Method for treating protein-containing compositions

Abstract

The present invention relates to a method for treating a protein-containing composition. The method comprises providing a protein-containing composition, wherein the protein comprises a milk protein and the milk protein comprises at least 90% by weight of beta-lactoglobulin. The composition is contacted with a transglutaminase to obtain a composition comprising a cross-linked protein. The method not only enhances the thermal stability, particularly in the presence of calcium, but also enhances the texture properties of such protein-containing compositions. The invention also relates to a cross-linked protein-containing composition obtainable or obtained by said process, to a process for the preparation of a dairy product or a substitute thereof with such a composition, and to the resulting dairy product or substitute thereof.

Description

Technical Field

[0001] The present invention relates generally to the field of protein-containing compositions and dairy products or substitutes thereof, wherein the milk protein consists mainly, preferably exclusively, of β-lactoglobulin. The invention also relates to methods for treating such protein-containing compositions and to methods for preparing such dairy products or substitutes thereof. Background Art

[0002] Milk is an edible liquid produced by the mammary glands of female mammals, such as cows. Milk contains micro- and macronutrients, including a variety of proteins. Milk proteins can be divided into two main categories: casein and whey protein. Casein accounts for the majority of milk protein (80%) and is divided into αS1-, αS2-, β- and κ-casein. Whey protein, which accounts for 20% of milk protein, generally includes β-lactoglobulin, α-lactalbumin, serum albumin and immunoglobulins.

[0003] The main protein in whey protein is beta-lactoglobulin. Beta-lactoglobulin is rich in leucine. Leucine is one of the 3 essential branched-chain amino acids used by skeletal muscle to provide energy during exercise. In addition, leucine is also a key amino acid involved in muscle anabolism. For these reasons, beta-lactoglobulin is gaining more and more interest from food companies, for example for its use in sports nutrition.

[0004] In standard milk ingredients such as milk, milk protein concentrates or whey protein concentrates, β-lactoglobulin is present in limited amounts because it is combined with other whey proteins and / or caseins. There is interest in producing food products, dairy products or substitutes thereof, including dairy beverages or substitutes thereof, in which the milk proteins consist predominantly (i.e. at least 90% by weight), preferably exclusively, of β-lactoglobulin.

[0005] However, the use of β-lactoglobulin alone or as the main milk protein in dairy products or substitutes thereof, in particular dairy beverages or substitutes, is associated with a number of disadvantages.

[0006] On the one hand, dairy products or substitutes thereof (especially dairy beverages or substitutes thereof) prepared with β-lactoglobulin as the main or only source of dairy protein exhibit thin texture and poor mouthfeel. The texture of such dairy products or substitutes thereof (especially dairy beverages or substitutes thereof) is unsatisfactory because they cannot exhibit a mouthfeel similar to that of dairy products (especially dairy beverages) based on conventional milk.

[0007] On the other hand, such dairy products or substitutes thereof (in particular dairy beverages or substitutes thereof) exhibit low thermal stability. This can be explained by the low thermal stability of β-lactoglobulin, in particular in the presence of calcium. When a heat treatment (such as UHT treatment) is applied at high temperature, in particular in the presence of calcium, the β-lactoglobulin of these dairy products or substitutes thereof tends to flocculate. For example, this can occur during the manufacturing process. Flocculation is undesirable. In fact, it impairs the technical functionality of β-lactoglobulin, for example by reducing their solubility. In addition, it adversely affects the organoleptic characteristics of the resulting dairy product or substitute thereof, for example by imparting an unpleasant uneven and granular texture. In addition, it may also adversely affect the processability of the dairy product or substitute thereof, because flocculation may lead to scaling and blocking of pipelines.

[0008] Therefore, there remains a need to prepare a composition comprising predominantly, preferably exclusively, beta-lactoglobulin as milk protein, while exhibiting improved thermal stability to elevated temperatures, particularly in the presence of calcium.

[0009] It would also be desirable for such compositions to impart a good texture, in particular an enhanced mouthfeel.

[0010] It is also desirable to prepare dairy products or alternatives thereof (especially dairy beverages or alternatives thereof) which comprise primarily, preferably exclusively, β-lactoglobulin as milk protein, while having improved thermal stability to high temperatures (especially in the presence of calcium) and good texture (especially enhanced mouthfeel).

[0011] Any reference to prior art documents in this specification is not to be taken as an admission that such prior art is well known or forms part of the common general knowledge in the art. Summary of the invention

[0012] The object of the present invention is to improve the prior art and in particular to provide a method, a composition containing cross-linked proteins and a dairy product or a substitute thereof which overcome the problems of the prior art and address the above needs, or at least provide a useful alternative.

[0013] Therefore, a first aspect of the present invention provides a method for treating a protein-containing composition, the method comprising the steps consisting of:

[0014] a) providing a protein-containing composition, wherein the protein of the protein-containing composition comprises milk protein, and wherein the milk protein of the protein-containing composition comprises at least 90% by weight

[0015] beta-lactoglobulin, wherein the protein-containing composition is a liquid,

[0016] b) contacting the protein-containing composition with at least one transglutaminase to obtain a composition containing cross-linked protein.

[0017] A second aspect of the present invention provides a cross-linked protein-containing composition, wherein the protein of the cross-linked protein-containing composition comprises milk protein, and wherein the milk protein of the cross-linked protein-containing composition comprises at least 90% by weight of β-lactoglobulin and is cross-linked.

[0018] The third aspect of the present invention provides a method for preparing a dairy product or a substitute thereof, wherein the protein of the dairy product or the substitute thereof comprises milk protein, and the milk protein of the dairy product or the substitute thereof comprises at least 90% by weight of β-lactoglobulin, the method comprising the steps consisting of:

[0019] a) treating a protein-containing composition according to the method of the first aspect of the invention to obtain a cross-linked protein-containing composition or providing a cross-linked protein-containing composition according to the second aspect of the invention,

[0020] b) mixing the composition comprising the cross-linked protein with calcium or a salt thereof to obtain a food composition,

[0021] c) homogenizing the food composition,

[0022] d) heat treating the food composition,

[0023] e) optionally, vaporizing the food composition,

[0024] f) Optionally, drying the food composition.

[0025] A fourth aspect of the present invention provides a dairy product or a substitute thereof, wherein the protein of the dairy product or the substitute thereof comprises milk protein, and wherein the milk protein of the dairy product or the substitute thereof comprises at least 90% by weight of β-lactoglobulin and is cross-linked.

[0026] It has been observed that crosslinking of β-lactoglobulin allows to improve the thermal stability of β-lactoglobulin when exposed to high temperatures, even in the presence of calcium. Furthermore, crosslinking of β-lactoglobulin allows to improve the textural properties. Indeed, crosslinked β-lactoglobulin confers an improved texture, in particular an enhanced mouthfeel, when used for the preparation of dairy products or substitutes thereof, such as dairy beverages or substitutes thereof.

[0027] Protein-containing compositions and dairy products or substitutes thereof comprising such cross-linked β-lactoglobulin as the main, preferably sole, source of milk protein have improved texture, in particular enhanced mouthfeel, and have improved thermal stability at high temperatures, even in the presence of calcium.

[0028] Those skilled in the art will more clearly understand these and other aspects, features and advantages of the present invention after reading the detailed description of the embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 are photographs showing the stability characteristics of the reference milk (A) without calcium of Example 5, the reference milk (B) containing calcium of Example 3, and the milk of the present invention (C) of Example 2 after UHT heat treatment using a rapid visco analyzer. The circles highlight the large-sized particles resulting from flocculation.

[0030] Figure 2 Differential scanning calorimetry measurements of an untreated BLG sample (see Example 7) are shown. The Y-axis corresponds to enthalpy (mW), and the x-axis corresponds to temperature (° C.). Curve A corresponds to the first measurement (ie, first scan), and curve B corresponds to the second measurement (ie, second scan).

[0031] Figure 3 Differential scanning calorimetry measurements of a cross-linked BLG sample (see Example 7) are shown. The Y-axis corresponds to enthalpy (mW), and the x-axis corresponds to temperature (° C.). Curve A corresponds to the first measurement (ie, first scan), and curve B corresponds to the second measurement (ie, second scan).

[0032] Figure 4 A photograph of comparative protein-containing composition A having a gelled texture obtained after heat treatment at 80°C for 1 hour is shown.

[0033] Figure 5 is a photograph showing the stability characteristics after UHT heat treatment using a rapid visco analyzer for Comparative Milk A. The circles highlight the large-sized particles resulting from flocculation.

[0034] Figure 6 The LDS page for measuring the molecular weight of the protein of the milk of the present invention of Example 2 is shown. Lane 9 corresponds to the LDS page result of the milk of the present invention of Example 2. Lane 11 corresponds to the LDS page result of the BLG sample only. Lane 12 corresponds to the LDS page result of the molecular weight marker. Arrows a to f indicate the bands of the molecular weight markers. a = α-lactalbumin (14.4 kDa), b = trypsin (20.1 kDa), c = carbonic anhydrase (30 kDa), d = ovalbumin (45 kDa), e = bovine serum albumin (66 kDa), f = phosphorylase B (97 kDa). Arrow β points to the band of β-lactoglobulin (18 kDa). DETAILED DESCRIPTION

[0035] As used in this specification, the words "include", "comprising", etc. should be interpreted as having an inclusive meaning as opposed to an exclusive or exhaustive meaning, that is, the meaning of "including but not limited to".

[0036] As used in this specification, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0037] Unless otherwise stated, all percentages in this specification are by weight where applicable.

[0038] Unless otherwise defined, all technical and scientific terms have and should be given the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0039] As used herein, the term "protein" refers to a polymeric form of amino acids of any length, which may include coded and non-coded amino acids, naturally occurring amino acids and non-naturally occurring amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones.

[0040] As used herein, the term "recombinant protein" refers to a protein produced in a cell (e.g., a recombinant host cell) of a species or type different from the species or type of cell in which the protein is naturally produced, or a protein produced in a cell (e.g., a recombinant host cell) at levels that do not naturally produce the protein.

[0041] As used herein, the term "non-transglutaminase proteins" refers to all proteins other than transglutaminase.

[0042] The term "milk protein" refers to animal milk protein (which may also be referred to as non-recombinant milk protein) and / or recombinant milk protein (which may also be referred to as non-animal milk protein).

[0043] The term "animal milk protein" or "non-recombinant milk protein" refers to a protein from mammalian milk, preferably bovine milk (ie, cow (Bos taurus) milk).

[0044] The term "recombinant milk protein" or "non-animal milk protein" refers to a milk protein produced in a cell (e.g., a recombinant host cell) of a different species or type than the species or type of cell in which the milk protein is naturally produced, or a milk protein produced in a cell (e.g., a recombinant host cell) at a level that does not naturally produce the protein. In other words, it relates to a protein that may be inherently present in mammalian milk, preferably bovine milk (i.e., domestic cow milk), but which is recombinantly produced (e.g., produced by a recombinant host cell).

[0045] As used herein, the term "recombinant host cell" refers to a host cell comprising a recombinant polynucleotide. Therefore, for example, a recombinant host cell can produce polynucleotides or proteins that are not present in a host cell of a natural (non-recombinant) form, or a recombinant host cell can produce polynucleotides or proteins at a level different from a host cell of a natural (non-recombinant) form. It should be understood that such terms refer not only to specific test cells, but also to the progeny of such cells. Because some modifications may occur in offspring due to mutations or environmental influences, such progeny may be different from parental cells, but are still included in the scope of the term "recombinant host cell" as used herein. Non-limiting examples of host cells include bacteria, yeast, fungal cells, plant cells, mammalian cells, or any other suitable host cells known in the art.

[0046] The term "polynucleotide" as disclosed herein refers to the sense and antisense strands of RNA, cDNA, genomic DNA, and the synthetic forms and mixed polymers described above. Polynucleotides may be chemically or biochemically modified, or may contain non-natural or derived nucleotide bases. Such modifications include, for example, tags, methylation, substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications such as uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates), charged bonds (e.g., phosphorothioates, phosphorodithioates), side moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralens), chelators, alkylating agents, and modified bonds (e.g., α-anomeric nucleic acids). Examples of modified nucleotides are known in the art (see, e.g., Malyshev et al., 2014. Nature 509:385; Li et al., 2014. J. Am. Chem. Soc. 136:826). Also included are synthetic molecules that mimic the ability of polynucleotides to bind to specified sequences via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, molecules in which peptide bonds replace phosphate bonds in the backbone of the molecule. Other modifications may include, for example, analogs in which the ribose ring contains a bridging moiety or other structure, such as modifications found in "locked" polynucleotides.

[0047] As used herein, the term "recombinant polynucleotide" refers to a polynucleotide that has been removed from its naturally occurring environment, a polynucleotide that is not associated with all or part of a polynucleotide adjacent to or adjacent to the polynucleotide when present in nature, a polynucleotide that is operably connected to a polynucleotide that is not connected in nature, a polynucleotide that is changed, or a polynucleotide that does not exist in nature. The term can be used, for example, to describe a cloned DNA isolate, or a polynucleotide comprising a chemically synthesized nucleotide analog. If a polynucleotide contains a non-naturally occurring genetic modification, it is also considered to be "recombinant". For example, if an endogenous polynucleotide contains (for example, by human intervention) an insertion, deletion or substitution of one or more nucleotides that are artificially introduced, it is considered to be a "recombinant polynucleotide". This modification can introduce point mutations, substitution mutations, deletion mutations, insertion mutations, missense mutations, frameshift mutations, duplication mutations, amplification mutations, translocation mutations or inversion mutations in a polynucleotide. The term includes polynucleotides in a recombinant host cell chromosome and polynucleotides that are not in a recombinant host cell chromosome (for example, polynucleotides contained in an episome). A recombinant polynucleotide in a recombinant host cell or organism may replicate using the in vivo cellular machinery of the recombinant host cell; however, such a recombinant polynucleotide, although subsequently replicated within the cell, is still considered recombinant for purposes of the present invention.

[0048] As used herein in the context of protein sequences, the term "identical" refers to residues in two sequences that are identical when compared for maximum correspondence. The length of sequence identity comparison can be in a fragment of at least 9 amino acid residues, at least 20 amino acid residues, at least 24 amino acid residues, at least 28 amino acid residues, at least 32 amino acid residues, or at least 36 or more amino acid residues. There are many different algorithms and methods well known to those skilled in the art that can be used to measure protein sequence identity. By way of example but not limitation, the identity percentage of two protein sequences can be calculated using CLUSTAL W (version 1.82, version 2), CLUSTAL omega, BLAST, EMBOSS matcher or MULTALIN.

[0049] The term "dairy substitute" refers to a food product in which the milk protein comprises, preferably consists only of, recombinant milk protein, in particular recombinant β-lactoglobulin, and generally simulates the texture and visual aspects of standard dairy products. More preferably, such a food product does not contain any animal-derived protein, but it may contain recombinant forms of said proteins derived from animals. Even more preferably, such a food product does not contain any animal-derived ingredients, except recombinant milk protein and recombinant forms of other proteins derived from animals. It may contain non-animal ingredients, such as plant ingredients.

[0050] The term "standard dairy products" refers to food products that are usually made from milk and / or ingredients derived from milk (e.g., cream), and the milk proteins consist only of animal milk proteins. They usually contain all types of milk proteins inherently present in milk, including casein and whey proteins.

[0051] The term "protein-containing composition" refers to a composition comprising proteins, including milk proteins. In the framework of the present invention, the milk proteins of the protein-containing composition comprise at least 90% by weight of β-lactoglobulin, preferably at least 95% by weight, more preferably at least 98% by weight of β-lactoglobulin. Even more preferably, the milk proteins of the protein-containing composition consist only of β-lactoglobulin.

[0052] The term "texturizer" refers to ingredients other than milk protein that contribute to viscosity. Examples include starches (e.g., tapioca starch, corn starch, rice starch, potato starch, cassava starch, corn flour, etc.), pectins, gums (e.g., locust bean gum, carob gum, guar gum, etc.), hydrocolloids (e.g., alginates, agar, etc.).

[0053] The term "oligomer of β-lactoglobulin" corresponds to a protein comprising, preferably consisting of, 2 to 10, preferably 2 to 4 β-lactoglobulins. In an embodiment, the β-lactoglobulins in the oligomer of β-lactoglobulin are cross-linked between molecules. In particular, the β-lactoglobulins in the oligomer of β-lactoglobulin are linked via a covalent bond, in particular via an isopeptide bond or an amide bond, between a γ-carboxamide group of a glutamine amino acid residue of a β-lactoglobulin and a free amine group of a lysine amino acid residue of another β-lactoglobulin.

[0054] In a first aspect, the present invention relates to a method for treating a protein-containing composition.

[0055] The method comprises the step a) of providing a protein-containing composition. The protein-containing composition comprises proteins. The proteins of the protein-containing composition comprise milk proteins. The protein-containing composition may have at least 2% by weight of milk proteins, in particular beta-lactoglobulin. In a preferred embodiment, the protein-containing composition may have 2% to 30% by weight, preferably 2% to 25% by weight, more preferably 6% to 21% by weight, most preferably 9% to 15% by weight of milk proteins, in particular beta-lactoglobulin. At this amount, the composition is a substantial source of milk proteins, in particular beta-lactoglobulin. Furthermore, this amount ensures sufficient substrate for an effective enzymatic reaction with the transglutaminase.

[0056] The protein-containing composition comprises protein. The protein of the protein-containing composition comprises milk protein. The milk protein of the protein-containing composition comprises at least 90 wt%, preferably at least 95 wt%, more preferably at least 98 wt% beta-lactoglobulin.

[0057] In a preferred embodiment, the milk protein of the protein-containing composition may consist only of β-lactoglobulin. In particular, the protein-containing composition does not contain any milk protein other than β-lactoglobulin. For example, the protein-containing composition does not contain casein.

[0058] In an embodiment, the non-transglutaminase protein of the protein-containing composition may comprise at least 90 wt%, preferably at least 95 wt%, more preferably at least 98 wt% beta-lactoglobulin.

[0059] In a more preferred embodiment, the non-transglutaminase protein of the protein-containing composition may consist only of β-lactoglobulin. In other words, the protein-containing composition does not comprise other proteins than β-lactoglobulin and transglutaminase.

[0060] In an embodiment, the β-lactoglobulin is an animal β-lactoglobulin and / or a recombinant β-lactoglobulin. The recombinant β-lactoglobulin may be at least 80% identical to the animal β-lactoglobulin present in mammalian milk, preferably bovine milk (i.e., domestic cow milk). Preferably, the recombinant β-lactoglobulin may be at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to the β-lactoglobulin present in mammalian milk, preferably bovine milk (i.e., domestic cow milk).

[0061] In an advantageous embodiment, the protein-containing composition is liquid. This enables the enzymatic reaction of step b) to be performed. Indeed, transglutaminase requires an aqueous liquid system to catalyze the cross-linking reaction.

[0062] In an embodiment, the protein-containing composition can be prepared by diluting a concentrated milk protein component into an aqueous liquid, preferably water. The aqueous liquid, preferably water, is added in an amount that enables the target milk protein content of the protein-containing composition to be achieved. The concentrated milk protein component may contain 60% to 100% by weight, preferably 75% to 98% by weight, more preferably 80% to 98% by weight of milk protein, in particular β-lactoglobulin. The milk protein of the concentrated milk protein component may contain at least 90% by weight, preferably at least 95% by weight, more preferably at least 98% by weight of β-lactoglobulin. Preferably, the milk protein of the concentrated milk protein component may consist only of β-lactoglobulin. More preferably, the protein of the concentrated milk protein component may consist only of β-lactoglobulin. The concentrated milk protein component may be a liquid or a powder. The concentrated milk protein component may be, for example, a β-lactoglobulin concentrate or a β-lactoglobulin isolate. The concentrated milk protein component is a concentrated source of β-lactoglobulin. This is advantageous for providing a protein-containing composition wherein the milk protein consists predominantly, preferably exclusively, of β-lactoglobulin and has a targeted amount of β-lactoglobulin.

[0063] In an embodiment, the protein-containing composition may have a total solids content of at least 2 wt%. In particular, the protein-containing composition may have a total solids content of 2 wt% to 30 wt%, preferably 2 wt% to 25 wt%, more preferably 6 wt% to 21 wt%, and most preferably 9 wt% to 15 wt%. The total solids (including protein) help optimize the enzymatic reaction with the transglutaminase by providing sufficient substrate for the enzymatic reaction.

[0064] The method further comprises the step b) of contacting the protein-containing composition with at least one transglutaminase to obtain a composition containing cross-linked protein.

[0065] Transglutaminases are a family of enzymes (EC 2.3.2.13) that catalyze the generation of covalent bonds between the γ-carboxamide group of a glutamine amino acid residue and the free amine group of a lysine amino acid residue present in a protein (such as β-lactoglobulin). When the bond is formed, ammonia is released. Covalent bonds are particularly isopeptide bonds or amide bonds. The formation of these covalent bonds between the γ-carboxamide group of a glutamine amino acid residue and the free amine group of a lysine amino acid residue is referred to as cross-linking. Cross-linking can be intramolecular, i.e. between a glutamine amino acid residue and a lysine amino acid residue of the same protein. Cross-linking can be intermolecular, i.e. between a glutamine amino acid residue from a first protein and a lysine amino acid residue from a second protein.

[0066] Among the transglutaminases that can be used in the process of the invention, mention may be made of the transglutaminase BDF commercialized by BDF ingredients (Spain). .

[0067] In an embodiment, in step b), the transglutaminase may be reacted with the protein-containing composition at a temperature of 40°C to 68°C, preferably 48°C to 65°C, for 20 minutes to 4 hours. In a preferred embodiment, in step b), the transglutaminase may be reacted with the protein-containing composition at a temperature of 40°C to 68°C, preferably 48°C to 65°C, for 1 hour to 4 hours. In a more preferred embodiment, in step b), the transglutaminase may be reacted with the protein-containing composition at a temperature of 40°C to 68°C, preferably 48°C to 65°C, for 1 hour to 2 hours. These time and temperature conditions help to optimize the enzymatic reaction with the transglutaminase while avoiding unwanted uncontrolled protein aggregation. Below this temperature range, the enzymatic reaction will be quite long, which is disadvantageous for industrial considerations. Above this temperature range, milk proteins (particularly β-lactoglobulin) and transglutaminase tend to undergo uncontrolled aggregation, resulting in undesirable large-sized protein aggregates. These protein aggregates may adversely affect the functionality of β-lactoglobulin. They may also impair the enzymatic reaction with transglutaminase. In particular, such uncontrolled protein aggregation produces a protein-containing composition that is unsuitable for the preparation of a dairy product or a substitute thereof, such as a dairy beverage or a substitute thereof. In particular, it may adversely affect the organoleptic properties of the resulting dairy product or a substitute thereof, for example by imparting an unpleasant inhomogeneous and grainy structure.

[0068] In another embodiment, the transglutaminase is contacted with the protein-containing composition in step b) wherein the weight ratio of milk protein to transglutaminase is from 20: 1 to 1000: 1, preferably from 20: 1 to 500: 1, more preferably from 25: 1 to 75: 1, most preferably from 30: 1 to 60: 1. This weight ratio ensures a good ratio between the enzyme and its substrate to promote an optimal enzymatic reaction with the transglutaminase.

[0069] It has been found that enzymatic treatment with transglutaminase improves the thermal stability of compositions wherein the milk proteins consist predominantly, preferably exclusively, of β-lactoglobulin. The transglutaminase treatment crosslinks the β-lactoglobulin. This results in β-lactoglobulins having improved thermal stability properties when exposed to high temperatures, even in the presence of calcium. They show no or limited flocculation when exposed to high temperatures, even in the presence of calcium.

[0070] Furthermore, the cross-linked β-lactoglobulin of the present invention imparts improved texture, in particular enhanced mouthfeel, when used in the preparation of dairy products or substitutes thereof, such as dairy beverages or substitutes thereof.

[0071] In other embodiments, the method may include the step of adding at least one fat component to the protein-containing composition before step b). The fat component according to the present invention can be any fat suitable for human consumption. The fat component can be a vegetable component, i.e. any fat suitable for human consumption derived from vegetable material. The example of vegetable fat is vegetable oil. The fat component can be an animal component, i.e. any fat suitable for human consumption derived from an animal. The example of animal fat is butter. Preferably, the fat component is a vegetable fat, particularly a vegetable oil. The vegetable oil can be selected from the group consisting of algae oil, almond oil, canola oil, coconut oil, corn oil, cottonseed oil, hazelnut oil, hemp seed oil, grape seed oil, olive oil, palm oil, peanut oil, rice bran oil, safflower oil, sesame seed oil, soybean oil, sunflower oil, walnut oil or a combination thereof. The vegetable oil can be a hydrogenated form or a non-hydrogenated form.

[0072] In particular, the protein-containing composition and the cross-linked protein-containing composition may comprise at least one fat component as described above. In particular, the protein-containing composition and the cross-linked protein-containing composition may comprise 0.5 wt.% to 7 wt.%, preferably 1 wt.% to 5 wt.%, more preferably 1 wt.% to 3 wt.% fat. In a preferred embodiment, the fat of the protein-containing composition may consist only of vegetable fat. Similarly, the fat of the cross-linked protein-containing composition may consist only of vegetable fat.

[0073] Features of compositions containing proteins may be applied to compositions containing cross-linked proteins, and vice versa.

[0074] In an embodiment, the method may comprise a step of partially or completely inactivating the transglutaminase within the composition containing the cross-linked protein after step b). Preferably, the inactivation step of the method may consist of completely inactivating the transglutaminase within the composition containing the cross-linked protein. This avoids further reactions of the transglutaminase, which may adversely affect the organoleptic properties of the composition containing the cross-linked protein.

[0075] The transglutaminase may be inactivated by any prior art method, such as by heat treatment, pH reduction or reduction of water activity. Preferably, the transglutaminase may be inactivated by heat treatment.

[0076] In an embodiment, after the enzyme treatment step b) or after the inactivation step (if any), the method may further comprise a step of drying the composition containing cross-linked proteins to obtain a cross-linked protein powder. The milk protein containing the cross-linked protein powder may comprise at least 90% by weight, preferably at least 95% by weight, more preferably at least 98% by weight of β-lactoglobulin. In a preferred embodiment, the milk protein containing the cross-linked protein powder may consist only of β-lactoglobulin. In an embodiment, the non-transglutaminase protein containing the cross-linked protein powder may comprise at least 90% by weight, preferably at least 95% by weight, more preferably at least 98% by weight of β-lactoglobulin. In a more preferred embodiment, the non-transglutaminase protein containing the cross-linked protein powder may consist only of β-lactoglobulin. Additional details about β-lactoglobulin are provided above. The drying step may be performed by drum drying, roller drying, spray drying or freeze drying. The drying step may also be performed by any other food drying technique well known in the art. Converting the composition into a powder may be advantageous because powders generally have good physicochemical stability and extended shelf life. The drying step concentrates the protein, including β-lactoglobulin. After drying, the resulting cross-linked protein-containing powder may comprise 20 to 80 wt%, preferably 30 to 50 wt%, of milk protein, in particular beta-lactoglobulin. In an embodiment, the method may comprise an evaporation step prior to the drying step.

[0077] In an embodiment, the cross-linked protein-containing powder may comprise at least one fat component as described above. In an embodiment, the cross-linked protein-containing powder may comprise 1 wt% to 25 wt%, preferably 4 wt% to 16 wt% fat. Preferably, the fat of the cross-linked protein-containing powder may consist only of vegetable fat.

[0078] In an embodiment, prior to step b) of contacting the protein-containing composition with at least one transglutaminase, the protein-containing composition is not heat treated at above 75° C., preferably above 80° C. In particular, prior to said step b), the protein-containing composition is not heat treated for at least 10 seconds, preferably at least 30 seconds, more preferably at least 1 minute.

[0079] In another embodiment, the cross-linked protein-containing composition is not heat-treated at a temperature of 70°C to 95°C, preferably 80°C to 95°C, immediately after step b) of contacting the protein-containing composition with at least one transglutaminase. In a preferred embodiment, the cross-linked protein-containing composition is not heat-treated at a temperature of 70°C to 95°C, preferably 80°C to 95°C, for 5 seconds to 15 minutes immediately after step b). For the sake of clarity, the cross-linked protein-containing composition may be further heat-treated when used or processed as an ingredient for the target application. For example, when used / processed in the preparation of a food product (particularly a dairy product or a substitute thereof), the cross-linked protein-containing composition may be heat-treated to extend the shelf life of the final food product. However, the method of the present invention for treating a protein-containing composition does not include a step of heat-treating the cross-linked protein-containing composition at a temperature of 70°C to 95°C, preferably 80°C to 95°C, immediately after step b). Cross-linking of milk proteins (particularly β-lactoglobulin) with transglutaminase is sufficient to significantly improve the thermal stability of the protein-containing composition, especially when exposed to high temperatures. No heat treatment is required to induce controlled denaturation of milk proteins after transglutaminase treatment to improve thermal stability before any subsequent heat treatment (including high temperature heat treatment) is applied. Thus, the process is simplified, but good thermal stability properties of the protein-containing composition are still ensured.

[0080] In an embodiment, the cross-linked protein-containing composition comprises an oligomer of β-lactoglobulin having at least 2 β-lactoglobulins, preferably 2 to 10 β-lactoglobulins, more preferably 2 to 4 β-lactoglobulins. In particular, the β-lactoglobulins in the oligomer of β-lactoglobulin are cross-linked, in particular intermolecularly cross-linked. Further details on the terms "cross-linked" and "intermolecularly cross-linked" are provided in the second aspect of the invention.

[0081] In an embodiment, the composition containing cross-linked proteins comprises proteins, in particular oligomers of β-lactoglobulin, having a molecular weight of x times the molecular weight of the β-lactoglobulin used in step a), wherein x is an integer and x is equal to or greater than 2. Preferably, the composition containing cross-linked proteins comprises proteins, in particular oligomers of β-lactoglobulin, having a molecular weight of 2 to 10 times, preferably 2 to 4 times, the molecular weight of the β-lactoglobulin used in step a). More preferably, the composition containing cross-linked proteins comprises proteins, in particular oligomers of β-lactoglobulin having a molecular weight of twice the molecular weight of the β-lactoglobulin used in step a), and / or proteins, in particular oligomers of β-lactoglobulin having a molecular weight of three times the molecular weight of the β-lactoglobulin used in step a), and / or proteins, in particular oligomers of β-lactoglobulin having a molecular weight of four times the molecular weight of the β-lactoglobulin used in step a).

[0082] In an embodiment, the composition comprising cross-linked proteins comprises proteins having a molecular weight of 16 kDa to 180 kDa, preferably 16 kDa to 74 kDa, more preferably 18 kDa to 72 kDa. In another embodiment, the composition comprising cross-linked proteins comprises proteins, in particular oligomers of β-lactoglobulin, having a molecular weight of 34 kDa to 180 kDa, preferably 34 kDa to 74 kDa, more preferably 36 kDa to 72 kDa.

[0083] In an embodiment, the cross-linked protein-containing composition comprises proteins (particularly oligomers of β-lactoglobulin with a molecular weight of 34 kDa to 38 kDa, preferably 36 kDa), and / or proteins (particularly oligomers of β-lactoglobulin with a molecular weight of 52 kDa to 56 kDa, preferably 54 kDa), and / or proteins (particularly oligomers of β-lactoglobulin with a molecular weight of 70 kDa to 74 kDa, preferably 72 kDa).

[0084] In a second aspect, the present invention relates to a composition comprising a cross-linked protein.

[0085] The cross-linked protein-containing composition comprises protein. The protein of the cross-linked protein-containing composition comprises milk protein. The milk protein of the cross-linked protein-containing composition comprises at least 90 wt%, preferably at least 95 wt%, more preferably at least 98 wt% of beta-lactoglobulin.

[0086] In a preferred embodiment, the milk protein of the cross-linked protein-containing composition may consist only of β-lactoglobulin.

[0087] In embodiments, the non-transglutaminase protein of the cross-linked protein-containing composition may comprise at least 90% by weight, preferably at least 95% by weight, more preferably at least 98% by weight of beta-lactoglobulin.

[0088] In a more preferred embodiment, the non-transglutaminase protein of the cross-linked protein-containing composition may consist solely of β-lactoglobulin.

[0089] The milk proteins (particularly β-lactoglobulin) of the composition containing cross-linked proteins are cross-linked. In particular, part of the glutamine and lysine amino acid residues of the milk proteins (particularly β-lactoglobulin) are cross-linked, i.e. linked by covalent bonds between the γ-carboxamide groups of the glutamine amino acid residues and the free amine groups of the lysine amino acid residues, in particular by isopeptide bonds or amide bonds. The milk proteins (particularly β-lactoglobulin) may be cross-linked at least intermolecularly, i.e. linked by covalent bonds between the γ-carboxamide groups of the glutamine amino acid residues of the first milk protein (particularly β-lactoglobulin) and the free amine groups of the lysine amino acid residues of the second milk protein (particularly the second β-lactoglobulin), in particular by isopeptide bonds or amide bonds. They may also be cross-linked intramolecularly, i.e. linked by covalent bonds between the γ-carboxamide groups of the glutamine amino acid residues of the same milk protein (particularly the same β-lactoglobulin) and the free amine groups of the lysine amino acid residues, in particular by isopeptide bonds or amide bonds.

[0090] As described in the first aspect of the present invention, the cross-linked β-lactoglobulin imparts improved texture when exposed to elevated temperatures and has enhanced thermostability properties, even in the presence of calcium.

[0091] The cross-linked protein-containing compositions of the present invention comprising the cross-linked β-lactoglobulin have improved thermal stability. In particular, they remain stable when exposed to high temperatures and show no or limited flocculation, even in the presence of calcium.

[0092] Furthermore, they impart an improved texture, in particular an enhanced mouthfeel, when used in the preparation of dairy products or alternatives thereof, such as dairy beverages or alternatives thereof.

[0093] Features of the protein-containing composition and the cross-linked protein-containing composition of the first aspect of the present invention may be applied to the cross-linked protein-containing composition of the second aspect of the present invention, and vice versa.

[0094] In a preferred embodiment, the cross-linked protein-containing composition is obtainable by or obtainable by the method according to the first aspect of the invention.

[0095] The cross-linked protein-containing composition may be a liquid or a powder. When it is a powder, the features of the cross-linked protein-containing powder provided in the first aspect of the present invention may be applicable to the cross-linked protein-containing composition of the second aspect of the present invention, and vice versa.

[0096] In an embodiment, the cross-linked protein-containing composition comprises an oligomer of β-lactoglobulin having at least 2 β-lactoglobulins, preferably 2 to 10 β-lactoglobulins, more preferably 2 to 4 β-lactoglobulins. In particular, the β-lactoglobulins in the oligomer of β-lactoglobulin are cross-linked, in particular intermolecularly cross-linked. Additional details on the terms "cross-linked" and "intermolecularly cross-linked" are provided above.

[0097] In an embodiment, the composition containing cross-linked proteins comprises proteins, in particular oligomers of β-lactoglobulin, whose molecular weight is x times the molecular weight of β-lactoglobulin, wherein x is an integer and x is equal to or greater than 2. Preferably, the composition containing cross-linked proteins comprises proteins, in particular oligomers of β-lactoglobulin, whose molecular weight is 2 to 10 times, preferably 2 to 4 times, the molecular weight of β-lactoglobulin. More preferably, the composition containing cross-linked proteins comprises proteins (in particular oligomers of β-lactoglobulin having a molecular weight twice that of β-lactoglobulin), and / or proteins (in particular oligomers of β-lactoglobulin having a molecular weight three times that of β-lactoglobulin), and / or proteins (in particular oligomers of β-lactoglobulin having a molecular weight four times that of β-lactoglobulin).

[0098] In an embodiment, the composition comprising cross-linked proteins comprises proteins having a molecular weight of 16 kDa to 180 kDa, preferably 16 kDa to 74 kDa, more preferably 18 kDa to 72 kDa. In another embodiment, the composition comprising cross-linked proteins comprises proteins, in particular oligomers of β-lactoglobulin, having a molecular weight of 34 kDa to 180 kDa, preferably 34 kDa to 74 kDa, more preferably 36 kDa to 72 kDa.

[0099] In an embodiment, the cross-linked protein-containing composition comprises proteins (particularly oligomers of β-lactoglobulin with a molecular weight of 34 kDa to 38 kDa, preferably 36 kDa), and / or proteins (particularly oligomers of β-lactoglobulin with a molecular weight of 52 kDa to 56 kDa, preferably 54 kDa), and / or proteins (particularly oligomers of β-lactoglobulin with a molecular weight of 70 kDa to 74 kDa, preferably 72 kDa).

[0100] In a third aspect, the present invention relates to a method for preparing a dairy product or a substitute thereof.

[0101] The dairy product or its substitute comprises protein. The protein of the dairy product or its substitute comprises milk protein. The milk protein of the dairy product or its substitute comprises at least 90% by weight, preferably at least 95% by weight, more preferably at least 98% by weight of beta-lactoglobulin. In a preferred embodiment, the milk protein of the dairy product or its substitute may consist only of beta-lactoglobulin. In particular, the dairy product or its substitute may not contain any milk protein other than beta-lactoglobulin. For example, the dairy product or its substitute may not contain casein.

[0102] In an embodiment, the milk protein of the dairy product consists only of animal milk protein, in particular animal β-lactoglobulin.In an embodiment, the milk protein of the dairy substitute product comprises, preferably consists only of, recombinant milk protein, in particular recombinant β-lactoglobulin.

[0103] In an embodiment, the non-transglutaminase protein of the dairy product or its substitute may comprise at least 90 wt%, preferably at least 95 wt%, more preferably at least 98 wt% beta-lactoglobulin.

[0104] In a more preferred embodiment, the non-transglutaminase protein of the dairy product or its substitute may consist only of β-lactoglobulin. In other words, the dairy product or its substitute does not contain other proteins than β-lactoglobulin and transglutaminase.

[0105] The dairy product or its substitute may have at least 0.5 wt% of milk protein, in particular β-lactoglobulin. In particular, the dairy product or its substitute may have 0.5 wt% to 15 wt%, preferably 1 wt% to 15 wt%, more preferably 2 wt% to 10 wt%, most preferably 2 wt% to 5 wt% of milk protein, in particular β-lactoglobulin.

[0106] In an embodiment, the dairy product or a substitute thereof may be selected from the group consisting of cream, creamer, condensed milk, milk powder, milk, flavored milk, a smoothie substitute, a milkshake, a powdered milk drink or a substitute thereof.

[0107] In a preferred embodiment, the dairy product or substitute thereof is a dairy beverage or substitute thereof.The dairy beverage or substitute thereof may be selected from the group consisting of creamer, milk, flavored milk, smoothie, milkshake, powdered milk beverage or substitute thereof.

[0108] The method comprises step a) of treating a protein-containing composition according to the method of the first aspect of the invention to obtain a cross-linked protein-containing composition, or step a) of providing a cross-linked protein-containing composition according to the second aspect of the invention.

[0109] In particular, cross-linked β-lactoglobulins (i.e. β-lactoglobulins comprised in the cross-linked protein-containing composition of the present invention) have improved properties. In particular, they impart an improved texture when exposed to high temperatures and have enhanced thermostability properties, even in the presence of calcium. For these reasons, they are advantageous for the preparation of dairy products or substitutes thereof.

[0110] In particular, dairy products or substitutes thereof prepared with such cross-linked β-lactoglobulin have an improved texture, in particular an enhanced mouthfeel, compared to dairy products or substitutes thereof prepared with untreated β-lactoglobulin. Furthermore, the dairy products or substitutes thereof remain stable when exposed to high temperatures and show no or limited flocculation, even in the presence of calcium.

[0111] The cross-linked protein-containing composition may be a liquid or a powder. If the cross-linked protein-containing composition is a powder, it may be reconstituted in an aqueous liquid, preferably water, to obtain a liquid cross-linked protein-containing composition prior to step b).

[0112] In an embodiment, the cross-linked protein-containing composition may be diluted in an aqueous liquid, preferably water, before step b) to achieve a target milk protein content. In particular, after dilution, the cross-linked protein-containing composition may have a milk protein content, in particular a β-lactoglobulin content, of at least 2 wt%, preferably 2 wt% to 30 wt%, more preferably 2 wt% to 15 wt%, most preferably 2 wt% to 10 wt%.

[0113] The method further comprises the step b) of mixing the composition comprising the cross-linked protein with calcium or a salt thereof. Examples of calcium salts include calcium carbonate, calcium chloride, calcium gluconate, calcium lactate, tricalcium phosphate, or a combination thereof.

[0114] In embodiments, the food composition and the resulting dairy product or alternative thereto may comprise 0.01 wt% to 2.5 wt%, preferably 0.05 wt% to 1.3 wt%, more preferably 0.1 wt% to 0.6 wt% calcium.

[0115] Calcium is a mineral naturally present in milk and is inherently present in dairy products. Therefore, in order to resemble dairy products, the presence of calcium in the dairy product or its substitute is extremely important. Despite the presence of significant amounts of calcium, the dairy product or its substitute of the present invention is heat stable and exhibits no or limited flocculation when exposed to high temperatures.

[0116] In a preferred embodiment, the cross-linked protein-containing composition may be further mixed with at least one texturizing agent in step b). In particular, the food composition and the resulting dairy product substitute or its substitute may contain 0.05 wt % to 2 wt %, preferably 0.1 wt % to 1 wt %, more preferably 0.1 wt % to 0.5 wt % of a texturizing agent.

[0117] In a further embodiment, the cross-linked protein-containing composition may be further mixed with at least one fat component and / or at least one sugar component in step b).

[0118] The fat component may be a fat ingredient as described in the first aspect of the invention. The food composition and the dairy product or its substitute may comprise 0.3 wt % to 33 wt % fat, preferably 0.3 wt % to 15 wt %, more preferably 1 wt % to 10 wt %, most preferably 1 wt % to 5 wt % fat. Preferably, the fat may consist only of vegetable fat.

[0119] Sugar component as used herein refers to the component that imparts or increases the sweetness of food products. The example of sugar component includes agave syrup, brown sugar, coconut sugar, maple syrup, rice syrup, oat syrup, corn syrup, dextrose, fructose, glucose, honey, invert sugar, maltose, molasses, sucrose, steviol glycosides and their mixture. Food composition and dairy product or its substitute can comprise 0 weight % to 15 weight % sugar component, preferably 0.1 weight % to 15 weight %, more preferably 1 weight % to 10 weight %, most preferably 1 weight % to 5 weight % sugar component.

[0120] In an additional embodiment, the composition containing the cross-linked protein may be further mixed with at least one aqueous liquid, preferably water, in step b).The food composition and the dairy product or its substitute may comprise 50% to 95% by weight, preferably 75% to 95% by weight of an aqueous liquid, preferably water.

[0121] In another embodiment, the composition containing the cross-linked protein may also be mixed with one or more additional food ingredients. The additional food ingredients may be any food ingredient known to those skilled in the art to be suitable for human consumption. In a particular embodiment, the additional food ingredient may be selected from the group consisting of vitamins, minerals other than calcium, fiber, flavoring agents, buffering agents, coloring agents, salts, or mixtures thereof.

[0122] The method further comprises a step c) of homogenizing the food composition. The homogenization step may be performed at a pressure of 50 to 700 bar, preferably 50 to 500 bar, more preferably 50 to 350 bar, most preferably 100 to 350 bar. The homogenization step may be performed at a temperature of 50 to 80°C, preferably 50 to 75°C, more preferably 55 to 70°C.

[0123] The method further comprises a step d) of heat treating the food composition. This step can mainly extend the shelf life of the dairy product or its substitute by limiting the development of undesirable microorganisms (including pathogenic microorganisms). It also helps to inactivate transglutaminase. In this case, no inactivation step is required in step a) of the method. The heat treatment can be carried out at a temperature of 70°C to 150°C for 3 seconds to 15 minutes, preferably at 95°C to 145°C for 3 seconds to 5 minutes. For example, the heat treatment can be an ultra-high temperature (i.e., UHT) treatment, an ultra-pasteurization treatment or a pasteurization treatment. Any food heat treatment technology known to those skilled in the art can be applied.

[0124] The dairy product of the invention remains stable throughout the process, including during the heat treatment step, even in the presence of calcium. The cross-linked state of the β-lactoglobulins improves their thermal stability, even in the presence of calcium.

[0125] The heat treatment step d) may be upstream or downstream of the homogenization step c).

[0126] In an embodiment, the method may further comprise a step of evaporating the food composition after step d). For example, this step may be required to prepare a condensed milk substitute or a substitute thereof.

[0127] In another embodiment, the method may further include a step of drying the food composition after step d) or after the evaporation step (if any). The drying step may be performed by drum drying, roller drying, spray drying or freeze drying. The drying step may also be performed by any other food drying technology well known in the art. For example, such a drying step may be required to prepare a powdered milk substitute, a powdered milk beverage substitute or a substitute thereof.

[0128] In an embodiment, after step a) and before step d), the composition containing cross-linked proteins is not heat treated at a temperature of 70°C to 95°C, preferably 80°C to 95°C. In a preferred embodiment, after step a) and before step d), the composition containing cross-linked proteins is not heat treated at a temperature of 70°C to 95°C, preferably 80°C to 95°C, for 5 seconds to 15 minutes. Cross-linking of milk proteins, in particular β-lactoglobulin, with transglutaminase is sufficient to significantly improve the thermal stability of the protein-containing composition and the resulting dairy product or its substitute before applying the thermal treatment of step d) to extend the shelf life of the final dairy product or its substitute. Prior to the thermal treatment of step d), an upstream thermal treatment inducing controlled denaturation of the milk proteins is not required to improve thermal stability.

[0129] In an embodiment, the dairy product or substitute thereof comprises an oligomer of β-lactoglobulin having at least 2 β-lactoglobulins, preferably 2 to 10 β-lactoglobulins, more preferably 2 to 4 β-lactoglobulins. In particular, the β-lactoglobulins in the oligomer of β-lactoglobulin are cross-linked, in particular intermolecularly cross-linked. Further details on the terms "cross-linked" and "intermolecularly cross-linked" are provided in the second aspect of the invention.

[0130] In an embodiment, the dairy product or its substitute comprises a protein, in particular an oligomer of β-lactoglobulin, whose molecular weight is x times the molecular weight of β-lactoglobulin, wherein x is an integer and x is equal to or greater than 2. Preferably, the dairy product or its substitute comprises a protein, in particular an oligomer of β-lactoglobulin, whose molecular weight is 2 to 10 times, preferably 2 to 4 times, the molecular weight of β-lactoglobulin. More preferably, the dairy product or its substitute comprises a protein (in particular an oligomer of β-lactoglobulin having a molecular weight twice that of β-lactoglobulin), and / or a protein (in particular an oligomer of β-lactoglobulin having a molecular weight three times that of β-lactoglobulin), and / or a protein (in particular an oligomer of β-lactoglobulin having a molecular weight four times that of β-lactoglobulin).

[0131] In an embodiment, the dairy product or its substitute comprises a protein having a molecular weight of 16 kDa to 180 kDa, preferably 16 kDa to 74 kDa, more preferably 18 kDa to 72 kDa. In another embodiment, the dairy product or its substitute comprises a protein, in particular an oligomer of β-lactoglobulin, having a molecular weight of 34 kDa to 180 kDa, preferably 34 kDa to 74 kDa, more preferably 36 kDa to 72 kDa.

[0132] In an embodiment, the dairy product or its substitute comprises protein (particularly oligomers of β-lactoglobulin with a molecular weight of 34 kDa to 38 kDa, preferably 36 kDa), and / or protein (particularly oligomers of β-lactoglobulin with a molecular weight of 52 kDa to 56 kDa, preferably 54 kDa), and / or protein (particularly oligomers of β-lactoglobulin with a molecular weight of 70 kDa to 74 kDa, preferably 72 kDa).

[0133] In a fourth aspect, the present invention relates to a method of preparing a dairy product or a substitute thereof.

[0134] The dairy product or its substitute can be a dairy product or its substitute as provided in the third aspect of the present invention. In particular, the features of the dairy product or its substitute provided in the third aspect of the present invention can also be applied to the dairy product or its substitute of the fourth aspect of the present invention, and vice versa.

[0135] In one embodiment, the dairy product or substitute thereof may be obtainable by or obtained by the method of the third aspect of the invention.

[0136] The dairy product or its substitute comprises protein. The protein of the dairy product or its substitute comprises milk protein. The milk protein of the dairy product or its substitute comprises at least 90 wt%, preferably at least 95 wt%, more preferably at least 98 wt% of beta-lactoglobulin.

[0137] In a preferred embodiment, the milk protein of the dairy product or its substitute may consist only of β-lactoglobulin. In particular, the dairy product or its substitute may not contain any milk protein other than β-lactoglobulin. For example, the dairy product or its substitute may not contain casein.

[0138] In an embodiment, the milk protein of the dairy product consists only of animal milk protein, in particular animal β-lactoglobulin.In an embodiment, the milk protein of the dairy substitute product comprises, preferably consists only of, recombinant milk protein, in particular recombinant β-lactoglobulin.

[0139] In an embodiment, the non-transglutaminase protein of the dairy product or its substitute may comprise at least 90 wt%, preferably at least 95 wt%, more preferably at least 98 wt% beta-lactoglobulin.

[0140] In a more preferred embodiment, the non-transglutaminase protein of the dairy product or its substitute may consist only of β-lactoglobulin. In other words, the dairy product or its substitute does not contain other proteins than β-lactoglobulin and transglutaminase.

[0141] The milk proteins (especially β-lactoglobulin) of the dairy product or its substitute are cross-linked. In particular, part of the glutamine and lysine amino acid residues of the milk proteins (especially β-lactoglobulin) are cross-linked. In particular, part of the glutamine and lysine amino acid residues of the milk proteins (especially β-lactoglobulin) are cross-linked, i.e. connected by covalent bonds between the γ-carboxamide groups of the glutamine amino acid residues and the free amine groups of the lysine amino acid residues, in particular by isopeptide bonds or amide bonds. The milk proteins (especially β-lactoglobulin) can be cross-linked at least intermolecularly, i.e. connected by covalent bonds between the γ-carboxamide groups of the glutamine amino acid residues of the first milk protein (especially β-lactoglobulin) and the free amine groups of the lysine amino acid residues of the second milk protein (especially the second β-lactoglobulin), in particular by isopeptide bonds or amide bonds. They may also be cross-linked intramolecularly, i.e. via covalent bonds, in particular via isopeptide bonds or amide bonds, between the γ-carboxamide groups of glutamine amino acid residues and the free amine groups of lysine amino acid residues of the same milk protein (in particular the same β-lactoglobulin).

[0142] The dairy product or its substitute may have at least 0.5 wt% of milk protein, in particular β-lactoglobulin. In particular, the dairy product or its substitute may have 0.5 wt% to 15 wt%, preferably 1 wt% to 15 wt%, more preferably 2 wt% to 10 wt%, most preferably 2 wt% to 5 wt% of milk protein, in particular β-lactoglobulin.

[0143] When the dairy product or its substitute is a powder, it may contain higher amounts of milk protein, especially β-lactoglobulin. In particular, it may contain 5 to 80 wt%, preferably 20 to 60 wt% of milk protein, especially β-lactoglobulin.

[0144] As described in the third aspect of the present invention, the dairy product or the alternative thereof may contain 0.01 wt% to 2.5 wt%, preferably 0.05 wt% to 1.3 wt%, more preferably 0.1 wt% to 0.6 wt% calcium.

[0145] The dairy products or substitutes thereof of the present invention comprising cross-linked β-lactoglobulin as the main, preferably only, source of milk protein have an improved texture, in particular an enhanced mouthfeel, compared to dairy products or substitutes thereof comprising untreated β-lactoglobulin as the main, preferably only, source of milk protein. Furthermore, the dairy products or substitutes thereof of the present invention have an improved thermal stability. In particular, they do not show flocculation or show limited flocculation at high temperatures, even in the presence of calcium.

[0146] In an embodiment, the dairy product or its substitute may further comprise calcium or its salt, at least one texturizing agent, at least one fat component, at least one sugar component, at least one aqueous liquid and / or at least one additional food ingredient as provided in the third aspect of the invention.

[0147] The amounts of calcium, texturizing agent, sugar component, fat and aqueous liquid in the dairy product or its substitute are provided in the third aspect of the invention.

[0148] In an embodiment, the dairy product or substitute thereof comprises an oligomer of β-lactoglobulin having at least 2 β-lactoglobulins, preferably 2 to 10 β-lactoglobulins, more preferably 2 to 4 β-lactoglobulins. In particular, the β-lactoglobulins in the oligomer of β-lactoglobulin are cross-linked, in particular intermolecularly cross-linked. Further details on the terms "cross-linked" and "intermolecularly cross-linked" are provided in the second aspect of the invention.

[0149] In an embodiment, the dairy product or its substitute comprises a protein, in particular an oligomer of β-lactoglobulin, whose molecular weight is x times the molecular weight of β-lactoglobulin, wherein x is an integer and x is equal to or greater than 2. Preferably, the dairy product or its substitute comprises a protein, in particular an oligomer of β-lactoglobulin, whose molecular weight is 2 to 10 times, preferably 2 to 4 times, the molecular weight of β-lactoglobulin. More preferably, the dairy product or its substitute comprises a protein (in particular an oligomer of β-lactoglobulin having a molecular weight twice that of β-lactoglobulin), and / or a protein (in particular an oligomer of β-lactoglobulin having a molecular weight three times that of β-lactoglobulin), and / or a protein (in particular an oligomer of β-lactoglobulin having a molecular weight four times that of β-lactoglobulin).

[0150] In an embodiment, the dairy product or its substitute comprises a protein having a molecular weight of 16 kDa to 180 kDa, preferably 16 kDa to 74 kDa, more preferably 18 kDa to 72 kDa. In another embodiment, the dairy product or its substitute comprises a protein, in particular an oligomer of β-lactoglobulin, having a molecular weight of 34 kDa to 180 kDa, preferably 34 kDa to 74 kDa, more preferably 36 kDa to 72 kDa.

[0151] In an embodiment, the dairy product or its substitute comprises protein (particularly oligomers of β-lactoglobulin with a molecular weight of 34 kDa to 38 kDa, preferably 36 kDa), and / or protein (particularly oligomers of β-lactoglobulin with a molecular weight of 52 kDa to 56 kDa, preferably 54 kDa), and / or protein (particularly oligomers of β-lactoglobulin with a molecular weight of 70 kDa to 74 kDa, preferably 72 kDa).

[0152] Those skilled in the art will appreciate that they are free to incorporate all features of the present invention disclosed herein. In particular, the features described for the product of the present invention can be combined with the method of the present invention, and vice versa. In addition, the features described for different embodiments of the present invention can be combined.

[0153] Furthermore, where known equivalents exist for specific features, such equivalents should be incorporated as if explicitly mentioned in this specification.Further advantages and features of the present invention will become apparent upon reference to the accompanying drawings and non-limiting examples.

[0154] Example

[0155] Example 1: Preparation of a composition containing cross-linked proteins according to the present invention

[0156] The cross-linked protein-containing composition is prepared using the process of the present invention. The cross-linked protein-containing composition comprises only beta-lactoglobulin as a milk protein source.

[0157] A β-lactoglobulin isolate (i.e., 92% by weight of β-lactoglobulin) was diluted with water. BLG-100 neutral (Arla Foods Ingredients)) was added until a protein content, i.e., a β-lactoglobulin content of 10% by weight was reached to obtain a liquid composition. 1% of a fat component was added to the liquid composition. The liquid composition was then treated with transglutaminase at 50°C for 1.5 hours to crosslink the β-lactoglobulin. The resulting crosslinked protein-containing composition was cooled in an ice bath and then stored before further use.

[0158] Example 2: Preparation of milk according to the present invention

[0159] Milk according to the present invention (hereinafter referred to as the present invention milk) was prepared using the cross-linked protein-containing composition obtained in Example 1. The milk contained only β-lactoglobulin as a milk protein source.

[0160] First, a milk mixture was prepared by mixing the ingredients in Table 1 below.

[0161] Element Amount (weight %) The composition containing cross-linked protein of Example 1 35％ Calcium carbonate 0.3％ Fat composition 3.5％ sugar 5％ Conditioning agent 0.5％ water 55.7％

[0162] Table 1

[0163] After mixing, the milk mixture was heat-treated at 145° C. for 5 seconds by UHT treatment, and then homogenized at 150 bar to obtain milk.

[0164] Example 3: Preparation of reference milk

[0165] The reference milk was prepared using the same method as provided in Example 2. It was also prepared using a formulation similar to that provided in Example 2. However, 35 wt % of the cross-linked protein-containing composition of Example 1 was replaced by 3.5 wt % of BLG-100 neutral (Arla Foods Ingredients) was used instead, which contains untreated β-lactoglobulin. Furthermore, the water content was adjusted accordingly, ie in the present case 87.2% by weight of water was added.

[0166] Example 4: Preparation of milk without a texturizing agent

[0167] The milk of the present invention without a texturizing agent was prepared in the same manner as provided in Example 2.

[0168] However, the formula did not contain a texturizing agent. Specifically, a milk mixture was prepared by mixing the ingredients of Table 2 below.

[0169] Element Amount (weight %) The composition containing cross-linked protein of Example 1 35％ Calcium carbonate 0.3％ Fat composition 3.5％ sugar 5％ water 56.2％

[0170] Table 2

[0171] Example 5: Preparation of reference milk without calcium

[0172] A reference milk without calcium was prepared similarly to the reference milk of Example 3.

[0173] The only difference was that the recipe of the calcium-free reference milk did not contain calcium. In particular, a milk mixture was prepared by mixing the ingredients of Table 3 below.

[0174]

[0175] Table 3

[0176] Example 6: Evaluation of the thermal stability of milk during UTH heat treatment in the presence of calcium

[0177] The thermal stability of three different milks was evaluated:

[0178] - the reference milk of Example 3 comprising calcium,

[0179] - the calcium-free reference milk of Example 5 which does not contain calcium, and

[0180] - The milk according to the invention of Example 2.

[0181] To study the thermal stability, milks were prepared with the recipes as mentioned in Examples 3, 5 and 2, respectively, but without homogenization and UHT treatment as provided in these Examples.

[0182] In particular, the obtained milks were heat treated with UHT heat treatment, which was performed using a rapid viscosity analyzer (RVA 4800, PerkinElmer, USA). First, 30 g of the different milks were poured into a concentric cylinder CC25-PR pressure cell. Then, the temperature was linearly increased from 50° C. to 140° C. (14° C. / min), kept at 140° C. for 1 min, and finally linearly decreased to 50° C. The milks were continuously stirred during the heat treatment.

[0183] The stability of the milk was assessed by visual inspection after heat treatment ( Figure 1 ). In particular, if the milk is unstable, particles visible to the naked eye can be observed. These particles are produced by flocculation.

[0184] The reference milk containing calcium underwent flocculation during UHT treatment and showed visible particles ( Figure 1 In contrast, the reference milk without calcium did not undergo flocculation and showed no visible particles ( Figure 1). Therefore, when calcium is present during UHT processing, β-lactoglobulin tends to flocculate. This flocculation will lead to an unpleasant texture in the milk, for example, an uneven texture, a perceptible grainy texture and a bad mouthfeel.

[0185] Such, the milk of the invention comprising calcium and in which the β-lactoglobulin is cross-linked does not undergo flocculation after UHT treatment. No particles ( Figure 1 ). Therefore, cross-linking of β-lactoglobulin before heat treatment at high temperature can enhance the thermal stability of β-lactoglobulin in the presence of calcium. Without being bound by theory, it is believed that cross-linking of glutamine amino acid residues is beneficial to reduce the interaction of free calcium ions with protein molecules. This will lead to enhanced thermal stability in the presence of calcium.

[0186] Example 7: Differential Scanning Calorimetry .

[0187] Differential scanning calorimetry was performed on the cross-linked protein-containing composition of Example 1 (hereinafter referred to as cross-linked BLG) and on an untreated protein-containing composition (hereinafter referred to as untreated BLG).

[0188] Cross-linked BLG samples were prepared as provided in Example 1. Untreated BLG samples were prepared by dissolving BLG-100 neutral (Arla Foods Ingredients) in water until a protein content, ie, β-lactoglobulin content of 10% was reached.

[0189] Cross-linked BLG and untreated BLG samples were evaluated. The evaluation was performed using a differential scanning calorimeter DSC3+ (Mettler Toledo, Switzerland). Two scans were performed using the following temperature program:

[0190] 1.25℃, 10min,

[0191] 2.25℃-180℃, 10K / min (first scan),

[0192] 3.180℃-25℃, -10K / min,

[0193] 4.25°C-180°C, 10K / min (second scan).

[0194] After differential scanning calorimetry, graphs are obtained showing the change in enthalpy (y-axis) as a function of temperature (x-axis). In these graphs, the starting temperature of the enthalpy peak shows the beginning of protein denaturation, the end temperature shows the end of denaturation, and the peak temperature describes at which temperature the maximum enthalpy is found to denature the protein.

[0195] The results are shown in Figure 2and Figure 3 In the second measurement, the enthalpy of the cross-linked BLG was changed compared to the untreated BLG. A small shift was observed for the cross-linked BLG, in particular an increase in the peak temperature maximum of 1.5 °C. This may indicate an improvement in the β-lactoglobulin thermal stability of the cross-linked BLG. A low or no enthalpy was observed in the second measurement.

[0196] Therefore, the differential scanning calorimetry data tend to confirm that cross-linking of β-lactoglobulins improves their thermal stability.

[0197] Example 8: Milk texture

[0198] Evaluate the texture, especially mouthfeel, of the following milks:

[0199] - the milk of the present invention according to Example 2,

[0200] - the reference milk of Example 3, and

[0201] - The milk according to the invention of Example 4 without a texturizing agent.

[0202] The mouthfeel of milk was evaluated after tasting by 12 trained tasters to assess dairy products, especially the texture of milk.

[0203] During tasting, it was noted that the reference milk had a thin texture and a poor mouthfeel. In contrast, the inventive milk and the milk without texturizer had an enhanced mouthfeel compared to the reference milk.

[0204] It is believed that cross-linking of β-lactoglobulin by transglutaminase contributes to the texture of milk comprising β-lactoglobulin exclusively as milk protein and improves its mouthfeel.

[0205] The milk of the invention has an enhanced mouthfeel compared to milk without a texturizing agent. It is therefore believed that the mouthfeel is further improved by adding a texturizing agent to achieve a texture more similar to standard milk.

[0206] Example 9: Effect of applying a heat treatment step before the transglutaminase treatment step (Comparative Milk A)

[0207] Prior art document D1 = Mariana Battaglin villas-boa et al., "The effect of transglutaminase-induced polymerization in the presence of cysteine ​​on beta-lactoglobulin antigenicity", international dairy journal, Elsevier applied science, Barking, GB.

[0208] In the present examples a protein treatment involving heat treatment prior to transglutaminase treatment as disclosed in prior art document D1 was evaluated.

[0209] To this end, a comparative protein-containing composition A was prepared by applying a heat treatment step prior to attempting an enzyme treatment with transglutaminase. Comparative protein-containing composition A comprised only beta-lactoglobulin as milk protein source.

[0210] A β-lactoglobulin isolate (i.e., 92% by weight of β-lactoglobulin) was diluted with water. BLG-100 neutral (Arla Foods Ingredients)) was added until the protein content, i.e., the β-lactoglobulin content was 10% by weight to obtain a liquid composition. 1% of a fat component was added to the liquid composition. The liquid composition was then heat treated at 80°C for 1 hour.

[0211] After heat treatment, the resulting protein-containing composition gels ( Figure 4 ). Such a protein-containing composition in gel form cannot be further processed with transglutaminase, since the transglutaminase cannot be dissolved in the composition, since no or only limited free water is available.

[0212] The obtained protein-containing composition was also used to prepare comparative milk A.

[0213] A milk mixture of comparative milk A was prepared by mixing the ingredients of Table 4 below.

[0214] Element Amount (weight %) Comparison of protein-containing composition A 35％ Calcium carbonate 0.3％ Fat composition 3.5％ sugar 5％ Conditioning agent 0.5％ water 55.7％

[0215] Table 4

[0216] In order to study the thermal stability, the obtained milk mixture of comparative milk A was heat treated with UHT heat treatment, which was carried out using a rapid viscosity analyzer (RVA 4800, PerkinElmer, USA). First, 30 g of the milk mixture was poured into a concentric cylinder CC25-PR pressure cell. Then, the temperature was linearly increased from 50° C. to 140° C. (14° C. / min), kept at 140° C. for 1 minute, and finally linearly decreased to 50° C. The milk mixture of comparative milk A was continuously stirred during the heat treatment.

[0217] The stability of the comparative milk A was evaluated by visual inspection after heat treatment ( Figure 5 ). In particular, if the milk is unstable, particles visible to the naked eye can be observed. These particles are produced by flocculation.

[0218] Comparative milk A showed a large amount of aggregated protein, which appeared as particles visible to the naked eye. This indicates that Comparative milk A is unstable.

[0219] Based on the above, applying heat treatment before transglutaminase treatment is disadvantageous because:

[0220] - heat treatment to produce a protein-containing composition in the form of a gel that is not or barely processable with transglutaminase,

[0221] - The obtained milk is not heat stable and exhibits an unsatisfactory grainy texture.

[0222] Example 10: LDS page test

[0223] Materials and methods

[0224] The samples of the milk of the present invention of Example 2 and the control samples were subjected to LDS-PAGE (lithium dodecyl sulfate-polyacrylamide gel electrophoresis) method for detecting the molecular weight of proteins (≥10 kDa).

[0225] The control samples are as follows:

[0226] - Samples with molecular weight markers of α-lactalbumin, β-lactoglobulin, trypsin, carbonic anhydrase, ovalbumin, bovine serum albumin, and phosphorylase B.

[0227] - Contains only uncross-linked animal β-lactoglobulin (BiPro, from Agropur Ingredients,

[0228] The sample was originally from Davisco Food International, hereinafter referred to as the BLG-only sample.

[0229] Clean the sample by filtration or centrifugation to obtain a defatted sample. 65 μL of the defatted sample was denatured with 25 μL of lithium dodecyl sulfate (LDS) solution at 95° C. and reduced with 10 μL of dithiothreitol (DTT).

[0230] The prepared samples were then denatured and heated at 95°C for 5 to 10 min, cooled to room temperature, and vortexed briefly.

[0231] The prepared samples are then loaded onto discontinuous NuPAGE 4% to 12% Bis-Tris gels using MES buffer at a near neutral pH. An electric field is applied to the gel according to the principle of electrophoresis. The proteins are concentrated in the stacking gel and separated according to their molecular weight in the separating gel. After separation, the gel is fixed and the protein bands are visualized by silver staining.

[0232] Control samples, samples with molecular weight markers (lane 12) and samples were prepared similarly as described.

[0233] result

[0234] The results are shown in Figure 6 Lane 9 corresponds to the LDS page result of the milk of the present invention in Example 2. Lane 11 corresponds to the LDS page result of the BLG sample only. Lane 12 corresponds to the LDS page result of the molecular weight marker.

[0235] It can be observed that cross-linking increases the molecular weight of the milk protein. Initially, β-lactoglobulin is 18 kDa, and after cross-linking the appearance of three new bands above 18 kDa can be seen. These molecular weights of these bands correspond to the molecular weights of dimers, trimers and tetramers of β-lactoglobulin. It can be noted that dimers are the predominant form over trimers and tetramers. Monomers are still present, but part of them have been converted into dimers, trimers and tetramers after cross-linking.

[0236] Although the present invention has been described by way of example, it will be appreciated that variations and modifications may be made without departing from the scope of the invention as defined in the claims.

Claims

1. A method for treating a protein-containing composition, the method comprising the steps of: a) providing a protein-containing composition, wherein the protein of the protein-containing composition comprises milk protein, and wherein the milk protein of the protein-containing composition comprises at least 90% by weight of beta-lactoglobulin, wherein the protein-containing composition is a liquid, b) contacting the protein-containing composition with at least one transglutaminase to obtain a cross-linked protein-containing composition.

2. The method for treating a protein-containing composition according to claim 1, wherein the protein-containing composition may have at least 2% by weight of milk protein, preferably 2% to 30% by weight of milk protein.

3. A method for treating a protein-containing composition according to claim 1 or 2, comprising the step of adding at least one fat component to the protein-containing composition before step b).

4. The method for treating a protein-containing composition according to any one of the preceding claims, wherein in step b) the transglutaminase is contacted with the protein-containing composition, wherein the weight ratio of milk protein to transglutaminase is from 20:1 to 1000:1, preferably from 20:1 to 500:1, more preferably from 25:1 to 75:

1.

5. The method for treating a protein-containing composition according to any one of the preceding claims, wherein in step b) the transglutaminase is reacted with the protein-containing composition at a temperature of 40°C to 68°C for 20 minutes to 4 hours.

6. A method for treating a protein-containing composition according to any one of the preceding claims, wherein the milk proteins, preferably non-transglutaminase proteins, of the protein-containing composition consist only of β-lactoglobulin.

7. A cross-linked protein-containing composition, wherein the protein of the cross-linked protein-containing composition comprises milk protein, and wherein the milk protein of the cross-linked protein-containing composition comprises at least 90% by weight of β-lactoglobulin and is cross-linked.

8. The cross-linked protein-containing composition according to claim 7, wherein the milk protein, preferably the non-transglutaminase protein of the cross-linked protein-containing composition consists only of β-lactoglobulin.

9. The cross-linked protein-containing composition according to claim 7 or 8, which can be obtained by the method according to any one of claims 1 to 6 or is obtained by the method according to any one of claims 1 to 6.

10. The cross-linked protein-containing composition according to any one of claims 7 to 9, comprising a protein with a molecular weight of 34 kDa to 38 kDa and / or a protein with a molecular weight of 52 kDa to 56 kDa and / or a protein with a molecular weight of 70 kDa to 74 kDa.

11. A method for preparing a dairy product or a substitute thereof, wherein the protein of the dairy product or the substitute thereof comprises milk protein, and the milk protein of the dairy product or the substitute thereof comprises at least 90% by weight of beta-lactoglobulin, the method comprising the steps consisting of: a) treating a protein-containing composition according to the method of any one of claims 1 to 6 to obtain a cross-linked protein-containing composition or providing a cross-linked protein-containing composition according to any one of claims 7 to 10, b) mixing the cross-linked protein-containing composition with calcium or a salt thereof to obtain a food composition, c) homogenizing the food composition, d) heat treating the food composition, e) optionally, evaporating the food composition, f) optionally, drying the food composition.

12. The method for preparing a dairy product or a substitute thereof according to claim 11, wherein in step b) the cross-linked protein-containing composition is further mixed with at least one texturizing agent.

13. The method for preparing a dairy product or a substitute thereof according to any one of claims 11 or 12, wherein in step b) the cross-linked protein-containing composition is further mixed with at least one fat component and / or at least one sugar component and / or at least one aqueous liquid.

14. A dairy product or a substitute thereof, wherein the protein of the dairy product or the substitute thereof comprises milk protein, and wherein the milk protein of the dairy product or the substitute thereof comprises at least 90% by weight of beta-lactoglobulin and is cross-linked.

15. The dairy product or substitute thereof according to claim 14, wherein the milk protein of the dairy product or substitute thereof consists only of β-lactoglobulin.

16. A dairy product or substitute thereof according to claim 14 or 15, obtainable by or obtained by a method according to any one of claims 11 to 13.

17. A method for preparing a dairy product or a substitute thereof according to any one of claims 11 to 13 or a dairy product or a substitute thereof according to any one of claims 14 to 16, wherein the dairy product or the substitute thereof is a dairy beverage or a substitute thereof, preferably the dairy beverage or the substitute thereof is selected from the group consisting of: creamer, milk, flavored milk, smoothie, milkshake, milk powder beverage or a substitute thereof.

18. A method for preparing a dairy product or a substitute thereof according to any one of claims 11 to 13 or 17 or a dairy product or a substitute thereof according to any one of claims 14 to 17, wherein the milk protein of the dairy product consists only of animal milk protein.

19. A method for preparing a dairy product or a substitute thereof according to any one of claims 11 to 13 or any one of claims 17 to 18 or a dairy product or a substitute thereof according to any one of claims 14 to 18, wherein the milk protein of the dairy substitute product comprises, preferably consists only of, recombinant milk protein.

20. A method for preparing a dairy product or a substitute thereof according to any one of claims 11 to 13 or any one of claims 17 to 19 or a dairy product or a substitute thereof according to any one of claims 14 to 19, comprising a protein having a molecular weight of 34 kDa to 38 kDa and / or a protein having a molecular weight of 52 kDa to 56 kDa and / or a protein having a molecular weight of 70 kDa to 74 kDa.

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