GB2511028A

GB2511028A - Nano emulsions, methods of forming the same and uses thereof

Info

Publication number: GB2511028A
Application number: GB1222829.2A
Authority: GB
Inventors: Weili Li; Christopher John Smith; Emma Jayne Derbyshire; Katie Elizabeth Lane
Original assignee: Manchester Metropolitan University
Current assignee: Manchester Metropolitan University
Priority date: 2012-12-18
Filing date: 2012-12-18
Publication date: 2014-08-27
Also published as: GB201222829D0

Abstract

A method for preparing a stable oil-in-water nano emulsion comprising: (a) combining one or more emulsifiers with oil and water to form a pre-mix; (b) forming a coarse emulsion from the pre-mix of step (a); and (c) forming a nano emulsion from said coarse emulsion, where the emulsifier(s) have an HLB (hydrophilic-lipophilic balance) value of 3-16, and step (c) comprises the use of ultrasound. The emulsifier may be a lipid soluble emulsifier with an HLB value of 3-5, and is preferably lecithin. The emulsifier may be a water soluble emulsifier with an HLB value of 12-16, and is preferably Tween-40 (RTM). Preferably, the method comprises the use of both a lipid soluble emulsifier and a water soluble emulsifier. The oil is preferably a long chain omega-3 polyunsaturated fatty acid (LC3PUFA). Also claimed is the use of an LC3PUFA containing emulsion in food products, nutritional supplements, pharmaceutical products, and cosmetic and body care products. Also claimed is a high yield method for extraction of an oil phase from an oil-in-water nano emulsion comprising the step of subjecting the nano emulsion to desiccation.

Description

Intellectual Property Office Application No. GB1222829.2 RTM Date:20 June 20t4 The following terms are registered trade marks and should be read as such wherever they occur in this document: Tween Intellectual Property Office is an operating name of the Patent Office www.ipo.govuk NANO EMULSIONS, METHODS OF FORMING THE SAME AND USES THEREOF

BACKGROUND

Technical Field

The present invention relates generally to the field of nano emulsions. More particularly, but not exclusively, the present invention concerns stable oil-in-water nano emulsions.

Description of the Related Art

An emulsion is a mixture of two or more liquids that are normally immiscible, wherein one liquid (a dispersed phase) is dispersed in the other (a continuous phase). As an example, oil and water can form either an oil-in-water emulsion where the oil is the dispersed phase and water is the continuous phase, or a water-in-oil emulsion where water is the dispersed phase and oil is the continuous phase.

Common emulsions are generally unstable and tend not to form spontaneously or easily, often requring energy input through shaking, stirring, homogenizing, or exposure to ultrasound. However, over time emulsions tend to revert to the stable state of the different IS phases and in some emulsions, for example an oil and vinegar emulsion, the two phases quickly separate unless shaken continuously.

However, when the droplet size of disperse phase of emulsion is reduced to micro and nano scale, they are generally considered to be more stable.

Nano emulsions and some micro emulsions are translucent and these are often confused with one another: typically light waves are scattered by droplets if their sizes exceed about one-quarter of the wavelength of incident light (390-750 nm), but if the droplet sizes in an emulsion are below this size the light can penetrate through the emulsion without being scattered, thereby appearing translucent. This is highly advantageous in many applications.

Micro emulsions form upon simple mixing of the components and do not require the high energy input generally used in the formation of ordinary emulsions and nano emulsions.

Instead, micro emulsions use emulsifiers to spontaneously create a thermodynamically stable emulsion, by solubilizing" oil molecules with a mixture of emulsifiers, co-emulsifiers, and co-solvents.

However, the stability of a micro emulsion is often easily compromised by changing conditions, such as dilution, heating, or changing pH levels. This makes micro emulsions difficult to use in certain applications, such as food and cosmetic applications. In addition, the emulsifer concentration in a micro emulsion is typically several times higher than that in a translucent nano emulsion and can significantly exceed the concentration of the dispersed phase. Since emulsifiers can present undesirable side effects, their presence is disadvantageous or prohibitive in many applications. The high proportion of emulsifier also significantly limits the proportion of a dispersed phase that a micro emulsion can carry.

In contrast to micro emulsions, nano emulsions are kinetically stable and are therefore, more resilient to changing conditions. Furthermore, nano emulsions usually exhibit optical transparency at higher droplet volume fractions, demonstrate beffer diffusive transport and increased shelf stability. They are therefore considered to be a far superior medium for encapsulating sensitive or volatile ingredients.

It has been observed that most nutrients function differently when incorporated into food matrixes rather than in bulk (Kris-Etherton et al., 2001). In particular, small droplets of nutrients can easily be transported through cell membranes, giving increased blood plasma and erythrocyte concentrations (Huang et al., 2010). Accordingly, the incorporation of nutrients into foods using nano emulsions has proved to be one of the best platforms to enhance oral bioavailability of phytochemicals making them popular in the food industry (Huang et al., 201 0).

Although it is known to use ultrasound to produce nano emulsions (W02005/051305, W02010/ JP60630 and PL1 61 051), none of the prior art to date has managed to achieve stable oil-in-water nano emulsion with a high oil content (higher than 30%).

It is therefore an object of the present invention to prepare stable oil-in-water nano emulsions with a higher oil content (higher than 30%) using ultrasound technology and optimum emulsifier composition for the delivery of nutrients.

Long chain omega-3 (ri-3) polyunsaturated fatty acids (LC3PUFA) have been linked to a spectrum of health benefits (Ruxton 2011). The main sources of LCSPUFA are currently marine originated making them unsuitable br vegetarians and giving rise to concerns about exposure to environmental contaminants and fish stock sustainability (Mozaffarian and Rimm 2006; Jenkins et aL, 2009). Algae are the primary producers of DHA in the food chain and recently, vegetarian algal based LG3FUFA oil has been developed to provide a viable vegetarian alternative to fish oil. The product offers similar benefits to fish oil and can therefore be used as an equivalent source for vegetarians, vegans and non-fish eaters.

So far, the oil has been approved for use in infant formula and in dietary supplements for adults including pregnant women. In addition, clinical trials have demonstrated algal oil-fortified foods represent bioequivalent and safe sources of OHA (Arterburn et a!., 2007; MoCowen eta!., 2010).

It is therefore, another object of the present invention to provide a stabe vegetarian LG3PUFA-containing oil-in-water nano emulsion for incorporation into a range of nutritional and body care products.

Once a new nano emulsion has been created, it is required to submit it to a variety of tests, including the test of oxidative stability of the nano emulsion. This is done by testing the oxidative state of the oil phase after a period of time and in a range of conditions, but the separation process can present diflioulties and achieving a high yield separation of the oil phase was problematic.

It is therefore, a further object of the present invention to provide a high yield method for extraction of the oil phase from an oil-in-water nano emulsion, primarily, but not exclusively, for testing purposes.

SUMMARY OF THE INVENTION

In a first aspect of the present invention there is provided a system for preparing a stable oil-in-water nano emulsion comprising the steps of: (a) combining one or more emulsifiers with oil and water to form a pre-mix; (b) lorming a coarse emulsion from the pre-mix of step (a); and (a) forming a nano emulsion from said coarse emulsion, wherein the emulsifier(s) have an HLB value (Hydrophilic-lipophilic balance) of between approximately 3 and approximately 16 and step (c) comprises the use of ultrasound.

With this arrangement, the emulsification of the emulsion ingredients is found to be surprisingly simple and efficient and the resultant nano emulsion appears to be remarkably stable even with high oil content. By high oil content', what is meant is an oil content of greater than approximately 30 wt% of the nano emulsion.

The emulsifier may comprise a lipid soluble emulsifier with an HLB value of approximately 3 to approximately 5. The emulsifier may comprise a water soluble emulsifier with an HLB value of approximately 12 to approximately 16.

Preferably, the method may comprise use of both a lipid soluble emulsifier with an HLB value of approximately 3 to approximately 10 and a water soluble emulsifier with an HLB value of approximately 10 to approximately 16.

Most preferably, the method may comprise use of a lipid soluble emulsifier with an HLB value of approximately 3 to approximately 5 and a water soluble emulsifier with an HLB value of approximately 12 to approximately 16.

Even more preferably, the method may comprise use of an emulsifier with an HLB value of approximately 4 and a water soluble emulsifier with an HLB value of approximately 15 to approximately 16.

Preferably, the emulsifier comprises lecithin and/or Tween 40. Alternatively, the emulsifier may comprise a combination of lecithin and Tween 40. The combination may comprise an approximate ratio of lecithin: Tween 40 of 1:1. Preferably, the emulsifier(s) is (are) in liquid form.

Preferably, the emulsifier comprises Tween 40 or a Tween 40/lecithin combination when a reduced droplet size takes priority, e.g. food products with a short expiry date. Preferably, ID however, the emulsifier comprises lecithin where longevity of the end product takes priority, e.g. in food products with a longer expiry date, nutritional supplements, topical body care products, etc. Preferably, the proportion of the emulsifier makes up approximately between 1.5 wt% and 7.5 wt% in the total pre-mix. Preferably still, the proportion of the emulsifier makes up approximately between 3 wt% and 6.5 wt% in the total pre-mix. More preferably, the proportion of the emulsifier makes up approximately between 4 wt% and 6.25 wt% in the total pre-mx. Most preferably, the proportion of the emulsifier makes up approximately 6 wt% in the total pre-mix.

Preferably, where two emulsifiers are used, the ratio is approximately 1:1.

Preferably, the proportion of the oil used comprises at least 20 wt%. Preferably still, the proportion of the oil used comprises at least 40 wt%. More preferably, the proportion of the oil used comprises at least 45 wt%, even more preferably, at least 50 wt%. Most preferably, the proportion of the oil used comprises approximately between 45 wt% and 55 wt%, but may be up to approximately 70 wt%.

Preferably, the oil comprises LCSPUFA-containing oil. More preferably, the oil comprises vegetarian LC3PUFA-containing oil. Therefore, the oil may comprise flaxseed oil.

Even more preferably, the oil comprises DHA (docosahexaenoic acid). Most preferably, the oil comprises algae oil.

Preferably, forming the coarse emulsion from the pre-mix comprises mixing the pre-mix.

Preferably, the pre-mix is hand mixed for approximately ID to 60 seconds, more preferably to 50 seconds, most preferably, 30 seconds.

Preferably, forming the coarse emulsion from the pre-mix further comprises a session in a water bath. Preferably, the pre-mix is placed in the water bath for approximately between 0.5 to 2 hours, more preferably, approximately between 0.75 to 1.5 hours, most preferably, approximately 1 hour. Preferably, the water bath is set at a temperature of approximately between 0C and 7OC, preferably still, approximately between 35C and 60C, more preferably, approximately between 4OG and 60C, and most preferably, at approximately 55C.

Preferably, forming the coarse emulsion from the pie-mix comprises further hand stirring of the pre-mix.

Preferably, forming the coarse emulsion from the pie-mix comprises a second session in the water bath as set out above.

Preferably, the pre-mix is subjected to high power mixing to form the coarse emulsion.

Preferably, forming a nano emulsion from said coarse emulsion comprises subjecting the coarse emulsion to ultrasound whilst placed in a cooling jacket. Preferably, a 24KHz sonicator is used.

Preferably, the coarse emulsion is subjected to ultrasound for approximately between 5 to 30 minutes, more preferably, approximately between 5 to 20 minutes, most preferably, approximately 10 minutes. Preferably, the sonicator is set at a power at least 30 amps, preferably still, at least 50 amps, more preferably, at least 70 amps, and most preferably, at approximately 100 amps.

Preferably, prior to step (a), the method comprises preparing a precursor agent. Preferably, the precursor agent comprises a mixture of said emulsifier with one other pre-mix component of oil or water. Preferably, the precursor agent comprises a mixture of said same oil and said same emulsifier. Preferably, the precursor agent comprises at least 40 wt% oil. Preferably still, the precursor agent comprises at least 50 wt% oil. More preferably, the precursor agent comprises at least 60 wt% oil. Most preferably, the precursor agent comprises at least 70 wt% oil. Preferably, the preparation of the precursor agent comprises mixing the emulsifier and the oil together. Preferably, the emulsifier is in liquid form.

Alternatively, the precursor agent comprises a mixture of said same emulsifier and water.

Preferably, the precursor agent comprises at least 40 wt% water. Preferably still, the precursor agent comprises at least 50 wt% water. More preferably, the precursor agent comprises at least 60 wt% water. Most preferably, the precursor agent comprises at least wt% water. Preferably, the preparation of the precursor agent comprises mixing the emulsifier and the water together. Preferably, the emulsifier is in liquid form.

Preferably, the precursor ingredients are placed into a water bath, most preferably after the mixing step.

Preferably, the water bath is set at a temperature of at approximately between 4C and 702G. Preferably still, the water bath is set at a temperature of at approximately between 35C and 60C. More preferably, the water bath is set at a temperature of at approximately between 402C and 6OC. Most preferably, the water bath is set at a temperature of at approximately between 552G.

Preferably, the precursor agent remains in water bath for approximately between 1 and 3 hours. Preferably still, the precursor agent remains in water bath for approximately ID between 1.5 and 2.5 hours. More preferably, the precursor agent remains in water bath for approximately between 1.75 and 2.25 hours. Most preferably, the precursor agent remains in water bath for approximately 2 hours.

Preferably, the proportion of the precursor agent comprises approximately between 1 wt% and 35 wt% relative to said target quantity. Preferably still, the proportion of the precursor agent comprises approximately between 5 wt% and 25 wt%. Most preferably, the proportion of the precursor agent comprises approximately between ID wt% and 20 wt%.

Preferably, the proportion of the water comprises approximately between 20 wt% and 75 wt%. Preferably still, the proportion of the water comprises approximately between 24 wt% and 74 wt%. More preferably, the proportion of the water comprises approximately between 40 wt% and 50 wt%. Most preferably, the proportion of the water comprises approximately 44 wt%.

A therapeutic agent may be dissolved in the oil of the pre-mix and/or the precursor agent.

The above combination of steps yielded a stable oil-in-water nano emulsion with an unexpectedly high % weight of oil, whilst (i) using only a low % weight of emulsifier and (ii) only requiring the use of ultrasound equipment once at the end of the process and only for a short period of time. The nano emulsion was observed to be stable at a range of temperatures (2C to 4OC) for significant periods of time (up to 28 days at 4OC). This system therefore, provides significant improvements over other previously used methods where often more than one ultrasound session is required for longer periods whilst only providing a low oil % weight emulsion.

Other significant advantages were also observed, namely that the system can easily and efficiently form the optional precursor agent, the pre-mix and the coarse emulsion conventional laboratory and industrial equipment. It is envisaged that the system can also be used to provide stable oil-in-water nano emulsions with high % weight of any oil (or a combination of oils) and using a variety of different emulsifiers at low % for a multitude of applications.

Furthermore, a nano emulsion with such a high % weight of a fairly non-descript oil could be used to carry greater amounts of therapeutic agent.

In a second aspect of the invention there is provided use of an LC3PUFA-containing and other oil soluble nutrients nano emulsion ir food products made according to the above system.

In a third aspect of the invention there is provided use of an LC3FLJFA-containing and other oil soluble nutrients nano emulsion in nutritional supplements made according to the above system.

In a fourth aspect of the invention there is provided use of an LG3PUFA-containing and other oil soluble nutrients nano emulsion in pharmaceutical products made according to the above system.

In a fifth aspect of the invention there is provided use of an LC3PUFA-containing nano emulsion in cosmetics and body care products made according to the above system.

It will be appreciated that the preferred features described in relation to the first aspect of the invention apply to the second to fifth aspects of the invention.

In a sixth aspect of the invention there is provided a high yield method for extraction of an oil phase from an oil-in-water nano emulsion comprising the step of subjecting the nano emulsion to desiccation.

With the above method clear oil separation from the nano emulsion at a high yield (over 10% of the total oil therein) was achieved, thereby allowing statistically significant oxidative stability testing. This method of extraction was found to be consistent in its effectiveness and far superior over other methods such as centrifugation and freezing! thawing cycles, which failed to separate the oil, were highly inconsistent or were very low yield.

In a seventh aspect of the invention there is provided a system for preparing a stable oil-in-water nano emulsion comprising the steps of: (a) preparing a precursor agent; (b) combining a proportion of the precursor agent with target quantities of water, an emulsifier and an oil to form a pre-mix; (c) forming a coarse emulsion from the pro-mix of step (b); and (d) forming a nano emulsion from said coarse emulsion, wherein the precursor agent comprises a mixture of said emulsifier withi one other premix component and step (d) comprises the use of ultrasound.

By target quantities what is meant is quantities that substantially amount to the quantity of nano emulsion that is desired. This may therefore be laboratory quantities or mass manufacture quantities or anything in between.

With the addition of a precursor agent comprising an emulsifier and one other pre-mix component, the emulsification of the target quantities of emulsion ingredients is found to be surprisingly simple and efficient and the resultant nano emulsion appears to be remarkably stable even with high oil content. By high oil content, what is meant is oil content of greater than approximately 30 wt% of the nano emulsion.

It is to be appreciated that the preferable features described in relation to the first aspect of the invention may also apply to the seventh aspect of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a better understanding of the invention, and to show how exemplary embodiments may be carried into effect, reference will now be made to the accompanying drawings in which: Figure 1 is a table of ingredient ratios for preparation of a nano emulson according to an exemplary embodiment of the invention; Figure 2 shows tabulated data of droplet measurement distributions for two nano emulsions made according to the exemplary embodiment of the invention; Figure 3 shows comparative graphical data ol droplet size distributions for a Flaxseed oil in a nano emulsion made with different emulsifiers after 10 minutes under ultrasound according to an embodiment of the invention; Figure 4 shows comparative graphical data of droplet size distributions for an Algae oil in a nano emulsion made with different emulsifiers after 10 minutes under ultrasound according to another exemplary embodiment of the invention; Figure 5 shows the experimental design process for obtaining optimal results for a nano emulsion according to another exemplary embodiment of the invention; Figure 6 shows graphical data of p-anisidine test results (demonstrating lipid oxidation) for a nano emulsion made with a lecithin emulsifier; Figure 7 shows graphical data of p-anisidine test results (demonstrating lipid oxidation) for a nano emulsion made with a Tween 40 emulsifier; Figure 8 shows graphical data of p-anisidine test results (demonstrating lipid oxidation) for a nano emulsion made with a combination of Tween 40 and lecithin emulsifiers; Figure 9 shows the experimental design process for obtaining optimal results for oil phase separation from a nano emulsion according to another embodiment of the invention; and Figure 10 shows the experimental design process for incorporating the nano emulsions into a yogurt product.

DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

According to an exemplary embodiment of the present invention there is provided a system for preparing a stable oil-in-water nano emulsion. The system comprises the steps of: a) combining one or more emulsifiers with oil and water to form a pre-mix; (b) forming a coarse emulsion from the pre-mix of step (a); and (c) forming a nano emulsion from said coarse emulsion, wherein the emulsifier(s) have an HLB value (Hydrophilic-lipophilic balance) of between approximately Sand approximately 16 and step (c) comprises the use of ultrasound.

The system was developed using both flaxseed oil and algae oil and a small selection of acceptable edible emulsifiers, although it will be appreciated that other oils and emulsifiers could be used.

As shown in Figure 5, various combinations of the following variables were tried in order to achieve a system for obtaining a stable oil-in-water nano emulsion with high %wt oil content: oil! water quantity, type of emulsifier, quantity of emulsifier, use of an optional precursor agent and ultrasound amplitude used on coarse emulsion.

In particular, 7 wt%, 15 wt%, 20 wt%, 50 wt% and 70 wt% of oil was tried in combination with 2 wt%, 4 wt%, 5 wt%, 6 wt% and 8 wt% of lecithin and Lecithin/Tween 40 emulsifiers in combination with the precursor agent to achieve the coarse emulsion. Furthermore, 7 wt%, wt%, 20 wt%, 50 wt% and 70 wt% of water was tried in combination with 2 wt%, 4 wt%, wt%, 6 wt% and 8 wt% of Tween 40 emulsifiers in combination with the precursor agent to achieve the coarse emulsion. A range of amplitudes of ultrasound were also tried: 30 amps, 50 amps, 70 amps and 100 amps, in order to generate the nano emulsion from the coarse emulsion.

For clarity, Tween 40 comprises polysorbate 40, polyoxyethylene (20) and sorbitan monopalmitate.

In summary, the optimal results were observed when using 6 wt% emulsifier and ultrasound at 100 amps to achieve stable 50 wt% oil in water nano emulsions. All three emulsifiers produced acceptable results, namely a stable nano emulsion, with adequate oxidative stability for an acceptable period of time.

PROTOCOL FOR THE PREPARATION OF A 50% OIL-IN-WATER NANO EMULSION a) Preparation of the precursor agent (optional) A precursor agent is generally used when a lipid soluble emulsifier is employed as one or the only emulsifier, e.g. with an HLB value of less than 10.

The precursor agent is prepared in advance made up of 70 wt% of oil and 30 wt% emulsifier. The oil and emulsifier are mixed together and then the mixture is placed into a water bath set at approximately 552G for approximately 2 hours.

b) Preparation of the pre-mix Where a precursor agent has been prepared and only a single emulsifier with an HLB value of less than 10 is being employed, the pre-mix is composed of: 20 wt% precursor agent; 36 IS wt% of further oil; and 44 wt% water.

In tho event that no procursor agont has beon preparod and an emulsifior with an HLB value of greater than 10 is being employed, the pre-mix is composed of: 6 wt% emulsifier with an HLB value of greater than 10; 50 wt% further oil; and 44 wt% water.

Where a precursor agent has been prepared and an emulsifier with an HLB value of less than 10 and an emulsifier with an HLB value of greater than 10 are both being employed, the pre-mix is composed of: 10 wt% precursor agent; 3 wt% emulsifier with an HLB value of greater than 10; 43 wt% further oil; and 44 wt% water.

c) Formation of the coarse emulsion The pre-mix is hand mixed for approximately 30 seconds before being placed in a water bath for a first session at approximately 55C for approximately 1 hour.

Following thus, the mix is hand mixed for a further approximately 30 seconds before being placed in a water bath for a second time at approximately 55C for approximately 1 hour.

After removal from the water bath, the mix is subjected to high power mixing to form the coarse emulsion.

d) Preparation of the nano emulsion The coarse emulsion is placed in a cooling jacket and a tip of a 24KHz sonicator is immersed (to a maximum depth of 45 mm) into the coarse emulsion. Ultrasound is commenced at approximately 100 amps for approximately 10 minutes.

In the event that the nano emulsion is to be used to deliver a non-oil therapeutic agent, prior to step (b), a therapeutic agent may be dissolved in the oil of the pre-mix and/or the precursor agent.

The nano emulsion is then suitable for incorporation into a product.

Specific examples of the various nano emulsions were produced according to these guidelines and subjected to oxidative stability testing (discussed in detail below).

EXAMPLE 1: Flaxseed oil-in-water nano emulsion Preparation of the nano emulsions a) An optional precursor agent was prepared as follows: PRECURSOR: 70 wt% of flaxseed oil and 30 wt% lecithin in liquid form.

The oil and the emulsifier were mixed together placed into a water bath set at approximately 55C for approximately 2 hours.

b) Three pre-mixes were prepared as follows: PRE-MIX 1: Adding approximately 20 g of the precursor to approximately 36 wt% of flaxseed oil and approximately 44 wt% deionised water (makes up a total of 50 wt% oil with 14% oil from the precursor).

PRE-MIX 2: Adding approximately 50 wt% of flaxseed oil, approximately 44 wt% deionised water and approximately 6 wt% of Tween 40.

PRE-MIX 3: Adding approximately ID g of the precursor to approximately 43 wt% of flaxseed oil, approximately 44 wt% deionised water and approximately 3 wt% of Tween 40 (makes up a total of 50 wt% oil with 7% oil from the precursor).

c) The formation of three coarse emulsions (COARSE 1 2 and 3) were prepared from each of the three pre-mixes by subjecting each pre-mix to hand mixing for approximately 30 seconds before being placed in a water bath for a first session at approximately 55C for approximately 1 hour.

Each mix was hand mixed for a further approximately 30 seconds before being placed in a water bath for a second time at approximately 552C for approximately 1 hour.

After removal from the water bath, the three mixes were homogenised (using a Silverson high power laboratory mixer) to form three coarse emulsions (COARSE 1, 2 and 3).

d) The three coarse emulsions were placed in a cooling jacket and a tip of a 24KHz sonicator (a Hielscher UP400 ultrasonic device) was immersed (to a maximum depth of mm) into each the coarse emulsion sample. Ultrasound was commenced at approximately 100 amps for approximately 10 minutes to obtain NANO 1, NANO 2 and NANO 3, respectively. During that period, the cooling jacket was agitated once per minute by hand to avoid hot spots.

The results The droplet measurement distributions for each of the three samples were tabulated (Figure 2) and plotted (Figure 3).

From Figure 3, it can be seen that the nano emulsion NANO 3 (the Tween4o/ Lecithin sample) gave the greatest proportion of droplet sizes under 500 nm (mean droplet diameter 205 nm), cosely followed by NANO 2 (the Tween 40 sample) (mean droplet diameter 208 nm).

NANO 1 (the lecithin sample) gave a significantly lower proportion of droplet sizes under 500 nm (mean droplet diameter 264 nm).

EXAMPLE 2: Algae oil-in-water nano emulsion Preparation of the nano emulsions a) An optional precursor agent was prepared as follows: PRECURSOR: 70 wt% of algae oil and 30 wt% lecithin in liquid form.

The oil and the emulsifier were mixed together placed into a water bath set at approximately 552C for approximately 2 hours.

b) Three pre-mixes were prepared as follows: FRE-MIX 1: Adding approximately 20 g of the precursor to approximately 36 wt% algal oil and approximately 44 wt% deionised water (makes up a total of 50 wt% oil with 14% oil from the precursor).

PIlE-MIX 2: Adding approximately 50 wt% algal oil to approximately 44 wt% deionised water and approximately 6 wt% of Tween 40.

FRE-MIX 3: Adding approximately 10 g of the precursor to approximately 43 wt% algal oil, approximately 44 wt% deionised water and approximately 3 wt% of Tween 40 (makes up a total of 50 wt% oil with 7% oil from the precursor).

c) The formation of three coarse emulsions (COARSE I 2 and 3) were prepared from each of the three pre-mixes by subjecting each pre-mix to hand mixing for approximately 30 seconds before being placed in a water bath for a first session at approximately 559C for approximately 1 hour.

Each mix was hand mixed for a further approximately 30 seconds before being placed in a water bath for a second time at approximately 559C for approximately 1 hour.

d) The three coarse emulsions were placed in a cooling jacket and a tip of a 24KHz sonicator (a Hielscher UP400 ultrasonic device) was immersed (to a maximum depth of mm) into each the coarse emulsion sample. Ultrasound was commenced at approximately 100 amps for approximately 10 minutes to obtain three nano emulsions: NANO I, NANO 2 and NANO 3, respectively.

The results The droplet measurement distributions for each of the three samples were tabulated (Figure 2) and plotted (Figure 4).

From Figure 4, it can be seen that the nano emulsion NANO 2 (the Tween 40 sample) gave the greatest proportion of droplet sizes under 500 nm (mean droplet diameter 182 nm), closely followed by NANO 3 (the Tween4o/ Lecithin sample) (mean droplet diameter 188 nm).

Again, NANO 1 (the lecithin sample) gave a significantly lower proportion of droplet sizes under 500 nm (mean droplet diameter 256 nm).

COMPARISON OF EXAMPLES 1 AND 2 The above results demonstrate that the optimal droplet size is best achieved by using either Tween 40 or a combination of lecithin and Tween 40 in the pre-mix. With a high proportion of small droplet sizes under 500 nm, the bioavailability of the nano emulsions is expected to be significantly improved.

PROTOCOL FOR TESTING OXIDATIVE STABILITY OF AN OIL-IN-WATER NANO

EMULSION

Lipids are susceptible to oxidative processes in the presence of external components such as light, heat, enzymes, metals, metalloproteins, and micro-organisms. When lipids oxidise they are often responsible for off-flavors. It can also contribute to the loss of essential amino acids, fat-soluble vitamins, and other bioactives. Therefore, lipid oxidaton is a major cause of food quality deterioration.

Lipid oxidation can be tested by checking for a p-anisidine value. The p-anisidine value (or p-Any) measures the content of aldehydes that are generated during the decomposition of hydroperoxides. The reaction of p-anisidine reagent with aldehydes under acidic conditions produces yellowish products that absorb at light at 350 nm. The colour is quantified to provide the p-Any, defined as the absorbance of a solution resulting from the reaction of 1 g of fat in isooctane solution (100 ml) with p-anisidine (0.25% in glacial acetc acid). The test correlates well with the amount of total volatile substances in the oil and so, is a reliable IS indicator of oxidative rancidity oils.

It is well recognised that p-Any should be lower than 20, if oils are to be declared fit for consumption. In general, the lower the p-Any, the better since it is an indication that there are less volatile components in the oil.

In order to assess oxidative stability of the oil in the nano emulsions over time, it was necessary to extract the oil phases at reguar intervals and compare it with the oxidative stability of the oil in normal circumstances and in a coarse emulsion.

Initially, difficulties were encountered during attempts to establish the oxidative stability of various nano emulsion systems. A number of alternative methods were attempted (Figure 9), but the majority of the methods were unsuccessful due to either there being no separation of the oil phase, or the yield was too low and/or too inconsistent to allow effective testing to be performed.

Subjecting the nano emulsion to heat for up to approximately 12 hours at temperatures of up to approximately 65C provided 100% oil separation, but unfortunately resulted in acceleration of oxidation, which yielded false oxidative stability results.

Ultimately, one method was identified that provided separation, consistent high yield of the oil phase and did not affect the oxidative stability.

a) Extraction of the oil phase Successful extraction of the oil phase has been shown to be consistent by subjecting the nano emulsion to desiccation in a glass desiccator.

Desiccation was carried out at cc, 20'C and 40t. Samples were then stored at cc for a minimum of 4 days to 7 days. It was found that at 20'C, the samples required desiccation for at least 2 days, with 40t providing the optimum desiccation temperature, around 24 hours to obtain an acceptable oil extraction yield for the oxidation test.

b) Oxidative testing of the oil phase Oxidative testing was carried out on algae oil samples at three preparation stages of the nano emulsions: bulk oil prior to any emulsification (as a control); oil phase extracted from a coarse emulsion (COARSE 1, 2 and 3); and oil phase extracted from a nano emulsion (NANO 1, 2 and 3): Oil from the three preparation stages was tested at 2 days, 9 days and 16 days.

Accordingly, on, or prior to each testing day, the algae oil phase was extracted from each of NANO 1, 2 and 3 and COARSE 1, 2 and 3. The six extracted algae oil phases were subjected to p-anisidine testing along with a bulk algae oil sample.

The results The results of the tests were plotted graphically on Figures 6, 7 and 8.

It can be seen by comparison of the Figures 6, 7 and 8 that with lecithin as the emulsifier, the algae oil has an oxidative stability over 16 days that closely mirrors that of bulk oil whether in a coarse emulsion (COARSE 1) or a nano emulsion (NANO 1). Up to 16 days, NANO 1 remains at a p-Any of around 4 or 5, whereas the bulk oil p-Any increases up to around Bat 16 days. Accordingly, lecithin as the emulsifier appears to provide a good level of stability for a significant period of time. The lecithin provided a lower proportion of droplet sizes under 500 nm (mean droplet diameter 256 nm) than the other two emulsifier systems, in other words, the other two systems provded smaller droplet sizes. It is thought that a nano-emulsion system using lecithin emulsifier would be advantageous where longevity of the end product takes priority over optimal bioavailability, e.g. in food products with a longer expiry date, nutritional supplements, topical body care products, etc. With a combination of Tween 40 and lecthin as the emulsifier, the algae oil has an oxidative stability over 16 days that departs significantly from that of bulk oil whether in a coarse emulsion (COARSE 3) or a nano emulsion (NANO 3). NANO 3 showed stability at a p-Any of around 5 up to 2 days and then slowly climbed up towards the p-Any 20 up to around 10 days. COARSE 3 appeared to remain stable for a while longer, where the p-Any rose steadily from around S to 20 between 2 and 13 days. Accordingly, the combination of Tween 40 and lecithin as the emulsifier appears to provide an acceptable level of stability for an acceptable period of time (10 days).

Finally, with a Tween 40 emulsifier, the algae oil has an oxidative stability over 9 days that mirrors that of bulk oil whether in a coarse emulsion (COARSE 2) or a nano emulsion (NANO 2) at a p-Any of below 5. However, after 9 days, both the COARSE 2 and NANO 2 demonstrated rapidly declining stability rising to a p-AnV of 20 by day 11 and up to a p-AnV of around 70 by day 16. Accordingly, the Tween 40 emulsifier appears to provide a good level of stability for an acceptable period of time (10 days).

COMPARISON OF OXIDATIVE STABILITY WITH BIOAVAILABILITY INDICATORS

Given the above results, lecithin as the emulsifier appears to provide a good level of stability for a significant period of time. Weighted against the droplet size results, where lecithin provided an acceptable proportion of droplet sizes under 500 nm (volume-surface mean diameter = 256 nm and weighted average mean diameter = 723 nm) than the other two emulsifier systems, it is thought that a nano-emulsion system using lecithin emulsifier would be advantageous where longevity of the end product takes priority over optimal bioavailability, e.g. in food products with a longer expiry date, nutritional supplements, topical body care products, etc. The combination of Tween 40 and lecithin as the emulsifier appears to provide an acceptable level of stability for an acceptable period of time (10 days). Again, when weighted against the droplet size results, where the combination of Tween 40 and lecithin as the emulsifier provided a good proportion of droplet sizes under 500 nm (volume-surface mean diameter = 188 nm and weighted average mean diameter = 328 nm), it is thought that a nano-emulsion system using a combination of Tween 40 and lecithin emulsifier would be advantageous where bioavailability takes priority over longevity, e.g. food products with a short expiry date.

The Tween 40 emulsifier appears to provide a good level of stability for an acceptable period of time (10 days). When we consider the droplet size results, where the Tween 40 emulsifier provided a good proportion of droplet sizes under 500 nm (volume-surface mean diameter = 182 nm and-weighted average mean diameter = 329 nm), it is thought that a nano-emulsion system using a Tween 40 emulsifier would also be advantageous where bioavailability perhaps took priority over longevity, e.g. food products with a short expiry date. However, it is thought that the closeness of the Tween 40 bioavailability results compared with a combination of Tween 40 and lecithin perhaps provides Tween 40 with an edge over the combination of Tween 40 and lecithin.

PRODUCT TESTING

EXAMPLE: Fortified yogurt product development and formulation In order to test the acceptability of a product that incorporated the nano emulsions, a range of fortified yogurt products were created and taste-tested.

Development and sensory analysis of 600mci/lOOg DHA dosage product The results of the tests can be seen in Figure 1 0.

Firstly, an amount of 3.428g of each of NANO 1, NANO 2 and NANO 3 (containing 600mg of omega-3 fatty acid docosahexaenoic acid (DHA) and 1.714g oil) were incorporated into bOg samples of plain (unflavoured) yogurt. This provided an unacceptable taste test.

Secondly, an amount of 3.428g of each of NANO 1, NANO 2 and NANO 3 (containing 600mg of omega-a fatty acid docosahexaenoic acid (DHA) and 1.7149 oil) were incorporated into 1 DOg samples of plain (unflavoured) yogurt with the addition of 1 3g of one of strawberry, raspberry, or chocolate milkshake flavouring. This provided an unacceptable taste test with a bitter aftertaste and presented issues with consistency. The strawberry flavouring presented the best result.

Finally, an amount of 3.428g of each of NANO I, NANO 2 and NANO 3 (containing 600mg of omega-3 fatty acid docosahexaenoic acid (DHA) and 1.714g oil) were incorporated into bOg samples of plain (unflavoured) yogurt with the addition of 13g of a natural strawberry flavouring and O.4g of a sweetening agent, Agave nectar. This provided an acceptable taste test with a similar consistency to other yogurt products.

Development and sensory analysis of 75Dm p1 1000 DHA dosage product The dosage was adjusted following a pilot trial (600mg DHA), which showed little change in blood levels. The dose was amended to 1500mg, which represented the oil manufacturer's guidelines.

Each of 96 trial participants was given lOg of three types of strawberry favoured and sweetened yogurt using the following formulations: Sample _________ Bulkoil Nanoemulsion Control Ingredient Percentage Total to make 2 litres Percentage Total to make 2 litres Percentage Total to make 2 litres Pouringyogurt 84.442 1688.84 82.3 1646 86.56 1731.2 Agave 13 260 13 260 13 260 Emulsion 0 0 4.285 85.7 0 0 Oil 2.142 42.84 0 0 0 0 Flavour 0.4 8 0.4 8 0.4 8 Total 99.984 1999.68 99.985 1999.7 99.96 1999.2 The "bulk oil" yogurt represented algae oil not as an emulsion, the "nano-emulsion" yogurt represented the use of the algae oil as a nano-emulsion according to the invention and the "control" yogurt represented the yogurt without an oil additive. Accordingly, a 200g sample of each of the "bulk oil" and the "nano-emulsion" yogurts both provided 1500mg of DHA.

Sensory analysis was conducted. The results of which are shown below and in Figure II: Sanlaic Bulk Nno Control an eala an Mcan ar Sand444 "ra 1.'2 5,35a 1.72 6, l' 1c Arearancn 5A 1.40 5.43 1.30 5.51 1.54 Flnvour 6,23 1% 3.65 2.00 6.31A 1.74 :s..sc' 1.61 4733 1.4 ¶.6 1.2 Cnng,stcncv 4.17 1.67 2.2 1.54 3% .55 Afiertaatcn 5.5' 7,73 3.46 3.8? 1.70 0a:craiiacnzptabiilx: 1.74 3,S9 L95.95-1.73 Data arc uceac,:tcd a r.rcarLa and atanjaxd 4c',aDfl4. thffcrct 1,'rtcra an ±3 garre an dcnolc anna: thatacc s,gnificanth diilu:e.atrc nO 1. .4p.46-66 [7: iJaing 1*n and Dc,caaag teat ar,d a Ec,nferanj can'ectcn at S pr cent (n9'S.

The results indicate that the panel of 96 volunteers did not detect significant differences between the "control", "bulk oil" enriched and "nano emulsion" enriched yogurts for appearance and consistency attributes.

1 0 The "bulk oil" enriched yogurt closely mirrored the "control" yogurt in the other attributes.

Further development is intended for the improvement of the aftertaste, texture and flavour of the "nano emulsion' enriched yogurt to increase the overall acceptability of an enriched yogurt product.

CONCLUDING REMARKS

Until the present invention a 50% oil nano emulsion system had not yet been developed for use in the food industry. With the nano emulsions of the present invention, a higher dosage of a given oil can be emulsified.

With regard to the specific flaxseed and algae oil examples, the nano emulsions provide at least acceptable bioavailability and good stability for satisfactory periods of time. These nano emulsions therefore, have the potential to be used in most liquid food systems, such as milk or soya, yogurt pots and drinks, salad dressings, ice cream and frozen yogurt. The nano emulsions using either oil present a suitable option for a LC3FUFA source to the vegetarian and vegan population.

It is also envisaged that the specific flaxseed and algae oil nano emulsions of the present invention may have a wider target market by being suitable for use in pharmaceuticals, nutraceuticals and topical body care products.

Finally, the system provided by the present invention is expected to be suitable for achieving stable, high oil % oil-in-water nano emulsions for a variety of commercial and industrial applications.

Although a few preferred embodiments have been shown and described, it will be appreciated by those skilled in the art that various changes and modifications might be made without departing from the scope of the invention.

Claims

CLAIMS1. A system for preparing a stable oil-in-water nano emulsion comprising the steps of: (a) combining one or more emulsifiers with oil and water to form a pre-mix; (b) forming a coarse emulsion from the pre-mix of step (a); and (c) forming a nano emulsion from said coarse emulsion, wherein the emulsifier(s) have an HLB value (Hydrophilic-lipophilic balance) of between approximately 3 and approximately 16 and step (c) comprises the use of ultrasound.
2. The system according to claim 1, wherein the emulsifier comprises a lipid soluble emulsifier with an HLB value of approximately 3 to approximately 5.
3. The system according to claim 1, wherein the emulsifier comprises a water soluble emulsifier with an HLB value of approximately 12 to approximately 16.
4. The system according to claim 1, wherein the method comprises use of both a lipid soluble emulsifier with an HLB value of approximately 3 to approximately S and a water soluble emulsifier with an HLB value of approximately 12 to approximately 16.

CD 15
5. The system according to claim 1, wherein the emulsifier comprises lecithin and/or Tween 40.
6. The system according to claim 4, wherein the emulsifier may comprise a combination of lecithin and Tween 40 In an approximate ratio of lecithin: Tween 40 of 1:1.
7. The system according to any one of claims 1 to 6, wherein the emulsifier(s) is (are) in liquid form.
8. The system according to any one of claims I to 7, wherein the proportion of the emulsifier makes up approximately between 1.5 wt% and 7.5 wt% in the total pre-mix.
9. The system according to any one of claims ito 8, wherein the proportion of the oil used comprises at least 20 wt%.
10. The system according to any one of claims Ito 9, wherein the oil comprises LC3PUFA-containing oil.
Ii. The system according to any one of claims Ito 10, wherein the oil comprises algae oil.
12. The system according to any one of claims I to ii, wherein forming the coarse emulsion from the pre-mix comprises mixing the pre-mix by hand for approximately 10 to 60 seconds.
13. The system according to claim 12, wherein forming the coarse emulsion from the pre-mix further comprises a session in a water bath for approximately between 0.5 to 2 hours at a temperature of approximately between 0°C and 70°C.
14. The system according to claim 13, wherein forming the coarse emulsion from the pre-mix comprises further hand stirring of the pre-mix.
15. The system according to any one of claims 13 or 14, wherein forming the coarse emulsion from the pre-mix comprises a second session in the water bath.
16. The system according to any one of claims I to 15, wherein the coarse emulsion is subjected to ultrasound for approximat&y between 5 to 30 minutes using a 24KHz sonicator set at a power at least 30 amps.
17. The system according to any one of claims 1 to 16, wherein prior to step (a), the method comprises preparing a precursor agent.
18. The system according to claim 17, wherein the precursor agent comprises a mixture of said emulsifier with one other pre-mix component of oil or water.
19. The system according to claim 18, whereIn the precursor agent comprises a mixture of said same oil and said same emulsifier.
20. The system according to any one of claims 18 or 19, wherein the precursor agent comprises at least 40 wt% oil. r
21. The system according to any one of claims 18 to 20, wherein the preparation of the precursor agent comprises mixing the emulsifier and the oil together.
22. The system according to any one of claims 17 or 18, wherein the precursor agent comprises a mixture of said same emulsifier and water.
23. The system according to claim 22, wherein the precursor agent comprises at least 40 wt% water.
24. The system according to any one of claims 22 or 23, wherein the preparation of the precursor agent comprises mixing the emulsifier and the water together.
25. The system according to any one of claims 17 to 24, wherein the precursor ingredients are placed into a water bath after the mixing step.
26. The system according to claim 25, wherein the water bath is set at a temperature of at approximately between 4°C and 70°C.
27. The system according to any one of claims 25 or 26, wherein the precursor agent remains in the water bath for approximately between I and 3 hours.
28. The system according to any one of claims 17 to 27, wherein a therapeutic agent is dissolved in the oil of the pre-mix and/or the precursor agent.
29. Use of an LC3PUFA-containing and other oil soluble nutrients nano emulsion in food products made according to the method of any one of claims 1 to 28.
30. Use of an LC3PUFA-containing and other oil soluble nutrients nano emulsion In nutritional supplements made according to the method of any one of claims 1 to 28.
31. Use of an LC3PUFA-containing and other oil soluble nutrients neno emulsion in pharmaceutical products made according to the method of any one of claims ito 28.
32. Use of an LC3PUFA-containing nano emulsion in cosmetics and body care products made according to the method of any one of claims ito 28.
33. A high yield method for extraction of an oil phase from an oil-in-water nano emulsion CD comprising the step of subjecting the nano emulsion to desiccation.iS
34. A system for preparing a stable oil-in-water nano emulsion comprising the steps of; (a) preparing a precursor agent; (b) combining a proportion of the precursor agent with target quantities of water, an emulsifier and an oil to form a pre-mix; (c) forming a coarse emulsion from the pre-mix of step (b); and (ci) forming a nano emulsion from said coarse emulsion, wherein the precursor agent comprises a mixture of said emulsifier with one other premix component and step (d) comprises the use of ultrasound,