12 3237± 131 716 423 AP 33 ±3 16± 4 644± 15 1951 4025 Quercetin 202± 20 78± 4 2227± 49 1102 2855 VAGA NC NC 4389±19 NC NC VAQ NC NC 1933+9 NC NC Data (mg/L) expressed as means of three experimental replicates with standard deviations (co maximum solubility was not attained, NC= solubility enhancement was not calculated) 5 VAGA= vanillic acid and gallic acid each 0.5%, VAQ= vanillic acid quercetin combination at 0.5% and 0.25% of nanoemulsion respectively, AP= ascorbyl palmitate Example 3: Adsorption of Gallic Acid into Fish Muscle Fresh fish was obtained from the local market. 10 Formation offood grade emulsions The formation of nanoemulsions involved two stages. Initially coarse emulsions were prepared with an Ultraturrax mixer. Ingredients (10% v/v canola oil, 90% V/V water and 5% W/V hydrated Hi-Cap) were mixed passed through the Ultraturrax mixer at 19000 rpm for I 15 min. The coarse emulsions were then passed through an air driven Microfluidizer (Model M 110 L Microfluidics, USA). The operating pressure was 95 MPa with three passes through the Microfluidizer.

- 23 Adsorption ofphytochemicals onto fish muscle The fresh fish was washed and cut into small pieces of approximate I cm x 1 cm dimensions using a sharp blade. Nanoemulsions were appropriately diluted in falcon tubes with Milli Q 5 water to a total volume of 20 mL, so as to achieve the required phytochemical concentration. Fish was added to these falcon tubes, with the weight of fish being taken to the nearest of I g per I mL diluted nanoemulsion/solvents. These falcon tubes were subjected to rotary shaking for 30 min to ensure sufficient contact between the nanoemulsion and fish muscles. Samples were drawn at time intervals of 0, 10, 20 and 30 min of this fish dip in the nanoemulsion 10 solutions. The amount of phytochemicals adsorbed onto the fish muscles was calculated by the loss of phytochemicals from dip solutions. The phytochemical used was Gallic acid. The adsorption of gallic acid from nanoemulsion to fish muscle is concentration dependent (Figure I). It was completely adsorbed at lower concentrations. Whereas, on increasing the 15 application concentration, there was some unadsorbed gallic acid left in the nanoemulsion. These findings are in accordance with a single similar reported study, where soluble grape polyphenols in water at a lower concentration were all removed from water completely after centrifuging with fish muscle (Pazos, Gallardo, Torres, & Medina, 2005). While 20 centrifugation might produce better adsorption, it is less practical for a commercial point of view. In our study we tried to test the system in a more realistic way of dipping the fish which offers many benefits, i.e. ease of application, simplicity, non destructiveness, and it offers better control over the amount delivered. Moreover, the assertion that the gallic acid is attached to the fish muscle is supported by the fact that a reasonable mass balance for the 25 gallic acid was achieved when it was extracted back by washing the fish muscle with solvents. It is also important to mention that achieving an exact mass balance is quite a difficult task because of the difficulty of the extraction, limited choices of solvents used lbr this methodology, degradation of the antioxidants, or the formation of conjugates, in some instances, with the fish muscles. 30 - 24 A four-fold increase in the uptake of gallic acid by fish muscle was observed after 30 min of dipping, when the nanoemulsion at 1200 ppm was compared to the control at 12000 ppm (Figure 2). One possible reason for this phenomenon is that water is not a good carrier of bioactives because of the multicomponent nature of food. This result presents valuable 5 information for enhancing the adsorption of gallic acid onto a fish surface. It is commonly assumed that a better aqueous or lipid solubility of a compound can provide better preservation effects, however, in this case gallic acid, which is highly water soluble, is better attached to the fish surface when it is part of a nanoemulsion. This might be due to the fact that nanoemulsions, having both hydrophilic and hydrophobic components, provide a better 10 attachment to the fish muscle. The partitioning coefficient and its relationship to solubility enhancement is a key measure in the proposed mechanism of solubility enhancement. Nanoemulsions consist of different regions such as lipid, water, and the adjoining of the two regions, an interface. The partition 15 coefficient is an important criterion for understanding the affinity of added compound to immiscible phases based on its hydrophilic and hydrophobic interactions (Bouchard elal 2002). The nanoemulsion developed in this research acted as a delivery system for a variety of phytochemicals with diverse log P values. The effect of log P on solubility has been presented in Figure 3. Solubility enhancement was observed for all compounds, except for 20 those compounds with very high oil and very high water solubility. The range of log P values resulting in solubility enhancement was from 0.9 to 3.2. Above or below this range the solubility enhancement was not observed. 25 Example 4: Adsorption of phytochemicals onto fish muscle The experiment of Example 3 was repeated with different phytochemicals including vanillic acid, quercetin. rutin, (+)-catechin, caffeic acid and vanillin. The results are shown in Table 7.

- 25 Vanillic acid is an example of a small phenolic acid. As the dip concentration of vanillic acid increased from 200 to 700 mg/L the adsorption of vanillic acid onto the fish muscle rose from 155 to 620 ig/g respectively. The residual concentration of vanillic acid in the dip solution decreased throughout the 30 minutes dip time, however the major drop was found in the 700 5 mg/L in the initial 10 minutes. A further increase in the applied concentration did not improve the adsorption of vanillic acid and it remained 600 pg/g. Quercetin is an example of a flavonoid with one of the highest antioxidant activity. Nanoemulsion containing quercetin as a dip medium exhibited a maximum drop in residual 10 quercetin concentration after an initial 10 minutes of fish dip followed by fractional changes over the next 20 minutes. Increasing the quercetin concentration in the nanoemulsion from 250 to 1000 mg/L resulted in a consistent rise in adsorption onto the fish. A maximum calculated adsorption of 615 pg/g was observed at a dip concentration of 1000 mg/L. 15 Rutin is glycoside of quercetin that exhibits numerous pharmacological properties. Data for rutin indicated a direct relationship of the calculated adsorbed rutin concentration to the concentration of rutin in the nanoemulsion for 30 minutes of dip time. A maximum calculated adsorption of 849 .g/g was observed for 1200 mg/L of rutin concentration in nanoemulsion as dip medium. 20 (+)-Catechin is a flavan-3-ol compound with a cis- configuration. (+)- Catechin showed a direct relationship between the adsorbed calculated concentration ( tg/g) and the concentration in the nanoemulsion. A maximum calculated adsorbed concentration 581 pg/g of catechin was observed at the 1000 mg/L. An aqueous control containing (+)- catechin 25 concentration of 1000 mg/L was also tested for its adsorption after 30 minutes of dipping. Complete (100%) adsorption (calculated) of catechin by fish muscle occurred after 20 minutes of the fish dip. Caffeic acid is a 3,4-dihydroxycinnamic acid widely found in plants and it has various 30 biological functions. Calculated adsorption of caffeic acid (ig/g) was found to be - 26 proportional to its concentration in the nanoemulsion for concentrations from 200-600 mg/L. Maximum value for the calculated adsorption (457 pig/g) of caffeic acid was found at nanoemulsion concentrations of 1000 mg/L. In the control experiment using water as solvent (1000 mg/L), 380 [tg/g caffeic acid was calculated to adsorb after 30 minutes of dipping the 5 fish. Vanillin is an example of food preservation and flavouring agent. Vanillin in nanoemulsion at 200-1200 mg/L and two aqueous controls at 200 and 500 mg/L showed a large decrease in the residual concentration in first 10 minutes. This decrease in residual concentration 10 continued slowly over the next 20 minutes A maximum calculated adsorption of about 548 pg/g was observed for the maximum vanillin concentration (1200 mg/L) after 30 minutes of dipping the fish. The aqueous solution of vanillin (controls at 200 and 500 mg/L) dip treatment resulted I18 15 and 294 pg/g respectively compared to nanoemulsion during the fish dipping for 30 minutes.

-27 In In n r'JN 10 -1 N r o + +1 f4 -- r 'l m In sH Lf (N4 m~ m +1 ' * +1 +1 +1 +1 (N H4 +1 +1 +1 +1 +1I CY) t.0 +1 In) N qzzr '-1 +1 +1 0 .0 00 In) 10 0 N In) r-I : -4 -4 -4c 00 rn * -:t m c 00 0 -I r- r-4 IZ o) my qT n 10 In M m M M I 10 r4 00 r14 1) cc mr (N N NM +11+ +1 0 + +1 +1 ( - 4- -4-1 +1 -4- +1 aY) 00 0 N In O) 0 +1 +1 I) 00 r~ ff -4 M% -4-4T r) f 0 TT r n r In (N +- +1 +1 +1 cc cc 00 M 4 If n 0 o +1 ZT 0o my cc r* ; +1 w~ w0 cc 4 c -4 In H- C- ( 9 -4 c c0 - +1 +1 +1 +1 +1 +1 +1 +1 +1 +1I +1 +1 - N N ( z 0 Lfl cc cc c r4 10 -z M (Nl In r-' M n 1 -4 " (N 10 '-1 (l N (N S +1 +1 +1 -4n ( ~ + N N, cc r- +1 + +1 -I-+1 +1 +1 CL c c C) N N- -4 0c o c cc m 0o 0 mc00 M- (N4 q:: N r, to -4 -I In 0 0 0 C: V m2 0 o E to I cco I n c In c (j c c 0 c c r4 0 U (N In N, - H r ;t 0c W4 H HU SC> 0. 0ui!1pale2-(+) uil~aflano U!II!UeA oo N " How -4. NN ~ . N ~I ou 3 +1 +1 + + I0 + +1 +1 +1 t +1 +1 +1 Q L m N- cc rI q 1a T +1 +1 +1 my 'zT zt 0) O Ln (N c 0 C M (Nlj 0 * cc N 0 N- c m~ 0 m -- q I~ N In ro M (N) m-z w m cn m z0 H- H-( 10 In N, 1 U') H 11- c- H 0 r - 4 -i r- N m M (N +1 + +1 +1 + +1 0 +1 I'D +1 +1 +1 +1 i-I +1 +1 +1 +1 In r r-' M cc * +1 N 0 41 c Nk10 mo 1-4 r- mccc '-4 1*M m r In In 10 m In m m (N 4 10 me cc In -i+1 +1 0 - m In cc rsj N '-4 +1 0 1 +1 +1 1-1 + -- 4 10 +1 +1 +1 +1 +1 +1 +1 C) + c- w o 0 . 0 +1 N, +1 c N- (Ni N cc 0 '1 rCD c r- N 0 100 C 40 m 0m (.m 0 mY my In r-4 10 In) In -4 (Ni In) N N -4 N In) 1-0 m n cc 0ccc0 0 1 T C+1 r4 rJ + +1 +c c1 In +1 In 4 r -4 +1 +1 0 0 1+1 m +1 +1 +1 + c +1 +1 +1 +1 +1 -1 0 ko 0 c cc0 m m cc0 (Dw- N m (N N N, r-i m InI N* 004 ccr)r- N H(N '-4 Nr c 0 0 )4c E r -4 (N - )-1im 10 ) '-4 (Nim 0cc0 m r-4 '-4 cc 0 1- (I0 m Nm (N 0 0c 0- U w HH .20 UoIufln PPe 3!II!UeA ppe 2Ialje: - 28 Example 5: Adsorption of combination of quercetin and vanillic acid onto fish muscle A combination of vanillin and quercetin in a nanoemulsion at a combined concentration of 205 mg/L was tested for its adsorption onto fish fillets after 30 minutes dip treatment. The 5 amount of quercetin in the original nanoemulsion was lower at 25 mg/L compared to vanillin at 180 mg/L. Quercetin was completely adsorbed onto fish fillet after 10 minutes of fish dip. Residual vanillin (89 mg/L) was found in nanoemulsion after 30 minutes of dip (Table 8). This indicated that 89 pg/g of vanillin should have been adsorbed onto fish fillet. This was confirmed by the re-extraction of 80 jig/g fish. 10 Table 8 Vanillin and quercetin in combined nanoemulsion dip solutions for 10-30 min. offish dip Initial Conc. Residual Concentration of dip solution (mg/L) (mg/L) 0 min. 10 min. 20 min. 30 min. Vanillin 179±10 153±5 126±9 89±17 Quercetin 25±3 0±0 0±0 0±0 Results expressed as average values of two experimental replicates. 15 Example 6: Demonstration of synergistic effects of the compounds. Providing a range of mixtures with synergistic effects. Introduction Trimethylamine (TMA) has been found to be closely related to the sensory scores of Fish (Baixas-Nogueras et al. 2002) and it is directly related to the post mortem age of Fish under 20 any set of storage conditions (Wong & Gill 1987). Moreover, TMA levels in fish are routinely used in industry as a index of freshness quality (Pena-Pereira, Lavilla & Bendicho 2010). Other offensive volatile compounds related to aerobic refrigerated storage are methanthiol, dimethyl disulfide and dimethyl trisulfide (Lindsay 1990). Fish freshness is also related to other volatile compounds, including thiols, short chain alcohols, carbonyl 25 compound and amines (Bdnd et al. 2001).

- 29 The nanoemulsion was shown to be effective in adsorbing preservatives onto fish. Further work was needed to test their efficacy in actually improving the quality of fish during storage. The effect of natural preservatives on TMA in fish was therefore tested. 5 Objectives To determine if the developed nanoemulsion preservation system was effective in maintaining the quality of fresh fish fillets by: 1. Determining if a model nanoemulsion delivery system was better than a water only 10 system in preventing the release of TMA from fish. 2. Choosing the most effective phytochemical treatments based on their potential to be suitable for industrial applications. Materials and methods 15 Chemicals: Standards of 2-hepatnone, and 2-phenyl ethyl alcohol were purchased from Sigma Chemical Co. USA. Trimethylamine (anhydrous, 99%) standard was purchased from Sigma-Aldrich Inc (USA). 2-Nonanone, (R)-(+)-limonene, ethyl butyrate and tridecane were purchased from Aldrich Chemical Co. USA. Ethyl caproate and myrcene were purchased from Fluka 20 Germany. Undecene was purchased from Polyscience (USA). The water used in all analysis deionised using a Milli-Q water system from Millipore Corporation (Massachusetts, USA). Methods: Sample preparation 25 Fresh barramundi fish fillets (Lates Calcarifer) were purchased from the retail display of a local market and stored on ice during transport to laboratory. Nanoemulsions stored at -80'C were thawed and diluted to the desired concentrations with deionised water; the level of dilution varied with the difference phytochemical due to solubility difference. Aqueous solutions of phytochemicals, where required, were freshly prepared in deionised water. Fish 30 was cut into small uniform cubical pieces (2 g) and dipped into the phytochemical solutions - 30 (1200 mg/L) in a beaker in the form of nanoemulsions or water, at a treatment ratio of 1:1 w/v ratio for 20 minutes. These beakers were occasionally shaken by hand for uniform mixing and contact with the fish muscle surface. Fish pieces were drained of excess solutions that were attached to muscles. The fish fillets (2 g) after treatment were placed in 25 mL glass 5 vials, and immediately crimp-capped (magnetic, Teflon lined rubber septum). Samples were either directly taken for the headspace analysis or were stored at -80'C prior to analysis. The -80'C stored samples were thawed at 4'C overnight before headspace analysis. The vials then were stored at 8'C and then subjected to headspace analysis over the 0, 3, 5, 7 and 10 days of storage. 10 Analysis Flasks containing samples were subjected to headspace analysis to determine odour compounds. TMA standards were run at 0-24 mg concentrations. TMA standards were added to isotonic water (0.9% NaCI) in tubes stored on ice. TMA peak areas were plotted against 15 these concentrations, and the sample concentrations were determined by plotting their peak areas against the calibration cruve. Similarly, pure standards were run for ethyl butyratC. undecane, 2-heptanone, tridecane, 2-nonane, dimethyl sulfide, nonanal, hexanal. 2-butanol. ethyl caproate. 3-methylbutanol and phenyl ethyl alcohol. Other compounds for which standards were not available, were identified using the MSD ChemStation data analysis 20 software (Agilent Technologies, Palo Alto, CA, USA) by comparing with the mass spectrum for the relevant compounds from the NIST Chemistry WebBook (Linstrorm el al. 2001). Solid Phase Micro Extraction (SPME) An SPME fibre of DVB/CAR/PDMS (divinylbenzene, carboxen, polydimethylsiloxane) 25 (50/30 pm, 23-Gauge) with an automated holder (Supelco Bellefonte, PA, USA) was used for analysis. It was connected to a multipurpose automated sampler from Gerstel Inc. (USA). For SPME analysis, thawed samples were placed in an autosampler maintained at 4'C. The Fibre was inserted into the glass vial headspace and incubated at 45'C. The SPME fibre was sampled for 45 minutes. Subsequently the fibre was inserted into the injector port of an 30 automated GC-MS and desorbed for 6.6 minutes.

- 31 Gas Chromatography-Mass Spectrometry (GC-MS) conditions Samples were analysed with a 6890N gas chromatograph equipped with an MSD 5975 mass spectrometric detector (Agilent Technologies, Palo Alto, CA, USA). The gas chromatograph 5 was fitted with a DB-WAX column (J&W Science, i.d. 253.00 pm, length 30.0 m, Film thickness 0.25 pm). The column liner (Agilent Technologies, Palo Alto, CA, USA) was borosilicate glass with a plug of resilanised glass wool (2-4 mm) at the tapered end to the column. Helium of ultra high purity (BOC Australia) was used as a carrier gas at a linear velocity of 56 cm/minutes and at a column flow rate of 2.4 mL/minutes The oven temperature 10 was started at 40'C for 2 minutes then increased at 50'C minutes" to 100 C. The temperature was again increased at 20 'C minutes" to 220 'C and held for 4 minutes The MS ion source was kept at 250 'C. Mass spectra in the electron impact (EI) mode were generated at 70 eV and collected from the range m/z 35 to 350 for scan runs. Data analysis was carried out with the help of the MSD ChemStation data analysis software (Agilent Technologies, 15 Palo Alto, CA, USA). Compounds were identified using standard and or by comparison with the mass spectral library. Statistical analysis Gallic acid samples were taken from three experimental replicates and results expressed as 20 the mean values with standard deviations. Results for screening experiments with phytochemicals resulted from two experimental replicates. Selected treatments from the screening trials were further repeated in quadruplicate. These results from quadruplet samples were analysed with Minitab 15 (Minitab Inc., USA) using a general linear model with the Dennett method. P values <0.05 were considered as significant. 25 Results and Discussion Gallic acid showed superior adsorption to the fish muscle when in nanoemulsion form than during aqueous delivery. Hence gallic acid was used as a model compound to compare the effect of its concentration and delivery form in suppressing the production of TMA. Suitable 30 concentrations selected from the model compound data were used for the dip medium for - 32 evaluation of other phytochemicals. Phytochemical treatments resulting in suppression of TMA were then selected as suitable for further repeat testing individually and in combinations. Phytochemical combinations were tested for possible synergistic relationship in activity. 5 From the literature, a maximum limit of TMA for eating acceptability is known to be specie dependent, however it is reported to be normally around 10-15 mg/100 g fish (Connell 1975: Dalgaard, Gram & Huss 1993). A maximum TMA limit for fresh barramundi has not been reported. However, for comparison purposes a TMA level of 10 mg/100 g fish was 10 considered as the cut off point for maximum acceptability during the present study. Example 7: Synergies of phytochemical combinations Synergy existed between both combinations of vanillic acid and BHA, and vanillic acid with quercetin. The total concentration of the nanoemulsions here was kept constant at 1200 mg/L 15 and these nanoemulsions were diluted with water or mixed in a way to preserve a 1:1 ratio of individual compounds. Vanillic acid and BHA in combination showed some form of synergy by keeping the TMA levels lower than for BHA alone and reducing TMA levels on day 10 compared to vanillic 20 acid alone (Figure 4). Similarly vanillic acid and quercetin showed synergy by resulting in lower TMA levels at day 3 compared to their individual treatments at day 3. Vanillic acid and quercetin maintained the TMA levels below 10 mg/1l00 g Fish for 10 and 7 days respectively. Both were able to keep the TMA level to organoleptically acceptable TMA levels throughout the storage, while a combination of both phytochemicals had a more pronounced effect at day 25 3 of storage. These findings suggested synergy might be accomplished through the correct selection of' different combinations of phytochemicals. Example 8: Plant extracts in aqueous and nanoemulsion form A large surge in TMA concentrations was observed in all extract treatments at day 3 (Figure 30 5) as was observed for the purified phytochemicals. This suggested that untreated ish stored at 8*C reached an organoleptically unacceptable level within the first three days. Similarly, for the blank nanoemulsion the useful life was 5 days. All plant extracts, irrespective of the form of delivery, resulted in higher TMA levels at day 3 compared to the blank nanoemulsions. However, subsequent TMA values for plant extracts in the nanoemulsion 5 form were lower than 10 mg/100 g. Apple extract in the form of nanoemulsion tended to reduce TMA levels in stored fish compared to tea and grape seed. Plant extracts (apple, grape seed and tea) in nanoemulsion form, in general, showed better prevention of TMA levels compared to their aqueous solutions (Figure 5). 10 Example 9: Phytochemicals with potential for industrial application TMA levels higher than 15 mg/100 g fish were generally observed in untreated fish on day 3 of storage at 8'C. Storage at 8'C would be regarded as an abusive temperature condition for untreated fish, and it is expected that fish would not be acceptable to eat after this time. Variable results in the suppression the TMA values were observed for different 15 phytochemical treatments. Selected phytochemicals from the earlier described screening experiments were therefore chosen for repetition at a concentration of 1200 mg/L. These analyses were done in quadruplicate for 3 days storage to account for the variation in TMA values. 20 TMA in untreated fish rose to 12 mg/100 g at day 3 of storage (Table 9). The initial average TMA values for the batch of fish as purchased for these replicated experiments was 4±0.10 mg/100 g fish. This suggests that this fish batch had reached an initial higher degree of spoilage than previously used fish samples. All other treatments reduced the level of TMA production in fish fillets. 25 Except for the vanillin nanoemulsion, all the treatments produced significantly (p < 0.05) lower concentrations of TMA than in the treated fish (Table 9). The most effective treatment to suppress TMA values in fish (Table 9) suggested there is a synergistic relationship between vanillin and -carotene in suppression of TMA production. Vanillin alone in the 30 nanoemulsion did not significantly (p < 0.05) affect the TMA levels. However, for p- -34 carotene, the TMA level, at day 3 was 4.7 mg/100 g, above the level produced by combination of vanillin and p-carotene in the nanoemulsions (1.5 mg/100 g). This 1.5 mg/100 g of TMA level at day 3 is even below the initial TMA value on day 0 (4 ± 0.10 mg/100 g fish) indicating that TMA has been removed from the system. 5 Similarly, the combination of P-carotene and curcumin, where the TMA concentration was 3.4 mg/100 g confirms the synergy achieved with combinations of phytochemicals when used in nanoemulsions. In the same manner curcumin alone in the nanoemulsion produced a TMA value (7.7 mg/100 g), that was double than that achieved by the combination of curcumin and 10 p-carotene. P-carotenes has therefore shown synergies, both with curcumin and vanillin. when used in dip solution based around nanoemulsions. Table 9 Effect of different phytochemicals loaded (1200 mg/L) nanoemulsions on the mean TMA levels in treated fish stored at 8 0 C for 3 days Treatments TMA levels (mg/100 g) Untreated (Control) 12.01 Vanillin in nanoemulsion 10.6 Vanillic acid 5.7* BHA 5.4*" 3 -carotene 4.7 * Quercetin 7.2* Vanillic acid and quercetin 5.7*a Vanillic acid and BHA 53*a Vanillic acid and p-carotene 5.8*" Vanillin and oregano oil 5.8*" Vanillin and curcumin 5.7* ' Vanillic acid and gallic acid 4.9*" Vanillin and p-carotene 1. 5 *ahcd I* bc B-carotene and curcumin 3.4* Curcumin 7.7* Vanillin in water 19.0 15 Note: Values with a, b, c and d were significantly (p < 0.05) less than the 25, 50, 75 and 100% blank nanoemulsion controls respectively. * values are significantly (p < 0.05) less than untreated sample - 35 Example 10: Effect of blank nanoemulsion on TMA levels in fish An important result from this work was that nanoemulsion without any phytochemical had an inhibitory effect on TMA production in fish during storage. To confirm this finding, different concentrations of the blank nanoemulsions were made by dilution with water to Final 5 concentrations of 25, 50, 75 and 100 (v/v %). Along with other treatments, these blank nanoemulsions were tested in quadruplicate for their effect on TMA levels in fish during storage at 8 0 C. Blank nanoemulsions at all dilution levels were found to significantly (p < 0.05) reduce TMA 10 level in fish fillets (Table 10) compared to the untreated fish. The TMA suppression capacity of blank nanoemulsion generally decreased on increasing the dilution level of these nanoemulsions. To separate the effects of nanoemulsion from that of the phytochernicals the results for each phytochemical were compared to the blank nanoemulsion. Table 10 shows the effect of different blank nanoemulsion concentration on TMA levels produced in fish. 15 Table 10 Effect of dilution of blank nanoemulsion used as dip solutions fbr fish on TMA levels on day 3 of storage Concentration of Nanoemulsion (%) TMA (mg/100 g) 0 (Untreated) 12.01 25 8.3 50 7.6 75 6.4 100 4.8 Results expressed as mean of quadruplicate experimental repeats 20 Fish when dipped in quercetin nanoemulsions and curcumnin nanoemulsions and stored at 8'C, developed TMA concentrations that were not significantly different (p < 0.05) from that produced using any dilution of the nanoemulsion (Table 10). Use of a combination of vanillin and p-carotene, produced a significantly (p < 0.05) reduced TMA compared with the use of the undiluted (100%) control of nanoemulsion. 25 - 36 The profound impact of nanoemulsion dilution levels on the suppression of TMA levels in treated fish suggests the suppression of TMA might be related to the concentration of HICAPTM 100 in these nanoemulsions. Possible reasons for this might be interaction and binding of TMA with HICAPTM 100 itself or partitioning of TMA into the nanoemulsion oil 5 phase. HICAPTM 100 can form films and acts as a barrier to oxygen, or it may also adsorb TMA and affect its release. All nanoemulsion treatments were made by dilution of stock nanoemulsion with water to 1200 mg/L concentration when used as a dip medium For fish. Since all the nanoemulsions loaded with different phytochemicals/plant extracts required different levels of dilutions based on the different solubilities, a comparison can be drawn 10 between effectiveness and the amount of HICAPTM 100 present in every treatment, irrespective of presence of other phytochemicals. Example 11: Use of Various oil types for formulation of nanoemulsion Introduction: 15 The aim of this experiment was to confirm whether different types of oil other than canola are equally efficient at producing stable nanoemulsions. Moreover, if a model compound (vanillic acid) could show similar solubilities into these nanoemulsions. Materials and Method: 20 Chemicals: The chemicals for the preparation of the nanoemulsions including gallic acid, tocopherol, vanillic acid, ascorbyl palmitate, quercetin, were purchased from Sigma-Aldrich (Steinheim. Germany). In addition R-(+)limonene was purchased from Australia Botanical Products Pty Ltd, while HI-CAPTM100, a hydrophilic modified corn starch (octenyl succinate starch) was 25 obtained from National Starch & Chemical (Australia). All other reagents were obtained from Sigma-Aldrich (Steinheim,Germany). Canola Oil (Crisco vegetable oil) was purchased from the local market. The water used in all analysis was deionised using a Milli-Q water system from Millipore Corporation (Massachusetts, USA).

- 37 Methodology: The formation of nanoemulsions involved two stages: (1) a coarse emulsion was made with a high shear Ultra-TurraxT25 homogenizer (IKA Instruments, Stantenin Breigan, Germany) to reduce the particle size and decrease the viscosity of the emulsion system for effective 5 subsequent processing The coarse emulsions were passed through an air driven Microfluidize r(ModelM- 11. Microfluidics, USA). This involved feeding the coarse emulsion through a 200mL Stainless Steel reservoir. The microfluidizer splits the liquid into two opposing channels each with a 10 stream pore size of 75 .tm in a ceramic interaction chamber where the two streams collide head on. The resultant amplification of pressure in the interaction chamber is 232 times the external applied air pressure for the device. Different pressures were tested ranging from 35 MPa to 95 MPa, while the number of passes through microfluidizer (cycles) ranged from I to 7. 15 The limit of solubility of phytochemicals in the nanoemulsion was measured by adding an excess of the phytochemical or plant extract powders to the oil and water before making the nanoemulsion. These nanoemulsions were then subjected to centrifugation (5000rpm for 10 minutes) so that the excess phytochemicals were removed and visible to assessment at the 20 bottom of the tube. The amount of phytochemicals left in the nanoemulsion, after centrifugation and consequent filtration with 0.45ptm nylon filter, was considered as the soluble level. The treated nanoemulsions were analysed for total phenol content using the Total Phenol Assay (Singleton & Rossi 1965). 25 Results: The results are shown in Figure 6. Discussion: Vanillic acid chosen in this study as a model compound of intermediate log P (1.42) value 30 can be successfully loaded into nanoemulsions made with different types of oils.

- 38 Example 12: Loading stage of bioactives Introduction: The aim of this experiment was to determine whether a model compound could be loaded into nanoemulsion after processing of nanoemulsion. This step is useful for application 5 development as different bioactives could be added to a blank nanoemulsion base. Materials and Method: Pre-mixing: Please refer to Example 11. 10 Post-mixing: Blank nanoemulsions were prepared and vanillic acid was added in excess afterwards with gentle shaking. Results: The results are shown in Figure 7. 15 Discussion: Vanillic acid readily mixed with the blank nanoemulsion and formed a suspension. Upon centrifugation excess vanillic acid separated out of solution as sediment but the amount left as dissolved in the nanoemulsion was essentially the same as the amount found when vanillic acid was added before homogenisation. It should be noted that these suspensions formed 20 before centrifugation can be still used for various applications to carry large amounts of bioactive compound in the suspension. The amount of suspensions made with these nanoemulsions could range from 10 to 15% w/v. This could also be noted that further improvement to this can be made by addition of extra energy post mixing. This energy could come from any source such as ultraturrax, sonication, homogenization, etc. 25 Example 13: Delivery of bioactives to different surfaces Introduction: Earlier in Examples 3-10 delivery of various bioactives to fish was established. This experiment aimed to test the adsorption onto further fresh food surfaces such as meat, fruits 30 and vegetables.

- 39 Material and Methods: All chemicals were of analytical grade and purchased from Sigma-Aldrich (Steinheim, Germany), including trichloroacetic acid and ammonium sulfate. Ethyl acetate was of 5 analytical grade and purchased from Merck (Darmstadt, Germany). Acetonitrile and methanol were of HPLC grade, and purchased from Merck (Darmstadt, Germany). The water used in all analysis was deionised using a Milli-Q water s ystern from Millipore Corporation (Massachusetts, USA). 10 Methodology Preparation of nanoemulsion Nanoemulsions were prepared using the method detailed in Example 11. Results: 15 The results are shown in Figure 8. Discussion: Data in Figure 8 shows vanillic acid can be delivered by the nanoemulsion and adsorbed to all the surfaces. However the amount of bioactive delivered to these surfaces varied. The 20 range of delivery of vanillic acid was from 300 4g/mL to 1000 pg/mL in potatoes and chicken respectively.

- 40 References Baixas-Nogueras, S. n., Bover-Cid, S., Veciana-NoguACs, T., & Vidal-Carou, M. C. (2002). Chemical and Sensory Changes in Mediterranean Hake (Merluccius mierluccius) under 5 Refrigeration (6a^'8 A*C) and Stored in Ice. Journal of Agricultural and Food Chemistry. 50(22), 6504-6510. doi: 10.1021/jfD25615p Bene et al. (2001) A new method of rapid determination of volatile substances: the SPME directed method: Part III. Determination of the freshness of fish. Sensor and Actuators B: 10 Chemical, 72(3); 204-7. Benjamin, Luis and Everett (2011), Static headspace analysis of volatile compounds released from beta lactoglobulin-solubilised emulsions, determined by phase ratio variation method. Food Research International, 44(1), 417-24. 15 Connell, (1975), Control of Fish Quality, Fishing News Books Ltd, London. Dalgaard, Gram & Huss, (1993), Spoilage and shelf-life of cod fillets packed in vacuum modified atmospheres, International Journal of Food Microbiology, 19(4), 283-94. 20 Garti, N., Spernath, A., Aserin, A., & Lutz, R. (2005). Nano-sized self-assemblies of nonionic surfactants as solubilization reservoirs and microreactors for food systems. Soft Matter, 1(3), 206-218. 25 Lindsay, R. C. (1990). Fish flavors. Food Reviews International. 6(4), 437 - 455. Lindstrom, Mallard, Standards & Technology, NIST Chemistry WebBook, National Institute of Standards and Technology, Gathersburg, MD.

-41 McClements, D. J. (2010). Emulsion Design to Improve the Delivery of Functional Lipophilic Components. Annual Review of Food Science and Technology - (nev in 2010). 1(1), 241-269. 5 Morita, K., Kubota, K., & Aishima, T. (2003). Comparison of aroma characteristics of 16 fish species by sensory evaluation and gas chromatographic analysis. Journal of the Science of Food and Agriculture, 83(4), 289-297. doi: 10.1002/jsfa.l 31 l Pazos, M., Gallardo, J. M., Torres, J. L., & Medina, 1. (2005). Activity of grape polyphenols 10 as inhibitors of the oxidation of fish lipids and frozen fish muscle. Food Chemistry 92(3)., 547-557. Pena-Pereira, F., Lavilla, I., & Bendicho, C. (2010). Colorimetric assay for determination of trimethylamine-nitrogen (TMA-N) in fish by combining headspace-single-drop 15 microextraction and microvolume UV-vis spectrophotometry. Food Chemistry. 119(1), 402 407. doi: DOI: 10.1016/j.foodchem.2009.07.038 Sagalowicz, L., & Leser, M. E. (2010). Delivery systems for liquid food products. Current Opinion in Colloid & Interface Science, 15(1-2), 61-72. 20 Selli, S., & Cayhan, G. G. (2009). Analysis of volatile compounds of wild gilthead sea bream (Sparus aurata) by simultaneous distillation-extraction (SDE) and GC-MS. Aficrochemicai Journal, 93(2), 232-235. 25 Theeshan, B., & Aruoma, A. L.-R. A. C. 0. I. (2004). Total phenol, flavonoid, proanthocyanidin and vitamin C levels and antioxidant activities of Mauritian vegetables. Journal ofthe Science of Food and Agriculture, 84(12), 1553-1561.

-42 Wong, K., & Gill, T. (1987). Enzymatic Determination of Trimethylarnine and Its Relationship to Fish Quality. Journal of Food Science, 52(1), 1-3. doi: 10.111 /j.1365 2621.1987.tbl3960.x

Claims (22)

1. A nanoemulsion composition comprising water, at least one edible oil and a modified starch emulsifier; wherein the average particle size of the dispersed oil phase in the 5 nanoemulsion is less than 500 nm and with the proviso that any emulsifier, other than the modified starch emulsifier, is present in an amount of less than 3% w/w of the nanoernulsion composition.

2. A nanoemulsion composition of claim 1 wherein the at least one edible oil is a plant 10 or animal oil or fat or mixtures thereof.

3. A nanoemulsion composition of claim I or claim 2 wherein the at least one edible oil is a plant oil. 15

4. A nanoemulsion composition of claim 3 wherein the plant oil is selected from canola oil, olive oil, safflower oil, peanut oil and soybean oil.

5. A nanoemulsion composition of any one of claims I to 4 wherein the modified starch emulsifier is an alkali metal C 5 -C, 2 alkenyl succinate substituted starch. 20

6. A nanoemulsion composition of claim 5 wherein the modified starch emulsifier is starch sodium-6-octenyl succinate.

7. A nanoemulsion composition of any one of claims I to 6 wherein the at least one 25 edible oil is present in an amount in the range of 5 % to 30 % v/v of the composition.

8. A nanoemulsion composition of claim 7 wherein the at least one edible oil is present in an amount of about 5 % to 20 % v/v of the composition. - 44

9. A nanoemulsion composition of any one of claims 1 to 8 wherein the modified starch emulsifier is present in an amount in the range of 2.5 % to 10 % w/v of the composition. 5

10. A nanoemulsion composition of claim 9 wherein the modified starch emulsifier is present in an amount in the range of 3 % to 7.5 % w/v of the composition.

11. A nanoemulsion composition of claim 10 wherein the modified starch emulsifier is present in an amount of about 5 % w/v of the composition. 10

12. A nanoemulsion composition of any one of claims I to 11, further comprising a bioactive compound.

13. A food preservative composition comprising the nanoemulsion of any one of claims I 1 5 to 12 and at least one food preserving compound.

14. A food preservative composition according to claim 13 wherein the food preserving compound is at least one antioxidant and/or antibacterial phytochemical. 20

15. A food preservative composition according to claim 14 wherein the phytochemical is selected from gallic acid, tacopherols, ascorbic acid and its esters, vanillic acid, quercetin, catechins, caffeic acid, vanillin, rutin, curcumin or a mixture thereof.

16. A food preservative composition according to any one of claims 13 to 15 wherein the 25 composition is further diluted with water.

17. A method of preserving food comprising exposing the food to a food preserving composition according to any one of claims 13 to 16. 30

18. A method according to claim 17 wherein the food is a perishable food. -45

19. A method according to claim 18 wherein the perishable food is fish, meat, fruit or vegetables. 5

20. A method according to any one of claims 17 to 19 wherein the exposing is dipping the food in the food preservative composition.

21. A method according to any one of claims 17 to 19 wherein the exposing is spraying the food preservative composition on the food. 10

22. A functional food comprising the nanoemulsion composition of any one of claims I to 12 and at least one vitamin, mineral, prebiotic, antioxidant, plant sterol or plant extract.