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, gelling agents, foaming agents, creaming agents, whipping 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 to increase 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 have been described for use in stabilising water-in-oil emulsions. These are essentially colloidal dispersions where water forms a discrete phase and oil 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 water-in-oil 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 water-in-oil emulsions that are liquids and solids include dressings (vinegar in oil), butter and coffee whitener respectively.

Most protein isolates that are useful for stabilising water-in-oil 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 hydrophilic portions of the proteins are understood to at least partially dissolve into the discrete phase, leaving the more hydrophobic 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 foaming properties (foaming properties being similar to emulsifying properties) of lupin proteins.

There remains a need for new protein isolates useful as water-in-oil emulsifiers in a variety of different applications, especially emulsifiers having functionality across a wide range of pH ranges.

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 a water-in-oil emulsion to form a food product, the composition including:

- lupin proteins in an amount of about 75 to 98 % by dry weight of the composition,

wherein about 60 to 70 % of the lupin proteins have a molecular weight of no more than about 30 kDa. In a further embodiment there is provided a composition useful as an emulsifier for stabilising a water-in-oil emulsion to form a food product, the composition including:

- protein; and

- lupin fibre in an amount of from about 2 to 25 % by dry weight of the composition.

In further embodiments there is provided a food product including oil or fat, water and a composition useful as an emulsifier for stabilising a water-in-oil emulsion according to the invention. In one embodiment the food product is provided in the form of a water-in- oil emulsion such as a salad dressing or like product, a butter or like product, or a coffee whitener or like product.

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

The present invention also relates to a process for producing a composition useful as an emulsifier for stabilising a water-in-oil emulsion, the process including:

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

- recovering the lupin proteins from the extract to form a composition wherein about 60 to 70 % of the lupin proteins have a molecular weight less than 30 kDa, thereby producing the composition useful as a water-in-oil emulsifier.

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 water-in-oil 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.

Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

In research leading to the invention, the inventors sought to provide improved water-in- oil emulsifiers and food products formed therefrom.

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

Advantageously the compositions of the invention described herein are capable of stabilising water-in-oil emulsions across a wide range of oil to water ratios, pH, type and concentration of salt and temperature conditions. The compositions of the present invention are particularly useful for stabilising water-in-oil emulsions over a wide pH and temperature range. Due to the stability of the emulsifier over a wide pH range, the emulsifier is less likely to precipitate or to cause other components of an emulsion to precipitate, coalesce or flocculate. This is particularly useful when pH-altering additives, such as flavours, are added to a water-in-oil emulsion containing the lupin protein isolate. Thus, a single emulsifier can be used to make a number of foods with different flavours and the stability of the emulsifier makes it possible to retain mouth-feel and/or other organoleptic properties in different flavour versions of the same product.

Additionally, the stability of the emulsifiers over a wide range of temperatures means that it is possible to use the emulsifiers to derive foams that can be treated with heat without destroying the foam structure.

A further advantage of the compositions of the invention described herein is that they 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 a water-in-oil emulsion to form a food product, the composition including:

- lupin protein in an amount of about 75 to 98 % by dry weight of the composition,

wherein about 60 to 70 % of the lupin proteins have a molecular weight of no more than about 30 kDa.

Typically the majority of the lupin proteins have a molecular weight no lower than about 8 kDa. In some embodiments proteins having a lower molecular weight (i.e. lower than 8 kDa) 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 aqueous phase in the oil phase (i.e. the aqueous phase forms a discrete phase and the oil 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 aqueous phase throughout the mixture. Phase separation may occur by as settling or synersis of the aqueous phase out of the oil phase. Minimising can mean any of reducing, diminishing, lessening, curtailing or decreasing phase separation.

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

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

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

- protein; and

- lupin fibre in an amount of from about 2 to 25 % by dry weight of the composition.

In these embodiments, the protein may be obtained from a range of sources. Preferably it is obtained from lupin. Preferably about 60 to 70 % of the lupin proteins have a molecular weight of no more than about 30 kDa. The inventors have also found that useful water-in-oil 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. 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 attached 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 and/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 proteins may have a molecular weight from about 17 to 20 kDa. Typically in the composition of the invention, about 30 to 35 % of the proteins may have a molecular weight from about 12 to 15 kDa. Typically in the composition of the invention, about 30 to 35% of the proteins may have a molecular weight from about 8 to 11 kDa.

In certain embodiments, the relative abundance of the molecular weight ranges of lupin proteins in the composition are as shown in Table 5. Table 5 discloses the relative abundance of protein species having the defined molecular weight ranges in the composition of the invention. This data is derived from size exclusion chromatography data as described in Figure 1. Table 6 discloses the relative abundance of protein species in a sample of the composition that has been treated to enable MALDI TOF analysis. Figure 2 describes the relative abundance of molecular ions in the MALDI TOF treated sample.

The proteins may include γ-conglutin and fragments thereof. Typically the fibre is soluble fibre. The soluble fibre is determined by AOAC Official Method 985.29 Total Dietary Fiber in Foods in AOAC Official Methods of Analysis 2005.

Typically the composition of the invention has an ANS hydrophobicity score of about 0.7 to 1.0 at pH 7.4.

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 hydrophobic 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 higher at more neutral pH partially reflecting the solubility and ionic charges present on 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 at. 2008 Pharma. Res. 25: 1487.

The composition is generally soluble in an aqueous solution having a pH of about 2 to 9.

In use, the composition may be applied as a liquid or as a powder.

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

In another embodiment there is provided a process for producing a composition useful as an emulsifier for stabilising a water-in-oil emulsion, the process including:

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

- recovering the lupin proteins from the extract to form a composition wherein about 60 to 70 % of the lupin proteins have a molecular weight less than 30 kDa, thereby producing the composition useful as a water-in-oil emulsifier. 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, thereby forming the composition useful as a water-in-oil emulsifier,

- recovering a fraction from the carbohydrase-treated slurry, said fraction including fibre and lupin proteins wherein about 60 to 70 % of the lupin proteins have a molecular weight of no more than about 30 kDa.

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 70 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, Mutabils 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 lower molecular weight proteins is useful as a water-in-oil 5 emulsifier, something that could not be observed when the lower molecular weight proteins are in composition with the higher molecular weight proteins.

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 I carbohydrase may be a pectinase, cellulase or hemicellulase. Preferably the

10 carbohydrase is an enzymatic composition of cellulase and pectinase.

As discussed previously, it may be desirable to split the glycans to remove as much carbohydrate as possible from the protein, thereby producing a protein composition having no or very little attached and/or entrapped sugars, carbohydrates and/or fibre.

Therefore, in one embodiment, most if not all of the proteins are provided without having

15 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 attached fibre during the j protein preparation process (e.g. in the alkali and/or enzyme treatment steps).

20 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 25 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 pH can be changed to, for example, between 7 and 8, preferably 7.5. The heat treatment can be carried out at, for example, about 90 ⁰ C for about 5 minutes.

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 lower molecular weight proteins in the enzyme-treated, protein-containing supernatant are separated from the higher 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, the lower molecular weight proteins can be recovered by any technique for recovering lower molecular weight proteins 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 a water-in-oil 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.

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 dressings (vinegar in oil), butter, coffee whitener 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.

The examples that follow are intended to illustrate but in no way limit the present invention.

Examples Characterisation of water-in-oil emulsifier composition

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

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

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

A sample water-in-oil 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 soli s asis. Example 2: Molecular level analysis data for the protein component of the wa term-oil composition

Example 2.1 Amino acid analysis

Cysteine analysis was performed using performic acid oxidation followed by 24hr 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 water-in-oil emulsifier composition

Samples were dissolved in 60 μm ACN/H ₂ O (50:50 v/v) 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 2500, 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 linear 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) was used as the 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: Physical characterisation of the water-in-oil emulsifier composition

Example 3.1: Solubility of water-in-oil emulsifier composition

Determination of Solubility

1. Make 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 7: Solubility data ranges for the water-in-oil emulsifier composition

Note: Solubility was determined at pH 6.5

Table 8: Typical solubility data for the composition

Note: The solubility measurements were taken at pH 6.5 Table 9: Typical solubility data for a 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 7 and 8 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 7 and 8.

Example 3.2: Determination of hydrophobicity value for water-in-oil emulsifier composition

ANS Method for determination of hvdrophobicitv

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) 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 0.0263g ANS in 5 mL of methanol. When the

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:

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.

Table 10: ANS Hydrophohicity range for the protein component of the composition

Table 11: Typical values for ANS Hydrophobicity for protein components of lupin, soy, dairy and beef sera (at pH 7.4)

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

Table 12: 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 low pH. Example 3.3: 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 water-in-oil emulsifier in water (100 ml_). 2. Mix using Sorvall homogeniserfor 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 13: Typical effect of re-hydration medium for protein on emulsion activity

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

There is no difference 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 water-in-oii 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 water- in-oil composition occurs when the ratio of oil to water is 3:5.

Figure 5

As shown in Figure 5, the water-in-oil emulsifier retains good emulsion activity over a wide pH range.

Table 14: Typical effect of freezing and thawing on emulsion activity

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

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

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

Formation of emulsions using water-in-oil emulsifier composition

Example 4: Non-dairy creamer

Table 15: Ingredients for the preparation of non-dairy creamer

Method for the preparation of non-dairy creamer

1. Rehydrate water-in-oil emulsifier composition in water.

2. Mix on high speed mixer.

3. Heat water-in-oil emulsifier composition and water to > 65 ⁰ C.

4. Mix in maltodextrin using high speed mixer. 5. Heat hydrogenated vegetable fat to > 65 ⁰ C.

6. Mix monoglyceride into vegetable fat.

7. Combine aqueous and fat phases and pass through 2 stage homogeniser.

8. Heat treat for liquid product or spray dry. Example 5: Mayonnaise

Table 16: Ingredients for the preparation of mayonnaise

Method for the preparation of mayonnaise

1. Combine water-in-oil emulsifier, lemon juice, mayonnaise flavours and vinegar at low speed.

2. Add oil continuously gradually increasing speed as emulsion is formed.

Example 6: Basic Meringues

Table 17: Ingredients for the preparation of foam using water-in-oil emulsifier

Method for the preparation of foam using water-in-oil emulsifier

1. Add water to powder ensuring that protein is fully hydrated

2. Whip at maximum speed for 5 minutes using Kenwood mixer.

3. Add sugar slowly while whipping till foam becomes glossy (approximately another 1.5 to 2 minutes).

4. Pipe onto greased, aluminum foiled oven trays.

5. Cook in 120 ⁰ C oven for approximately 1 hour (dependent on size and number of meringues put in the oven).

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

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

Example 7.1: Modification using Enzymes

Proteases which break the protein, peptide or amino acid linkages e.g.: exo-proteases can be used to change the protein structure of the water-in-oil 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 water-in-oil 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 water-in-oil 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 water-in-oil 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. All of these different combinations constitute various alternative aspects of the invention.