Use of beta-lactoglobulin for modulating protein GELS and compositions

Abstract

The present invention relates to the use of a β-lactoglobulin to modulate the gel firmness of a protein composition, by addition of extraneous β-lactoglobulin to the composition.

Description

[0001] USE OF BETA-LACTOGLOBULIN FOR MODULATING PROTEIN GELS AND COMPOSITIONS

[0002] Field of the invention

[0003] The present invention relates to the use of a p-lactoglobulin to modulate the gel firmness of a protein composition, by addition of extraneous p-lactoglobulin to the composition

[0004] Background of the invention

[0005] With a growing world population and energy, water and other resources becoming scarcer, the food industry is challenged to produce more food using less resources. An important contribution to overcoming this challenge is argued to be the protein transition; a shift from production and consumption of animal-based products to plant-based products. The past decade, significant progress has been made in utilizing and optimizing proteins from plant sources, but despite these efforts, food formulators often still face challenges with plant protein functionality and nutrition. More recently, a new source of alternative proteins has emerged using precision fermentation. While precision fermentation has long been utilized in the production of food ingredients such as enzymes and vitamins, its application has recently evolved to encompass the production of bulk proteins from animal origin, like dairy proteins. These proteins have the same functionality and nutrition as their animal-based counterparts, but with a typically lower environmental footprint. Dairy proteins from precision fermentation may be used either as standalone ingredient, or added to plant- and animal dairy-based formulations to improve functionality, nutrition and sustainability.

[0006] One of the challenges of using plant-based proteins is in the field of gel structures. In food products, many, gel structures are usually formed with the aid of or by animal derived products such as gelatine or egg protein. Examples of animal-free proteins that are nowadays routinely used in food products are pea, fava, soy, and rice protein. Non-covalent interaction, such as hydrogen bonds, hydrophobic- and electrostatic interactions, and covalent interaction, such as disulfide bonds, are features that determine important properties, such as gel strength or - firmness, water distribution, and elastic modulus, in the formation of heat-gels (See e.g. Wu et al, 2024).

[0007] While it may be a challenge to produce food product gels comprising animal-free protein without the use of animal-derived gelation agents, there is a need for an animal-free gelation agent that can enhance the gel firmness of animal-free protein compositions.

[0008] Summary of the invention

[0009] The invention relates to the use of a p-lactoglobulin to modulate the gel firmness of a protein composition, by addition of extraneous p-lactoglobulin to the composition

[0010] The invention further relates to a composition for heat set gelation comprising a protein and an extraneous p-lactoglobulin, wherein the gel firmness is increased after heat set gelation of the composition compared to the gelled composition not comprising the extraneous p-lactoglobulin.

[0011] The invention further relates to a method for gelation of a composition comprising: a. providing a protein composition, b. adding extraneous p-lactoglobulin to the composition, c. gelling the composition.

[0012] The invention further relates to a gelled composition obtainable by the method according to the invention, or to a gelled composition comprising an extraneous p-lactoglobulin, wherein the gel firmness is increased compared to a gelled composition to which no extraneous p-lactoglobulin is added.

[0013] Description of the invention

[0014] The inventors have established that p-lactoglobulin has surprising modulating properties. This is convenient since there are several challenges for protein comprising compositions, especially for compositions comprising animal-free proteins and for animal-free protein compositions. These challenges include gel firmness. The inventors have provided a solution to low firmness of animal-free protein compositions post heating. Non-animal gels using animal-free gelling agents, such as animal- free egg replacers, often suffer from low gel-firmness after heating of the egg replacer or a food product comprising such egg replacer. It has been demonstrated by the inventors that p-lactoglobulin can be used to enhance the firmness of a protein composition.

[0015] Accordingly, in a first aspect, there is provided for the use of a p-lactoglobulin to modulate the gel firmness of a protein composition, by addition of extraneous p-lactoglobulin to the composition.

[0016] In the embodiments herein, meaning all embodiment of all aspects herein, the term “protein composition” is referred to as “protein composition according to the invention”, which term is interchangeably used with the term “protein composition” or plainly “composition”.

[0017] The person skilled in the art will comprehend that p-lactoglobulin is a protein itself, albeit not a plant protein. In the embodiments herein, when the term protein is used, such as when discussing the ratio of protein to extraneous p-lactoglobulin, the protein is to be regarded as the protein fraction excluding the extraneous p-lactoglobulin, unless explicitly stated that the extraneous p-lactoglobulin is included. In the embodiments herein, the term “extraneous p-lactoglobulin” means that p-lactoglobulin is added as a separate fraction irrespective of a protein fraction of the composition that may comprise p-lactoglobulin as an intrinsic compound; the intrinsic p-lactoglobulin is thus part of the protein fraction excluding the extraneous p-lactoglobulin. In the embodiments herein, when the term p-lactoglobulin is used, it is to be construed as extraneous p-lactoglobulin; intrinsic p-lactoglobulin is only intended if the explicit term "intrinsic p-lactoglobulin” is used.

[0018] In the embodiments herein, the term “protein” is to be construed according to the definition according to the general knowledge in the technical field and as summarized in the Definitions section herein.

[0019] In the embodiments herein, the term “gel firmness” also referred to as “gel strength” is to be construed as generally known in the field, relating to the linear viscoelastic properties of a gel, depicted as G’ (g). The person skilled in the art knows how to determine gel firmness. A preferred technique to determine gel firmness is herein set forth in the examples. In the embodiments herein, modulation of gel firmness can be an increase or a decrease in gel firmness. In the embodiments herein, modulation of gel firmness preferably is increase of gel firmness.

[0020] In the embodiments herein, gelation preferably is accomplished by heat.

[0021] Accordingly, in the embodiments herein, the modulation of the gel firmness may be an increase in gel firmness. Preferably, the modulation of the gel firmness is an increase in gel firmness after heat set gelation of the composition.

[0022] In the embodiments herein, the increase in gel firmness may be any significant increase in gel-firmness compared to a gelled composition to which no extraneous p-lactoglobulin is added. The gel firmness may be increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3- fold, 4-fold 5-fold 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or at least 2 log increased compared to a gelled composition to which no extraneous p-lactoglobulin is added. The term “a gelled composition to which no extraneous p-lactoglobulin is added” is construed in all embodiments herein as a corresponding composition which comprises otherwise the same components but only differs in its components in that no extraneous p-lactoglobulin is added.

[0023] The person skilled in the art will comprehend that the protein composition may, proteins apart from the extraneous p-lactoglobulin, comprise more than one protein. The protein composition may comprise two or more distinct proteins apart from the extraneous p-lactoglobulin, such as two, three, four, five, six or seven proteins. The protein composition may be a complex composition comprising many proteins, proteins apart from the extraneous p-lactoglobulin, such as a raw protein extract from a plant. For some proteins, the capability of forming disulfide bridges may be relevant for gel firmness. Accordingly, in the embodiments herein, at least one of the proteins in the composition, apart from the extraneous p-lactoglobulin, may be capable of forming disulfide bridges. In the embodiments herein the protein capable of forming disulfide bridges may comprise at least two, such as at least two, three, four, five, six, seven, eight, nine or at lest 10 amino acids comprising a free thiol group. In the embodiments herein, an amino acid comprising a fee thiol group is preferably a cysteine.

[0024] In the embodiments herein, the protein, or a mixture of proteins, may be comprised of fully native protein, or fully denatured protein and mixes in any ration there between, such as 10% w / w : 90 w / w denatured and 90% w / w native : 10% w / w denatured. The person skilled in the art will comprehend that when a protein or a mixture of protein is denatured to such an extend that there is a large amount of aggregates in the composition, it will be hard to produce a gel from such protein. Accordingly, the protein or protein composition preferably comprises little aggregates.

[0025] In the embodiments herein, at least part of the protein in the composition may be a plant protein. The part of the protein, excluding the extraneous p-lactoglobulin, that is plant protein may be at least 1 %, such as 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%,

[0026] 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 4%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%,

[0027] 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%,

[0028] 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,

[0029] 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%; 100% meaning that all protein in the composition, excluding the extraneous p-lactoglobulin, is plant protein. In the embodiments herein, a more preferred p-lactoglobulin is a p-lactoglobulin that is not of an animal source, a p-lactoglobulin not isolated and / or purified from milk, whey or the like. Accordingly, a more preferred composition according to the invention is a protein composition that comprises a p- lactoglobulin that is not of an animal source.

[0030] In the embodiments herein, an even more preferred composition is a protein composition that does not comprise any compounds from an animal source, i.e. an animal-free protein composition. Such animal- free protein composition preferably comprises at most 0.1 % (w / w) compounds from animal origin, more preferably at most 0.01 % (w / w), at most 0.001 % (w / w), even more preferably at most 0.0001 % (w / w). Most preferably no compounds from animal origin are detectable in an animal-free protein composition. A specifically preferred protein composition according to the invention is devoid of lactose. Accordingly, in the embodiments herein, the protein composition may be devoid of, or may have low amounts of, compounds that occur in animal-derived p-lactoglobulin sources. Examples of such compounds are a- lactalbumin, bovine serum albumin, casein, glycomacropeptide, immunoglobulins, lactoferrin, and lactose. A protein composition that is devoid of compounds that occur in animal- derived p-lactoglobulin sources may be advantageous for applications wherein such components are undesirable or detrimental, for example for allergenic, health, or nutritional considerations. The amount of any of a-lactalbumin, bovine serum albumin, casein, glycomacropeptide, immunoglobulins, lactoferrin, or lactose in a protein composition according to the invention may be at most 1 % (w / w), such as between 0.0001 % and 0.5% (w / w), between 0.001 and 0.2% (w / w), or between 0.01 and 0.1 % (w / w), based on the total weight of the protein composition. The amount of any of a-lactalbumin, bovine serum albumin, casein, glycomacropeptide, immunoglobulins, lactoferrin, or lactose in a protein composition according to the invention may be at most 1 % (w / w), such as at most 0.1 % (w / w), at most 0.01 % (w / w), at most 0.001 % (w / w), or most preferably at most 0.0001 % (w / w).

[0031] In an embodiment, the ratio of p-lactoglobulin : a-lactalbumin (w / w) may be from 10,000 to 2, from 5,000 to 5, or from 1 ,000 to 10.

[0032] In an embodiment, the ratio of p-lactoglobulin : bovine serum albumin (w / w) may be from 10,000 to 5, from 5,000 to 10, or from 1 ,000 to 50.

[0033] In an embodiment, the ratio of p-lactoglobulin : glycomacropeptide (w / w) may be from 10,000 to 5, from 5,000 to 10, or from 1 ,000 to 50.

[0034] In an embodiment, the ratio of p-lactoglobulin : lactoferrin (w / w) may be from 100,000 to 10, from 10,000 to 50, or from 1 ,000 to 100.

[0035] In the embodiments herein, the composition may be in any relevant form, such as an aqueous composition or an non-aqueous composition. Preferably, the composition according to the invention is an aqueous composition. Such aqueous composition may be any aqueous composition as long as it comprises at least 10% w / w water, a protein and the extraneous p-lactoglobulin, the latter both as defined herein. Examples of aqueous compositions are, but are not limited to, butter, oleogels, emulsions such as mayonnaise, coffee creamers, and whipping cream. An aqueous composition according to the invention may also be a beverage, such as a ready to drink (RTD) protein beverage and / or a clear water protein beverage, preferably an animal-free protein beverage. The term “beverage” is herein interchangeably used with the term ’’drink”. Examples of protein beverages are, but are not limited to, Protein Drink, Protein Shake, Whey Protein Drink, Plant-Based Protein Drink, Meal Replacement Shake, Recovery Protein Drink, High-Protein Beverage, Protein-Enriched Drink, Diet Protein Shake, Medical Nutrition Shake, Nutritional Supplement Shake, Protein Shot, Protein Booster Shot, Whey Protein Shot, Protein Energy Shot

[0036] In the embodiments herein, the pH of the aqueous composition may be between pH 3 and pH 9, such as pH 3, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1 , 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or pH 9.0, preferably between pH 3 and pH 6 or more preferably between pH 6 and pH 9, or between pH 7.0 and 8.5. In the embodiments herein, the pH of the composition may be adjusted using food-safe acidic or basic additives. In the embodiments herein, the pH of the protein containing food product may be adjusted to about pH 3 to about pH 8, for example about pH 3.3 to about pH 8, about pH 4 to about pH 8, about pH 4 to about pH 7, or about pH 4 to about pH 6.8, or about pH 5 to about pH 7, or about pH 5 to about pH 6.8. In the embodiments herein, the pH of the protein containing food product may be adjusted to about pH 6.8. pH is preferably measured by equilibrating samples to 25°C and measuring using a pH probe (e.g. EC620132, Thermo Scientific) after calibrating using standards at pH 4, 7, and 10 (e.g. Pronalys, LabServ). Other methods of measuring pH will be apparent to the person skilled in the art.

[0037] In the embodiments herein, the composition may be a food product, meaning that the composition is an edible product. The food product may be any food product known to the person skilled in the art. In the embodiments herein, the food product may be a fermented food, a yoghurt, a soup, a sauce, a bar, a gel, a foam, a nutritional formulation, a beverage, a beverage whitener, a cheese, a dairy tofu, a food emulsion or a dessert. In the embodiments herein, the food product may be a yoghurt, drinking yoghurt, a bar, a gel, a foam, a nutritional formulation, medical food, dairy beverage, a product that requires the protein to form a heat-set gel, an acid protein fortified beverage, a jelly drink, a protein water, a foam, a heat-set foam extruded food product, or a food emulsion. In the embodiments herein, the food product may be considered suitable for those on a vegan diet. In the embodiments herein, composition may be the a liquid nutritional composition, including a medical beverage and a non-medical beverage. A beverage may include a sports beverage, dairy beverage, or a yoghurt beverage.

[0038] In the embodiments herein, the food product may have one or more characteristics of a dairy food product. In the embodiments herein, the food product has one or more characteristics of a dairy food product selected from the group comprising: appearance, consistency, firmness, organoleptic properties, density, stiffness, structure, viscosity, texture, elasticity, storage stability, heat stability, acidheat stability, coagulation, binding, leavening, aeration, foaming capacity, foam stability, foam overrun, behaviour when whipped, creaminess, emulsification. In the embodiments herein, the organoleptic properties may be, bit are not limited to, taste, aroma, mouthfeel, in-mouth creaminess, appearance, colour, grittiness, sandiness, and smoothness.

[0039] In the embodiments herein, the food product may contain nutrients that include vitamins and minerals. The recommended daily requirements of vitamins and minerals can be specified for various population subgroups. See for instance, Dietary Reference Intakes: RDA and Al for vitamins and elements, United States National Academy of Sciences, Institute of Medicine, Food and Nutrition Board (2010) tables recommended intakes for infants 0-6, 6-12 months, children 1-3, and 4-8 years, adults males (6 age classes), females (6 age classes), pregnant (3 age classes) and lactating (3 age classes). Concentrations of essential nutrients in the liquid nutritional composition can be tailored in the exemplary serve size for a particular subgroup or medical condition or application so that the nutrition and ease of delivery requirements can be met simultaneously. Nutrient content can be assessed using analytical methods known in the art, including but not limited to AOAC International reference methods AOAC 990.03 and AOAC 992.15, electrophoresis (e.g., SDS-PAGE), liquid column chromatography, immunochemical tests, or on-chip electrophoresis (e.g., using the Agilent Protein 80 kit and the Agilent 2100 Bioanalyzer) for determination of type and / or content of proteins and amino acids. Alternatively, chemical / biological attributes can be calculated from the nutrient contents of ingredients.

[0040] In the embodiments herein, the food product may be administered to a subject to maintain or increase muscle protein synthesis, maintain or increase muscle mass, prevent or increase loss of muscle mass, maintain or increase growth, prevent or decrease muscle catabolism, prevent or treat cachexia, prevent or treat sarcopenia, increase rate of glycogen resynthesis, modulate blood sugar levels, increase insulin response to raised blood glucose concentration, increase satiety, increase satiation, increase food intake, increase calorie intake, improve glucose metabolism, increase rate of recovery following surgery, increase rate of recovery following injury, increase rate of recovery following exercise, increase sports performance, and / or provide nutrition.

[0041] In the embodiments herein, the food product may comprise at least about 0.1 % fat by weight, such as about 0.1 %, or about 0.5%, or about 1 %, or about 3%, or about 5%, or about 10% fat by weight. In various embodiments, the protein containing food product may comprise from about 0.1 % to 40% fat by weight, and useful ranges may be selected from between any of these values (for example, from about 0.1 % to about 40%, or about 0.5% to about 40%, or about 1 % to about 40%, or about 3% to about 40%, or about 5% to about 40%, or about 10% to about 40%, or about 15% to about 40%, or about 20% to about 40%, or about 0.1 % to about 35%, or about 0.5% to about 35%, or about 1 % to about 35%, or about 3% to about 35%, or about 5% to about 35%, or about 10% to about 35%, or about 15% to about 35%, or about 20% to about 35%, or about 0.1 % to about 30%, or about 0.5% to about 30%, or about 1 % to about 30%, or about 3% to about 30%, or about 5% to about 30%, or about 10% to about 30%, or about 15% to about 30%, or about 20% to about 30%, or about 0.1 % to about 20%, or about 0.5% to about 20%, or about 1 % to about 20%, or about 3% to about 20%, or about 5% to about 20%, or about 10% to about 20%, or about 15% to about 20%).

[0042] In embodiments herein, the food product may comprise at least about 0.1 % carbohydrate by weight, such as about 0.1%, or about 0.5%, or about 1 %, or about 3%, or about 5%, or about 10% fat by weight. In various embodiments, the protein containing food product may comprise from about 0.1 % to 40% carbohydrate by weight, and useful ranges may be selected from between any of these values (for example, from about 0.1 % to about 40%, or about 0.5% to about 40%, or about 1 % to about 40%, or about 3% to about 40%, or about 5% to about 40%, or about 10% to about 40%, or about 15% to about 40%, or about 20% to about 40%, or about 0.1 % to about 35%, or about 0.5% to about 35%, or about 1 % to about 35%, or about 3% to about 35%, or about 5% to about 35%, or about 10% to about 35%, or about 15% to about 35%, or about 20% to about 35%, or about 0.1 % to about 30%, or about 0.5% to about 30%, or about 1 % to about 30%, or about 3% to about 30%, or about 5% to about 30%, or about 10% to about 30%, or about 15% to about 30%, or about 20% to about 30%, or about 0.1 % to about 20%, or about 0.5% to about 20%, or about 1 % to about 20%, or about 3% to about 20%, or about 5% to about 20%, or about 10% to about 20%, or about 15% to about 20%). Advantageously, the method of the first aspect allows for the preparation of a low-sugar or sugar-free food product comprising a fermentatively derived p-lactoglobulin. Low-sugar or sugar-free refers to from 0.01 % to 10% sugar by weight of the food product, such as from 0.01 % to 9%, or from 0.05% to 8%, or from 0.1 % to 6%, or from 0.2% to 5%, or from 0.5% to 4%.

[0043] In embodiments herein, the food product, may comprise at least about 10 kcal per 100 ml. of the food product. In various embodiments, the protein containing food product may comprise from about 10 to about 400 kcal per 100 mL of the food product, and useful ranges may be selected from between any of these values (for example, from about 10 to about 400, 10 to about 350, or about 10 to about 300, or about 10 to about 300, or about 10 to about 250, or about 10 to about 200, or about 10 to about 150, or about 10 to about 100, or about 50 to about 400, or about 50 to about 350, or about 50 to about 300, or about 50 to about 300, or about 50 to about 250, or about 50 to about 200, or about 50 to about 150, or about 50 to about 100, or about 100 to about 400, or about 100 to about 350, or about 100 to about 300, or about 100 to about 300, or about 100 to about 250, or about 100 to about 200, or about 100 to about 150, or about 150 to about 400, or about 150 to about 350, or about 150 to about 300, or about 150 to about 300, or about 150 to about 250, or about 200 to about 400, or about 200 to about 350, or about 200 to about 300, or about 200 to about 350).

[0044] In the embodiments herein, the composition may comprise at least 0.1 % (w / w) of total protein including the extraneous p-lactoglobulin, such as at least 0.2% (w / w), 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%,

[0045] 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%,

[0046] 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%,

[0047] 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,

[0048] 91 %, 92%, 93%, 94%s, 95%, 96%, 97%, 98%, 99%, or 100% (w / w) of total protein, including the extraneous p-lactoglobulin, in view of the total weight of the composition.

[0049] In the embodiments herein, in the composition, the ratio of protein to extraneous p-lactoglobulin may be within any useful range. The ratio may be within the range from 99 : 1 (w / w) to 1 : 99 (w / w). The ratio may be within the range from 99 : 1 (w / w) to 5 : 95 (w / w), such as from 98 : 2(w / w), from 97 ; 3(w / w), from 96 : 4(w / w), from 95 : 5 (w / w), from 90 : 10 (w / w), from 80 : 20 (w / w), from 75 : 25, from 70 : 30 (w / w), from 65 : 35, from 60 : 40 (w / w), from 50 : 50 (w / w), from 40 : 60 (w / w), from 30 : 70 (w / w), from 20 : 80 (w / w), from 10 : 90 (w / w) and from 5 : 95 (w / w). A preferred ratio is within the range from 95 : 5 (w / w) to 60 : 40 (w / w).

[0050] In the embodiments herein, the ratio of plant protein to extraneous p-lactoglobulin may be within any useful range. The ratio may be within the range from 99 : 1 (w / w) to 1 : 99 (w / w).The ratio may be within the range from 99 : 1 (w / w) to 5 : 95 (w / w), such as from 98 : 2 (w / w), from 97 : 3 (w / w), from 96 : 4 (w / w), from 95 : 5 (w / w), from 90 : 10 (w / w), from 80 : 20 (w / w), from 75 : 25, from 70 : 30 (w / w), from 65 : 35, from 60 : 40 (w / w), from 50 : 50 (w / w), from 40 : 60 (w / w), from 30 : 70 (w / w), from 20 : 80 (w / w), from 10 : 90 (w / w) and from 5 : 95 (w / w). A preferred ratio is within the range from 95 : 5 (w / w) to 60 : 40 (w / w).

[0051] In the embodiments herein, the plant protein in the composition may be any plant protein known to the person skilled in the art to be suitable in a composition according to the invention. In the embodiments herein, the plant protein in the composition may be a legume protein, a cereal protein and / or a nut protein. In the embodiments herein, the plant protein in the composition may comprise soy protein, rice protein, lupine protein, chickpea protein, cowpea protein, lentil protein, pea protein, fava protein, and / or protein from nuts, such as almond and cashew.

[0052] In the embodiments herein, the composition may comprise a metal salt, preferably an edible salt, preferably a mineral salt. Mineral salts are known to the person skilled in the art. Metal salts are known to the person skilled in the art and include organic metal salts and inorganic metal salts.

[0053] In the embodiments herein, the metal salt, preferably a mineral salt, in the composition may comprise: a. a cation which is a monovalent, bivalent or trivalent metal ion, such as sodium, potassium, calcium, magnesium, manganese, iron, molybdenum, chromium, copper, zinc, chromium, and selenium, preferably: calcium, potassium, sodium and / or magnesium; and b. an anion which preferably can sequester metal cations, such as phosphate, citrate, tartrate, sulphate, nitrate, and carbonate, preferably phosphate and / or citrate.

[0054] The person skilled in the art knows that salts may be anhydrous or hydrous, i.e. a salt may or may not contain water molecules in its crystalline form. The person skilled in the art knows how to compensate for the difference in weight between an anhydrous and a hydrated salt. The weight percentages herein refer to anhydrous salt, i.e. the slat without any water molecules.

[0055] Preferred salts are selected from the group consisting of: calcium carbonate, calcium chloride, tricalcium citrate, ferrous citrate, ferric phosphate, ferrous sulphate, magnesium carbonate, magnesium citrate, magnesium sulphate, zinc acetate, zinc citrate, zinc sulphate, copper(ll) hydroxide, copper(ll) citrate, copper(ll) sulphate, manganese gluconate, manganese(ll) glycerophosphate, manganese(ll) sulphate, potassium carbonate, potassium chloride, potassium acetate, sodium acetate, sodium bicarbonate, trisodium citrate, dipotassium phosphate, dimagnesium phosphate, disodium phosphate, monosodium phosphate, sodium propionate, monosodium tartrate, and disodium tartrate.

[0056] The amount of salts in the composition may be any amount used in compositions known in the art, such as, but not limited to, between 0.05% w / v and 5% w / v, such as between 0.1 % w / v and 3% w / v, such as between 0.1 % w / v and 2% w / v, or 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1 % w / v, 0.2%, 0.3%, 0.4%,

[0057] 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1 .1 %, 1.2%, 1.3%, 1.4%, 1 .5%, 1.6%, 1.7%, 1.8%, 1 .9%, 2.0%,

[0058] 2.1 %, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1 %, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%,

[0059] 3.7%, 3.8%, 3.9%, 4.0%, 4.1 %, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, or 5.0% w / v.

[0060] In the embodiments herein, the composition may be a liquid composition or a solid composition. A liquid composition according to the invention may be any liquid composition as long as it comprises a protein and the extraneous p-lactoglobulin, both as defined herein. For the avoidance of doubt, a liquid composition does not have to comprise water. A solid composition according to the invention may be any solid composition as long as it comprises a protein and the extraneous p-lactoglobulin, both as defined herein. A solid composition may be a powder composition. The person skilled in the art knows that solid / powder compositions may comprise traces of water. Herein, a solid composition may typically comprise up to 6% water. A preferred composition is a liquid composition.

[0061] In the embodiments herein, the extraneous p-lactoglobulin may be used as an egg replacer or a meat replacer, preferably an egg replacer that is used as a gelling agent.

[0062] In the embodiments herein, the p-lactoglobulin may be any p-lactoglobulin. p-lactoglobulin is the major whey protein in the milk of many mammals. In bovine milk it accounts for approximately 10 - 15% of total milk proteins and about 50 - 54% of whey protein.

[0063] Bovine p-lactoglobulin is expressed as a precursor protein comprising a 16 amino acid N-terminal signal peptide (referred to herein and elsewhere as the "full-length" p-lactoglobulin protein), which is cleaved to form a mature 162 amino acid protein. There are two primary variants of bovine p-lactoglobulin - variants A and B and a lesser variant - variant C. Sequences for both the mature and full-length forms of bovine p-lactoglobulin variants A, B and C, and wild-type full length and mature forms of p- lactoglobulin from other species are presented in Table 1.

[0064] Preferred p-lactoglobulins include proteins comprising an amino acid sequence having at least about 70% sequence identity to the sequence of a wild-type (native) p-lactoglobulin (either full length or mature p-lactoglobulin lacking a signal sequence, but preferably the mature sequence), but particularly any wild-type bovine, ovine, caprine, buffalo, equine, donkey or reindeer p-lactoglobulin sequence, including any sequence of SEQ ID NO: in Table 1 . In some embodiments the amino acid sequence of such variants may comprise a truncation or an elongation at the N-terminus and / or the C-terminus relative to the wild-type sequence, for example, truncations or elongations of from about 1 to about 20 amino acids, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, variants or modified p-lactoglobulin proteins may contain from 1 to 20 amino acid insertions, deletions, and / or substitutions (collectively) with respect to the wild-type sequence. Such proteins may be referred to herein as "elongated p-lactoglobulin proteins". In some embodiments the variants or modified p-lactoglobulin proteins may comprise one or more post-translational modifications that differ to a wild-type p-lactoglobulin protein, including glycosylation and or phosphorylation at one or more residues. An N-terminal elongation may have a sequence comprising or consisting of EA, or two or more repeats of EA, for example three or more repeats of EA, four or more repeats of EA, or five or more repeats of EA. For example, the N-terminal elongation may have a sequence comprising or consisting of EA, EAEA (SEQ ID NO: 22), EAEAEA (SEQ ID NO: 23), EAEAEAEA (SEQ ID NO: 24), EAEAEAEAEA (SEQ ID NO: 25, REAEAM (SEQ ID NO: 26), REAEAEAM (SEQ ID NO: 27), or REAEAEAEAM (SEQ ID NO: 28), KREAEAM (SEQ ID NO: 29), KREAEAEAM (SEQ ID NO: 30), or KREAEAEAEAM (SEQ ID NO: 31).

[0065] In the embodiments herein, the p-lactoglobulin may be a plurality of recombinant p-lactoglobulin proteins heterogeneous in amino acid sequence, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18 or 20 recombinant p -lactoglobulin proteins of differing amino acid sequence and / or of differing elongation.

[0066] The p-lactoglobulins, elongated p-lactoglobulins and mixes thereof as set forward in WO2022 / 269549 are preferred p-lactoglobulins and are herein incorporated by reference. Accordingly, the recombinant p-lactoglobulins comprising or consisting of an amino acid sequence having at least about 70% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 61 , 71 , 72, 73, and 74 as set forward in WO2022 / 269549 are preferred p-lactoglobulins herein.

[0067] In the embodiments herein, a specifically preferred p-lactoglobulin is an elongated p-lactoglobulin wherein at least 10%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the relative amount of p-lactoglobulin has an EAEA N-terminal elongation,

[0068] In the embodiments herein, a specifically preferred p-lactoglobulin is an elongated p-lactoglobulin wherein at least 3%, preferably at least 5%, 10%, 15%, 20%, 25%, 30%, or 35% of the relative amount of p-lactoglobulin has an EA N-terminal elongation,

[0069] In the embodiments herein, a specifically preferred p-lactoglobulin is an elongated p-lactoglobulin wherein at least 40% of the relative amount of p-lactoglobulin has an EAEA N-terminal elongation and / or wherein at least 3% of the relative amount of p-lactoglobulin has an EA N-terminal elongation,

[0070] In the embodiments herein, a specifically preferred p-lactoglobulin is an elongated p-lactoglobulin wherein at least 45% of the relative amount of p-lactoglobulin has an EAEA N-terminal elongation and / or wherein at least 5% of the relative amount of p-lactoglobulin has an EA N-terminal elongation,

[0071] As set forth in WO2022 / 269549, the p-lactoglobulins may comprise a truncation in the N-terminal part of the protein, such as a p-lactoglobulin lacking an L, LI, LIV, LIVT, LIVTQ or LIVTQT. Such truncation at the N-terminal part of the protein may be present together with the EA or EAEA N-terminal elongation. In the embodiments herein, the p-lactoglobulin protein may comprise non-native post-translational modification modulating e.g. the glycosylation and / or phosphorylation of the p-lactoglobulin, as set forward in WO2020219596A1 , which is herein incorporated by reference.

[0072] Accordingly, the p-lactoglobulin may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from T4, T6, T18, S21 , S27, S30, S36, T49, T76, T97, S110, S116, T125, S150, N152, and T154 of Bos taurus p-lactoglobulin, and having non-native glycosylation on one or more of such amino acid residues. The p-lactoglobulin may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises amino acid residue N 152 of Bos taurus p-lactoglobulin, and having non-native N-glycosylation on such amino acid residue. The p-lactoglobulin may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from T4, T6, T18, S21 , S27, S30, S36, T49, T97, SI 10, SI 16, T125, S150, and T154 of Bos taurus p-lactoglobulin, and having non-native O- glycosylation on one or more of such amino acid residues. The p-lactoglobulin may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from T4, T6, T18, Y20, S21 , S27, S30, S36, Y42, T49, T76, T97, Y99, Y102, SI 10, SI 16, T125, S150, and T154 of Bos taurus p-lactoglobulin, and having non-native phosphorylation on one or more of such amino acid residues. The p-lactoglobulin may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from K8, K14, R40, K47, K60, K69, K70, K75, K77, K83, K91 , K100, K101 , R124, K135, K138, K141 , and R148 of Bos taurus p-lactoglobulin, and having non-native methylation on one or more of such amino acid residues. The p-lactoglobulin may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from C66, C106, C119, C121 , and C160 of Bos taurus p-lactoglobulin, and having non-native palmitoylation on one or more of such amino acid residues, p-lactoglobulin may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from K8, K14, K47, K60, K69, K70, K75, K77, K83, K91 , K100, K101 , K135, K138, and K141 of Bos taurus p-lactoglobulin, and having non- native sumoylation on one or more of such amino acid residues. The p-lactoglobulin may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from C66, C106, C119, C121 , and C160 of Bos taurus p-lactoglobulin, and having non-native nitrosylation on one or more of such amino acid residues. The p-lactoglobulin may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from Y20, Y42, Y99, and Y102 of Bos taurus p-lactoglobulin, and having non-native tyrosine nitration on one or more of such amino acid residues. The p-lactoglobulin may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from F151 of Bos taurus p-lactoglobulin, and having non-native glypiation on such amino acid residue. The p-lactoglobulin may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from C160 of Bos taurus p-lactoglobulin, and having non-native farnesylation on such amino acid residue. The p- lactoglobulin may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from C160 of Bos taurus p-lactoglobulin, and having non- native geranylgeranylation on such amino acid residue.

[0073] In the embodiments herein, the p-lactoglobulin may have an attenuated or essentially eliminated allergenicity, such e.g. the recombinant milk protein as set forward in WO2021168343, which is herein incorporated by reference. Accordingly, the recombinant p-lactoglobulins comprising or consisting of an amino acid sequence having at least about 70% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, and 10 of WO2021168343 are preferred p- lactoglobulins herein.

[0074] It will be evident that the p-lactoglobulin may be a naturally occurring p-lactoglobulin, or may be a recombinant p-lactoglobulin. A natural p-lactoglobulin may be added as such or may be part of a composition distinct from the composition according to the invention, such as whey. A preferred p-lactoglobulin is a recombinant p-lactoglobulin as set forth herein. A preferred recombinant p-lactoglobulin is one selected from the group consisting of: a. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as said forth in any one of SEQ ID NO: 1 to 22 and 32 and 33; b. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 1 , wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 1 , and wherein, preferably, the additional charged amino acid is not an N-terminal EA, EAEA, EAEAEA, EAEAEAEA, or R; c. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 3, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 3, and wherein, preferably, the additional charged amino acid is not an N-terminal EA, EAEA, EAEAEA, EAEAEAEA, or R; d. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 5, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 5; e. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 7, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 7; f. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 9, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 9; g. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 11 , wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 11 ; h. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 13, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 13; i. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 15, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 15; j. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 17, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 17; k. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 19, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 19; and l. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 21 , wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 21 .

[0075] In the embodiments herein, the p-lactoglobulin comprising at least one additional charged amino acid may have a surface charge (^-potential) of at least 12mV at a pH of at most 4 and / or has a surface charge (^-potential) of at most -12mV at a pH of at least 6.

[0076] In the embodiments herein, the p-lactoglobulin comprising at least one additional charged amino acid may have a surface charge ( -potential)of at most -12mV at a pH of at least 5.5 and / or has a surface charge (^-potential) of at most -8mV at a pH of at least 5.

[0077] In the embodiments herein, the p-lactoglobulin comprising at least one additional charged amino acid may have enhanced physical stability compared to the p-lactoglobulin selected from the group consisting of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, and 21 that has the highest sequence identity with the p-lactoglobulin with the at least one additional charged amino acid.

[0078] In the embodiments herein, in the p-lactoglobulin the additional charged amino acids may be selected from the group consisting of Glutamic acid, Aspartic acid, Arginine, Lysine and Histidine.

[0079] In the embodiments herein, in the p-lactoglobulin the additional charged amino acid may be located between the N-terminal amino acid and the C-terminal amino acid of the p-lactoglobulin .

[0080] In the embodiments herein, in the p-lactoglobulin at least one of the additional charged amino acid may be located at the surface of the mature p-lactoglobulin .

[0081] In a second aspect, there is provided for a composition for heat set gelation comprising a protein and an extraneous p-lactoglobulin, wherein the gel firmness is increased after heat set gelation of the composition compared to the gelled composition not comprising the extraneous p-lactoglobulin.

[0082] In this aspect, the features are preferably the features of the first aspect. A composition for heat set gelation is a composition that is to be gelled by administration of heat. In the embodiments of this aspect, the composition is preferably the protein composition as set forth in the first aspect herein. In the embodiments of this aspect, the p-lactoglobulin is preferably the p-lactoglobulin as set forth in the first aspect herein.

[0083] In the embodiments of this aspect, the increase in gel firmness may be any significant increase in gelfirmness compared to a gelled composition to which no extraneous p-lactoglobulin is added. The gel firmness may be increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold 5-fold 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or at least 2 log increased compared to a gelled composition to which no extraneous p-lactoglobulin is added. In a third aspect, there is provided for a method for gelation of a composition comprising: a. providing a protein composition, b. adding extraneous p-lactoglobulin to the composition, c. gelling the composition.

[0084] In this aspect, the features are preferably those of the first aspect. In the embodiments of this aspect, the composition is preferably the protein composition as set forth in the first aspect herein. In the embodiments of this aspect, the p-lactoglobulin is preferably the p-lactoglobulin as set forth in the first aspect herein.

[0085] In the embodiments of this aspect, gelling of the composition may be performed by any means known in the art. A preferred means is heat set gelation, such as set forth in the examples herein. Accordingly, in the embodiments of the aspect, the gelling is preferably achieved by heating the composition.

[0086] In a fourth aspect, there is provided for a gelled composition obtainable by the method according to the third aspect, or a gelled composition comprising an extraneous p-lactoglobulin , wherein the gel firmness is increased compared to a gelled composition to which no extraneous p-lactoglobulin is added. Accordingly, there is provided for a gelled composition obtainable by the method according to the third aspect.

[0087] In addition, there is provided for a gelled composition comprising an extraneous p-lactoglobulin , wherein the gel firmness is increased compared to a gelled composition to which no extraneous p- lactoglobulin is added

[0088] In this aspect, the features are preferably those of the first aspect. In the embodiments of this aspect, the composition is preferably the protein composition as set forth in the first aspect herein. In the embodiments of this aspect, the p-lactoglobulin is preferably the p-lactoglobulin as set forth in the first aspect herein.

[0089] In the embodiments of this aspect, the increase in gel firmness may be any significant increase in gelfirmness compared to a gelled composition to which no extraneous p-lactoglobulin is added. The gel firmness may be increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold 5-fold 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or at least 2 log increased compared to a gelled composition to which no extraneous p-lactoglobulin is added.

[0090] Table 1 : Overview of sequences

[0091] Figure legends

[0092] Figure 1. Synergy factors of the respective percentages of pea protein isolate (PPI), fava bean protein isolate (FBPI) and egg white protein isolate (EWPI) combined with extraneous p-lactoglobulin to a total of 100%. Synergy factors > 1 (exceeding the dashed line) indicate a synergistic effect in terms of gel firmness.

[0093] Definitions

[0094] "Sequence identity" is herein defined as a relationship between two or more amino acid (peptide, polypeptide, or protein) sequences or two or more nucleic acid (nucleotide, polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one peptide or polypeptide to the sequence of a second peptide or polypeptide. In a preferred embodiment, identity or similarity is calculated over the whole SEQ ID NO as identified herein. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991 ; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988).

[0095] Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include e.g. the GCG program package (Devereux, J., et al., NucleicAcids Research 12 (1): 387 (1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). The well-known Smith Waterman algorithm may also be used to determine identity.

[0096] Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: BLOSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992); Gap Penalty: 12; and Gap Length Penalty: 4. A program useful with these parameters is publicly available as the "Ogap" program from Genetics Computer Group, located in Madison, Wl. The aforementioned parameters are the default parameters for amino acid comparisons (along with no penalty for end gaps).

[0097] Preferred parameters for nucleic acid comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: matches=+10, mismatch=0; Gap Penalty: 50; Gap Length Penalty: 3. Available as the Gap program from Genetics Computer Group, located in Madison, Wis. Given above are the default parameters for nucleic acid comparisons.

[0098] Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alaninevaline, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to ser; Arg to lys; Asn to gin or his; Asp to glu; Cys to ser or ala; Gin to asn; Glu to asp; Gly to pro; His to asn or gin; He to leu or val; Leu to ile or val; Lys to arg; gin or glu; Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trp to tyr; Tyr to trp or phe; and, Val to ile or leu.

[0099] A “nucleic acid molecule” or “polynucleotide” (the terms are used interchangeably herein) is represented by a nucleotide sequence. A “polypeptide” is represented by an amino acid sequence. A “nucleic acid construct” is defined as a nucleic acid molecule which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acids which are combined or juxtaposed in a manner which would not otherwise exist in nature. A nucleic acid molecule is represented by a nucleotide sequence. Optionally, a nucleotide sequence present in a nucleic acid construct is operably linked to one or more control sequences, which direct the production or expression of the peptide or polypeptide in a cell or in a subject.

[0100] “Operably linked” is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the nucleotide sequence coding for the polypeptide of the invention such that the control sequence directs the production / expression of the peptide or polypeptide of the invention in a cell and / or in a subject. “Operably linked” may also be used for defining a configuration in which a sequence is appropriately placed at a position relative to another sequence coding for a functional domain such that a chimeric polypeptide is encoded in a cell and / or in a subject.

[0101] “Expression” is construed as to include any step involved in the production of the peptide or polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification and secretion.

[0102] A “control sequence” is defined herein to include all components which are necessary or advantageous for the expression of a polypeptide. At a minimum, the control sequences include a promoter and transcriptional and translational stop signals. Optionally, a promoter represented by a nucleotide sequence present in a nucleic acid construct is operably linked to another nucleotide sequence encoding a peptide or polypeptide as identified herein.

[0103] The term "transformation" refers to a permanent or transient genetic change induced in a cell following the incorporation of new DNA (i.e. DNA exogenous to the cell). When the cell is a bacterial cell, as is intended in the present invention, the term usually refers to an extrachromosomal, self-replicating vector which harbors a selectable antibiotic resistance.

[0104] An “expression vector” may be any vector which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of a nucleotide sequence encoding a polypeptide of the invention in a cell and / or in a subject. As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes or nucleic acids, located upstream with respect to the direction of transcription of the transcription initiation site of the gene. It is related to the binding site identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites, and any other DNA sequences, including, but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one skilled in the art to act directly or indirectly to regulate the amount of transcription from the promoter. Within the context of the invention, a promoter preferably ends at nucleotide -1 of the transcription start site (TSS).

[0105] A “polypeptide” or “protein" as used herein refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A polypeptide is comprised of consecutive amino acids. The term "polypeptide" encompasses naturally occurring or synthetic molecules.

[0106] The term “recombinant polypeptide” or “recombinant protein” as used herein refers to a polypeptide that is produced in a cell of a different species or type as compared to the species or type of cell that produces the polypeptide in nature, or that is produced in a cell at a level at which it is not produced in nature.

[0107] The term "heterogeneous" as used herein with reference to a plurality of recombinant proteins means that the plurality of recombinant proteins comprises at least two or two or more, three or more, four or more, five or more, six or more, or seven or more proteins of differing amino acid sequence.

[0108] The term "mature" as used herein with reference to a protein refers to the protein, or amino acid sequence of the protein, after cleavage of the signal sequence. The term "full length" as used herein with reference to a protein refers to the protein, or amino acid sequence of the protein, comprising the signal sequence. Examples of mature and full-length proteins are provided in Table 1 herein.

[0109] The term "wild-type" as used herein with reference to proteins or polynucleotides refers to a protein or polynucleotide having an amino acid or nucleotide sequences that is the same as that expressed naturally. This term includes all naturally occurring variants of a particular protein, for example, all naturally occurring variants of p-lactoglobulin. Furthermore, this term includes both full length proteins and mature proteins and polynucleotides that encode wild-type full length and mature protein. The term is generally synonymous with the term "native".

[0110] Sequence identity herein of a polynucleotide, polynucleotide construct or of a polypeptide is preferably at least 70%. Preferably at least 70% is defined as preferably at least 70%, more preferably at least 71 %, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81 %, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, more preferably at least 98%, more preferably at least 99%, or most preferably 100% sequence identity. In case of 100% sequence identity, the polynucleotide or polypeptide has exactly the sequence of the depicted SEQ ID NO:. Sequence identity is preferably determined over the entire length of the subject sequence.

[0111] The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The skilled person is capable of identifying such erroneously identified bases and knows how to correct for such errors.

[0112] In this document and in its claims, the verbs "to comprise", “to contain”, and its conjugations are used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, the verb “to consist of’ may be replaced by “to consist essentially of’ meaning that a product or a composition or a nucleic acid molecule or a peptide or polypeptide of a nucleic acid construct or vector or cell as defined herein may comprise additional component(s) than the ones specifically identified; the additional component(s) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 10% of the value.

[0113] All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

[0114] Unless otherwise indicated each embodiment as described herein may be combined with another embodiment as described herein.

[0115] Unless stated otherwise, reference to such sources of information is not to be construed, in any jurisdiction, as an admission that such sources of information are prior art or form part of the common general knowledge in the art.

[0116] Further embodiments of the invention

[0117] 1. Use of a p-lactoglobulin to modulate the gel firmness of a protein composition, by addition of extraneous p-lactoglobulin to the composition.

[0118] 2. Use according to embodiment 1 , wherein the modulation of the gel firmness is an increase in gel firmness after heat set gelation of the composition.

[0119] 3. Use according to embodiment 2, wherein the gel firmness is increased with at least 5% compared to a gelled composition to which no extraneous p-lactoglobulin is added.

[0120] 4. Use according to any one of embodiments 1 to 3, wherein the composition comprises two or more distinct proteins apart from the extraneous p-lactoglobulin.

[0121] 5. Use according to any one of embodiments 1 to 4, wherein at least one of the proteins in the composition is capable of forming disulfide bridges.

[0122] 6. Use according to embodiment 5, wherein the protein capable of forming disulfide bridges comprises at least two amino acids comprising a free thiol group, preferably cysteines.

[0123] 7. Use according to any one of embodiments 1 to 6, wherein at least part of the protein in the composition is a plant protein.

[0124] 8. Use according to any one of embodiments 1 to 7, wherein the composition is an animal-free protein composition.

[0125] 9. Use according to any one of embodiments 1 to 8, wherein the composition is an aqueous composition.

[0126] 10. Use according to any one of embodiments 1 to 9, wherein the composition is a food product.

[0127] 11. Use according to any one of embodiments 1 to 10, wherein the composition comprises at least 0.1 % (w / w) of total protein including the extraneous p-lactoglobulin.

[0128] 12. Use according to any one of embodiments 1 to 11 , wherein the ratio of protein to extraneous p- lactoglobulin is within the range from 99 : 1 (w / w) to 1 : 99 (w / w).

[0129] 13. Use according to any one of embodiments 1 to 12, wherein the protein, or plant protein, comprises soy protein, rice protein, canola protein, lupine protein, chickpea protein, cowpea protein, lentil protein, pea protein, fava protein, and / or protein from nuts, such as almond and cashew.

[0130] 14. Use according to any one of embodiments 1 to 13, wherein the composition comprises a metal salt, preferably a mineral salt.

[0131] 15. Use according to any one of embodiments 1 to 14, wherein the extraneous p-lactoglobulin is used as an egg replacer or a meat replacer.

[0132] 16. Use according to any one of embodiments 1 to 15, wherein the p-lactoglobulin is one or more selected from the group consisting of: a. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as said forth in any one of SEQ ID NO: 1 to 22 and 32 and 33; b. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 1 , wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 1 , and wherein, preferably, the additional charged amino acid is not an N-terminal EA, EAEA, EAEAEA, EAEAEAEA, or R; c. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 3, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 3, and wherein, preferably, the additional charged amino acid is not an N-terminal EA, EAEA, EAEAEA, EAEAEAEA, or R; d. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 5, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 5; e. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 7, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 7; f. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 9, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 9; g. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 11 , wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 11 ; h. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 13, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 13; i. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 15, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 15; j. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 17, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 17; k. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 19, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 19; and l. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 21 , wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 21 .

[0133] 17. Use according to any one of embodiments 1 to 16, wherein the p-lactoglobulin has a surface charge (^-potential) of at least 12mV at a pH of at most 4 and / or has a surface charge (^-potential) of at most -12mV at a pH of at least 6.

[0134] 18. Use according to any one of embodiments 1 or 17, wherein the p-lactoglobulin has a surface charge ( -potential)of at most -12mV at a pH of at least 5.5 and / or has a surface charge ( - potential) of at most -8mV at a pH of at least 5.

[0135] 19. Use according to any one of embodiments 1 to 18, wherein p-lactoglobulin has enhanced physical stability compared to the p-lactoglobulin selected from the group consisting of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, and 21 that has the highest sequence identity with the p- lactoglobulin with the at least one additional charged amino acid.

[0136] 20. Use according to any one of embodiments 1 to 19, wherein the p-lactoglobulin is a recombinant p-lactoglobulin.

[0137] 21. Composition for heat set gelation comprising a protein and an extraneous p-lactoglobulin, wherein the gel firmness is increased after heat set gelation of the composition compared to the gelled composition not comprising the extraneous p-lactoglobulin. 22. Composition according to embodiment 21 , wherein the gel firmness is increased with at least 5% compared to the gelled composition to which no extraneous p-lactoglobulin is added.

[0138] 23. Composition according to embodiment 21 or 22, wherein the composition comprises two or more distinct proteins apart from the extraneous p-lactoglobulin.

[0139] 24. Composition according to any one of embodiments 21 to 23, wherein at least one of the proteins in the composition is capable of forming disulfide bridges.

[0140] 25. Composition according to any one of embodiments 21 to 24, wherein the protein capable of forming disulfide bridges comprises at least two amino acids comprising a free thiol group, preferably cysteines.

[0141] 26. Composition according to any one of embodiments 21 to 25, wherein at least part of the protein in the composition is a plant protein.

[0142] 27. Composition according to any one of embodiments 21 to 26, wherein the composition is an animal-free protein composition.

[0143] 28. Composition according to any one of embodiments 21 to 27, wherein the composition is an aqueous composition.

[0144] 29. Composition according to any one of embodiments 21 to 28, wherein the composition is a food product.

[0145] 30. Composition according to any one of embodiments 21 to 29, wherein the composition comprises at least 0.1 % (w / w) of total protein including the extraneous p-lactoglobulin.

[0146] 31 . Composition according to any one of embodiments 21 to 30, wherein the ratio of protein to extraneous p-lactoglobulin is within the range from 99 : 1 (w / w) to 1 : 99 (w / w).

[0147] 32. Composition according to any one of embodiments 21 to 31 , wherein the protein, or plant protein, comprises soy protein, rice protein, canola protein, lupine protein, chickpea protein, cowpea protein, lentil protein, pea protein, fava protein, and / or protein from nuts, such as almond and cashew.

[0148] 33. Composition according to any one of embodiments 21 to 32, wherein the composition comprises a metal salt, preferably a mineral salt.

[0149] 34. Composition according to any one of embodiments 21 to 33, wherein the extraneous p-lactoglobulin is used as an egg replacer or a meat replacer.

[0150] 35. Composition according to any one of embodiments 21 to 34, wherein the p-lactoglobulin is one or more selected from the group consisting of: a. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as said forth in any one of SEQ ID NO: 1 to 22 and 32 and 33; b. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 1 , wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 1 , and wherein, preferably, the additional charged amino acid is not an N-terminal EA, EAEA, EAEAEA, EAEAEAEA, or R; c. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 3, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 3, and wherein, preferably, the additional charged amino acid is not an N-terminal EA, EAEA, EAEAEA, EAEAEAEA, or R; d. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 5, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 5; e. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 7, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 7; f. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 9, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 9; g. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 11 , wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 11 ; h. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 13, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 13; i. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 15, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 15; j. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 17, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 17; k. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 19, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 19; and l. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 21 , wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 21 .

[0151] 36. Composition according to any one of embodiments 21 to 35, wherein the p-lactoglobulin has a surface charge (^-potential) of at least 12mV at a pH of at most 4 and / or has a surface charge ( - potential) of at most -12mV at a pH of at least 6.

[0152] 37. Composition according to any one of embodiments 21 to 36, wherein the p-lactoglobulin has a surface charge ( -potential)of at most -12mV at a pH of at least 5.5 and / or has a surface charge (^-potential) of at most -8mV at a pH of at least 5.

[0153] 38. Composition according to any one of embodiments 21 to 37, wherein p-lactoglobulin has enhanced physical stability compared to the p-lactoglobulin selected from the group consisting of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, and 21 that has the highest sequence identity with the p-lactoglobulin with the at least one additional charged amino acid.

[0154] 39. Method for gelation of a composition comprising: a. providing a protein composition, b. adding extraneous p-lactoglobulin to the composition, c. gelling the composition.

[0155] 40. Method for gelation of a composition according to embodiment 39, wherein gelling is achieved by heating the composition.

[0156] 41 . Method according to embodiment 39 or 40, the protein composition comprises two or more distinct proteins excluding the extraneous p-lactoglobulin.

[0157] 42. Method according to any one of embodiments 39 to 41 , wherein at least one of the proteins in the composition is capable of forming disulfide bridges.

[0158] 43. Method according to any one of embodiments 39 to 42, wherein the protein capable of forming disulfide bridges comprises at least two amino acids comprising a free thiol group, preferably cysteines.

[0159] 44. Method according to any one of embodiments 39 to 43, wherein at least part of the protein in the composition is a plant protein. 45. Method according to any one of embodiments 39 to 44, wherein the composition is an animal- free protein composition.

[0160] 46. Method according to any one of embodiments 39 to 45, wherein the composition is an aqueous composition.

[0161] 47. Method according to any one of embodiments 39 to 46, wherein the composition is a food product.

[0162] 48. Method according to any one of embodiments 39 to 47, wherein the composition comprises at least 0.1 % (w / w) of total protein including the extraneous p-lactoglobulin.

[0163] 49. Method according to any one of embodiments 39 to 48, wherein the ratio of protein to extraneous p-lactoglobulin is within the range from 99 : 1 (w / w) to 1 : 99 (w / w).

[0164] 50. Method according to any one of embodiments 39 to 49, wherein the protein, or plant protein, comprises soy protein, rice protein, canola protein, lupine protein, chickpea protein, cowpea protein, lentil protein, pea protein, fava protein, and / or protein from nuts, such as almond and cashew.

[0165] 51 . Method according to any one of embodiments 39 to 50, wherein the composition comprises a metal salt, preferably a mineral salt.

[0166] 52. Method according to any one of embodiments 39 to 51 , wherein the extraneous p-lactoglobulin is used as an egg replacer or a meat replacer.

[0167] 53. Method according to any one of embodiments 39 to 52, wherein the p-lactoglobulin is one or more selected from the group consisting of: a. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as said forth in any one of SEQ ID NO: 1 to 22 and 32 and 33; b. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 1 , wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 1 , and wherein, preferably, the additional charged amino acid is not an N-terminal EA, EAEA, EAEAEA, EAEAEAEA, or R; c. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 3, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 3, and wherein, preferably, the additional charged amino acid is not an N-terminal EA, EAEA, EAEAEA, EAEAEAEA, or R; d. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 5, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 5; e. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 7, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 7; f. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 9, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 9; g. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 11 , wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 11 ; h. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 13, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 13; i. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 15, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 15; j. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 17, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 17; k. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 19, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 19; and l. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 21 , wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 21 . 54. Method according to any one of embodiments 39 to 53, wherein the p-lactoglobulin has a surface charge (^-potential) of at least 12mV at a pH of at most 4 and / or has a surface charge (^-potential) of at most -12mV at a pH of at least 6.

[0168] 55. Method according to any one of embodiments 39 to 54, wherein the p-lactoglobulin has a surface charge ( -potential)of at most -12mV at a pH of at least 5.5 and / or has a surface charge ( - potential) of at most -8mV at a pH of at least 5.

[0169] 56. Method according to any one of embodiments 39 to 55, wherein p-lactoglobulin has enhanced physical stability compared to the p-lactoglobulin selected from the group consisting of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, and 21 that has the highest sequence identity with the p- lactoglobulin with the at least one additional charged amino acid.

[0170] 57. Method according to any one of embodiments 39 to 56, wherein the p-lactoglobulin is a recombinant p-lactoglobulin. 8. Gelled composition obtainable by the method according to any of embodiment 39 to 57, or a gelled composition comprising an extraneous p-lactoglobulin, wherein the gel firmness is increased with at least 5% compared to a gelled composition to which no extraneous p-lactoglobulin is added.

[0171] 59. Gelled composition according to embodiment 58, wherein the composition comprises two or more distinct proteins apart from the extraneous p-lactoglobulin.

[0172] 60. Gelled composition according to embodiment 58 or 59, wherein at least one of the proteins in the composition is capable of forming disulfide bridges.

[0173] 61 . Gelled composition according to any one of embodiments 58 to 60, wherein the protein capable of forming disulfide bridges comprises at least two amino acids comprising a free thiol group, preferably cysteines.

[0174] 62. Gelled composition according to any one of embodiments 58 to 61 , wherein at least part of the protein in the composition is a plant protein.

[0175] 63. Gelled composition according to any one of embodiments 58 to 62, wherein the composition is an animal-free protein composition.

[0176] 64. Gelled composition according to any one of embodiments 58 to 63, wherein the composition is an aqueous composition.

[0177] 65. Gelled composition according to any one of embodiments 58 to 64, wherein the composition is a food product.

[0178] 66. Gelled composition according to any one of embodiments 58 to 65, wherein the composition comprises at least 0.1 % (w / w) of total protein including the extraneous p-lactoglobulin.

[0179] 67. Gelled composition according to any one of embodiments 58 to 66, wherein the ratio of protein to extraneous p-lactoglobulin is within the range from 99 : 1 (w / w) to 1 : 99 (w / w). 68. Gelled composition according to any one of embodiments 58 to 67, wherein the protein, or plant protein, comprises soy protein, rice protein, canola protein, lupine protein, chickpea protein, cowpea protein, lentil protein, pea protein, fava protein, and / or protein from nuts, such as almond and cashew.

[0180] 69. Gelled composition according to any one of embodiments 58 to 68, wherein the composition comprises a metal salt, preferably a mineral salt.

[0181] 70. Gelled composition according to any one of embodiments 58 to 69, wherein the extraneous p-lactoglobulin is used as an egg replacer or a meat replacer.

[0182] 71 . Gelled composition according to any one of embodiments 58 to 70, wherein the p-lactoglobulin is one or more selected from the group consisting of: a. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as said forth in any one of SEQ ID NO: 1 to 22 and 32 and 33; b. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 1 , wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 1 , and wherein, preferably, the additional charged amino acid is not an N-terminal EA, EAEA, EAEAEA, EAEAEAEA, or R; c. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 3, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 3, and wherein, preferably, the additional charged amino acid is not an N-terminal EA, EAEA, EAEAEA, EAEAEAEA, or R; d. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 5, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 5; e. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 7, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 7; f. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 9, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 9; g. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 11 , wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 11 ; h. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 13, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 13; i. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 15, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 15; j. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 17, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 17; k. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 19, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 19; and l. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 21 , wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 21 .

[0183] 72. Gelled composition according to any one of embodiments 58 to 71 , wherein the p-lactoglobulin has a surface charge (^-potential) of at least 12mV at a pH of at most 4 and / or has a surface charge (^-potential) of at most -12mV at a pH of at least 6.

[0184] 73. Gelled composition according to any one of embodiments 58 to 72, wherein the p-lactoglobulin has a surface charge ( -potential)of at most -12mV at a pH of at least 5.5 and / or has a surface charge (^-potential) of at most -8mV at a pH of at least 5.

[0185] 74. Gelled composition according to any one of embodiments 58 to 73, wherein p-lactoglobulin has enhanced physical stability compared to the p-lactoglobulin selected from the group consisting of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, and 21 that has the highest sequence identity with the p-lactoglobulin with the at least one additional charged amino acid.

[0186] 75. Gelled composition according to any one of embodiments 58 to 74, wherein the p-lactoglobulin is a recombinant p-lactoglobulin. Examples

[0187] The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. In this specification, where reference has been made to external sources of information, including patent specifications and other documents, this is generally for the purpose of providing a context for discussing the features of the present invention.

[0188] Example 1 : Modulation of gel firmness

[0189] The inventors have provided a solution to low firmness of egg replacers post heating. Non-animal egg replacers often suffer from low gel-firmness after heating of the egg replacer or a food product comprising such egg replacer. It has been demonstrated by the inventors that extraneous p- lactoglobulin can be used to enhance the firmness of non-aqueous protein compositions such as egg replacer formulations, especially animal-free egg replacer formulations such as plant-based formulation comprising e.g. pea-, soy- and / or fava-protein.

[0190] Example 2: Heat-set gels comprising plant- and animal-derived proteins and (extraneous) B- lactoqlobulin

[0191] Materials and Methods

[0192] Fava bean protein isolate (Tendra protein isolate) was obtained from Cosun and albumin egg white (84.1 % w / w protein) was obtained from Special Ingredients. Split peas were purchases from SmaakGenot B.V. N-Ethylmaleimide (NEM) was purchased from Thermo Fisher Scientific, p-lactoglobulin was prepared as described in example 1 of WO 2022 / 269549.

[0193] Extraction of pea protein isolate

[0194] Split peas were ground using a Retsch ZM 200 (Retsch GmbH, Haan, Germany) operated at 6000 rpm and combined with a 75 pm sieve. The resulting flour was stored in hermetically sealed 1 kg bags, and kept refrigerated until further use. As a first step in protein extraction, the pea flour was added to RO water in a 1 : 10 ratio and dispersed using an overhead stirrer. After 10 minutes of stirring, the pH was adjusted to 7.0 using 1 M NaOH, and the dispersion was stirred for another two hours. The dispersion was centrifuged at 10,000 g for 30 min, to separate the insoluble material (i.e., starch, cell wall material) in the pellet from the soluble protein in the supernatant. The supernatant was collected and kept in the refrigerator. The next day, the supernatant was adjusted to pH 8.0 by adding 1 M NaOH, filtered using a filter paper, and subsequently concentrated four times using ultrafiltration with a Repligen KrosFlo® KR2i Tangential Flow Filtration (TFF) system (Repligen Corporation, Waltham, MA, USA) equipped with a Repligen TangenX™ SIUS PD 0.1 m2(LP) HyS 100 kDa single-use filtration cassette. The retentate with the globulin fraction was collected and lyophilized, resulting in a globulin-rich pea protein isolate, labelled PPI. Heat-set gelation and determination of synergistic effects

[0195] Pea protein isolate (PPI), fava bean protein isolate (FBPI), egg white protein isolate (EWPI) and p-lactoglobulin were dissolved in RO water in concentrations of 15 w / w % protein. Additionally, mixtures of p-lactoglobulin and plant- and animal-derived protein were prepared in weight-to-weight ratios of 20 : 80, 50 : 50 and 80 : 20 maintaining a final protein concentration of 15 w / w % for each mixture. The pH of the solutions were adjusted to pH 7 (±0.05) with 1 M NaOH or HCI. The stock solutions were then diluted to 10 w / w % protein and 150 pl of a 25 w / w % NaCI solution was added to make it representative of ionic strength conditions in a real food system.

[0196] The linear viscoelastic properties of the samples were evaluated using small amplitude oscillatory shear (SAOS). The 10 w / w% protein solutions were transferred to a sand-blasted concentric cylinder geometry (CC17) and mineral oil was added on top of the sample to prevent solvent evaporation. The samples were held at 20 °C for 5 minutes, then the temperature was increased to 90 °C with a rate of 3 °C per minute, held at 90 °C for 30 minutes, cooled down to 20 °C with a rate of 3 °C per minute and held at 20 °C for 30 minutes. The strain amplitude was set at 1 %, the oscillation frequency at 1 Hz and the G’ was recorded. The final G’ value was used to express gel firmness.

[0197] To determine whether the combination of extraneous p-lactoglobulin and plant- and animal-derived protein was synergistic in terms of gel firmness, the measured G’ value of a mixture was divided by the sum of the G’ contributions of each protein corrected for their fraction in the mixture, as shown in Eq. 1.

[0198] Synergy factor = - -G' mixture -Eq 1f BLG-G BLG f plant protein- G ' plant protein

[0199] A value greater than 1 indicated that extraneous p-lactoglobulin and plant- and animal-derived protein show synergy in terms of gel firmness, while a value smaller than 1 indicated that extraneous p- lactoglobulin and plant and animal-derived protein show an antagonistic effect in terms of gel firmness.

[0200] Evaluating the role of disulfide bonding in gelation

[0201] To evaluate the importance of disulfide bonding for gelation of a specific protein, or mixture of proteins, the samples were dissolved in a 20 mM N-ethylmaleimide (NEM) solution, blocking free thiol groups and inhibiting the formation of new disulfide bonds (Lei et al., 2016). After addition of NEM, the gelation behavior of the 10 w / w % protein solution was tested in the same way as described in previous section. The final G’ value of the gels with and without NEM were compared. If NEM addition led to different gelling behavior and a substantial final G’ reduction, this indicated that the protein relies on disulfide bonding for gelation.

[0202] Results and Conclusion

[0203] Figure 1 depicts mixtures of PPI, FBPI and EWPI with extraneous p-lactoglobulin in different ratios. For PPI no synergy was observed at any of the ratios tested. FBPI showed synergies at a ratio of 80 : 20 and 50 : 50 (FBPI: p-lactoglobulin) and EWPI showed synergy at a ratio 20 : 80 (EWPI : p-lactoglobulin). The other ratios of EWPI could not be measured reliably, due to suspected (micro)phase separation, causing the gel to exhibit heterogeneity in both composition and material properties.

[0204] Whether a protein relies on disulfide bonding for gelation is likely a key predictor of synergy, as observed in mixtures of FBPI and EWPI with p-lactoglobulin Previous studies have indicated that disulfide bonding plays a minor role during heat-set gelation of pea proteins (O’Kane et al., 2004; Sun & Arntfield, 2012), while it plays a major role for egg protein (Xu et al., 2024) and p-lactoglobulin (Otte et al., 2000). Limited research has been conducted on the role of disulfide bonding upon gelation of fava bean proteins, which is why we evaluated this ourselves using the thiol block agent N-ethylmaleimide (NEM). We found that addition of the thiol blocking agent NEM to 10 w / w % FBPI solutions pre-gelation resulted in a G’ reduction from 1.5 kPa (without NEM) to 0.8 kPa (with NEM). In a 50 : 50 mixture of FBPI and extraneous p-lactoglobulin, we observed a reduction from 7.1 kPa (without NEM) to 1 .0 kPa (with NEM). This indicates that disulfide blonding plays an important role for both the gelation of fava bean protein alone, and for a mixture of FBPI and extraneous p-lactoglobulin.

[0205] Preliminary results of ongoing research suggest that also for a pea protein isolate, which typically comprises few free thiol groups that can form disulfide bridges, synergy in gel strength can be achieved when extraneous p-lactoglobulin is added.

[0206] References

[0207] Lei, Z., Chen, X. D., & Mercade-Prieto, R. (2016). Effect of N-Ethylmaleimide as a Blocker of Disulfide Crosslinks Formation on the Alkali-Cold Gelation of Whey Proteins. PLOS ONE, 77(10), e0164496. https: / / doi.org / 10.1371 / journal.pone.0164496

[0208] O’Kane, F. E., Happe, R. P, Vereijken, J. M., Gruppen, H., & van Boekel, M. A. J. S. (2004). Heat- induced gelation of pea legumin: Comparison with soybean glycinin. Journal of Agricultural and Food Chemistry, 52(16), 5071-5078. https: / / doi.org / 10.1021 / jf035215h

[0209] Otte, J., Zakora, M., & Qvist, K. B. (2000). Involvement of Disulfide Bonds in Bovine p-Lactoglobulin B Gels Set Thermally at Various pH. Journal of Food Science, 65(3), 384-389. https: / / doi.Org / 10.1111 / j.1365-2621 ,2000.tb16012.x

[0210] Sun, X. D., & Arntfield, S. D. (2012). Molecular forces involved in heat-induced pea protein gelation: Effects of various reagents on the rheological properties of salt-extracted pea protein gels. Food Hydrocolloids, 28(2), 325-332. https: / / doi.Org / 10.1016 / j.foodhyd.2011.12.014

[0211] Xu, Y, Yang, J., Wu, M„ Lei, S„ Yin, P„ Yin, Q„ Zhu, T., Wang, Q„ Zhao, X., Jin, D„ Liu, R., Ge, Q„ & Yu, H. (2024). Effect of N-ethylmaleimide as a blocker of disulfide bonds formation on the properties of different protein-emulsion MP composite gels. Food Chemistry: X, 24, 101831 . https: / / doi.Org / 10.1016 / j.fochx.2024.101831

Claims

CLAIMS1. Use of a p-lactoglobulin to modulate the gel firmness of a protein composition, by addition of extraneous p-lactoglobulin to the composition.

2. Use according to claim 1 , wherein the modulation of the gel firmness is an increase in gel firmness after heat set gelation of the composition.

3. Use according to claim 2, wherein the gel firmness is increased with at least 5% compared to a gelled composition to which no extraneous p-lactoglobulin is added.

4. Use according to any one of claims 1 to 3, wherein the composition comprises two or more distinct proteins apart from the extraneous p-lactoglobulin.

5. Use according to any one of claims 1 to 4, wherein at least one of the proteins in the composition is capable of forming disulfide bridges.

6. Use according to claim 5, wherein the protein capable of forming disulfide bridges comprises at least two amino acids comprising a free thiol group, preferably cysteines.

7. Use according to any one of claims 1 to 6, wherein at least part of the protein in the composition is a plant protein.

8. Use according to any one of claims 1 to 7, wherein the composition is an animal-free protein composition.

9. Use according to any one of claims 1 to 8, wherein the composition is an aqueous composition.

10. Use according to any one of claims 1 to 9, wherein the composition is a food product.

11. Use according to any one of claims 1 to 10, wherein the composition comprises at least 0.1 % (w / w) of total protein including the extraneous p-lactoglobulin.

12. Use according to any one of claims 1 to 11 , wherein the ratio of protein to extraneous p- lactoglobulin is within the range from 99 : 1 (w / w) to 1 : 99 (w / w).

13. Use according to any one of claims 1 to 12, wherein the protein, or plant protein, comprises soy protein, rice protein, canola protein, lupine protein, chickpea protein, cowpea protein, lentil protein, pea protein, fava protein, and / or protein from nuts, such as almond and cashew.

14. Use according to any one of claims 1 to 13, wherein the composition comprises a metal salt, preferably a mineral salt.

15. Use according to any one of claims 1 to 14 wherein the p-lactoglobulin is one or more selected from the group consisting of: a. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as said forth in any one of SEQ ID NO: 1 to 22 and 32 and 33;b. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 1 , wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 1 , and wherein, preferably, the additional charged amino acid is not an N-terminal EA, EAEA, EAEAEA, EAEAEAEA, or R; c. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 3, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 3, and wherein, preferably, the additional charged amino acid is not an N-terminal EA, EAEA, EAEAEA, EAEAEAEA, or R; d. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 5, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 5; e. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 7, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 7; f. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 9, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 9; g. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 11 , wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 11 ; h. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 13, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 13; i. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 15, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 15; j. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 17, wherein the p-lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 17; k. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 19, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 19; and l. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 21 , wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 21 .

16. Use according to any one of claims 1 to 15, wherein the p-lactoglobulin has a surface charge ( - potential) of at least 12mV at a pH of at most 4 and / or has a surface charge (^-potential) of at most -12mV at a pH of at least 6.

17. Use according to any one of claims 1 or 16, wherein the p-lactoglobulin has a surface charge ( - potential)of at most -12mV at a pH of at least 5.5 and / or has a surface charge (^-potential) of at most -8mV at a pH of at least 5.

18. Use according to any one of claims 1 to 17, wherein p-lactoglobulin has enhanced physical stability compared to the p-lactoglobulin selected from the group consisting of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, and 21 that has the highest sequence identity with the p-lactoglobulin with the at least one additional charged amino acid.

19. Use according to any one of claims 1 to 18, wherein the p-lactoglobulin is a recombinant p-lactoglobulin.

20. Composition for heat set gelation comprising a protein and an extraneous p-lactoglobulin, wherein the gel firmness is increased after heat set gelation of the composition compared to the gelled composition not comprising the extraneous p-lactoglobulin.

21. Composition according to claim 20, wherein the gel firmness is increased with at least 5% compared to the gelled composition to which no extraneous p-lactoglobulin is added.

22. Composition according to claim 20 or 21 , wherein the composition comprises two or more distinct proteins apart from the extraneous p-lactoglobulin.

23. Composition according to any one of claims 20 to 22, wherein at least one of the proteins in the composition is capable of forming disulfide bridges.

24. Composition according to any one of claims 20 to 23, wherein the protein capable of forming disulfide bridges comprises at least two amino acids comprising a free thiol group, preferably cysteines.

25. Composition according to any one of claims 20 to 24, wherein at least part of the protein in the composition is a plant protein.

26. Composition according to any one of claims 20 to 25, wherein the composition is an animal-free protein composition.

27. Composition according to any one of claims 20 to 26, wherein the composition is an aqueous composition.

28. Composition according to any one of claims 20 to 27, wherein the composition is a food product.

29. Composition according to any one of claims 20 to 28, wherein the composition comprises at least 0.1 % (w / w) of total protein including the extraneous p-lactoglobulin.

30. Composition according to any one of claims 20 to 29, wherein the ratio of protein to extraneous p-lactoglobulin is within the range from 99 : 1 (w / w) to 1 : 99 (w / w).

31. Composition according to any one of claims 20 to 30, wherein the protein, or plant protein, comprises soy protein, rice protein, canola protein, lupine protein, chickpea protein, cowpea protein, lentil protein, pea protein, fava protein, and / or protein from nuts, such as almond and cashew.

32. Composition according to any one of claims 20 to 31 , wherein the composition comprises a metal salt, preferably a mineral salt.

33. Composition according to any one of claims 20 to 32, wherein the p-lactoglobulin is one or more selected from the group consisting of: a. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as said forth in any one of SEQ ID NO: 1 to 22 and 32 and 33; b. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 1 , wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 1 , and wherein, preferably, the additional charged amino acid is not an N-terminal EA, EAEA, EAEAEA, EAEAEAEA, or R; c. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 3, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 3, and wherein, preferably, the additional charged amino acid is not an N-terminal EA, EAEA, EAEAEA, EAEAEAEA, or R; d. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 5, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 5;e. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 7, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 7; f. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 9, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 9; g. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 11 , wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 11 ; h. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 13, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 13; i. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 15, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 15; j. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 17, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 17; k. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 19, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 19; and l. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 21 , wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 21 .

34. Composition according to any one of claims 29 to 33, wherein the p-lactoglobulin has a surface charge (^-potential) of at least 12mV at a pH of at most 4 and / or has a surface charge (^-potential) of at most -12mV at a pH of at least 6.

35. Composition according to any one of claims 20 to 34, wherein the p-lactoglobulin has a surface charge ( -potential)of at most -12mV at a pH of at least 5.5 and / or has a surface charge ( - potential) of at most -8mV at a pH of at least 5.

36. Composition according to any one of claims 20 to 35, wherein p-lactoglobulin has enhanced physical stability compared to the p-lactoglobulin selected from the group consisting of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, and 21 that has the highest sequence identity with the p- lactoglobulin with the at least one additional charged amino acid.

37. Method for gelation of a composition comprising: a. providing a protein composition, b. adding extraneous p-lactoglobulin to the composition, c. gelling the composition.

38. Method for gelation of a composition according to claim 37, wherein gelling is achieved by heating the composition.

39. Method according to claim 37 or 38, the protein composition comprises two or more distinct proteins excluding the extraneous p-lactoglobulin.

40. Method according to any one of claims 37 to 39, wherein at least one of the proteins in the composition is capable of forming disulfide bridges.

41. Method according to any one of claims 37 to 40, wherein the protein capable of forming disulfide bridges comprises at least two amino acids comprising a free thiol group, preferably cysteines.

42. Method according to any one of claims 37 to 41 , wherein at least part of the protein in the composition is a plant protein.

43. Method according to any one of claims 37 to 42, wherein the composition is an animal-free protein composition.

44. Method according to any one of claims 37 to 43, wherein the composition is an aqueous composition.

45. Method according to any one of claims 37 to 44, wherein the composition is a food product.

46. Method according to any one of claims 37 to 45, wherein the composition comprises at least 0.1 % (w / w) of total protein including the extraneous p-lactoglobulin.

47. Method according to any one of claims 37 to 46, wherein the ratio of protein to extraneous p- lactoglobulin is within the range from 99 : 1 (w / w) to 1 : 99 (w / w).

48. Method according to any one of claims 37 to 47, wherein the protein, or plant protein, comprises soy protein, rice protein, canola protein, lupine protein, chickpea protein, cowpea protein, lentil protein, pea protein, fava protein, and / or protein from nuts, such as almond and cashew.

49. Method according to any one of claims 37 to 48, wherein the composition comprises a metal salt, preferably a mineral salt.

50. Method according to any one of claims 37 to 49, wherein the p-lactoglobulin is one or more selected from the group consisting of: a. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as said forth in any one of SEQ ID NO: 1 to 22 and 32 and 33; b. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 1 , wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 1 , and wherein, preferably, the additional charged amino acid is not an N-terminal EA, EAEA, EAEAEA, EAEAEAEA, or R; c. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 3, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 3, and wherein, preferably, the additional charged amino acid is not an N-terminal EA, EAEA, EAEAEA, EAEAEAEA, or R; d. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 5, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 5; e. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 7, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 7; f. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 9, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 9; g. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 11 , wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 11 ; h. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 13, wherein the p-lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 13; i. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 15, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 15; j. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 17, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 17; k. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 19, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 19; and l. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 21 , wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 21 .51 . Method according to any one of claims 37 to 50, wherein the p-lactoglobulin has a surface charge (^-potential) of at least 12mV at a pH of at most 4 and / or has a surface charge (^-potential) of at most -12mV at a pH of at least 6.

52. Method according to any one of claims 37 to 51 , wherein the p-lactoglobulin has a surface charge ( -potential)of at most -12mV at a pH of at least 5.5 and / or has a surface charge (^-potential) of at most -8mV at a pH of at least 5.

53. Method according to any one of claims 37 to 52, wherein p-lactoglobulin has enhanced physical stability compared to the p-lactoglobulin selected from the group consisting of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, and 21 that has the highest sequence identity with the p-lactoglobulin with the at least one additional charged amino acid.

54. Method according to any one of claims 37 to 53, wherein the p-lactoglobulin is a recombinant p-lactoglobulin.

55. Gelled composition obtainable by the method according to any of claim 37 to 54, or a gelled composition comprising an extraneous p-lactoglobulin, wherein the gel firmness is increased with at least 5% compared to a gelled composition to which no extraneous p-lactoglobulin is added.

56. Gelled composition according to claim 55, wherein the composition comprises two or more distinct proteins apart from the extraneous p-lactoglobulin.

57. Gelled composition according to claim 55 or 56, wherein at least one of the proteins in the composition is capable of forming disulfide bridges.

58. Gelled composition according to any one of claims 55 to 57, wherein the protein capable of forming disulfide bridges comprises at least two amino acids comprising a free thiol group, preferably cysteines.

59. Gelled composition according to any one of claims 55 to 58, wherein at least part of the protein in the composition is a plant protein.

60. Gelled composition according to any one of claims 55 to 59, wherein the composition is an animal-free protein composition.61 . Gelled composition according to any one of claims 55 to 60, wherein the composition is an aqueous composition.

62. Gelled composition according to any one of claims 55 to 61 , wherein the composition is a food product.

63. Gelled composition according to any one of claims 55 to 62, wherein the composition comprises at least 0.1 % (w / w) of total protein including the extraneous p-lactoglobulin.

64. Gelled composition according to any one of claims 55 to 63, wherein the ratio of protein to extraneous p-lactoglobulin is within the range from 99 : 1 (w / w) to 1 : 99 (w / w).

65. Gelled composition according to any one of claims 55 to 64, wherein the protein, or plant protein, comprises soy protein, rice protein, canola protein, lupine protein, chickpea protein, cowpea protein, lentil protein, pea protein, fava protein, and / or protein from nuts, such as almond and cashew.

66. Gelled composition according to any one of claims 55 to 65, wherein the composition comprises a metal salt, preferably a mineral salt.

67. Gelled composition according to any one of claims 55 to 66, wherein the p-lactoglobulin is one or more selected from the group consisting of: a. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as said forth in any one of SEQ ID NO: 1 to 22 and 32 and 33; b. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 1 , wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 1 , and wherein, preferably, the additional charged amino acid is not an N-terminal EA, EAEA, EAEAEA, EAEAEAEA, or R; c. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 3, wherein the p-lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 3, and wherein, preferably, the additional charged amino acid is not an N-terminal EA, EAEA, EAEAEA, EAEAEAEA, or R; d. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 5, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 5; e. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 7, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 7; f. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 9, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 9; g. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 11 , wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 11 ; h. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 13, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 13; i. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 15, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 15; j. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 17, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 17; k. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 19, wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 19; andI. a p-lactoglobulin having an amino acid sequence that has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 21 , wherein the p- lactoglobulin comprises at least one additional charged amino acid compared to p- lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 21 .

68. Gelled composition according to any one of claims 55 to 67, wherein the p-lactoglobulin has a surface charge (^-potential) of at least 12mV at a pH of at most 4 and / or has a surface charge ( - potential) of at most -12mV at a pH of at least 6.

69. Gelled composition according to any one of claims 55 to 68, wherein the p-lactoglobulin has a surface charge ( -potential)of at most -12mV at a pH of at least 5.5 and / or has a surface charge (^-potential) of at most -8mV at a pH of at least 5.

70. Gelled composition according to any one of claims 55 to 69, wherein p-lactoglobulin has enhanced physical stability compared to the p-lactoglobulin selected from the group consisting of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, and 21 that has the highest sequence identity with the p-lactoglobulin with the at least one additional charged amino acid.

71. Gelled composition according to any one of claims 55 to 70, wherein the p-lactoglobulin is a recombinant p-lactoglobulin.

Images