Field of the invention

The invention relates to protein extracts, concentrates and isolates, especially those obtainable from plant sources that are useful in the manufacture of foods and food products, especially as emulsifiers, creaming agents and the like.

Background of the invention

Vegetable and plant proteins have been used for some time in the manufacture of food products. In some applications these sources of proteins are used for nothing more than increasing the nutritive quality of food products. For example, soy protein may be added to meat products to increase protein content.

In other applications the protein sources are added to modify a quality or characteristic of a food product, or otherwise to provide a quality or characteristic to a mixture of ingredients which then leads to the formation of a food product. Examples of the latter are protein sources that are used as emulsifiers, gelling agents, thickeners, whipping agents, creaming agents and whitening agents.

Vegetable protein isolates and concentrates have been described for use in stabilising oil-in-water emulsions. These are essentially colloidal dispersions where oil forms a discrete phase and water forms a continuous phase. Depending on factors including relative amount of oil to water, the type of oil, the amount of solids, polysaccharides, pH, ionic strength and type and concentration of salt, these oil-in-water emulsions may be provided in the form of a liquid, a semi liquid in the form of a gel or paste, or a solid. Examples of oil-in-water emulsions that are liquids, semi liquids and solids include milk, low fat mayonnaise and cheese, respectively.

Most protein isolates that are useful for stabilising oil-in-water emulsions work by unfolding at the interface of the oil and water phases to provide the requisite degree of hydrophobicity for stabilising the emulsion. The hydrophobic portions of the proteins are understood to at least partially dissolve into the discrete phase, leaving the more aqueous soluble portions to dissolve into the continuous phase.

The degree of unfolding and surface hydrophobicity may be enhanced by thermal denaturation of the protein prior to emulsification, by chemical modification of the protein or by enzymatic modification of the protein. The latter are understood to increase an entangled network of protein molecules at the interface of the oil and water phases.

Studies to date have generally found that thermal denaturation of lupin proteins is the most effective modification for improving emulsifying properties of lupin proteins.

Many of the lupin isolates or concentrates obtained to date are formed by de-oiling in hexane and these compositions tend to contain insoluble fibre or unpleasant flavours.

There remains a need for new protein isolates or concentrates useful as oil in water emulsifiers and in other food applications such as a dispersant or surfactant.

Further, given current consumer demand for low fat foods, there is also a need for oil in water emulsifiers for stabilising low fat emulsions, especially for stabilising low fat dairy products, mayonnaises, salad dressings, low fat sauces such as hollandaise sauce, low fat confectionery fillings and low fat chocolate-like products.

Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and regarded as relevant by a person skilled in the art.

Summary of the invention

The invention seeks to improve or at least to minimise one or more of the above problems or limitations and in one embodiment provides a composition useful as an emulsifier for stabilising an oil-in-water emulsion to form a food product, the composition including: - lupin proteins in an amount of about 70 to 98 % by dry weight of the composition,

wherein about 85 to 95 % of the lupin proteins have a molecular weight greater than 20 kDa.

In a further embodiment there is provided a composition useful as an emulsifier for stabilising an oil-in-water emulsion to form a food product, the composition including:

- lupin proteins in an amount of about 70 to 98% by dry weight of the composition,

said lupin proteins being substantially free of attached fibre.

In other embodiments there is provided a composition useful as an emulsifier for stabilising an oil-in-water emulsion to form a food product, the composition produced by a process including:

- contacting an extract of lupin proteins with a carbohydrase in conditions for removal of carbohydrate from the lupin proteins;

- recovering lupin proteins from the extract to form a composition wherein about 85- 95% of the lupin proteins have a molecular weight greater than 20 kDa.

In other embodiments there is provided a composition useful as an emulsifier for stabilising an oil-in-water emulsion to form a food product, the composition produced by a process including:

- providing an extract of lupin proteins;

- precipitating lupin proteins from the extract at a pH of about 5 to about 6;

- recovering the precipitated proteins. In further embodiments there is provided a food product including oil or fat, water and a composition useful as an emulsifier for stabilising an oil-in-water emulsion. In one embodiment the food product is provided in the form of an oil-in-water emulsion such as a milk, cheese or like product, an ice cream, or mayonnaise, salad dressing, sauce, confectionery filling or like product. In one embodiment the food product is a low fat food product.

In another embodiment the food product is provided in the form of a creaming agent, a dispersant or a surfactant.

In yet further embodiments there is provided a process for forming an emulsifier for stabilising an oil-in-water emulsion including:

- contacting an extract of lupin proteins with a carbohydrase in conditions for removal of carbohydrate from the lupin proteins;

- recovering lupin proteins from the extract to form a composition wherein about 85- 95% of the lupin proteins have a molecular weight greater than 20 kDa.

In another embodiment there is provided a process for forming an emulsifier for stabilising an oil in water emulsion including:

- providing an extract of lupin proteins;

- precipitating lupin proteins from the extract at a pH of about 5 to about 6;

- recovering the precipitated proteins.

Brief description of the drawings

Figure 1 : Size exclusion profile of the composition of the invention.

Figure 2: Matrix Assisted Laser Desorption/lonisation - Time of Flight (MALDI-TOF) profile of the composition of the invention. Figure 3: Typical effect of different ratios of oil to water on the emulsion activity of the composition.

Figure 4: Typical effect of different ratios of water to oil on the emulsion activity of the composition.

Figure 5: Typical effect of pH on emulsion activity of the oil-in-water emulsifier.

Detailed description of the embodiments

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

In research leading to the invention, the inventors sought to provide improved oil in water emulsifiers, and in particular emulsifiers for stabilising low fat oil-in-water emulsions and food products formed therefrom.

The inventors have surprisingly found that lupin protein extracts, concentrates or isolates that predominantly consist of proteins having a higher molecular weight are useful for stabilising oil-in-water emulsions, and in particular for stabilising oil in water emulsions having a low oil content relative to water.

Stabilisation of oil-in-water emulsions is particularly important when these emulsions are used in low fat products which require stability to maintain quality during their shelf life.

Thus the compositions described herein find particular application in the production of low fat food products formed from low fat containing oil-in-water emulsions. Advantageously the compositions of the invention described herein are capable of stabilising oil-in-water emulsions across a wide range of oil to water ratios, pH, salt and temperature conditions.

A further advantage is that the emulsifier compositions of the invention described herein is that these emulsifying characteristics do not require additional thermal denaturation or chemical or enzymatic modification.

Thus in one embodiment there is provided a composition useful as an emulsifier for stabilising an oil-in-water emulsion to form a food product, the composition including:

- lupin proteins in an amount of about 70 to 98 % by dry weight of the composition,

wherein about 85 to 95 % of the lupin proteins have a molecular weight greater than 20 kDa.

Typically the majority of the lupin proteins have a molecular weight no greater than about 250 kDa. In some embodiments proteins having a greater molecular weight (i.e greater than 25OkDa) may be provided in a low relative abundance.

An emulsion is generally understood as a mixture of two or more immiscible liquids, e.g. oil and water, in which one liquid forms a discrete phase and another a continuous phase. Liquids are said to be immiscible if they are not able to mix, in any proportions, to form a homogenous solution. Generally, a solution is homogenous if the liquids that make up the solution are uniformly dispersed throughout the solution.

The compositions of the present invention are capable of stabilising mixtures of aqueous solutions and oils by forming a suspension of the oil phase in the aqueous phase (i.e. the oil phase forms a discrete phase and the aqueous phase forms a continuous phase), thereby preventing or minimising phase separation of the oil phase from the aqueous phase and increasing the homogeneity of the mixture. This results in an even distribution of the oil phase throughout the mixture. Phase separation may occur by the coalescence of the discrete oil droplets into larger droplets or creaming, where the oil migrates to the surface of the aqueous phase. Minimising can mean any of reducing, diminishing, lessening, curtailing or decreasing phase separation.

An oii-in-water emulsion is generally understood as meaning an emulsion wherein oil forms the discrete phase and water forms the continuous phase.

A low fat oil-in-water emulsion is generally understood as meaning an oil-in-water emulsion in a low-fat product.

An emulsifier is generally understood as meaning a substance which stabilizes an emulsion by increasing its kinetic stability. In particular, once an emulsion has been established, the emulsifier minimises phase separation so that the relevant discrete and continuous phases forming the emulsion are maintained.

In embodiments described herein, the composition includes lupin protein whereby about 70 to 98 % of the dry weight of the composition is lupin protein. Preferably the composition includes lupin proteins in an amount of about 75 to 98 %, 80 to 98 %, 85 to 98 %, or 90 to 98 %.

The inventors have also found that useful oil-in-water emulsifiers can be provided by removing attached and/or entrapped fibre from lupin proteins. An example of attached fibre is O- and N- linked carbohydrates. The removal of fibre improves the emulsion activity.

Thus in another embodiment there is provided a composition useful as an emulsifier for stabilising an oil-in-water emulsion to form a food product, the composition including:

- lupin proteins in an amount of about 70 to 98 % by dry weight of the composition,

said lupin proteins being substantially free of attached fibre.

In one embodiment, about 85 to 95% of the lupin proteins have a molecular weight greater than 20 kDa.

In these embodiments, most if not all of the proteins are provided without having sugars or carbohydrates or fibre attached to them and/or entrapped within them. It will be clear that not all attached and/or entrapped fibre may be removed and that some residual fibre may remain. The residual fibre, carbohydrates and/or sugars may be present due to, for example, incomplete digestion of the fibre during the preparation of the protein composition (discussed in more detail below). However, it will also be clear that it is desirable to remove as much attached and/or entrapped carbohydrate as possible from the protein, thereby producing a protein composition having no or very little attached or entrapped sugars, carbohydrates and/or fibre. Therefore, in the context of the present invention, "substantially free" refers to compositions that have no attached and/or entrapped fibre, or if the fibre is present, it is only present incidentally e.g. by incomplete digestion.

Typically in the composition of the invention about 30 to 35 % of the lupin proteins have a molecular weight from about 25 to 55 kDa.

Typically about 25 to 30 % of the lupin proteins have a molecular weight from about 60 to 80k Da.

Typically about 25 to 40 % of the lupin proteins have a molecular weight from about 200 to 250 kDa.

In certain embodiments, the relative abundances of the molecular weight range of lupin protein in the composition are as shown in Table 5 or Table 6.

The lupin proteins may include α- and β-conglutin and fragments thereof.

Typically the composition further includes lipid in an amount of from about 10 to 30 % by dry weight of the composition.

About 70 to 80 % of the lipids may contain unsaturated fatty acid chains.

About 80 to 90 % of the fatty acid chains in the lipid are C18 (i.e. having 18 carbon atoms). Typically the lipid is lupin lipid.

One particular advantage of the composition is that the lipid component of the composition is stable, In particular, the lipid within the composition has been observed to have a shelf life of up to 12 months. In certain embodiments, the stability of the lipid component is believed to facilitate maintenance of oil-in-water emulsions. In particular, without wanting to be bound hypothesis, it is believed that the lipid component may dissolve into the discrete oil phase and in doing so be particularly effective for stabilising an oil-in-water emulsion over a longer term, thereby providing a product with improved shelf life.

Another advantage is that further to use as an emulsifier, the composition of the invention derived from lupin forms a useful source of plant-derived lipid having long shelf life for use in food technology applications where addition of lipid is required to fulfil a nutritional or functional need.

Typically, the composition of the invention has an ANS hydrophobicity value of about 0.75 to 0.85 at pH 7.4. Without wanting to be bound by hypothesis, it is believed that the relatively high hydrophobicity of the compositions of the invention means that the compositions are more likely to dissolve into an oil phase than aqueous phase, thereby stabilising oil-in-water emulsions, and in particular low fat oil-in-water emulsions.

An ANS hydrophobicity value or score is a measure of the hydrophobicity of a given protein- containing composition. The value represents the degree of binding of ANS dye to hydrophobicity regions of proteins. The ANS value is influenced by the solubility of the composition in the given solvent in which the ANS hydrophobicity assay is conducted. As exemplified in the Examples further herein, the ANS value for the composition of the invention is lower at more acidic pH reflecting the limited solubility of the composition at acidic pH. It will be understood that the ANS value is a measure of hydrophobicity of the composition of the invention as it exists after the treatment that is provided to the composition in the ANS assay steps as in Example 3. The ANS hydrophobicity assay is also described in Howe A et al. 2008 Pharma. Res. 25: 1487- 1499. The composition is generally partially insoluble in an aqueous solution having a pH of about 5.5. It is more soluble below pH 5.2 and above pH 5.8.

In one embodiment the relative abundance of the amino acids in the composition of the invention is as described in Table 10 herein.

Typically the composition of the invention does not include a fermentation product such as lactic acid, or lecithin.

The composition according to the invention may be produced by a number of processes. In one embodiment the composition is produced by a process including:

- contacting an extract of lupin proteins with a carbohydrase in conditions for removal of carbohydrate from the lupin proteins.

- recovering lupin proteins from the extract to form a composition wherein about 85 to 95 % of the lupin proteins have a molecular weight greater than 20 kDa.

Typically the carbohydrase is a pectinase cellulase and/ or hemicellulase.

In one embodiment the process includes the following steps:

- alkali treatment of a lupin protein-containing slurry; and thereafter

- carbohydrase treatment of the protein containing component of the slurry,

- recovering a fraction from the carbohydrase-treated slurry, said fraction including lupin proteins wherein about 85 to 95 % of the lupin proteins have a molecular weight greater than 20 kDa.

In another embodiment, the process includes the following steps:

- providing an extract of lupin proteins; - precipitating lupin proteins from the extract at a pH of about 5 to about 6;

- recovering the precipitated proteins.

The extract of lupin proteins may be provided in the form of a lupin protein-containing slurry.

The lupin protein-containing slurry may be formed from milling of de-hulled lupins to form a slurry. Prior to milling, the de-hulled lupins may have been steeped in water. The water may be heated to temperatures of no more than about 7O ⁰ C. In certain embodiments the lupins have not been de-oiled with an organic solvent such as hexane for removing lupin oils or fats, although in other embodiments de-oiling is possible.

The lupin protein-containing slurry generally has a pH in the range of 8 to 9, preferably about 8.4. The adjustment of the pH may be provided by an alkali, NaOH being one example. At the completion of alkali treatment, a protein-containing supernatant and fibre-containing pellet is formed. The protein-containing supernatant may then be separated from the fibre-containing pellet. This can be done by, for example, decanting the supernatant from the pellet.

Typically the lupins are of genus Lupinus. Particularly preferred species are Angustifolius, Luteus, Mutablis and other low alkaloid varieties of species including Albus. In one embodiment the lupin is not a pea, especially not a member of genus Pisum.

While not wanting to be bound by hypothesis, the inventors believe that the protein- containing supernatant is a mixture of higher and lower molecular weight proteins and residual lupin fibre, the latter remaining after alkaline treatment.

The inventors recognised that by treating this fibre component of the protein-containing supernatant, it then becomes possible to separate the lower molecular weight proteins from the higher molecular weight proteins. From this, the inventors were able to observe that the composition having higher molecular weight proteins is useful as an oil-in-water emulsifier. The treatment according to the invention involves the use of fibre-hydrolysing enzymes. In one preferred embodiment, the fibre-hydrolysing enzyme is a carbohydrase. The carbohydrase may be a pectinase, celfulase or hemicellulase. Preferably the carbohydrase is an enzymatic composition of cellulase and pectinase.

As discussed previously, it is desirable to split glycans to remove as much carbohydrate as possible from the protein, thereby producing a protein composition having no or very little attached or entrapped sugars, carbohydrates and/or fibre. Therefore, in a preferred embodiment, most if not all of the proteins are provided without having sugars or carbohydrates or fibre attached to them and/or entrapped within them. It will be clear that not all fibre may be removed and that some residual fibre may remain. The residual fibre, carbohydrates and/or sugars may be present due to, for example, incomplete digestion of the attached fibre during the protein preparation process (e.g. in the alkali and/or enzyme treatment steps).

In one embodiment the carbohydrase treatment digests soluble fibre. The fibre may or may not be attached to protein prior to digestion.

The quantity of enzyme used is dependent on the specific activity of the enzyme at the pH and temperature of the incubation period.

For example the treatment might be designed to be completed in one hour with the enzymic reaction being carried out at a pH range of from about 6 to 8.

Generally the enzymic reaction is completed when the protein-containing supernatant becomes less turbid.

After the completion of the enzymic reaction it may be desirable to deactivate the enzymes. This can be done by, for example, changing the pH of the supernatant and/or heat treatment of the supernatant.

The inventors have found that the decrease in specific gravity of the soluble protein fraction enables better separation of protein fractions through a clarifier which uses centrifugal force to separate the insoluble from the soluble protein fractions. At completion of the enzyme reaction, the higher molecular weight proteins in the enzyme-treated, protein-containing supernatant are separated from the lower molecular weight proteins. This can be achieved by adjusting the pH of the supernatant to the isoelectric point of the higher molecular weight proteins in the supernatant to precipitate the higher molecular weight proteins. The pH range may be from about 5 to 6, preferably about 5.5.

After precipitation of higher molecular weight proteins, these higher molecular weight proteins can be recovered by any technique that enables the precipitated proteins to be separated from lower molecular weight protein of less than about 30 kDa, including centrifugation and/or membrane filtration.

The recovered proteins can also be further treated by, for example, adjusting the pH, washing and/or spray drying.

In certain embodiments there is provided a food product or ingredient for formation of a food product including water, and oil and/or fat and an emulsifier composition of the invention, as previously discussed. The food product may be provided in the form of an oil-in-water emulsion, or it may be homogenised to create an emulsion. In the latter embodiments the emulsion may be stabilised by a composition as described above.

Preferably the food product or ingredient is a low fat product formed that contains an oil- in water emulsion.

Particularly useful oils for use in a food product or ingredient of the invention are those that are edible, e.g. animal and plant oils.

Examples of food products or ingredients according to the invention include dairy and related products, mayonnaises, salad dressings, sauces, confectionery fillings and the like.

In use, the composition may be applied in the form of a liquid or a powder for formation of a food product or ingredient for forming a food product, which requires the formation of an oil-in-water emulsion. The examples that follow are intended to illustrate but in no way limit the present invention.

Examples Characterisation of oil-in-water emulsifier composition

Example 1: Chemical analysis data of the major components of the oil-in-water composition

Table 1: Chemical analysis data of the major components of the composition according to the invention showing the range of values

Note: DSB refers to composition calculated on a dry solids basis.

A sample oil-in-water emulsifier composition according to the invention was analysed and found to be composed of the following:

Table 2: Typical chemical analysis data for the major components of the composition

Note: DSB refers to composition calculated on a dry solids basis. Example 2: Molecular level analysis data for the protein component of the oil in water composition

Example 2.1 Amino acid analysis

Cysteine analysis was performed using performic acid oxidation followed by 24 hr acid hydrolysis with 6 mol/kg HCI at 110 ⁰ C. For tryptophan analysis samples underwent 24 hr base hydrolysis in 5 mol/kg NaOH at 110 ⁰ C. After hydrolysis all amino acids were analysed using the Waters AccQTag Ultra chemistry. Samples were analysed in duplicate and results are expressed as an average.

Table 3: Amino acid analysis for lupin protein

Table 4: Typical amino acid composition for serum albumin, soy protein and lupin protein

Example 2.2: Size exclusion chromatographic analysis of the protein component of the composition

Samples were analysed by the altered size exclusion high performance liquid chromatography method of Batey, I. L., Gupta, R. B., MacRitchie, F., 1999. Use of size exclusion high performance liquid chromatography in the study of wheat flour proteins- an improved chromatographic procedure, Cereal Chem. 68, 207. In this method the flour (10 mg) was mixed with 1 ml 0.5 % SDS phosphate buffer and sonicated for 15 sec, ensuring that the sample was completely dispersed within the first five seconds. The mix was then centrifuged for 10 min at 17,000 x g and the supernatant was filtered through a 0.45 μm PVDF filter. A Phenomenex BIOSEP-SEC 4000 column was used for the analysis. A running time of 10 min was used (flow rate 2 mL/min) instead of the Standard 35 min run (0.5 mL/min). The eluent used was aqueous acetonitrile (ACN) buffer (0.05 % trifluoroactic acid (TFA) in water and 0.05 % in ACN). The proteins were detected at a wavelength of 214 nm.

Table 5: Size exclusion chromatographic analysis of the molecular size range for the protein component of the composition

Example 2.3: MALDI-TOF analysis data of protein component of oil-in-water emulsifier composition

Samples were dissolved in 60 μm ACN/H ₂ O (v/v, 50:50) containing 0.05 % v/v TFA for 1 hour. Sample preparation was carried out according to the dried droplet method using sinapinic acid (SA) as matrix. (Kussmann, M., E. Nordhoff, H. Rahbek-Nielsen, S. Haebel, M. Rossel-Larsen, L. Jakobsen, J. Gobom, E. Mirgorodskaya, A. Kroll- Kristensen, L. Palm and P. Roepstorff, 1997, MALDI-MS sample preparation techniques designed for various peptide and protein analytes, J. Mass Spectrom. 32:593) The matrix solution was prepared by dissolving SA in ACN/H ₂ O (50:50 v/v) with 0.05 % v/v TFA at a concentration of 10 mg/mL. A sample/ matrix solution mixture (1 :10 v/v) was deposited (2 μL) on to a 96-sample MALDI probe tip, and dried at room temperature.

MALDI-TOF mass spectrometric experiments were carried out on a Voyager DE-PRO TOF mass spectrometer (Applied Biosystems, Foster City, CA, USA) equipped with UV nitrogen laser (337 nm). The instrument was used with the following parameters: laser intensity 2 500, mass range 50-100 kDa, acceleration voltage 25 kV, grid voltage 93% guide wire 0.2 %, delay time 850 ns. Spectra were obtained in positive (inear ion mode and were averaged from 50 laser shots to improve the S/N level. All the samples were automatically accumulated in a random pattern over the sample spot to provide the final spectrum. Human transferrin (79 549 Da) as external standards for mass assignment.

Table 6: MALDI - TOF analysis data of the molecular size ranges for the protein component of the composition

Example 3: Characterisation of lipid component of oil-in-water emulsifier composition

Table 7: Lipid analysis ranges for oil-in water-emulsifier

Table 8: Typical lipid composition for oil-in-water emulsifier

Table 9: Fatty acid ranges for oil-in-water emulsifier Table 10: Typical fatty acid composition for oil-in-water emulsifier

Example 4: Physical characterisation of the oil-in-water emulsifier composition

Example 4.1: Solubility of oil-in-water emulsifier composition

Determination of Solubility

1. Prepare a 2 % solution (using 10 g of powder) of each sample to be tested.

2. Leave hydrating for an hour.

3. Place the solution in the blender (Sunbeam) and blend for 1 minute at the lowest speed.

4. Centrifuge at 2000 RCF for 5 mins.

5. Measure the supernatant.

6. Determine the solids in the supernatant and moisture of the powder. 7. Calculate solubility as:

Solubility (%) = (Solids in Supernatant x 100)/Total solids

Table 11: Solubility data ranges for the oil-in-water emulsifier composition

Note: Solubility was determined at pH 6.5

Table 12: Typical solubility data for the composition

Note: The solubility measurements were taken at pH 6.5 Table 13: Typical solubility data fora range of prior art proteins

Note: The solubility measurements were determined at the pH of the product

It will be understood that the solubilities determined for the samples shown in Tables 11 and 12 are exemplary of the compositions according to the invention defined herein. Solubility is dependent on processing steps for manufacture of the composition including for example the parameters for drying. Accordingly, the composition of the invention may have solubilities outside the ranges generally shown in Tables 11 and 12.

Example 4.2: Determination of hydrophobicity value for oil-in-water emulsifier composition

ANS Method for determination of hvdrophobicity

The principle of the method is measuring the binding of a fluorescent dye (ANS) to the hydrophobic regions of the proteins. The resulting values are inversely proportional to hydrophobicity thus a lower value indicates a greater hydrophobicity.

1. Prepare Buffer 1 : 0.1 mol/kg phosphate buffer from KH ₂ PO ₄ and Na ₂ HPO ₄ pH 7.4. 2. Prepare a stock protein solution: 0.05 % (m/m) protein solution in Buffer 1 (40 mL).

3. Prepare diluted protein solutions: a. 0.05 %: stock solution b. 0.04 %: 8 mL stock solution and 2 mL Buffer 1 (dilution factor: 1.25)

5 c. 0.033 %: 6.66 mL stock solution and 23.34 mL Buffer 1 (dilution factor: 1.5) d. 0.025 %: 5 mL stock solution and 5 mL Buffer 1 (dilution factor: 2) e. 0.01 %: 2 mL stock solution and 8 mL Buffer 1 (dilution factor: 5)

4. Use protein standard(s): k-casein and/or soy isolate.

5. Prepare the ANS-reagent by dissolving 0.0263 g ANS in 5 mL of methanol. When the *0 ANS is dissolved , use nitrogen flow to degas the solution.

6. Prepare the solution for measurement under N ₂ -flow by adding 20 μL ANS-reagent to each diluted protein solution. These solutions have to precipitate just before measurement.

7. Measure the molecular size using:

15 Spectrophotometer: JASCO FP-920 Fluorescence Spectrophotometer Excitation wavelength: 370 nm Measuring wavelength: 480 nm

8. Evaluate ANS-hydrophobicity (S ₀ ) as the slope of the fitted line in the graph of emitted value versus protein concentration. 0

Table 14: ANS Hydrophobicity range for the protein component of the composition

Table 15: Typical values for ANS Hydrophobicity for protein components of lupin, soy, dairy and beef sera (atpH 7.4)

The oil-in-water emulsifier is more hydrophobic than soy protein and much more hydrophobic than sodium caseinate and bovine serum albumin.

Table 16: Typical values for the effect ofpH on the ANS Hydrophobicity Score

The pH at which the ANS Hydrophobicity is measured influences the charge on the amino acids and the solubility of the proteins. The proteins are more hydrophobic at the lower pH. Example 4.3: Emulsion activity tests

Method for the determination of emulsion activity (using concentration of 3.6 % by weight)

1. Prepare a 7 % concentration by weight of oil-in-water emulsifier in water (100 mL). 2. Mix using Sorvall homogeniser for 10 sees (maximum speed).

3. Add 100 mL of vegetable oil.

4. Mix for 1 min on maximum speed.

5. Take approximately 40 g and centrifuge for 10 mins (maximum speed).

6. Measure emulsification activity (EA):

EA (%) = (emulsified layer (top) (cm} x 100)/ Total layer (cm)

Table 17: Typical effect of re-hydration medium for oil-in-water emulsifier on emulsion activity

Note: Standard error for the emulsion activity test +/- 2.5%

There is no difference at this concentration in emulsion activity irrespective of whether the protein is re-hydrated in oil or water.

Figure 3

As shown in Figure 3, the highest emulsion activity for a 3.6% concentration of the oil- in-water composition occurs when the ratio of oil to water is 5:3.

Figure 4

As shown in Figure 4, the highest emulsion activity for a 3.6% concentration of oil-in- water composition occurs when the ratio of water to oil is 3:5.

Figure 5

As shown in Figure 5, the oil-in-water emulsifier is affected by the pH with higher emulsions forming above and below pH 5.5.

Table 18: Typical effect of freezing and thawing on emulsion activity

Note: Standard error for the emulsion activity test +/- 2.5%

Note: Concentration of oil-in-water emulsifier composition was 3.6% by weight

The oil-in-water emulsifier does not lose emulsion activity after repeated freezing and thawing.

Table 19: Typical effect of chilling on emulsion activity of oil-in-water emulsifier

The oil-in-water emulsifier does not lose emulsion activity after chilling.

Formation of emulsions using oil-in-water emulsifier composition

Example 5: Icecream

Method for the preparation of icecream

1. Heat the water to 45 ⁰ C.

2. Once heated, add the dry ingredients to the water whilst mixing constantly. 3. Add the oil.

4. Add the sugar plus CREMODAN ^® SE SE 709VEG.

5. Heat the mixture until it reaches 80 ⁰ C mixing continuously.

6. Homogenise mixture 190/40. 7. Store the ice cream mix overnight at 4 ⁰ C.

8. Churn the mix for 30 mins using the ice cream maker.

9. Leave in the freezer to harden.

Example 6: Meat emulsion for sliceable sandwich meat (Laboratory model meat mix)

Method for the preparation of the meat emulsion

1. Mix Part A ingredients at speed 1 for 15 minutes using Hobart mixer. 2. Blend together all dry ingredients in Part B and add to Part A. Add iced water and mix at speed 1 for 15 minutes. 3. Spoon fill test cups with required mixture, pushing mixture down into cup to expel air.

4. Cover each cup and place in 75 ⁰ C water bath for 20 minutes.

5. Cool in ice water. 6. Refrigerate.

Example 7: Enhancement of the functional performance of the base oil-in-water emulsifier

The following methods can be used to improve performance of the oil-in-water emulsifier.

Example 7.1: Modification using Enzymes

Proteases which break the protein, peptide or amino acid linkages eg: exo-proteases can be used to change the protein structure of the oil-in-water emulsifier. In certain embodiments the composition of the invention is one that has not been digested with an endopeptidase or related protease.

Example 7.2: Modification using Heat Treatment

Heating to temperatures greater than 90 ⁰ C can change the agglomeration of the proteins and alter the performance characteristics of the oil-in-water emulsifier.

Example 7.3: Modification using mechanical means

Processes that use high shear and increased pressure such as using a homogeniser can structurally change the oil-in-water emulsifier by reducing the size of aggregates.

Spray drying parameters including spray nozzle size, feed inlet pressures and instantising processes can affect the aggregation and hence solubility and performance of the oil-in-water emulsifier. Example 7.4: Modification using chemical agents

Chemical agents that change the covalent and non-covalent bonds between amino acids in the proteins can change the structure of the protein. The conformational change influences the surface characteristics of the proteins. Examples of chemical agents include carboxylic anhydrides, ionic salts and redox reagents.

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. AH of these different combinations constitute various alternative aspects of the invention.