Use of a β-lactoglobulin to enhance the physical stability of a protein composition

Abstract

The present invention relates to a method to increase the physical stability of a β-lactoglobulin and to a β-lactoglobulin with increased physical stability. The present invention further relates to the use of an extraneous β-lactoglobulin to enhance the physical stability of a composition comprising a protein and the extraneous β-lactoglobulin.

Description

[0001] Use of a B-lactoqlobulin to enhance the physical stability of a protein composition

[0002] Field of the invention

[0003] The present invention relates to a method to increase the physical stability of a p-lactog lobulin and to a p-lactoglobulin with increased physical stability. The present invention further relates to the use of an extraneous p-lactoglobulin to enhance the physical stability of a composition comprising a protein and the extraneous p-lactoglobulin.

[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] Protein compositions such as ready to drink (RTD) beverages, typically require to be stored at ambient temperatures. To keep them shelf stable from a microbiological perspective they need to undergo a temperature treatment such as high or ultra-high temperature (UHT) treatment. The downside of such treatment is that it may cause physical instability, particularly as a result of protein coagulation (i.e., the protein unfolds and aggregates extensively causing the formation of clumps or coagulates). The effect sometimes leads to protein deposition on the heat exchanger surface, typically referred to as fouling. Protein coagulates may give a protein composition such as an RTD an undesirable appearance and are physically unstable since they will sediment rapidly. In addition, equipment used to process such protein compositions will suffer from fouling and need to be cleaned more often than when fouling does not occur.

[0007] Altogether, there remains a need to increase the physical stability of a protein and of a protein composition, especially of and for aqueous protein compositions, such as protein beverages, especially of animal-free protein beverages.

[0008] Summary of the invention

[0009] The present invention relates to a method to increase the physical stability of a p-lactoglobulin and to a p-lactoglobulin with increased physical stability.

[0010] The invention further relates to the use of an extraneous p-lactoglobulin to enhance the physical stability of a composition comprising a protein and the extraneous p-lactoglobulin. The invention further relates to a composition comprising a protein and further comprising an extraneous p-lactoglobulin,

[0011] The invention further relates to a process for the preparation of such composition.

[0012] The invention further relates to a process comprising a heat treatment of such composition.

[0013] The invention further relates to processing equipment comprising such composition.

[0014] Description of the invention

[0015] The inventors have established that when mixing protein with extraneous p-lactoglobulin, the physical stability of such protein composition is enhanced.

[0016] Accordingly, in a first aspect, there is provided for the use of an extraneous p-lactoglobulin to enhance the physical stability of a composition comprising a protein and the extraneous p-lactoglobulin.

[0017] In the embodiments herein, meaning all embodiment of all aspects herein, the term “composition” is referred to as “composition according to the invention”, which term is interchangeably used with the term “composition” or “protein composition”..

[0018] 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.

[0019] In the embodiments herein, the term “protein” is referred to as “protein according to the invention”, which term is interchangeably used with the term “protein”. A protein herein 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.

[0020] In the embodiments herein, enhanced physical stability may results in enhanced features such as increased resistance to sedimentation, decreased turbidity, decreased cloudiness, and decreased formation of lumps in the composition. A preferred feature of enhanced physical stability is an increase in heat coagulation time of the composition, thus enhanced heat stability of the composition. Accordingly, the enhanced physical stability may result in an increase in heat coagulation time of the composition and thus the enhanced physical stability being enhanced heat stability of the composition, herein also referred to as enhanced thermal stability. Specifically, the inventors have demonstrated that p-lactoglobulin, such as extraneous p-lactoglobulin, can be used to enhance the heat coagulation time of aqueous protein compositions, such as protein beverages, especially of animal-free protein beverages. In the embodiments herein, the heat coagulation time is thus used as a measure for the physical stability of a protein or of a composition; the enhanced physical stability being enhanced heat stability of the protein or composition, herein synonymous with the term “reduced tendency to aggregate of the protein or composition”. The term ‘‘heat coagulation time” is well-known in the field (see e.g. McSweeney et al, 2004, Food Hydrocolloids, Volume 18, Issue 1 ,109-125) and is the time between applying heat to the composition and the onset of the formation of visible protein particles, also called “flocs”. For determining heat coagulation time, heat may be applied to a protein or a composition while the protein or composition is in a borosilicate heat-stable glass tube which tube is immersed in an oil bath. The heat coagulation time can be depicted in any unit of time such as seconds, minutes and hours. A preferred assay to assess heat coagulation time of a protein or of a composition is the assay set forth in the examples herein. A preferred analysis time is 1800 seconds. The person skilled in the art will comprehend that when the heat coagulation time of a single protein is determined, the protein will be in a solution, such as an aqueous, buffered solution.

[0021] In the embodiments herein, the heat coagulation time of a protein or composition may be increased by any significant amount, such as increased by at least 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or preferably at least 10%, such as increased by at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or increased by at least 10- fold, 20-fold, 30-fold, 40-fold or 50-fold. Most preferably, the heat coagulation time is increased infinitely, meaning that no coagulation is observed during the assay or in the RTD.

[0022] 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%,

[0023] 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 4%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%,

[0024] 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,

[0025] 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,

[0026] 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.

[0027] In the embodiments herein, the increased heat coagulation time may reduce fouling of equipment used to process the composition.

[0028] In the embodiments herein, the processing of the composition may comprise a heat treatment.

[0029] Fouling of equipment may occur when a protein composition is processed in any type of equipment. When during or shortly before the processing said protein composition is heated above denaturation temperature, the decreased physical stability of the composition may result in fouling of the equipment when protein aggregates deposit on the surfaces of the equipment.

[0030] In the embodiment herein, the reduction of fouling of equipment may be at least 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, 100%, at least 2-foled, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or at least 10-fold. The reduced fouling of the equipment may result in having to clean the equipment less frequently within a certain timeframe, such as at least 2-fold less, 3-fold less, 4-fold less or at least 5- fold less. In the embodiments herein, the heat treatment may be any type of heat treatment known to the person skilled in the art, such as e.g. in Tadini and Gut, Front. Nutr., 26 April 2022, Sec. Nutrition and Food Science Technology, Volume 9; and Ashurst and Hargitt, Woodhead Publishing Series in Food Science, Technology and Nutrition, Soft Drink and Fruit Juice Problems Solved, Woodhead Publishing, 2009. which are herein incorporated by reference. Heat treatment are typically used to inactivate microbes such as bacteria, yeasts and moulds. A moderate heat treatment will inactivate the microbes to such extent that the amount of microorganisms in the product is below a safe threshold during a prolonged period of time. More rigid heat treatment will render the product sterile. After heat treatment, the composition is preferably quickly cooled down to 7°C or below such 6, 5, 4, 3, or2°C. Examples of such treatment are, but are not limited to:

[0031] Flash pasteurization, which is typically performed at a temperature of between 75 - 85°C for a time of between 1 - 4 minutes, or at a temperature of between 90 - 96°C for a time of between 30 and 90 seconds,

[0032] Hot filling, which is typically performed at a temperature of between 83 - 88°C for a time of between 30 - 90 seconds, or at a temperature of between 92 - 95°C for a time of between 10 - 15 seconds,

[0033] Tunnel pasteurization which is typically performed at a temperature of between 72 - 80°C for a time of between 5 - 20 minutes,

[0034] High temperature treatment which is typically performed at a temperature of between 105 - 115°C for a time of between 30 seconds - 4 minutes

[0035] Ultra high temperature treatment which is typically performed at a temperature of between 130 - 150°C, such between 135 - 140 °C for a time of between 1 - 9 seconds.

[0036] Hot filling or hot fill method is a typical packaging technique used in the beverage industry, particularly for acid or acidified foods and beverages. In this process, the product is typically heated to a temperature between 70°C and 93°C and then filled into containers. The filled containers are sealed and typically held at the fill temperature for about 10 minutes to achieve product sterility. This method ensures the elimination of unwanted microorganisms and preserves the product’s quality. Beverages with a pH less than 4.5 are typically pasteurized before hot filling to neutralize any microbiological activity. After sealing, the containers are typically quickly cooled to maintain product and vitamin integrity. The hot fill method can extend the shelf life of beverages up to six months, provided that the packaging materials used are resistant to the process conditions.

[0037] Further examples of heat treatments are:

[0038] Thermization where the composition is typically heated to 63 - 65 °C for about 15 seconds, a time / temperature combination that does not inactivate the phosphatase enzyme. To prevent aerobic spore-forming bacteria from multiplying after thermization, the composition is typically rapidly chilled to 4 °C or below

[0039] Low temperature, long time (LTLT) treatment where the composition is typically heated to 63 °C for about 30 minutes High temperature short time (HTST) treatment where the composition is typically heated to 72 - 75 °C for about 15 - 20 seconds or to >80°C for about 1 - 5 seconds.

[0040] Ultra pasteurization where the composition is typically heated to 125 - 138 °C for about 2 - 4 seconds

[0041] Sterilization in container where the composition is preferably after hot filling into clean containers, sterilized at 1 15 - 120 °C for about 20 to 30 minutes.

[0042] The person skilled in the art will comprehend that the temperature and times set forth here above can be varied as desired to achieve a desired heat load.

[0043] In the embodiments herein, in addition to the extraneous p-lactoglobulin, intrinsic p-lactoglobulin may be present in the composition. This means that the protein, or at least a part of the protein comprises p-lactoglobulin as an intrinsic compound; intrinsic meaning that the protein fraction in its natural occurring form comprises p-lactoglobulin. An example of a protein fraction that comprises intrinsic p- lactoglobulin is whey protein. Another example is milk.

[0044] In the embodiments herein, in the composition, the extraneous p-lactoglobulin may be any p- lactoglobulin known to the person skilled in the art. The p-lactoglobulin may be a non-recombinant or a recombinant 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. 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 less common 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.

[0045] 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 wildtype 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 amino acid A. 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 A, 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), REAEAEAEAM (SEQ ID NO: 28), KREAEAM (SEQ ID NO: 29), KREAEAEAM (SEQ ID NO: 30), or KREAEAEAEAM (SEQ ID NO: 31). In the embodiments herein, the p-lactoglobulin may be a mixture of heterogenous p-lactoglobulin proteins. 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. In a non-limiting embodiment, the p-lactoglobulin may comprise a native p- lactoglobulin and a p-lactoglobulin having an N-terminal elongation of the amino acid A. In a non-limiting embodiment, the p-lactoglobulin may comprise a native p-lactoglobulin and a p-lactoglobulin having an N-terminal elongation of the amino acids EAE. In a non-limiting embodiment, the p-lactoglobulin may comprise a p-lactoglobulin having an N-terminal elongation of the amino acid A and a p-lactoglobulin having an N-terminal elongation of the amino acids EAE.

[0046] The 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. In the embodiments herein, the p-lactoglobulin may comprise non-native post- translational modification modulating e.g. the glycosylation and / or phosphorylation of the p- lactoglobulin, as set forward in WO2020219596A1 and US2022211061 , which are herein incorporated by reference.

[0047] 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. 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, 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- lactoglobu lin 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.

[0048] In the embodiments herein, the p-lactoglobulin protein may have an attenuated or essentially eliminated allergenicity, such e.g. the p-lactoglobulin as set forward in WO2021168343 and US2023106635, which 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, and 10 of WO2021168343 and US2023106635 are preferred p-lactoglobulins herein.

[0049] 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. 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-lactoglobu lin 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).

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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%,

[0055] 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%,

[0056] 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,

[0057] 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,

[0058] 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.

[0059] 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).

[0060] 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).

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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. 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%,

[0067] 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%,

[0068] 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%,

[0069] 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.

[0070] 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.

[0071] In the embodiments herein, the liquid composition may be 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

[0072] In the embodiments herein, the pH of the aqueous composition may be between pH 3 and pH 8, 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., 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, or pH 8.0 preferably between pH 3 and pH 4 or between pH 6 and pH 8.

[0073] In a second aspect, there is provided for a composition comprising a protein and further comprising an extraneous p-lactoglobulin. In this aspect, the features are preferably the features of the first aspect. The composition of this aspect is herein referred to as the “composition according to the second aspect”. This means inter alia that the features as set forth for the composition of the first aspect are preferably also the features of the composition according to the second aspect.

[0074] In the composition according to the second aspect, the protein is a plant protein as set forth elsewhere herein. In the composition according to the second aspect, the ratio between p-lactoglobulin and other protein present in the composition 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).

[0075] In the composition according to the second aspect, the p-lactoglobulin may be a p-lactoglobulin as set forth in the seventh aspect herein. Accordingly, the p-lactoglobulin may be a p-lactoglobulin consisting of an amino acid sequence selected from the group consisting of:

[0076] - 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;

[0077] - 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;

[0078] - 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;

[0079] - 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;

[0080] - 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;

[0081] - 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 ;

[0082] - 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; - 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;

[0083] - 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;

[0084] - 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,

[0085] - 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.

[0086] In the composition according to the second aspect, 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.

[0087] In the composition according to the second aspect, 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.

[0088] In the composition according to the second aspect, 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. In the composition according to the second aspect, the enhanced physical stability of the p-lactoglobulin may result in an increase in heat coagulation time of the p-lactoglobulin and / or wherein the enhanced physical stability results in an increase in heat-induced opacity time of the p-lactoglobulin or of a composition comprising the p-lactoglobulin .

[0089] In the composition according to the second aspect, the heat coagulation time and / or the heat-induced opacity time of the p-lactoglobulin or of a composition comprising the p-lactoglobulin may be increased by at least 10%.

[0090] In the composition according to the second aspect, the p-lactoglobulin comprising at least one additional charged amino acid may be a recombinant p-lactoglobulin .

[0091] In the composition according to the second aspect, the p-lactoglobulin comprising at least one additional charged amino acid may be an animal-free protein.

[0092] In the composition according to the second aspect, 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. In the composition according to the second aspect, 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 .

[0093] In the composition according to the second aspect, in the p-lactoglobulin at least one of the additional charged amino acid may be located at the surface of the mature p-lactoglobulin .

[0094] In the composition according to the second aspect, the p-lactoglobulin may be a recombinant p- lactoglobu lin as set forth elsewhere herein.

[0095] In the composition according to the second aspect, the composition may further comprise a metal salt as set forth elsewhere herein, preferably a mineral salt as set forth elsewhere herein.

[0096] In the composition according to the second aspect, the metal salt, preferably a mineral salt, may comprise: a. a cation which is a monovalent, bivalent or trivalent metal ion such as sodium, potassium, calcium, magnesium, manganese, iron, molybdenum, copper, zinc, chromium, and selenium, preferably: calcium, potassium, sodium and / or magnesium, as described elsewhere herein, and b. an anion which preferably can sequester metal cations, such as phosphate, citrate, tartrate, sulphate, nitrate, and carbonate, preferably phosphate and / or citrate, as described elsewhere herein.

[0097] In the composition according to the second aspect, the amount of salt in the composition may be 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%, 0.5%, 0.6%, 0.7%,

[0098] 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%, 2.1 %, 2.2%, 2.3%,

[0099] 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%, 3.7%, 3.8%, 3.9%,

[0100] 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.

[0101] In a third aspect, there is provided for processing equipment comprising a composition according to the first or second aspect, or comprising a p-lactoglobulin according to the seventh aspect. Such processing equipment may be any processing equipment known to the person skilled in the art. In this aspect, the features are preferably those of the first and / or second aspect.

[0102] In a fourth aspect, there is provided for a method forthe production of a composition according to the first aspect or second aspect, comprising: a. providing the protein and the extraneous p-lactoglobulin, and optional other ingredients oi the composition, and b. mixing the protein and the extraneous p-lactoglobulin and optionally with the other ingredients.

[0103] In this aspect, the features are preferably those of the first, second and / or third aspect.

[0104] In a fifth aspect, there is provided for a process for heat treatment of a composition according to the first or second aspect, comprising a step of applying a heat treatment to the composition. The heat treatment may be any heat treatment known to the person skilled in the art and is preferably a heat treatment as set forth elsewhere herein. In this aspect, the features are preferably those of the first, second, third and / or fourth aspect.

[0105] The inventors have further established that the introduction of an additional charged amino acid into a p-lactoglobulin, and / or the exchange of a non-charged amino acid within a p-lactoglobulin for a charged amino acid, enhances the physical stability, specifically the heat stability of the molecule. The inventors hypothesize that this phenomenon is applicable to other proteins as well, such as other milk proteins like lactoferrin, alpha-lactalbumin, and non-milk proteins such as serum albumin, and immunoglobulins. Accordingly, in a sixth aspect, there is provided for a method for enhancing the physical stability of a protein, selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, said method comprising the introduction of an additional charged amino acid into the p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, or immunoglobulin, preferably p-lactoglobulin, and / or the exchange of a non-charged amino acid within the protein such as a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, for a charged amino acid. In this aspect, the features are preferably the features of the first and second aspect herein, The method is herein referred to as a method according to the invention. In this aspect, the features are preferably those of the first, second, third, fourth and / or fifth aspect. In this aspect, the protein, selected from a p-lactoglobulin lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, are collectively referred to as a protein according to the sixth aspect. Accordingly, there is provided for a method for enhancing the physical stability of a p-lactoglobulin, said method comprising the introduction of an additional charged amino acid into the p-lactoglobulin, and / or the exchange of a non-charged amino acid within the p-lactoglobulin, for a charged amino acid. In addition, there is provided for a method for enhancing the physical stability of a lactoferrin, alpha-lactalbumin, said method comprising the introduction of an additional charged amino acid into the lactoferrin, alpha-lactalbumin, and / or the exchange of a noncharged amino acid within the lactoferrin, alpha-lactalbumin, for a charged amino acid. In addition, there is provided for a method for enhancing the physical stability of an immunoglobulin, said method comprising the introduction of an additional charged amino acid into the immunoglobulin, and / or the exchange of a non-charged amino acid within the immunoglobulin, for a charged amino acid.

[0106] When a non-charged amino acid is replaced with a charged amino acid, it is herein construed that molecule contains, after replacement, an additional charged amino acid. It is within the scope of the invention that one or more charged amino acids are introduced into the protein according to the sixth aspect, preferably a p-lactoglobulin, by either introduction as such or by replacement of one or more non- charged amino acids by a charged amino acid.

[0107] In the embodiments herein, in the method according to the invention, the enhanced physical stability preferably comprises or is an increase in heat coagulation time and / or heat-induced opacity time of the protein according to the sixth aspect, preferably a p-lactoglobulin, or of a composition comprising the protein according to the sixth aspect, preferably a p-lactoglobulin, thus enhanced heat stability of the protein according to the sixth aspect, preferably a p-lactoglobulin, or of a composition comprising the protein according to the sixth aspect, preferably a p-lactoglobulin. Accordingly, in the method according to the invention, the enhanced physical stability preferably comprises or is an increase in heat coagulation time, and thus the enhanced physical stability being enhanced heat stability, herein also referred to as enhanced thermal stability.

[0108] In the method according to the invention, the enhanced physical stability preferably comprises an increase in heat-induced opacity time of the protein according to the sixth aspect, preferably p-lactoglobulin, and thus the enhanced physical stability being enhanced heat stability, herein also referred to as enhanced thermal stability. In the embodiments herein, the heat-induced opacity time is used as a measure for physical stability of the protein according to the sixth aspect, preferably a p-lactoglobulin, or of a composition comprising the protein according to the sixth aspect, preferably a p-lactoglobulin. The term ‘‘heat-induced opacity time” is well-known in the field and is the time between applying heat to the protein according to the sixth aspect, preferably a p-lactoglobulin, or composition comprising the protein according to the sixth aspect, preferably a p-lactoglobulin, and the onset of the formation of opacity, also referred to as opaqueness. For determining the heat-induced opacity time, heat may be applied to the protein according to the sixth aspect, preferably a p-lactoglobulin, or composition while the protein according to the sixth aspect, preferably a p-lactoglobulin, or composition is in a borosilicate heat-stable glass tube which tube is immersed in an oil bath. The heat-induced opacity time can be depicted in any unit of time such as seconds, minutes and hours. A preferred assay to assess heat-induced opacity time of a composition is the assay set forth in the examples herein. A preferred analysis time is 1800 seconds.

[0109] In the embodiments herein, the heat coagulation time and / or heat-induced opacity time of the protein according to the sixth aspect, preferably a p-lactoglobulin, or composition comprising the protein according to the sixth aspect, preferably a p-lactoglobulin, may be increased by any significant amount, such as increased by at least 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or preferably at least 10%, such as increased by at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or increased by at least 10-fold, 20-fold, 30-fold, 40-fold or 50-fold. Most preferably, the heat coagulation time is increased infinitely, meaning that no coagulation is observed during the assay or in the RTD.

[0110] In the embodiments herein, the heat coagulation time of the protein according to the sixth aspect, preferably a p-lactoglobulin, or composition comprising the protein according to the sixth aspect, preferably a p-lactoglobulin, may be increased by any significant amount, such as increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or preferably at least 10%, such as increased by at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or increased by at least 10-fold, 20-fold, 30-fold, 40-fold or 50-fold. Most preferably, the heat coagulation time is increased infinitely, meaning that no coagulation is observed during the assay or in the RTD.

[0111] In the embodiments herein, the heat-induced opacity time of the protein according to the sixth aspect, preferably a p-lactoglobulin, or composition comprising the protein according to the sixth aspect, preferably a p-lactoglobulin, may be increased by any significant amount, such as increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or preferably at least 10%, such as increased by at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or increased by at least 10-fold, 20-fold, 30-fold, 40-fold or 50-fold. Most preferably, the heat coagulation time is increased infinitely, meaning that no coagulation is observed during the assay or in the RTD.

[0112] In the embodiments herein, the additional charged amino acid or amino acids are preferably selected from the group consisting of Glutamic acid (E), Aspartic acid (D), Arginine (R), Lysine (K) and Histidine (H). A preferred charged amino acid is Glutamic acid. Another preferred charged amino acid is Aspartic acid. Another preferred charged amino acid is Arginine. Another preferred charged amino acid is Lysine. Another preferred charged amino acid is Histidine.

[0113] It is within the scope of the invention that a combination of additional charged amino acids is introduced in the protein according to the sixth aspect, preferably a p-lactoglobulin. In the embodiments of the invention, the one or more charged amino acids may be introduced together with a non-charged amino acid, such as e.g. “EA” or repeats of “EA”, such as “EAEA”.

[0114] In the embodiments herein, it is preferred that at least one of the one or more additional charged amino acid is located at the surface of the mature protein according to the sixth aspect, preferably a p-lactoglobulin. Preferably, two, three, four five, or all of the additional charged amino acids are located at the surface of the of the mature protein according to the sixth aspect, preferably a p-lactoglobulin.

[0115] The person skilled in the art knows how to predict whether an amino acid will be at the surface of a protein or not.

[0116] In the embodiments herein, the ^-potential of the protein according to the sixth aspect, preferably a p-lactoglobulin, after introduction of the additional charged amino acid into the p-lactoglobulin, and / or the exchange of the non-charged amino acid within the protein according to the sixth aspect, preferably a p-lactoglobulin, for a charged amino acid, is preferably altered by at least 0.5mV in a pH range of 2 to 4 and / or in a pH range of 5 to 7.

[0117] More preferably, the ^-potential is altered by at least 1 mV, 1.5mV, 2mV, 3mV, 4mV, 5mV, 6mV, 7mV, 8mV, 9mV, 10mV, 11 mV, or at least 12mV in a pH range of 2 to 4 and / or in a pH range of 5 to 7.

[0118] More preferably, the ^-potential is altered by at least 1 mV, 1.5mV, 2mV, 3mV, 4mV, 5mV, 6mV, 7mV, 8mV, 9mV, 10mV, 11 mV, or at least 12mV in a pH range of 2 to 4 and in a pH range of 5 to 7.

[0119] More preferably, the ^-potential altered by at least 0.5mV, 1 mV, 1 ,5mV, 2mV, 3mV, 4mV, 5mV, 6mV, 7mV, 8mV, 9mV, 10mV, 11 mV, or at least 12mV in a pH range of 2 to 4.

[0120] More preferably, the ^-potential altered by at least 0.5mV, 1 mV, 1 ,5mV, 2mV, 3mV, 4mV, 5mV, 6mV, 7mV, 8mV, 9mV, 10mV, 11 mV, or at least 12mV in a pH range of 5 to 7,

[0121] In the embodiments herein, the protein according to the sixth aspect, preferably a p-lactoglobulin, from which the physical stability is to be enhanced by introduction of an additional charged amino acid into the protein according to the sixth aspect, preferably a p-lactoglobulin, and / or by exchange of a noncharged amino acid within the protein according to the sixth aspect, preferably a p-lactoglobulin, for a charged amino acid, may be any protein according to the sixth aspect, preferably a p-lactoglobulin, known to the person skilled in the art. The protein according to the sixth aspect, preferably a p-lactoglobulin, may be a naturally occurring protein according to the sixth aspect, preferably a p-lactoglobulin. The protein according to the sixth aspect, preferably a p-lactoglobulin, may be a recombinant protein according to the sixth aspect, preferably a p-lactoglobulin. The protein according to the sixth aspect, preferably a p-lactoglobulin, may be an animal-free protein according to the sixth aspect, preferably a p-lactoglobulin. Preferably, when the protein according to the sixth aspect is a p-lactoglobulin, the p-lactoglobulin is a p-lactoglobulin selected from the group consisting of the p-lactoglobulins having a sequence as set forth in any one of SEQ ID NO:s 1 to 21. A more preferred p-lactoglobulin is a p-lactoglobulin with the sequence as set forth in SEQ ID NO: 1 , SEQ ID NO: 3, or SEQ ID NO: 5. A more preferred p-lactoglobulin is a p-lactoglobulin with the sequence as set forth in SEQ ID NO: 1. A further more preferred p-lactoglobulin is a p-lactoglobulin with the sequence as set forth in SEQ ID NO: 3. A further more preferred p-lactoglobulin is a p-lactoglobulin with the sequence as set forth in SEQ ID NO: 5.

[0122] In the embodiments herein, the method comprises production of the protein according to the sixth aspect, preferably a p-lactoglobulin, with enhanced physical stability by culture, preferably by fermentation, more preferably by precision fermentation. Preferably, the method is a recombinant method wherein the protein according to the sixth aspect, preferably a p-lactoglobulin, is expressed in a host cell of choice and is expressed from an expression construct. In the expression construct, the encoding sequence of the protein according to the sixth aspect, preferably a p-lactoglobulin, can be modified such that an additional charged amino acid is introduced or that a non-charged amino acid is replaced by a charged amino acid. Such technology is known to the person skilled in the art. In the embodiments herein, host cells of choice may be, but are not limited to fungi, such as filamentous fungi, as well as bacteria, yeast, algae, plant, insect, and mammalian cells. The host cell may be a yeast cell selected from the list consisting of Pichia pastoris (also known as Komagataella phaffii), Kluyveromyces lactis, Saccharomyces cerevisiae, Schizosaccharomyces pombe , Yarrowia lipolytica , Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia methanoiica, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous, and Candida albicans species. In some embodiments, the yeast cell may be a Saccharomycete. The host cell may be a fungal cell selected from the list consisting of Aspergillus spp. and Trichoderma spp. The host cell may be selected from Pichia pastoris, Kluyveromyces lactis and Aspergillus niger. The host ell may be selected from Pichia pastoris, Kluyveromyces lactis , Saccharomyces cerevisiae and Aspergillus niger. The host cell may be selected from Pichia pastoris and Aspergillus niger. The host cell may be selected from Pichia pastoris, Kluyveromyces lactis and Saccharomyces cerevisiae. The host cell may be bacterial host cell such as Lactococcus lactis, Bacillus subtilis or Escherichia coli. Other host cells include bacterial host such as, but not limited to, Lactococci sp., Lactococcus lactis, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus megaterium, Brevibacillus choshinensis, Mycobacterium smegmatis, Rhodococcus erythropolis and Corynebacterium glutamicum, Lactobacilli sp., Lactobacillus fermentum, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus plantarum and Synechocystis sp. 6803. A most preferred host is Pichia pastoris.

[0123] In the embodiments herein, the protein according to the sixth aspect, preferably a p-lactoglobulin, with enhanced physical stability preferably is an animal-free protein according to the sixth aspect, more preferably an animal-free a p-lactoglobulin. In a seventh aspect, there is provided for a protein with enhanced physical stability, selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, or an immunoglobulin, with enhanced physical stability, according to the sixth aspect, obtainable or obtained by the method according to the sixth aspect.

[0124] Preferably, in this seventh aspect, there is provided for a p-lactoglobulin obtainable by a method according to the sixth aspect or a p-lactoglobulin selected from the group consisting of:

[0125] - an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% 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 the additional charged amino acid is not an N-terminal EA, EAEA, EAEAEA, EAEAEAEA, or R;

[0126] - an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% 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 the additional charged amino acid is not an N-terminal EA, EAEA, EAEAEA, EAEAEAEA, or R;

[0127] - an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% 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;

[0128] - an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% 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;

[0129] - an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% 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;

[0130] - an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% 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 ;

[0131] - an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% 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;

[0132] - an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 15, wherein the p-lactog lobulin 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;

[0133] - an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% 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;

[0134] - an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% 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,

[0135] - an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% 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 .

[0136] In this aspect, the features are preferably the features of the first, second or sixth aspect herein. The protein with enhanced physical stability selected from a p-lactoglobulin lactoferrin, alpha-lactalbumin, serum albumin, or an immunoglobulin, preferably a p-lactoglobulin, with enhanced physical stability according to this aspect is herein referred to as the protein, preferably p-lactoglobulin, according to the invention.

[0137] In the embodiments herein, the protein, preferably a p-lactoglobulin, according to the invention comprising at least one additional charged amino acid preferably 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. In the embodiments herein, the protein, preferably a p-lactoglobulin, comprising at least one additional charged amino acid preferably has a surface charge (^-potential) of at least 12mV at a pH of at most 4. In the embodiments herein, the protein, preferably a p-lactoglobulin, comprising at least one additional charged amino acid preferably has a surface charge (^-potential) of at most -12mV at a pH of at least 6. In the embodiments herein, the protein, preferably a p-lactoglobulin, comprising at least one additional charged amino acid preferably has a surface charge (^-potential) of at least 12mV at a pH of at most 4 and has a surface charge (^-potential) of at most -12mV at a pH of at least 6.

[0138] In the embodiments herein, the protein, preferably a p-lactoglobulin, according to the invention comprising at least one additional charged amino acid preferably 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. In the embodiments herein, the protein, preferably a p-lactoglobulin, comprising at least one additional charged amino acid preferably has a surface charge K-potential)of at most -12mV at a pH of at least 5.5. In the embodiments herein, the protein, preferably a p-lactoglobulin, comprising at least one additional charged amino acid preferably has a surface charge (^-potential) of at most -8mV at a pH of at least 5. In the embodiments herein, the p-lactoglobulin comprising at least one additional charged amino acid preferably has a surface charge ( -potential)of at most -12mV at a pH of at least 5.5 and has a surface charge (^-potential) of at most -8mV at a pH of at least 5.

[0139] In the embodiments herein, the p-lactoglobulin according to the invention comprising at least one additional charged amino acid, preferably 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. The person skilled in the art will comprehend that the comparison is to be made between a p-lactoglobulin according to the invention and a corresponding p-lactoglobulin from the list hereinabove that has the closest similarity to the p-lactoglobulin according to the invention.

[0140] In the embodiments herein, in the protein, preferably a p-lactoglobulin, according to the invention comprising at least one additional charged amino acid, the enhanced physical stability preferably results in an increase in heat coagulation time of the protein, preferably a p-lactoglobulin, or of a composition comprising the protein, preferably a p-lactoglobulin, and / or in an increase in heat-induced opacity time of the p-lactoglobulin or of a composition comprising the protein, preferably p a -lactoglobulin.

[0141] In the embodiments herein, in the protein, preferably a p-lactoglobulin, according to the invention comprising at least one additional charged amino acid, the enhanced physical stability preferably results in an increase in heat coagulation time and in an increase in heat-induced opacity time of the protein, preferably a p-lactoglobulin, or of a composition comprising the protein, preferably a p-lactoglobulin.

[0142] In the embodiments herein, in the protein, preferably a p-lactoglobulin, according to the invention comprising at least one additional charged amino acid, the enhanced physical stability preferably results in an increase in heat coagulation time of the protein, preferably a p-lactoglobulin, or of a composition comprising the protein, preferably a p-lactoglobulin.

[0143] In the embodiments herein, in the protein, preferably a p-lactoglobulin, according to the invention comprising at least one additional charged amino acid, the enhanced physical stability preferably results in an increase in heat-induced opacity time of the protein, preferably a p-lactoglobulin, or of a composition comprising the protein, preferably a p-lactoglobulin.

[0144] In the embodiments herein, the heat coagulation time and / or heat-induced opacity time of the protein, preferably a p-lactoglobulin, according to the invention or composition comprising the protein, preferably a p-lactoglobulin, may be increased by any significant amount, such as increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or preferably at least 10%, such as increased by at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or increased by at least 10-fold, 20-fold, 30-fold, 40-fold or 50-fold. Most preferably, the heat coagulation time is increased infinitely, meaning that no coagulation is observed during the assay or in the RTD.

[0145] In the embodiments herein, the heat coagulation time of the protein, preferably a p-lactoglobulin, or composition comprising the protein, preferably a p-lactoglobulin, may be increased by any significant amount, such as increased by at least 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or preferably at least 10%, such as increased by at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or increased by at least 10-fold, 20-fold, 30-fold, 40-fold or 50-fold. Most preferably, the heat coagulation time is increased infinitely, meaning that no coagulation is observed during the assay or in the RTD.

[0146] In the embodiments herein, the heat-induced opacity time of the protein, preferably a p-lactoglobulin, or composition comprising the protein, preferably a p-lactoglobulin, may be increased by any significant amount, such as increased by at least 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or preferably at least 10%, such as increased by at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or increased by at least 10-fold, 20-fold, 30-fold, 40-fold or 50-fold. Most preferably, the heat coagulation time is increased infinitely, meaning that no coagulation is observed during the assay or in the RTD.

[0147] In the embodiments herein, the protein, preferably a p-lactoglobulin, according to the invention comprising at least one additional charged amino acid may be a recombinant protein, preferably a p-lactoglobulin.

[0148] In the embodiments herein, the protein, preferably a p-lactoglobulin, according to the invention comprising at least one additional charged amino acid preferably is an animal-free protein, preferably an animal-free p-lactoglobulin.

[0149] In the embodiments herein, in the protein, preferably a p-lactoglobulin, according to the invention comprising at least one additional charged amino acid, the additional charged amino acid or amino acids may be selected from the group consisting of Glutamic acid (E), Aspartic acid (D), Arginine (R), Lysine (K) and Histidine (H). A preferred charged amino acid is Glutamic acid. Another preferred charged amino acid is Aspartic acid. Another preferred charged amino acid is Arginine. Another preferred charged amino acid is Lysine. Another preferred charged amino acid is Histidine.

[0150] It is within the scope of the invention that a combination of additional charged amino acids is present in the protein, preferably a p-lactoglobulin, according to the invention. In the embodiments of the invention, the one or more charged amino acids may be introduced together with a non-charged amino acid.

[0151] In the embodiments herein, in the protein, preferably a p-lactoglobulin, according to the invention comprising at least one additional charged amino acid, the additional charged amino acid is preferably located between the N-terminal amino acid and the C-terminal amino acid of the protein, preferably a p-lactoglobulin.

[0152] In the embodiments herein, in the protein, preferably a p-lactoglobulin, according to the invention comprising at least one additional charged amino acid, at least one of the one or more additional charged amino acids is preferably located at the surface of the mature protein, preferably a p-lactoglobulin. Preferably, two, three, four five, or all of the additional charged amino acids are located at the surface of the mature protein, preferably a p-lactoglobulin.

[0153] In an eight aspect, there is provided for the use of one or more charged amino acids to enhance the physical stability of a protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, by introduction of the one or more charged amino acids into the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, and / or by exchange of one or more non-charged amino acids within the protein selected from a p-lactoglobulin, lactoferrin, alpha- lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, for the one or more charged amino acids. In this aspect, the features are preferably the features of the first, second, sixth or seventh aspect herein.

[0154] In the embodiments of this aspect, the enhanced physical stability preferably results in an increase in heat coagulation time of the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, or of a composition comprising the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin and / or in an increase in heat-induced opacity time of the n, preferably a p-lactoglobulin, or of a composition comprising the protein selected from a p- lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, thus enhanced physical stability preferably is enhanced heat stability of the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin , or of a composition comprising the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin , herein also referred to as enhanced thermal stability.

[0155] In the embodiments of this aspect, the enhanced physical stability preferably results in an increase in heat coagulation time and in an increase in heat-induced opacity time of the protein selected from a p- lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, or of a composition comprising the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin.

[0156] In the embodiments of this aspect, the enhanced physical stability preferably results in an increase in heat coagulation time of the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, or of a composition comprising the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin.

[0157] In the embodiments of this aspect, the enhanced physical stability preferably results in an increase in heat-induced opacity time of the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, or of a composition comprising the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin.

[0158] In the embodiments of this aspect, the heat coagulation time and / or heat-induced opacity time of the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, or composition comprising the protein selected from a p- lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, may be increased by any significant amount, such as increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or preferably at least 10%, such as increased by at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or increased by at least 10-fold, 20-fold, 30-fold, 40-fold or 50-fold. Most preferably, the heat coagulation time is increased infinitely, meaning that no coagulation is observed during the assay or in the RTD. In the embodiments of this aspect, the heat coagulation time of the protein selected from a p- lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, or composition comprising the protein selected from a p-lactoglobulin, lactoferrin, alphalactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, may be increased by any significant amount, such as increased by at least 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or preferably at least 10%, such as increased by at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or increased by at least 10- fold, 20-fold, 30-fold, 40-fold or 50-fold. Most preferably, the heat coagulation time is increased infinitely, meaning that no coagulation is observed during the assay or in the RTD.

[0159] In the embodiments of this aspect, the heat-induced opacity time of the protein selected from a p- lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, or composition comprising the protein selected from a p-lactoglobulin, lactoferrin, alphalactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, may be increased by any significant amount, such as increased by at least 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or preferably at least 10%, such as increased by at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or increased by at least 10- fold, 20-fold, 30-fold, 40-fold or 50-fold. Most preferably, the heat coagulation time is increased infinitely, meaning that no coagulation is observed during the assay or in the RTD.

[0160] In the embodiments of this aspect, the additional charged amino acid or amino acids are preferably selected from the group consisting of Glutamic acid (E), Aspartic acid (D), Arginine (R), Lysine (K) and Histidine (H). A preferred charged amino acid is Glutamic acid. Another preferred charged amino acid is Aspartic acid. Another preferred charged amino acid is Arginine. Another preferred charged amino acid is Lysine. Another preferred charged amino acid is Histidine.

[0161] It is within the scope of this aspect that a combination of additional charged amino acids is introduced in the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin. In the embodiments of the invention, the one or more charged amino acids may be introduced together with a non-charged amino acid, such as e.g. “EA” or repeats of “EA”, such as “EAEA”.

[0162] In the embodiments of this aspect, at least one of the one or more additional charged amino acids is preferably located at the surface of the mature protein selected from a p-lactoglobulin, lactoferrin, alphalactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin. Preferably, all of the additional charged amino acids are located at the surface of the mature protein selected from a p- lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin.

[0163] In the embodiments of this aspect, the ^-potential of the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, after introduction of the one or more charged amino acids into the protein selected from a p- lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, and / or the exchange of the one or more non-charged amino acid within the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, for the one or more charged amino acid, is preferably altered by at least 0.5mV in a pH range of 2 to 4 and / or in a pH range of 5 to 7.

[0164] In the embodiments of this aspect, the ^-potential of the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, after introduction of the one or more charged amino acids into the protein selected from a p- lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, and / or the exchange of the one or more non-charged amino acid within the p-lactoglobulin for the one or more charged amino acid, is preferably altered by at least 0.5mV in a pH range of 2 to 4 and in a pH range of 5 to 7.

[0165] More preferably, the ^-potential is altered by at least 1 mV, 1.5mV, 2mV, 3mV, 4mV, 5mV, 6mV, 7mV, 8mV, 9mV, 10mV, 11 mV, or at least 12mV in a pH range of 2 to 4 and / or in a pH range of 5 to 7.

[0166] More preferably, the ^-potential is altered by at least 1 mV, 1.5mV, 2mV, 3mV, 4mV, 5mV, 6mV, 7mV, 8mV, 9mV, 10mV, 11 mV, or at least 12mV in a pH range of 2 to 4 and in a pH range of 5 to 7.

[0167] More preferably, the ^-potential altered by at least 0.5mV, 1 mV, 1 ,5mV, 2mV, 3mV, 4mV, 5mV, 6mV, 7mV, 8mV, 9mV, 10mV, 11 mV, or at least 12mV in a pH range of 2 to 4.

[0168] More preferably, the ^-potential altered by at least 0.5mV, 1 mV, 1 ,5mV, 2mV, 3mV, 4mV, 5mV, 6mV, 7mV, 8mV, 9mV, 10mV, 11 mV, or at least 12mV in a pH range of 5 to 7,

[0169] In the embodiments of this aspect, the p-lactoglobulin from which the physical stability is to be enhanced by introduction of the one ore more charged amino acids into the p-lactoglobulin, and / or by exchange of one or more non-charged amino acid within the p-lactoglobulin for the one or more charged amino acids, preferably is a p-lactoglobulin selected from the group consisting of the p-lactoglobulins having a sequence as set forth in any one of SEQ ID NO:s 1 to 21 .

[0170] In the embodiments of this aspect, the p-lactoglobulin from which the physical stability is to be enhanced by introduction of the one ore more charged amino acids into the p-lactoglobulin, and / or by exchange of one or more non-charged amino acid within the p-lactoglobulin for the one or more charged amino acids, preferably is a p-lactoglobulin having an amino acid sequence as set forth in SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, preferably, the p-lactoglobulin to be modified has the amino acid sequence as set forth in SEQ ID NO: 1 or in SEQ ID NO: 3.

[0171] In the embodiments of this aspect, the use preferably comprises production of the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, with enhanced physical stability by culture, preferably by fermentation, more preferably by precision fermentation. Culture, fermentation and precision fermentation are preferably as described in the sixth aspect herein.

[0172] In the embodiments of this aspect, the protein selected from a p-lactoglobulin, lactoferrin, alphalactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin with enhanced physical stability preferably is an animal-free protein selected from a p-lactoglobulin, lactoferrin, alphalactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin. Such animal-free protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, 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 selected from a p-lactoglobulin , lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin. A specifically preferred protein selected from a p- lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, according to the invention is devoid of lactose. Accordingly, in the embodiments herein, the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, may be devoid of, or may have low amounts of, compounds that occur in animal-derived protein sources, preferablyp-lactoglobulin sources. Examples of such compounds for a p-lactoglobulin composition are a-lactalbumin, bovine serum albumin, casein, glycomacropeptide, immunoglobulins, lactoferrin, and lactose. A derived protein selected from a p- lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, that is devoid of compounds that occur in animal-derived protein selected from a p- lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a 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 p-lactoglobulin 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 p-lactoglobulin. The amount of any of a-lactalbumin, bovine serum albumin, casein, glycomacropeptide, immunoglobulins, lactoferrin, or lactose in a p-lactoglobulin 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).

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] Table 1 : Overview of sequences

[0178] Figure legends

[0179] Figure 1 depicts the ^-potential (y-axis) versus the pH (x-axis). Depicted are the ^-potentials of a p-lactoglobulin according to the invention comprising 91% of a p-lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 32 and comprising 7% of a p-lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 33 and of a naturally occurring p-lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 1 . It is clear that the p-lactoglobulin according to the invention has a significantly lower ^-potential within the pH range of 5 to 7 compared to naturally occurring p-lactoglobulin.

[0180] Figure 2 depicts the heat-induced opacity time (HOT; y-axis) versus the pH (x-axis) as a measure of physical stability.

[0181] Depicted are the heat-induced opacity time of a p-lactoglobulin according to the invention comprising 91% of a p-lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 32 and comprising

[0182] 7% of a p-lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 33 and of a naturally occurring p-lactoglobulin. It is clear that the p-lactoglobulin according to the invention has a significantly higher heat-induced opacity time at pH lower than 4 and at pH higher than 6, when compared to naturally occurring p-lactoglobulin.

[0183] Definitions

[0184] "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).

[0185] 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.

[0186] 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).

[0187] 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.

[0188] 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; lie to leu or val; Leu to ile or val; Lys to arg; gin or glu; Met to leu or ile; Phe to met, leu ortyr; Ser to thr; Thrto ser; Trp to tyr; Tyr to trp or phe; and, Val to ile or leu.

[0189] 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.

[0190] “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.

[0191] “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.

[0192] 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.

[0193] 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.

[0194] 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).

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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".

[0200] 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.

[0201] 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.

[0202] 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.

[0203] All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

[0204] Unless otherwise indicated each embodiment as described herein may be combined with another embodiment as described herein.

[0205] 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.

[0206] Further embodiments of the invention

[0207] 1. Use of an extraneous p-lactoglobulin to enhance the physical stability of a composition comprising a protein and the extraneous p-lactoglobulin.

[0208] 2. Use according to embodiment 1 , wherein the enhanced physical stability results in an increase in heat coagulation time of the composition.

[0209] 3. Use according to embodiment 1 or 2, wherein the heat coagulation time is increased by at least 10%.

[0210] 4. Use according to any one of embodiments 1 to 3, wherein at least part of the protein in the composition is a plant protein.

[0211] 5. Use according to any one of embodiments 1 to 4, wherein the composition is an animal-free protein composition. 6. Use according to any one of embodiments 1 to 5, wherein the increased heat coagulation time reduces fouling of equipment used to process the composition.

[0212] 7. Use according to embodiment 6, wherein the processing of the composition comprises a heat treatment.

[0213] 8. Use according to any one of embodiments 1 to 7, wherein, in addition to the extraneous p-lactoglobulin, intrinsic p-lactoglobulin is present in the composition.

[0214] 9. Use according to any one of embodiments 1 to 8, wherein the extraneous p-lactoglobulin is a recombinant p-lactoglobulin.

[0215] 10. Use according to any one of embodiments 1 to 9, wherein the composition comprises at least 0.1 % (w / w) of total protein including the extraneous p-lactoglobulin.

[0216] 11. Use according to any one of embodiments 1 to 10, wherein the ratio of protein to extraneous p- lactoglobulin is within the range from 99 : 1 (w / w) to 1 : 99 (w / w).

[0217] 12. Use according to any one of embodiments 1 to 11 , wherein the plant protein is a legume protein, a cereal protein and / or a nut protein.

[0218] 13. Use according to any one of embodiments 1 to 12, wherein the 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.

[0219] 14. Use according to any one of embodiments 1 to 13, wherein the composition comprises a metal salt, preferably a mineral salt.

[0220] 15. Use according to embodiment 14, wherein the salt, preferably a mineral salt, comprises: a. a cation which is a monovalent, bivalent or trivalent metal ion such as sodium, potassium, calcium, magnesium, manganese, iron, molybdenum, 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.

[0221] 16. Use according to any one of embodiments 1 to 15, wherein the composition is a liquid composition or a solid composition.

[0222] 17. Use according to embodiment 16, wherein the liquid composition is an aqueous composition.

[0223] 18. Use according to embodiment 17, wherein the pH of the aqueous composition is between pH 3 and pH 8, preferably between pH 3 and pH 4 or between pH 6 and pH 8.

[0224] 19. Use according to embodiment 17 or 18, wherein the aqueous composition is a ready-to-drink beverage and / or a clear protein water. A composition comprising a protein and further comprising an extraneous p-lactoglobulin, and wherein the p-lactoglobulin consists of an amino acid sequence selected from the group consisting of:

[0225] - 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;

[0226] - 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;

[0227] - 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;

[0228] - 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;

[0229] - 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;

[0230] - 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 ;

[0231] - 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;

[0232] - 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; - 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;

[0233] - 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,

[0234] - 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.

[0235] 21 . A composition according to embodiment 20, wherein the p-lactoglobulin comprising at least one additional charged amino acid 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.

[0236] 22. A composition according to embodiment 20 or 21 , wherein the p-lactoglobulin comprising at least one additional charged amino acid 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.

[0237] 23. A composition according to any one of embodiments 20 to 22, wherein the p-lactoglobulin comprising at least one additional charged amino acid 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.

[0238] 24. A composition according to embodiment 23, wherein the enhanced physical stability of the p-lactoglobulin results in an increase in heat coagulation time of the p-lactoglobulin and / or wherein the enhanced physical stability results in an increase in heat-induced opacity time of the p-lactoglobulin or of a composition comprising the p-lactoglobulin.

[0239] 25. A composition comprising at least one additional charged amino acid according to embodiment 24, wherein the heat coagulation time and / orthe heat-induced opacity time ofthe p-lactoglobulin or of a composition comprising the p-lactoglobulin is increased by at least 10%.

[0240] 26. A composition according to any one of embodiments 20 to 25, wherein the p-lactoglobulin comprising at least one additional charged amino acid is a recombinant p-lactoglobulin.

[0241] 27. A composition according to any one of embodiments 20 to 26, wherein the p-lactoglobulin comprising at least one additional charged amino acid is an animal-free protein. 28. A composition according to any one of embodiments 20 to 27, wherein in the p-lactoglobulin the additional charged amino acids are selected from the group consisting of Glutamic acid, Aspartic acid, Arginine, Lysine and Histidine.

[0242] 29. A composition according to any one of embodiments 20 to 28, wherein in the p-lactoglobulin the additional charged amino acid is located between the N-terminal amino acid and the C-terminal amino acid of the p-lactoglobulin.

[0243] 30. A composition according to any one of embodiments 20 to 29, wherein in the p-lactoglobulin, at least one of the additional charged amino acid is located at the surface of the mature p-lactoglobulin.

[0244] 31. A composition according to any one of embodiments 20 to 30, wherein the protein is a plant protein.

[0245] 32. A composition according to any one of embodiments 20 to 31 , wherein the ratio between p-lactoglobulin and other protein present in the composition preferably is within the range from 99 : 1 (w / w) to 1 : 99 (w / w)

[0246] 33. A composition according to any one of embodiments 20 to 32, wherein the p-lactoglobulin is a recombinant p-lactoglobulin.

[0247] 34. A composition according to any one of embodiments 20 to 33, further comprising a metal salt, preferably a mineral salt.

[0248] 35. A composition according to embodiment 34, wherein the wherein the metal salt, preferably a mineral salt, comprises: a. a cation which is a monovalent, bivalent or trivalent metal ion such as sodium, potassium, calcium, magnesium, manganese, iron, molybdenum, 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.

[0249] 36. A composition according to embodiment 34 or 35, wherein the amount of salt in the composition is 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%,

[0250] 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%,

[0251] 2.0%, 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%,

[0252] 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%, or 5.0% w / v.

[0253] 37. Processing equipment comprising a composition according to any one of embodiments 20 to

[0254] 36, or of a composition as defined in anyone of embodiments 1 to 19. 38. A method for the production of a composition according to any one of embodiments 20 to 36, comprising: a. providing the protein and the extraneous p-lactoglobulin, and optional other ingredients of the composition, and b. mixing the protein and the extraneous p-lactoglobulin and optionally with the other ingredients.

[0255] 39. A process for heat treatment of a composition according to any one of embodiments 20 to 36, or of a composition as defined in anyone of embodiments 1 to 19, comprising the step of applying a heat treatment to the composition.

[0256] 40. A method for enhancing the physical stability of a protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, said method comprising the introduction of an additional charged amino acid into the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, and / or the exchange of a non-charged amino acid within the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, for a charged amino acid, wherein the enhanced physical stability preferably is enhanced heat stability.

[0257] 41. A method according to embodiment 40, wherein the enhanced physical stability comprises an increase in heat coagulation time and / or heat-induced opacity time of the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, or of a composition comprising the protein selected from a p- lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, and wherein the increase in heat coagulation time and / or heat-induced opacity time of the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, or of a composition comprising the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, is at least 5%.

[0258] 42. A method according to embodiment 40 or 41 , wherein the increase in heat coagulation time and / or heat-induced opacity time of the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, or of a composition comprising the protein selected from a p-lactoglobulin, lactoferrin, alphalactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, is at least 10%.

[0259] 43. A method according to any one of embodiments 40 to 42, wherein the additional charged amino acids are selected from the group consisting of Glutamic acid, Aspartic acid, Arginine, Lysine and Histidine. A method according to anyone of embodiments 40 to 43, wherein the additional charged amino acid is located at the surface of the mature protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin. A method according to any one of embodiments 40 to 44, wherein the ^-potential of the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, after introduction of the additional charged amino acid into the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, and / or the exchange of the noncharged amino acid within the protein selected from a p-lactoglobulin, lactoferrin, alphalactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, for a charged amino acid, is altered by at least 0.5mV in a pH range of 2 to 4 and / or in a pH range of 5 to 7. A method according to any one of embodiments 40 to 45, wherein the p-lactoglobulin from which the physical stability is to be enhanced by introduction of an additional charged amino acid into the p-lactoglobulin, and / or by exchange of a non-charged amino acid within the p-lactoglobulin for a charged amino acid, is a p-lactoglobulin selected from the group consisting of the p-lactoglobulins having a sequence as set forth in any one of SEQ ID NO:s 1 to 21 . A method according to embodiment 46, wherein the p-lactoglobulin from which the physical stability is to be enhanced by introduction of an additional charged amino acid into the p-lactoglobulin, and / or by exchange of a non-charged amino acid within the p-lactoglobulin for a charged amino acid, is a p-lactoglobulin having an amino acid sequence as set forth in SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, preferably as set forth in SEQ ID NO: 1 or in SEQ ID NO: 3. A method according to any one of embodiments 40 to 47, wherein the method comprises production of the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, with enhanced physical stability by culture, preferably by fermentation, more preferably by precision fermentation. A method according to any one of embodiments 40 to 48, wherein the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, with enhanced physical stability is an animal-free p-lactoglobulin. A protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin obtainable by a method according to any one of embodiments 40 to 40, or a p-lactoglobulin selected from the group consisting of:

[0260] - 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 the additional charged amino acid is not an N-terminal EA, EAEA, EAEAEA, EAEAEAEA, or R; - 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 the additional charged amino acid is not an N-terminal EA, EAEA, EAEAEA, EAEAEAEA, or R;

[0261] - 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;

[0262] - 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;

[0263] - 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;

[0264] - 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 ;

[0265] - 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;

[0266] - 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;

[0267] - 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;

[0268] - 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,

[0269] - 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. A p-lactoglobulin comprising at least one additional charged amino acid according to embodiment 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. A p-lactoglobulin comprising at least one additional charged amino acid according to embodiment 50 or 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. A p-lactoglobulin comprising at least one additional charged amino acid according to any one of embodiments 50 to 52, wherein the 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. A protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, comprising at least one additional charged amino acid according to any one of embodiments 50 to 53, wherein the enhanced physical stability results in an increase in heat coagulation time of the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, and / or wherein the enhanced physical stability results in an increase in heat- induced opacity time of the protein selected from a p-lactoglobulin, lactoferrin, alphalactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, or of a composition comprising the protein selected from a p-lactoglobulin, lactoferrin, alphalactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin. A protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably p-lactoglobulin, comprising at least one additional charged amino acid according to embodiment 54, wherein the heat coagulation time and / or the heat-induced opacity time of the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, or of a composition comprising the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, is increased by at least 10%. A protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, according to any one of embodiments 50 to 55, wherein the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, comprising at least one additional charged amino acid is a recombinant protein selected from a p-lactoglobulin, lactoferrin, alphalactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin. 57. A protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, according to any one of embodiments 50 to 56, wherein the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, comprising at least one additional charged amino acid is an animal-free protein.

[0270] 58. A protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, according to any one of embodiments 50 to 57, wherein the additional charged amino acids are selected from the group consisting of Glutamic acid, Aspartic acid, Arginine, Lysine and Histidine.

[0271] 59. A protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, according to any one of embodiments 50 to 58, wherein the additional charged amino acid is located between the N-terminal amino acid and the C-terminal amino acid of the protein selected from a p-lactoglobulin, lactoferrin, alphalactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin.

[0272] 60. A protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, according to any one of embodiments 50 to 59, wherein at least one of the additional charged amino acid is located at the surface of the mature protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin.

[0273] 61 . Use of one or more charged amino acids to enhance the physical stability of a protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, by introduction of the one or more charged amino acids into the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, and / or by exchange of one or more non-charged amino acids within the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin forthe one or more charged amino acids, wherein the enhanced physical stability preferably is enhanced heat stability.

[0274] 62. Use according to embodiment 60, wherein the enhanced physical stability comprises an increase in heat coagulation time and / or heat-induced opacity time of the p-lactoglobulin and wherein the increase in heat coagulation time and / or heat-induced opacity time of the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, or of a composition comprising the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, is at least 5%.

[0275] 63. Use according to embodiment 61 or 62, wherein the increase in heat coagulation time and / or heat-induced opacity time of the protein selected from a p-lactoglobulin, lactoferrin, alphalactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, is at least 10%. Use according to any one of embodiments 61 to 63, wherein the one or more charged amino acids are selected from the group consisting of Glutamic acid, Aspartic acid, Arginine, Lysine and Histidine. Use according to anyone of embodiments 61 to 64, wherein the one or more charged amino acids are located at the surface of the mature protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin. Use according to any one of embodiments 61 to 65, wherein the ^-potential of the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, after introduction of the one or more charged amino acids into the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, and / or the exchange of the one or more non-charged amino acid within the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, for the one or more charged amino acid, is altered by at least 0.5mV in a pH range of 2 to 4 and / or in a pH range of 5 to 7. Use according to any one of embodiments 61 to 66, wherein the p-lactoglobulin from which the physical stability is to be enhanced by introduction of the one ore more charged amino acids into the p-lactoglobulin, and / or by exchange of one or more non-charged amino acid within the p-lactoglobulin for the one or more charged amino acids, is a p-lactoglobulin selected from the group consisting of the p-lactoglobulins having a sequence as set forth in any one of SEQ ID NO:s 1 to 21. Use according to embodiment 67, wherein the p-lactoglobulin from which the physical stability is to be enhanced by introduction of the one ore more charged amino acids into the P-lactoglobulin, and / or by exchange of one or more non-charged amino acid within the p-lactoglobulin for the one or more charged amino acids, is a p-lactoglobulin having an amino acid sequence as set forth in SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, preferably, the p-lactoglobulin to be modified has the amino acid sequence as set forth in SEQ ID NO: 1 or in SEQ ID NO: 3. Use according to any one of embodiments 61 to 68, wherein the use comprises production of the protein selected from a p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, with enhanced physical stability by culture, preferably by fermentation, more preferably by precision fermentation. Use according to any one of embodiments 61 to 69, wherein the protein selected from a p- lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, and an immunoglobulin, preferably a p-lactoglobulin, with enhanced physical stability is an animal-free p-lactoglobulin, lactoferrin, alpha-lactalbumin, serum albumin, or immunoglobulin, preferably a p-lactoglobulin. Examples

[0276] 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.

[0277] Example 1 : Demonstration of increased physical stability of protein composition by the addition of extraneous H-lactocilobulin

[0278] In this example, it is demonstrated that the addition of extraneous p-lactoglobulin enhances the physical stability of the protein composition as measured by an increase in heat coagulation time of the protein composition.

[0279] Materials

[0280] Pea protein concentrate (NutralysS85XF) was obtained from Roquette, Fava protein isolate was obtained from Cosun, Soy protein isolate was obtained from Vitablend. p-lactoglobulin from Vivici, (extensively described in WO2022 / 269549), Whey protein isolate (WPI) obtained from Bipro. dipotassium phosphate (E340) and tri-calcium citrate (E333) were obtained from Laboratoriumdiscounter. Heat coagulation time was determined using an oil heating bath (Hettich Benelux ESP) and temperature-resistant glass test tubes with silicone bungs.

[0281] Sample Preparation p-lactoglobulin, WPI and plant protein samples were solubilized in reversed osmosis (RO) water in various concentrations, (extraneous) p-lactoglobulin - WPI combinations were prepared at 10 w / w% protein and p-lactoglobulin - plant protein combinations at 5 w / w% samples. Protein was solubilized under continuous stirring using a magnetic stirrer for 1 hour at room temperature. In the case of protein samples with salts, tri-calcium citrate, and di-potassium phosphate were added in concentrations of 100, 150, and 200 mg / 100 mL each, and stirred until they were dispersed or solubilized. The pH of the protein solutions was adjusted to 7.0 ± 0.05, using 0.1 M HCI or 0.1 M NaOH.

[0282] Measurement of heat stability

[0283] For heat stability measurements, protein samples of 2.5 mL each were placed in glass test tubes, sealed by silicone bungs, and immersed in an oil bath that was thermostatically controlled at 140°C. The rocking movement was set to a speed of 5. The heat coagulation time (HCT) was defined as the time between placing the sample in the oil bath and the onset of coagulation. All samples were measured in duplicate. The method has been adopted from McSweeney et al. (2004).

[0284] Results and discussion

[0285] The heat coagulation time (HCT) of plant proteins was used as a measure for physical stability of the compositions and was recorded without adding any salts, and then with the addition of salts in the concentrations of 100, 150, and 200 mg / 100 ml. Poor heat stability was observed when salts had been added. However, when a part of the plant protein was substituted with p-lactoglobulin in the ratios of 80 plant protein : 20 p-lactoglobulin and 60 plant protein : 40 p-lactoglobulinat 5 w / w% protein concentration, the physical stability of the plant protein composition improved in the presence of salts. The effect of p-lactoglobulin was most pronounced in soy protein solutions, where the HCT increased from 35 to 1800 seconds with 20% substitution. Although less pronounced, p-lactoglobulin also improved the physical stability of pea and fava bean protein solutions. With higher salt inclusion levels, the overall physical stability decreased, but substitution of p-lactoglobulin remained an effective adjustment to improve physical stability. The percentage increase of the individual HCT of plant proteins is depicted in Table 2.

[0286] The heat coagulation time (HCT) of dairy proteins was used as a measure for physical stability of the compositions and was recorded without adding any salts, as well as with the addition of salts in the concentrations of 200 mg / 100 ml. Poor heat stability was observed when salts had been added. However, when a part of the whey protein was substituted with p-lactoglobulin in the ratios of 90:10, 80:20, 70:30, 60:40, and 50:50 at 10 w / w % protein concentration, the heat stability of the whey protein composition improved in the presence of higher salt levels. The percentage increase of the individual HCT of dairy protein is depicted in Table 3.

[0287] Table 2: Heat coagulation time (HCT) of plant protein compositions with and without extraneous p-lactoglobulin (BLG)

[0288] Table 3: Heat coagulation time of dairy protein compositions with and without extraneous P-lactoglobulin (BLG)

[0289] Example 2: Demonstration of increased physical stability of B-lactoglobulin according to the invention compared to naturally occurring (3-lacto lobulin.

[0290] In this example, it is demonstrated that a p-lactoglobulin according to the invention has enhanced physical stability.

[0291] Materials

[0292] A p-lactoglobulin according to the invention was used comprising 91% of a p-lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 32 and comprising 7% of a p-lactoglobulin with the amino acid sequence as set forth in SEQ ID NO: 33. A naturally occurring p-lactoglobulin was used. Naturally occurring p-lactoglobulin can be isolated from milk and is also commercially available from e.g. Sigma (www.sigmaaldrich.com / NL / en / product / sigma / l3908).

[0293] Sample Preparation

[0294] The p-lactoglobulins were solubilized in reversed osmosis (RO) water in a concentration of 5% w / w and the pH of the protein solutions was adjusted to the desired pH ± 0.05, using 0.1 M HCI or 0.1 M NaOH.

[0295] Measurement of heat stability

[0296] For heat stability measurements, protein samples of 2.5 mL each were placed in glass test tubes, sealed by silicone bungs, and immersed in an oil bath that was thermostatically controlled at 140°C. The rocking movement was set to a speed of 5. The heat-induced opacity time (HOT) was defined as the time between placing the sample in the oil bath and the onset of opacity. All samples were measured in duplicate.

[0297] Results and discussion

[0298] The heat-induced opacity time (HOT) was used as a measure for physical stability of p-lactoglobulins. The results are depicted in Figure 2.

[0299] It was clearly demonstrated that the p-lactoglobulin according to the invention has a significantly higher heat-induced opacity time at pH lower than 4 and at pH higher than 6, when compared to naturally occurring p-lactoglobulin.

[0300] Example 2: Modulation of coagulation rate

[0301] Ready to drink (RTD) beverages, typically require to be stored at ambient temperatures. To keep them shelf stable from a microbiological perspective they need to undergo high or ultra-high temperature (UHT) treatment. The downside of such treatment is that it may cause physical instability, particularly as a result of protein coagulation (i.e., the protein unfolds and aggregate extensively causing the formation of clumps or coagulates). The effect is sometimes also referred to as fouling. Protein coagulates may give an RTD an undesirable appearance and are physically unstable since they will sediment rapidly. The inventors have provided a solution to this problem. It has been demonstrated by the inventors that extraneous p-lactoglobulin can be used to enhance the heat stability of aqueous protein compositions, such as protein beverages, especially of animal-free protein beverages.

Claims

CLAIMS1. Use of an extraneous p-lactoglobulin to enhance the physical stability of a composition comprising a protein and the extraneous p-lactoglobulin, wherein the enhanced physical stability is enhanced heat stability.

2. Use according to claim 1 , wherein the enhanced physical stability results in an increase in heat coagulation time of the composition of at least 5%.

3. Use according to claim 2, wherein the heat coagulation time is increased by at least 10%.

4. Use according to any one of claims 1 to 3, wherein at least part of the protein in the composition is a plant protein.

5. Use according to any one of claims 1 to 4, wherein the composition is an animal-free protein composition.

6. Use according to any one of claims 1 to 5, wherein the extraneous p-lactoglobulin is a recombinant p-lactoglobulin.

7. Use according to any one of claims 1 to 6, wherein the composition comprises at least 0.1 % (w / w) of total protein including the extraneous p-lactoglobulin.

8. Use according to any one of claims 1 to 7, wherein the ratio of protein to extraneous p- lactoglobulin is within the range from 99 : 1 (w / w) to 1 : 99 (w / w).

9. Use according to any one of claims 1 to 8, wherein the 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.

10. Use according to any one of claims 1 to 9, wherein the composition comprises a metal salt, preferably a mineral salt.

11. A composition comprising a protein and further comprising an extraneous p-lactoglobulin, wherein the protein preferably is a plant protein and wherein the p-lactoglobulin consists of an amino acid sequence selected from the group consisting of:- 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;- 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;- 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;- 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;- 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;- 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 ;- 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;- 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;- 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;- 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,- 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.

12. A composition according to claim 11 , wherein the ratio between p-lactoglobulin and other protein present in the composition preferably is within the range from 99 : 1 (w / w) to 1 : 99 (w / w)13. A composition according to claim 11 or 12, wherein the p-lactoglobulin is a recombinant p-lactoglobulin.

14. A composition according to any one of claims 11 to 13, further comprising a metal salt, preferably a mineral salt.

15. A composition according to claim 14, wherein the metal salt, preferably a mineral salt, comprises: a. a cation which is a monovalent, bivalent or trivalent metal ion such as sodium, potassium, calcium, magnesium, manganese, iron, molybdenum, 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.

16. A method for enhancing the physical stability of a p-lactoglobulin, said method comprising the introduction of an additional charged amino acid into the p-lactoglobulin, and / or the exchange of a noncharged amino acid within the p-lactoglobulin for a charged amino acid, wherein the enhanced physical stability preferably is enhanced heat stability.

17. A method according to claim 16, wherein the enhanced physical stability comprises an increase in heat coagulation time and / or heat-induced opacity time of the p-lactoglobulin or of a composition comprising the p-lactoglobulin and wherein the increase in heat coagulation time and / or heat-induced opacity time of the p-lactoglobulin or of a composition comprising the p-lactoglobulin is at least 5%.

18. A method according to claim 16 or 17, wherein the increase in heat coagulation time and / or heat- induced opacity time of the p-lactoglobulin or of a composition comprising the p-lactoglobulin is at least 10%.

19. A method according to any one of claims 16 to 18, wherein the additional charged amino acids are selected from the group consisting of Glutamic acid, Aspartic acid, Arginine, Lysine and Histidine.

20. A method according to anyone of claims 16 to 19, wherein the additional charged amino acid is located at the surface of the mature p-lactoglobulin.21 . A method according to any one of claims 16 to 20, wherein the ^-potential of the p-lactoglobulin after introduction of the additional charged amino acid into the p-lactoglobulin, and / or the exchange of the non-charged amino acid within the p-lactoglobulin for a charged amino acid, is altered by at least 0.5mV in a pH range of 2 to 4 and / or in a pH range of 5 to 7.

22. A method according to any one of claims 16 to 21 , wherein the p-lactoglobulin from which the physical stability is to be enhanced by introduction of an additional charged amino acid into the p-lactoglobulin, and / or by exchange of a non-charged amino acid within the p-lactoglobulin for acharged amino acid, is a p-lactoglobulin selected from the group consisting of the p-lactoglobulins having a sequence as set forth in any one of SEQ ID NO:s 1 to 21 .

23. A method according to claim 22, wherein the p-lactoglobulin from which the physical stability is to be enhanced by introduction of an additional charged amino acid into the p-lactoglobulin, and / or by exchange of a non-charged amino acid within the p-lactoglobulin for a charged amino acid, is a p-lactoglobulin having an amino acid sequence as set forth in SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, preferably as set forth in SEQ ID NO: 1 or in SEQ ID NO: 3.

24. A method according to any one of claims 16 to 23, wherein the method comprises production of the p-lactoglobulin with enhanced physical stability by culture, preferably by fermentation, more preferably by precision fermentation.

25. A method according to any one of claims 16 to 24, wherein the p-lactoglobulin with enhanced physical stability is an animal-free p-lactoglobulin.

26. A p-lactoglobulin obtainable by a method according to any one of claims 16 to 25, ora p-lactoglobulin selected from the group consisting of:- 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 the additional charged amino acid is not an N-terminal EA, EAEA, EAEAEA, EAEAEAEA, or R;- 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 the additional charged amino acid is not an N-terminal EA, EAEA, EAEAEA, EAEAEAEA, or R;- 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;- 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;- 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;- 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 ;- 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;- 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;- 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;- 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,- 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 .

27. A p-lactoglobulin comprising at least one additional charged amino acid according to claim 26, 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.

28. A p-lactoglobulin comprising at least one additional charged amino acid according to claim 26 or 27, 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.

29. A p-lactoglobulin comprising at least one additional charged amino acid according to any one of claims 26 to 28, wherein the 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.

30. A p-lactoglobulin comprising at least one additional charged amino acid according to claim 29, wherein the enhanced physical stability results in an increase in heat coagulation time of the p-lactoglobulin and / or wherein the enhanced physical stability results in an increase in heat-induced opacity time of the p-lactoglobulin or of a composition comprising the p-lactoglobulin.

31. A p-lactoglobulin comprising at least one additional charged amino acid according to claim 30, wherein the heat coagulation time and / or the heat-induced opacity time of the p-lactoglobulin or of a composition comprising the p-lactoglobulin is increased by at least 10%.

32. A p-lactoglobulin according to any one of claims 26 to 31 , wherein the p-lactoglobulin comprising at least one additional charged amino acid is a recombinant p-lactoglobulin.

33. A p-lactoglobulin according to any one of claims 26 to 32, wherein the p-lactoglobulin comprising at least one additional charged amino acid is an animal-free protein.

34. A p-lactoglobulin according to any one of claims 26 to 33, wherein the additional charged amino acids are selected from the group consisting of Glutamic acid, Aspartic acid, Arginine, Lysine and Histidine.

35. A p-lactoglobulin according to any one of claims 26 to 34, wherein the additional charged amino acid is located between the N-terminal amino acid and the C-terminal amino acid of the p-lactoglobulin.

36. A p-lactoglobulin according to any one of claims 26 to 35, wherein at least one of the additional charged amino acid is located at the surface of the mature p-lactoglobulin.

37. Use of one or more charged amino acids to enhance the physical stability of a p-lactoglobulin by introduction of the one or more charged amino acids into the p-lactoglobulin, and / or by exchange of one or more non-charged amino acids within the p-lactoglobulin for the one or more charged amino acids, wherein the enhanced physical stability preferably is enhanced heat stability.

38. Use according to claim 37, wherein the enhanced physical stability comprises an increase in heat coagulation time and / or heat-induced opacity time of the p-lactoglobulin, and wherein the increase in heat coagulation time and / or heat-induced opacity time of the p-lactoglobulin or of a composition comprising the p-lactoglobulin is at least 5%.

39. Use according to claim 37 or 38, wherein the increase in heat coagulation time and / or heat-induced opacity time of the p-lactoglobulin is at least 10%.

40. Use according to any one of claims 37 to 39, wherein the one or more charged amino acids are selected from the group consisting of Glutamic acid, Aspartic acid, Arginine, Lysine and Histidine.

41. Use according to anyone of claims 37 to 40, wherein the one or more charged amino acids are located at the surface of the mature p-lactoglobulin.

42. Use according to any one of claims 37 to 41 , wherein the ^-potential of the p-lactoglobulin after introduction of the one or more charged amino acids into the p-lactoglobulin, and / or the exchange of the one or more non-charged amino acid within the p-lactoglobulin for the one or more charged amino acid, is altered by at least 0.5mV in a pH range of 2 to 4 and / or in a pH range of 5 to 7.

43. Use according to any one of claims 37 to 42, wherein the p-lactoglobulin from which the physical stability is to be enhanced by introduction of the one ore more charged amino acids into the p-lactoglobulin, and / or by exchange of one or more non-charged amino acid within the p-lactoglobulin for the one or more charged amino acids, is a p-lactoglobulin selected from the group consisting of the p-lactoglobulins having a sequence as set forth in any one of SEQ ID NO:s 1 to 21 .

44. Use according to claim 43, wherein the p-lactoglobulin from which the physical stability is to be enhanced by introduction of the one ore more charged amino acids into the p-lactoglobulin, and / or by exchange of one or more non-charged amino acid within the p-lactoglobulin for the one or more charged amino acids, is a p-lactoglobulin having an amino acid sequence as set forth in SEQ ID NO: 1 , SEQ IDNO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, preferably, the p-lactoglobulin to be modified has the amino acid sequence as set forth in SEQ ID NO: 1 or in SEQ ID NO: 3.

45. Use according to any one of claims 37 to 44, wherein the use comprises production of the p-lactoglobulin with enhanced physical stability by culture, preferably by fermentation, more preferably by precision fermentation.

46. Use according to any one of claims 37 to 45, wherein the p-lactoglobulin with enhanced physical stability is an animal-free p-lactoglobulin.

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