Egg analogue product based on soy and canola protein

Background

Over the last decade or so, the increasing awareness towards global issues such as food security, environmental sustainability (Aiking & de Boer, 2020), animal welfare and the health-related hazards caused by excessive consumption of animal products, has led to a growth in the proportion of consumers following flexitarian, vegetarian and vegan diets (Derbyshire, 2016; Melina, Craig, & Levin, 2016). This trend has steered the focus of several food companies towards the development of products based on plant protein ingredients, including those derived from oilseeds, pulses, cereals, tubers and nuts. However, when compared to animal products, commercially available plant-based foods typically display lower nutritional value, often in terms of both macro- and micronutrients, as well as poorer organoleptic and functional properties.

In addition to milk, meat and fish analogues, plant-based egg analogues have recently been introduced to the market. Available in both fluid and semi-solid formats, the manufacture of these products requires a thorough understanding of the composition, structure and functionality of chicken egg, and involves a complex interplay of formulation and processing in order to achieve and preserve the desired properties. Egg analogues can be converted into a soft solid-like material upon heat treatment Therefore, in order for this product concept to be successful, it is of paramount importance to identify plant proteins, or blends thereof, with the desired emulsifying and gelling properties. With regards to the gelling properties, it is particularly challenging to identify plant proteins with denaturation temperatures similar to those of the major egg proteins. Indeed, most plant proteins denature at higher temperatures, thus resulting in the derived egg analogues requiring higher temperatures and/or longer cooking times to obtain gels with texture similar to that of chicken egg.

Summary of the invention

The present application describes a superior egg analogue product with a short ingredient list and which performs like real chicken egg in final applications.

In a first aspect, the invention relates to a vegan egg analogue product, said product comprising an emulsion having between 5 to 20 wt%, preferably between 8 to 13 wt% plant protein, between 5 to 30 wt%, preferably between 10 to 15 wt% vegetable fat, optionally between 0 to 5 wt%, preferably between 0.1 to 2 wt% polysaccharides, and between 40 to 90 wt% water, wherein said plant protein preferably comprises a combination of soy protein and canola protein, and wherein said emulsion has a fat to protein ratio between 0.3 to 5, preferably between 0.5 to 3, preferably between 0.6 to 2, preferably between 0.65 to 0.75, or most preferably about 0.70.

In a second aspect, the invention relates to a method of making an egg analogue product, said method comprising: a) Shear mixing of water and plant proteins, preferably a combination of soy and canola proteins, to form a plant protein dispersion; b) Optional addition and shear mixing of one or more polysaccharides, colouring, flavouring and preservative agents; c) Addition of a vegetable oil and emulsification, either by high pressure homogenization or high shear mixing, to form an emulsion; d) Optional pH adjustment of the emulsion to values between 6.0 to 9.0, preferably between 7.0 to 9.0; e) Pasteurization or sterilization either before or after emulsification via indirect heating or direct steam injection.

In a third aspect, the invention relates to an egg analogue product, preferably a liquid egg analogue product, for use as a whole egg substitute in pan cooking applications, sauces and baked products.

Brief description of the figures

Figure 1: Volume-weighted ( D[4,3] ) and surface weighted ( D[3, 2]) mean particle diameter measured with and without SDS (a), viscosity (b), cooking time (c) and yield (d) of Pl prototypes (see Table 5 and 6).

Figure 2: Volume-weighted ( D[4,3] ) and surface weighted ( D[3, 2]) mean particle diameter measured with and without SDS (a), viscosity (b), cooking time (c) and yield (d) of P2 prototypes (see Table 5 and 6).

Figure 3: Volume-weighted ( D[4,3] ) and surface weighted ( D[3, 2]) mean particle diameter measured with and without SDS (a), viscosity (b), cooking time (c) and yield (d) of P3 prototypes (see Table 5 and 6).

Figure 4: Volume-weighted ( D[4,3] ) and surface weighted ( D[3, 2]) mean particle diameter measured with and without SDS (a), viscosity (b), cooking time (c) and yield (d) of P4 prototypes without pH adjustment to 7.5 before heat treatment (see Table 5 and 6). Figure 5: Volume-weighted (D[4,3]) and surface weighted (D[3,2]) mean particle diameter measured with and without SDS (a), viscosity (b), cooking time (c) and yield (d) of P4 prototypes with pH adjusted to 7.5 before heat treatment (see Table 5 and 6).

Figure 5: Volume-weighted ( D[4,3]) and surface weighted ( D[3, 2]) mean particle diameter measured with and without SDS (a), viscosity (b), cooking time (c) and yield (d) of P5 prototypes (see Table 5 and 6).

Figure 7: Volume-weighted ( D[4,3]) and surface weighted ( D[3, 2]) mean particle diameter measured with and without SDS (a), viscosity (b), cooking time (c) and yield (d) of P6 prototypes (see Table 5 and 6).

Figure 8: Volume-weighted ( D[4,3]) and surface weighted ( D[3, 2]) mean particle diameter measured with and without SDS (a), viscosity (b), cooking time (c) and yield (d) of P7 prototypes (see Table 5 and 6).

Embodiments of the invention

The invention relates to a vegan egg analogue product, said product comprising an emulsion having plant protein, vegetable fat, and water, wherein said plant protein preferably comprises a combination of soy protein and canola protein.

The invention relates to a vegan egg analogue product, said product comprising an emulsion having plant protein, vegetable fat, optionally polysaccharides, and water, wherein said plant protein preferably comprises a combination of soy protein and canola protein.

The invention relates to a vegan egg analogue product, said product comprising an emulsion having between 5 to 20 wt% plant protein, between 5 to 30 wt% vegetable fat, optionally between 0 to 5 wt% polysaccharides, and between 40 to 90 wt% water, wherein said plant protein preferably comprises a combination of soy protein and canola protein.

The invention relates to a vegan egg analogue product, said product comprising an emulsion having between 5 to 20 wt% plant protein, between 5 to 30 wt% vegetable fat, optionally between 0 to 5 wt% polysaccharides, and between 40 to 90 wt% water, wherein said plant protein preferably comprises a combination of soy protein and canola protein, and wherein said emulsion has a fat to protein ratio between 0.3 to 5.

In preferred embodiments, said product comprises an emulsion having between 8 to 13 wt% plant protein. In preferred embodiments, said product comprises an emulsion having between 5 to 15 wt% vegetable fat.

In preferred embodiments, said product comprises an emulsion having optionally between 0.1 to 2 wt% polysaccharides.

In preferred embodiments, said emulsion has a fat to protein ratio between 0.5 to 3.

In preferred embodiments, said emulsion has a fat to protein ratio is between 0.6 to 2, or between 0.65 to 0.75, or about 0.70.

In some embodiments, the soy protein is a soy protein concentrate or a soy protein isolate and contributes between 10 to 90 wt% of the total protein in the emulsion.

In preferred embodiments, the soy protein is a soy protein concentrate or a soy protein isolate and contributes between 20 to 60 wt% of the total protein in the emulsion.

In some embodiments, the canola protein is a canola protein concentrate or a canola protein isolate and contributes between 10 to 90 wt% of the total protein in the emulsion.

In preferred embodiments, the canola protein is a canola protein concentrate or a canola protein isolate and contributes between 40 to 80 wt% of the total protein in the emulsion.

In preferred embodiments, the soy protein contributes between 20 to 60 wt. % of the total protein of the emulsion, and the canola protein contributes between 40 to 80 wt% of the total protein in the emulsion.

In some embodiments, the ratio of canola albumin to canola globulin ranges from 100:0 to 0:100.

In preferred embodiments, the ratio of canola albumin to canola globulin ranges from 80:20 to 20:80. In preferred embodiments, the ratio of canola albumin to canola globulin ranges from 60:40 to 40:60. In some embodiments, the canola protein is a mixture of two canola proteins, for example two canola protein isolates.

One canola protein isolate may be enriched in albumin. One canola protein isolate may be enriched in globulin.

In some embodiments, said emulsion has a pH range between 6.0 to 9.0.

In some embodiments, said emulsion has a pH range between 7.0 to 9.0.

In preferred embodiments, said emulsion has a pH range between 6.5 to 8.5.

In preferred embodiments, said emulsion has a pH range between 7.5 to 8.5.

In some embodiments, the pH of the emulsion is adjusted using alkali agents selected from potassium carbonate, potassium hydroxide, potassium bicarbonate, sodium hydroxide, sodium carbonate, sodium bicarbonate, ammonium hydroxide, ammonium carbonate, ammonium bicarbonate or combinations thereof.

In preferred embodiments, the pH of the emulsion is adjusted using potassium carbonate. In some embodiments, the polysaccharide is non-ionic, including, but not limited to, guar gum, fenugreek gum, locust bean gum and konjac glucomannan, or a combination thereof.

In some embodiments, the polysaccharide is ionic, including, but not limited to, xanthan gum, K- carrageenan, L-carrageenan and methylcellulose, or a combination thereof.

In some embodiments, the emulsion further comprises coloring and/or flavoring agents.

In some embodiments, the emulsion further comprises one or more natural preservatives.

In some embodiments, the emulsion has a volume-weighted mean particle diameter less than 30 microns, as determined by static light scattering.

In preferred embodiments, the emulsion has a volume-weighted mean particle diameter less than 10 microns, as determined by static light scattering.

In some embodiments, the emulsion has a volume-weighted oil droplet diameter less than 30 microns as determined by static light scattering in the presence of a denaturing agent.

In preferred embodiments, the emulsion has a volume-weighted oil droplet diameter less than 10 microns, as determined by static light scattering in the presence of a denaturing agent.

In preferred embodiments, the emulsion has a volume-weighted oil droplet diameter less than 2 microns, as determined by static light scattering in the presence of a denaturing agent.

In some embodiments, the emulsion has a viscosity of less than 2000 mPa.s, as determined by rotational rheometry after 30 minutes at a rotational speed of 160 rpm and 25°C.

In preferred embodiments, the emulsion has a viscosity of less than 1000 mPa.s, as determined by rotational rheometry after 30 minutes at a rotational speed of 160 rpm and 25°C.

In preferred embodiments, the emulsion has a viscosity of less than 800 mPa.s, as determined by rotational rheometry after 30 minutes at a rotational speed of 160 rpm and 25°C.

In preferred embodiments, the emulsion has a viscosity of between 400 to 800 mPa.s, as determined by rotational rheometry after 30 minutes at a rotational speed of 160 rpm and 25°C.

In some embodiments, the emulsion has between 50 and 100% of the cooking performance of a real egg, for example upon heat treatment and scrambling in a pan.

The invention further relates to a method of making a vegan egg analogue product, said method comprising: a) Shear mixing of water and plant proteins, preferably a combination of soy and canola proteins, to form a plant protein dispersion; b) Optional addition and shear mixing of one or more polysaccharides, colouring, flavouring and preservative agents; c) Addition of a vegetable oil and emulsification, either by high pressure homogenization or high shear mixing, to form an emulsion; d) Optional pH adjustment of the plant protein dispersion or the emulsion to values between 6.0 to 9.0, preferably between 7.0 to 9.0; e) Pasteurization or sterilization either before or after emulsification via indirect heating or direct steam injection.

In some embodiments, mixing in step (a) is by shear mixing.

In some embodiments, steps (a) to (d) are performed at a temperature ranging from 20 to 70°C.

In some embodiments, step € is performed in a high shear mixer, in a tubular heat exchanger or in a plate heat exchanger, at temperatures ranging from 60 to 150°C for times ranging from 30 minutes to 1 second.

In some embodiments, the heating time decreases with increasing temperature.

In preferred embodiments, said emulsion has a fat to protein ratio between 0.5 to 3.

In preferred embodiments, said emulsion has a fat to protein ratio between 0.6 to 2, or between 0.65 to 0.75, or about 0.70.

In some embodiments, the soy protein is a soy protein concentrate or a soy protein isolate and contributes between 10 to 90 wt% of the total protein in the emulsion.

In preferred embodiments, the soy protein is a soy protein concentrate or a soy protein isolate and contributes between 20 to 60 wt% of the total protein in the emulsion.

In some embodiments, the canola protein is a canola protein concentrate or a canola protein isolate and contributes between 10 to 90 wt% of the total protein in the emulsion.

In preferred embodiments, the canola protein is a canola protein concentrate or a canola protein isolate and contributes between 40 to 80 wt% of the total protein in the emulsion.

In preferred embodiments, the soy protein contributes between 20 to 60 wt. % of the total protein of the emulsion, and the canola protein contributes between 40 to 80 wt% of the total protein in the emulsion.

In some embodiments, the ratio of canola albumin to canola globulin ranges from 100:0 to 0:100.

In preferred embodiments, the ratio of canola albumin to canola globulin ranges from 80:20 to 20:80. In preferred embodiments, the ratio of canola albumin to canola globulin ranges from 60:40 to 40:60. In some embodiments, the canola protein is a mixture of two canola proteins, for example two canola protein isolates.

One canola protein isolate may be enriched in albumin. One canola protein isolate may be enriched in globulin.

In preferred embodiments, said emulsion has a pH range between 6.5 to 8.5.

In preferred embodiments, said emulsion has a pH range between 7.5 to 8.5. In some embodiments, the pH of the emulsion is adjusted using alkali agents selected from potassium carbonate, potassium hydroxide, potassium bicarbonate, sodium hydroxide, sodium carbonate, sodium bicarbonate, ammonium hydroxide, ammonium carbonate, ammonium bicarbonate or combinations thereof.

In preferred embodiments, the pH of the emulsion is adjusted using potassium carbonate.

In some embodiments, the polysaccharide is non-ionic, including, but not limited to, guar gum, fenugreek gum, locust bean gum and konjac glucomannan, or a combination thereof.

In some embodiments, the polysaccharide is ionic, including, but not limited to, xanthan gum, K- carrageenan, L-carrageenan and methylcellulose, or a combination thereof.

In some embodiments, the emulsion further comprises coloring and/or flavoring agents.

In some embodiments, the emulsion further comprises one or more natural preservatives.

In some embodiments, the emulsion has a volume-weighted mean particle diameter less than 30 microns, as determined by static light scattering.

In preferred embodiments, the emulsion has a volume-weighted mean particle diameter less than 10 microns, as determined by static light scattering.

In some embodiments, the emulsion has a volume-weighted oil droplet diameter less than 30 microns as determined by static light scattering in the presence of a denaturing agent.

In preferred embodiments, the emulsion has a volume-weighted oil droplet diameter less than 10 microns, as determined by static light scattering in the presence of a denaturing agent.

In preferred embodiments, the emulsion has a volume-weighted oil droplet diameter less than 2 microns, as determined by static light scattering in the presence of a denaturing agent.

In some embodiments, the emulsion has a viscosity of less than 2000 mPa.s, as determined by rotational rheometry after 30 minutes at a rotational speed of 160 rpm and 25°C.

In preferred embodiments, the emulsion has a viscosity of less than 1000 mPa.s, as determined by rotational rheometry after 30 minutes at a rotational speed of 160 rpm and 25°C.

In preferred embodiments, the emulsion has a viscosity of less than 800 mPa.s, as determined by rotational rheometry after 30 minutes at a rotational speed of 160 rpm and 25°C.

In some embodiments, the emulsion has between 50 and 100% of the cooking performance of a real egg, for example upon heat treatment and scrambling in a pan.

The invention further relates to an egg analogue product, preferably a liquid egg analogue product, produced by a method according to the invention.

The invention further relates to an egg analogue product according to the invention for use as a whole egg substitute in pan cooking applications, sauces and baked products. Detailed description of the embodiments

Egg analogue product

The egg analogue product is vegan. The egg analogue product can be a liquid, paste, semi-solid or solid. Preferably, the egg analogue product is a liquid. Preferably, the egg analogue product comprises water.

Plant protein

The egg analogue product comprises an emulsion having plant protein. The plant protein can be a combination of a legume protein and an oilseed protein. The protein can be flour, concentrate, or isolate, preferably an isolate. Preferably, the legume protein is a soy protein, preferably a soy protein isolate. Preferably, the oilseed protein is a canola protein, preferably a canola protein isolate, or one or more canola protein isolates.

In one embodiment, the egg analogue product comprises an emulsion having at least 5 wt%, at least 6 wt%, at least 7 wt%, at least 8 wt%, at least 9 wt%, or at least 10 wt% plant protein. The egg analogue product preferably comprises an emulsion having up to 20 wt%, up to 19 wt%, up to 18 wt% or up to 17 wt% plant protein. The egg analogue product preferably comprises an emulsion having between 5 to 20 wt% plant protein, between 6 to 19 wt% plant protein, between 7 to 18 wt% plant protein, or about 11 to 16 wt% plant protein, or about 12 wt% plant protein or about 15 wt% plant protein, preferably about 12 wt% plant protein.

Vegetable fat

In the context of the present invention, the term fat refers to triglycerides. Fats which are generally encountered in their liquid form are commonly referred to as oils. In the present invention the terms oils and fats are interchangeable. The vegetable fat can be liquid or solid. Preferably, the vegetable fat is liquid. The vegetable fat can be, for example, canola oil, olive oil, sunflower oil. The vegetable fat is preferably canola oil.

In one embodiment, the egg analogue product comprises an emulsion having at least 5 wt% vegetable fat, at least 6 wt% vegetable fat, at least 7 wt% vegetable fat, at least 8 wt% vegetable fat, at least 9 wt% vegetable fat, at least 10 wt% vegetable fat, at least 11 wt% vegetable fat, at least 12 wt% vegetable fat, or at least 13 wt% vegetable fat. The egg analogue product preferably comprises an emulsion having up to 26 wt% vegetable fat, up to 27 wt% vegetable fat, up to 28 wt% vegetable fat, up to 29 wt% vegetable fat, or up to 30 wt% vegetable fat.

Egg analogue product recipes In one embodiment, the product comprises an emulsion having about 4 wt% soy protein isolate. In one embodiment, the product comprises an emulsion having about 8 wt% canola protein isolate. In one embodiment, the product comprises an emulsion having about 15 wt% vegetable fat. In one embodiment, the emulsion has a pH of equal or greater than 7.5. In one embodiment, the emulsion has a fat to protein ratio of about 1.25. In one embodiment, the canola protein isolate fraction is made up of about 2.4 wt% albumin enriched canola protein isolate and about 5.6 wt% globulin enriched canola protein isolate. In one embodiment, the canola protein isolate fraction is made up of about 4 wt% albumin enriched canola protein isolate and about 4 wt% globulin enriched canola protein isolate. In one embodiment, the product comprises an emulsion having about 4 wt% soy protein isolate, about 8 wt% canola protein isolate, about 15 wt% vegetable fat, has a pH equal or greater than 7.5, has a fat to protein ratio of about 1.25, wherein the canola protein isolate fraction is made up of about 2.4 wt% albumin enriched canola protein isolate and about 5.6 wt% globulin enriched canola protein isolate.

In one embodiment, the product comprises an emulsion having about 4 wt% soy protein isolate, about 8 wt% canola protein isolate, about 15 wt% vegetable fat, has a pH equal or greater than 7.5, has a fat to protein ratio of about 1.25, wherein the canola protein isolate fraction is made up of about 4 wt% albumin enriched canola protein isolate and about 4 wt% globulin enriched canola protein isolate.

In a preferred embodiment, the product comprises an emulsion having about 12 wt% plant protein isolate, preferably soy protein isolate and canola protein isolate, about 12 wt% vegetable fat, has a pH equal or greater than 7.5, has a fat to protein ratio of about 0.70, wherein the canola protein isolate fraction is made up of about 4 wt% albumin enriched canola protein isolate and about 4 wt% globulin enriched canola protein isolate.

In a preferred embodiment, the product comprises an emulsion having about 4 wt% soy protein isolate, about 8 wt% canola protein isolate, about 12 wt% vegetable fat, has a pH equal or greater than 7.5, has a fat to protein ratio of about 0.70, wherein the canola protein isolate fraction is made up of about 4 wt% albumin enriched canola protein isolate and about 4 wt% globulin enriched canola protein isolate.

Use as egg replacer

In some embodiments, the product can be used as a replacement for whole eggs, egg yolks, or egg whites in food products. In some embodiments, the food products can be baked goods such as but not limited to cakes, brownies, cookies, pancakes, pastries, pies, tarts, and scones. In some embodiments, the product can be used as a replacement for eggs or egg parts in other products such as but not limited to pasta, noodles, meatloaf, burgers, custards, sauces, ice cream, mayonnaise, and/or salad dressings. The product can be used in many culinary applications, for example for aerating (e.g. in sponge cakes, souffles, pavlova), binding (e.g. in hamburgers, patties, omelettes, quenelles), clarifying (e.g. in stocks, consomme soups, aspic), coating (e.g. fried or deep fried foods, such as fish, meats, chicken and vegetables), enriching (e.g. cakes, puddings, pasta, egg-nog drinks), garnishing (e.g. consomme royal, consomme celestine), glazing (e.g. bread and bread rolls, duchesse potatoes), or for thickening (e.g. soups, custards).

Definitions

When a product or emulsion is described herein in terms of wt%, this means a mixture of the ingredients on a wet basis, unless indicated otherwise.

As used herein, the term "about" is understood to refer to numbers in a range of numerals, for example the range of -30% to +30% of the referenced number, or -20% to +20% of the referenced number, or -10% to +10% of the referenced number, or -5% to +5% of the referenced number, or -1% to +1% of the referenced number. All numerical ranges herein should be understood to include all integers, whole or fractions, within the range.

As used herein, the term "analogue" is considered to be an edible substitute of a substance in regard to one or more of its major characteristics. An "egg analogue" as used herein is a substitute of egg in the major characteristics of purpose and usage. Preferably, the egg analogue product is an analogue of chicken egg. Preferably, the egg analogue is a substitute of chicken egg and is statistically similar, or has to within 60 - 100% to the texture of chicken egg, for example statistically similar rubbery, or moist, or chewy, or compact, or crumbly texture of chicken egg.

As used herein, the term "vegan" refers to an edible composition which is entirely devoid of animal products, or animal derived products, for example eggs, milk, honey, fish, and meat.

As used herein, the term "vegetarian" relates to an edible composition which is entirely devoid of meat, poultry, game, fish, shellfish or by-products of animal slaughter.

As used herein, the term "liquid" relates to a product which has a viscosity below 2200 mPa.s-1 at 25°C under 160 rpm shear.

As used herein, the term polysaccharide relates to a type of carbohydrate. A polysaccharide is a polymer comprising chains of monosaccharides that are joined by glycosidic linkages. Polysaccharides are also known as glycans. By convention, a polysaccharide consists of more than ten monosaccharide units. Polysaccharides may be linear or branched. They may consist of a single type of simple sugar (homopolysaccharides) or two or more sugars (heteropolysaccharides). The main functions of polysaccharides are structural support, energy storage, and cellular communication. Examples of polysaccharides include carrageenan, cellulose, hemicellulose, chitin, chitosan, glycogen, starch, dextrin (starch gum), hyaluronic acid, polydextrose, inulin, beta-glucan, pectin, psyllium husk mucilage, beta-mannan, carob, fenugreek, guar gum tara gum, konjac gum or glucomannan, gum acacia (llrabic), karaya, tragacanth, arabinoxylan, gellan, xanthan, agar, alginate, methylcellulose, carboxymethlylcelulose, hydroxypropyl methylcellulose, microfibrilated cellulose, microcrystalline cellulose.

As used herein, a "protein isolate" comprises at least 70 wt% protein, more preferably at least 80 wt% protein, or about 87 wt.% protein, or about 91.5 wt% protein.

Soy protein isolate may comprise about 87 wt% protein, about 3.5 wt% fat, about 3.5 wt% fiber.

Albumin enriched canola protein isolate may comprise about 91 wt% protein, about 0.2 wt% fat, about 5.9 wt% fiber.

Globulin enriched canola protein isolate may comprise about 92 wt% protein, about 2.9 wt% fat, about 4.3 wt% fiber.

The albumin enriched canola protein isolate and/or the globulin enriched canola protein isolate may have substantially the same macronutrient composition, amino acid profile, and/or mineral profile as described in Tables 1 to 4 herein.

Those skilled in the art will understand that they can freely combine all features of the present invention disclosed herein. In particular, features described for the compositions of the present invention may be combined with the method or uses of the present invention and vice versa. Further, features described for different embodiments of the present invention may be combined. Where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred to in this specification.

Further advantages and features of the present invention are apparent from the figures and nonlimiting examples.

Examples

Example 1

Composition of emulsion ingredients

The soy protein ingredient used was a commercially available soy protein isolate referred to hereinafter as SPI. The canola protein ingredients used included commercially available protein isolates enriched in canola albumin (water-soluble plant protein fraction) and canola globulin (saltsoluble plant protein fraction), referred to hereinafter as CPI 1 and CPI 2, respectively, wherein the term "enriched" indicates a relative to the total protein concentration of the specific plant protein fraction >60%. The vegetable oil used was a commercially available canola oil. pH adjustment of the emulsions was performed using commercially available potassium carbonate, potassium hydroxide or sodium hydroxide.

The proximate composition of the protein ingredients was determined using the standard methods of the Association of Analytical Chemists (AOAC). Total nitrogen was determined by the Kjeldahl method and a nitrogen-protein conversion factor of 6.25 was used to calculate the protein concentration of the protein ingredients. Moisture concentration was determined by oven drying at 103°C for 5 hours. Ash concentration was determined by dry ashing in a muffle furnace at 500°C for 5 hours. Fat concentration was determined by acid hydrolysis using a Hydrotherm (Gerhardt Analytical Systems, Kbnigswinter, Germany) followed by extraction with petroleum ether using a Soxtherm (Gerhardt Analytical Systems, Kbnigswinter, Germany. Total dietary fiber was determined using the enzymatic kit K-TDFR (Megazyme, Bray, Co. Wicklow, Ireland). Total carbohydrate (excluding fibre) was calculated by difference (100 - sum of protein, moisture, ash, fat and fibre). The proximate composition of the protein ingredients is reported in Table 1.

The amino acid profile was determined by acid hydrolysis followed by ion-exchange chromatography (IEC). Tryptophan concentration was determined by alkaline hydrolysis followed by IEC. The amino acid profile of the protein ingredients is reported in Table 2 and 3.

The mineral profile was determined using inductively coupled plasma-emission spectroscopy (ICP-ES). Phytic acid concentration was determined using the enzymatic kit K-PHYT (Megazyme, Bray, Co. Wicklow, Ireland). The mineral profile of the protein ingredients is reported in Table 4.

Table 1: Macronutrient composition (g/100 g) of the soy protein isolate (SPI), albumin-enriched (CPI 1) and globulin-enriched (CPI 2) canola protein isolates. Table 2: Non-essential amino acid profile (g/100 g) of the soy protein isolate (SPI), albumin-enriched (CPI 1) and globulin-enriched (CPI 2) canola protein isolates. Table 3: Essential amino acid profile (g/100 g) of the soy protein isolate (SPI), albumin-enriched (CPI 1) and globulin-enriched (CPI 2) canola protein isolates. Table 4: Mineral profile (mg/100 g) of the soy protein isolate (SPI), albumin-enriched (CPI 1) and globulin-enriched (CPI 2) canola protein isolates. Example 2

Preparation of the prototypes

Preparation of the prototype products was performed using pilot scale equipment as follows: SPI, CPI 1 and CPI 2 were hydrated in demineralized water which had been preheated to 65°C, under continuous shear mixing using a Stephan Mixer UMSK 24 E (Stephan Food Service Equipment GmbH, Hameln, Germany) operating at 750 rpm for 15 minutes. Canola oil was added and preparation of the emulsions involved pre-homogenization by increasing the rotational speed of the mixing blades of the Stephan Mixer to 1500 rpm for 10 minutes followed by homogenization. Homogenization was performed either by further increasing the rotational speed of the blades to 3000 rpm for 10 minutes or using a Panther NS3006L (GEA Niro Soavi SpA, Parma, Italy) two-stage valve homogenizer (APV GEA Niro Soavi S.p.A., Italy) at various first and second stage pressures. Homogenization was performed either before or after the heat treatment (i.e., upstream or downstream homogenization). Heat treatment of the product was carried out either in the Stephan Mixer or using a HT320 HTST/UHT System (OMVE, Utrecht, Netherlands) equipped with a tubular heat exchanger, via either direct steam injection or indirect heating. The pH of some of the emulsions (or pre-emulsions) was adjusted to a value corresponding to 7.5 or 8.0 at 20°C using 2M potassium carbonate, potassium hydroxide or sodium hydroxide before the heat treatment.

The recipe and the processing conditions used to prepare the prototypes are shown in Table 5 and 6, respectively. The rationales for the preparation of the different prototype groups are the following:

• Pl: Investigate the influence of the SPI concentration

• P2: Investigate the influence of the homogenization method and heat treatment

• P3: Investigate the influence of the homogenization pressure

• P4: Investigate the influence of the CPI 1 to CPI 2 ratio and pH adjustment to 7.5

• P5: Investigate the influence of the alkali agent used for pH adjustment

• P6: Investigate the influence of the oil concentration

• P7: Investigate the influence of pH adjustment from 7.5 to 8.0 Table 5: Recipes of the prototypes prepared (highlighted in bold is the best performing prototype). Table 6: Processing conditions and equipment used to prepare the prototypes (highlighted in bold is the best performing prototype).

Particle size of the prototypes was analyzed after 16 hours of storage at 4°C by static light scattering using a Mastersizer 3000 (Malvern Instruments Ltd, Malvern, Worcestershire, UK) equipped with a Reverse Fourier lens with an effective confocal length of 300 mm, a He-Ne red light source (633 nm) and a LED blue light source (470 nm). Particle and dispersant refractive indices of 1.47 and 1.33, respectively, were selected. The emulsions were added dropwise to the Hydro SM sample dispersion unit containing demineralized water until a laser obscuration of 10% (± 0.5%) was reached. Results were calculated using the Mie theory and presented as volume-weighted mean particle diameter (D[4,3]) and surface-weighted mean particle diameter (D[3,2]). For the analysis of the oil droplet size, the emulsions were diluted 1:10 in a 1% solution of the denaturing agent sodium dodecyl sulfate (SDS) before particle size analysis so as to break non-covalent interactions between oil droplets.

Viscosity of the prototypes was analyzed after 16 h of storage at 4 °C by rotational rheometry using a RVA 4500 (PerkinElmer, Inc., Waltham, Massachusetts, USA). Before the analysis, the prototypes were stored at room temperature for 1 h. A continuous shear test was performed for 30 min at a rotational speed of 160 rpm and 25°C. The viscosity value obtained after 30 min was recorded.

The performance of the prototype products upon cooking in a pan heated using an Elegance 58105 induction hob (UNOLD AG, Hockenheim, Germany) and scrambling with a cooking stick was defined by means of two parameters, that is the yield and cooking time. The pan was pre-heated at 1600 W for 30 seconds before pouring the prototypes (200 g).

The yield is an indicator of the amount of water evaporated upon cooking and is defined as the weight of the prototypes after cooking expressed as a percentage of their weight before cooking. The cooking time is defined as the time required for the prototypes to achieve a texture similar to that of scrambled chicken eggs.

Example 3

Influence of SPI concentration

Figure 1 shows the influence of the SPI concentration on the physicochemical properties of the emulsions and their cooking performance.

For the same protein (12%) and canola oil (15%) concentrations, homogenization (80 + 30 bars, upstream) and heat treatment (80°C x 30 min, indirect, Stephan Mixer), decreasing the SPI concentration from 6 to 4% had no significant influence on the cooking performance, which was inferior compared to that of chicken egg in terms of both cooking time and yield, but promoted a significant decrease in viscosity, thus making the emulsion more pourable (see P1A vs P1B).

Example 4

Influence of homogenization method and heat treatment

Figure 2 shows the influence of the homogenization method and heat treatment on the physicochemical properties of the emulsions and their cooking performance. For the same recipe and heat treatment (86°C x 30 min, indirect, Stephan Mixer), switching from high shear mixing (3000 rpm x 10 mins, upstream) to high pressure homogenization (120 + 30 bars, upstream) had no significant influence on the physicochemical properties and cooking performance of the emulsion (see P2A vs P2B).

For the same recipe, homogenization (120 + 30 bars, upstream) and heating protocol (86°C x 30 min, Stephan Mixer), switching from indirect to direct heating had no significant influence on the cooking performance but promoted a significant decrease in viscosity, thus making the emulsion more pourable (see P2B vs P2C).

For the same recipe, homogenization (120 + 30 bars, upstream) and heating method (indirect), switching to a protocol consisting of a higher temperature/shorter time (i.e., from 86°C x 30 min to 90°C x 10 min, Stephan Mixer) promoted an increase in viscosity, thus making the emulsion less pourable, but had no major influence on the cooking performance (see P2B vs P2D).

For the same recipe and homogenization (120 + 30 bars, upstream), increasing the heating temperature from 86-90°C (for 10-30 min, direct and indirect, Stephan Mixer) to 96°C (for 2 min, direct, Stephan Mixer) promoted an increase in particle size and viscosity but had no major influence on the cooking performance (see P2B-P2D vs P2E).

The cooking performance of the P2 prototypes was inferior compared to that of chicken egg.

Example 5

Influence of homogenization pressure

Figure 3 shows the influence of the homogenization pressure on the physicochemical properties of the emulsions and their cooking performance.

For the same recipe and heat treatment (80°C x 1 min, indirect, tubular heat exchanger), increasing the homogenization pressure (from 40 + 20 bars to 80 + 40 bars, downstream) promoted a decrease in particle size and degree of oil droplet flocculation, and it had no significant influence on viscosity and cooking performance, the latter being similar to that of chicken egg (see P3A vs P3B).

Example 6

Influence of CPI 1 to CPI 2 ratio and pH adjustment to 7.5

Figure 4 shows the influence of the ratio of the albumin-enriched (CPI 1) to globulin-enriched (CPI 2) canola protein isolates on the physicochemical properties of the emulsions and their cooking performance without pH adjustment (pH 6.6-6.8).

For the same protein concentration (12%), SPI concentration (4%), canola oil concentration (15%), homogenization (3000 rpm x 10 min, downstream) and heat treatment (80°C x 30 min, indirect, Stephan Mixer), increasing the relative concentration of canola albumin promoted a decrease in particle size and viscosity, and variously influenced the cooking performance. The lowest viscosity, shortest cooking time and highest yield were provided by P4E.

Figure 5 shows the influence of the ratio of the albumin-enriched (CPI 1) to globulin-enriched (CPI 2) canola protein isolates upon pH adjustment to 7.5 using K2CO3 on the physicochemical properties of the emulsion and its cooking performance.

For the same protein concentration (12%), SPI concentration (4%), canola oil concentration (15%), homogenization (3000 rpm x 10 min, downstream) and heat treatment (80°C x 30 min, indirect, Stephan Mixer), increasing the relative concentration of canola albumin variously affected the physicochemical properties of the emulsion and its cooking performance. The shortest cooking time and highest yield were provided by P4D, both being similar to that of chicken egg. P4D was overall the best performing prototype among those investigated. P4F also performed well. pH adjustment to 7.5 using K2CO3 invariably promoted a decrease in particle and oil droplet size and improvement in the cooking performance.

Example 7

Influence of alkali agent used for pH adjustment

Figure 6 shows the influence of the alkali agent used for pH adjustment to 7.5 on the physicochemical properties of the emulsions and their cooking performance.

For the same protein concentration (12%), SPI concentration (4%), canola oil concentration (15%), homogenization (3000 rpm x 10 min, downstream) and heat treatment (80°C x 30 min, indirect, Stephan Mixer), switching from K2CO3 to KOH or NaOH for pH adjustment to 7.5 promoted a marked increase in particle size and viscosity, thus making the emulsion less pourable, and negatively affected the cooking performance, both in terms of cooking time and yield.

Example 8

Influence of oil concentration

Figure 7 shows the influence of oil concentration on the physicochemical properties of the emulsions and their cooking performance.

For the same protein concentration (12%), SPI concentration (4%), homogenization (50 + 20 bars, downstream) and heat treatment (80°C x 30 min, indirect, Stephan Mixer), decreasing the oil concentration from 20 to 8% promoted a decrease in particle size, had no significant influence on oil droplet size and resulted in a major decrease in viscosity. Interestingly, it had no influence on cooking performance but promoted an improvement in terms of texture of the cooked product, specifically in terms of springiness and firmness, as determined during an internal tasting session (10 participants, data not shown), P6D being the closest to scrambled chicken egg among the P6 protoypes.

Example 9

Influence of pH adjustment from 7.5 to 8.0

Figure 8 shows the influence of pH adjustment from 7.5 to 8.0 on the physicochemical properties of the emulsions and their cooking performance.

For the same protein concentration (12%), SPI concentration (4%), canola oil concentration (8%), homogenization (50 + 20 bars, downstream) and heat treatment (80°C x 30 min, indirect, Stephan Mixer), pH adjustment from 7.5 to 8.0 had no influence on particle size, promoted a decrease in oil droplet size and a slight increase in viscosity, while it had no influence on the cooking performance. However, it promoted a further improvement in the texture of the cooked product, specifically in terms of springiness and firmness, as determined during an internal tasting session (10 participants, data not shown). P7B was the best compromise between physicochemical properties, cooking performance and texture of the cooked product, this prototype being the closest to scrambled chicken egg among all the prototypes investigated.