Crystallization of β-lactoglobulin using multiple protein feeds

Abstract

The present invention relates to an improved method for producing a beta-lactoglobulin (BLG) composition by crystallization of BLG. The invention utilizes at least two different BLG-containing protein feeds, which are mixed to prepare a supersaturated protein solution in which BLG is crystallized. One of the protein feeds must have a pH of at least 5.6 and one of the protein feeds must have a pH of at most 5.4.

Description

[Technical field]

[0001] The present invention relates to an improved method for producing a beta-lactoglobulin (BLG) composition by crystallization of BLG. The invention utilizes at least two different BLG-containing protein feeds, which are mixed to prepare a supersaturated protein solution in which BLG is crystallized. One of the protein feeds must have a pH of at least 5.6 and one of the protein feeds must have a pH of at most 5.4. [Background technology]

[0002] The concept of milk protein fractionation is well known in the art and has been developed over the past few decades into a series of techniques for preparing compositions enriched with various milk protein species, each with specific properties and characteristics.

[0003] The isolation of β-lactoglobulin (BLG) from milk serum or whey has been the subject of many publications and typically involves multiple separation steps, and often chromatographic techniques, to arrive at a purified β-lactoglobulin product.

[0004] WO2018 / 115520 describes industrial-scale separation of BLG from a protein solution by crystallization in salting mode and in the pH range of 5-6. However, WO2018 / 115520 does not disclose preparing a supersaturated whey protein solution by mixing BLG-containing type A protein feed(s) with type B protein feed(s) as defined by the present invention.

[0005] WO2020 / 002422 describes the production of alpha-lactalbumin enriched whey protein products by industrial scale removal of BLG from a whey protein solution using BLG crystallization in salting mode at a pH range of 5-6. However, WO2020 / 002422 does not disclose the preparation of a supersaturated whey protein solution by mixing BLG-containing type A protein feed(s) with type B protein feed(s) as defined by the present invention.

[0006] de Jongh et al. (Mild Isolation Procedure Discloses New Protein Structural Properties of β-Lactoglobulin, J Dairy Sci., vol. 84(3), 2001, pp. 562-571) described the purification of BLG from fresh milk by low temperature acid coagulation of casein and subjecting the resulting acid whey to a combination of affinity chromatography (DEAE Sepharose) and gel permeation chromatography. The resulting BLG composition was stated to contain 0.985 g β-lactoglobulin per g protein.

[0007] Slack et al. (Journal of Food Processing and Preservation, vol. 10, 1986, pp. 19-30) explored a different approach and prepared a BLG-enriched precipitate by adjusting demineralized acid and sweet whey to pH 4.65 and separating the precipitate that formed by centrifugation and decantation. The resulting precipitate pellet was described as relatively insoluble and contained significant amounts of protein impurities in additional BLG. No crystal formation was observed. It should be noted that the BLG precipitate that may form at pH 4.65 is not BLG crystals.

[0008] Palmer (Crystalline Globulin from Cow's Milk, J. Biol. Chem., Vol. 104, pp. 359-372) reported a laborious and time-consuming process for producing protein crystals based on acid whey, using multiple sequences of salting-out precipitation of unwanted proteins, pH adjustments and dialysis to remove other unwanted proteins. Finally, when a highly purified BLG solution was obtained, BLG was crystallized. The process lasted more than 12 days and required the addition of toluene. Thus, the procedure disclosed in Palmer is not compatible with safe food production and provides a product that is obviously inedible.

[0009] Aschaffenburg et al. (Improved Method for the Preparation of Crystalline beta-Lactoglobulin and alpha-Lactalbumin from Cow's Milk, Bioch., vol. 65, 1957, pp. 273-277) disclose an improved process over that of Palmer's process, which allows the preparation of beta-lactoglobulin crystals in the order of days instead of weeks. However, the improved method still requires the removal of unwanted proteins before crystallization and further uses toluene for crystallization, which makes it incompatible with safe food production.

[0010] JP H10 218755 A discloses the preparation of a cosmetic composition containing a melanogenesis inhibitor, which contains BLG as an active ingredient. The document further suggests that BLG can be isolated, for example, by the following process: hydrochloric acid is added to milk, casein is precipitated, followed by filtration to obtain whey; the pH of the whey is adjusted to 6.0, ammonium sulfate is added in a half-saturated amount; the precipitated protein is removed by salting out, and the filtrate is recovered; the filtrate is saturated with ammonium sulfate, and the precipitated protein is recovered; the recovered protein is redissolved in water, dialyzed at pH 5.2, the crystals are separated, and β-lactoglobulin is prepared at a rate of about 1.8 g from 1 L of whey. However, the general process steps of the proposed process described in JP H10 218755 A are insufficient to lead to the formation of BLG crystals. Thus, the document does not include any disclosure of the crystallization of BLG or the feasibility of BLG crystals.

[0011] US 2 790 790 discloses a process for the precipitation of proteins from solutions, more particularly for the fractional precipitation of relatively uncoupled proteins from aqueous solutions by using sodium chloride as a precipitant. It is suggested that the process is useful for isolating BLG by NaCl-induced precipitation at pH 3.6-3.8. In Example II of the document, it is suggested that the NaCl-precipitate can be dialyzed in the usual manner, resulting in the formation of crystalline β-lactoglobulin. However, US 2 790 790 does not indicate that the formation of BLG crystals at pH 3.6-3.8 is actually possible, and does not contain any reference to the meaning of "the usual manner" of dialyzing the BLG precipitate. Thus, the document does not contain any disclosure of the crystallization of BLG or the feasibility of BLG crystals. Summary of the Invention

[0012] It has previously been discovered that beta-lactoglobulin (BLG) can be efficiently isolated on an industrial scale by crystallization (see PCT Application WO2018 / 115520), a method of crystallization that requires the handling and manipulation of large amounts of supersaturated protein solutions.

[0013] The inventors have found that large-scale implementation of WO2018 / 115520 often leads to undesired crystallization of BLG before crystallization is intended in the process. This is problematic, for example, when BLG crystallization occurs in membrane filtration units or other equipment that is sensitive to the presence of particulate matter.

[0014] The inventors have now discovered that isolation of BLG by crystallization can be improved by preparing a supersaturated protein solution by mixing two or more BLG-containing protein feeds that have no or only low levels of BLG supersaturation by themselves, but which when combined provide a higher degree of supersaturation. One of these protein feeds should have a pH of at least 5.6, more preferably at least 6.0, and the other should have a pH of at most 5.4, more preferably at most 5.0. Combined, the two feeds provide a supersaturated BLG protein solution with a pH close to 5.5, which the inventors have found to be the optimal pH for BLG crystallization yield.

[0015] The step of combining the protein feeds is preferably performed by blending the feeds and does not require the use of equipment sensitive to protein crystallization, thus the invention reduces or even completely avoids the risk of premature, unwanted BLG crystallization, which can lead to clogging and breakdown of the associated process equipment.

[0016] A schematic diagram of the formation of a supersaturated protein solution is shown in FIG. 1, where one or more type B protein feed(s) (having a pH of at most 5.4, and preferably below) are mixed with one or more type A protein feed(s) (having a pH of at least 5.6, and preferably above) in an amount sufficient to provide an initial protein solution that has a pH in the range of 5-6 and is supersaturated with respect to BLG.

[0017] The inventors have found this approach to be highly advantageous for large-scale crystallization of BLG, since individual type A and type B protein feeds can be generated with no or very limited risk of premature (and therefore unwanted) BLG crystallization. However, as soon as type A protein feed(s) and type B feed(s) are mixed, a supersaturated initial protein solution is formed.

[0018] Figure 2 shows diagrammatically how the yield of BLG crystallization depends on the pH of the protein solution in which crystallization is to occur. The inventors have found that the optimum pH is around 5.5. Thus, by mixing the more acidic type B protein feed(s) with the less acidic type A protein feed(s), an initial protein solution that is supersaturated with respect to BLG is obtained. If the type A protein feed(s) and / or type B protein feed(s) are slightly supersaturated with respect to BLG, the initial protein solution must be even more supersaturated.

[0019] Guided by the disclosures of this application and WO2018 / 115520 (hereby incorporated for all purposes), one skilled in the art can prepare type A protein supply(s) and type B protein supply(s) and mix them to provide the initial protein solution described herein.

[0020] Thus, one aspect of the invention relates to a method for preparing an edible composition comprising beta-lactoglobulin (BLG), preferably in crystallized and / or isolated form, comprising the steps of: a) preparing an initial protein solution comprising BLG, said initial protein solution being supersaturated with respect to BLG and having a pH in the range of 5-6; b) crystallizing BLG in a supersaturated, initial protein solution, preferably in salting-out mode, thereby obtaining a BLG crystal-containing solution; and c) optionally separating the BLG crystals from the remaining liquid of the BLG crystal-containing solution; Step a) comprises preparing an initial protein solution by combining, preferably mixing, one or more type A protein feed(s) with one or more type B protein feed(s), And, - "Type A protein feed" is defined as a protein feed that contains BLG and has a pH of at least 5.6, and - "Type B protein feed" is defined as a protein feed that contains BLG and has a pH of at most 5.4. [Brief description of the drawings]

[0021] [Figure 1] Referring to step a) of the method, type A protein feed(s) are mixed with type B protein feed(s) to prepare an initial protein solution. [Diagram 2] 1 is a schematic representation of how the pH of the A type protein feed(s), B type protein feed(s) and initial protein solution are positioned relative to the optimal pH for isolation of BLG by crystallization. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] As mentioned above, one aspect of the invention relates to a method for preparing an edible composition comprising beta-lactoglobulin (BLG), preferably in crystallized and / or isolated form, the method comprising the steps of: a) preparing an initial protein solution comprising BLG, said initial protein solution being supersaturated with respect to BLG and having a pH in the range of 5-6; b) crystallizing BLG in a supersaturated, initial protein solution, preferably in salting-out mode, thereby obtaining a BLG crystal-containing solution; and c) optionally separating the BLG crystals from the remaining liquid of the BLG crystal-containing solution; Step a) involves preparing an initial protein solution by combining, preferably mixing, one or more type A protein feed(s) with one or more type B protein feed(s).

[0023] In the context of the present invention, the term "edible composition" relates to a composition that is safe for human consumption, is used as a food ingredient, and does not contain problematic amounts of toxic components such as toluene or other undesirable organic solvents.

[0024] The terms "edible composition comprising beta-lactoglobulin" and "edible BLG composition" are used interchangeably herein.

[0025] BLG is the most prominent protein in bovine whey and milk serum and exists in several genetic variants, the predominant ones in cow's milk, labeled A and B. BLG is a lipocalin protein and can bind many hydrophobic molecules, suggesting a role in their transport. BLG has also been shown to be able to bind iron via siderophores, which may play a role in combating pathogens. Homologs of BLG are lacking in human breast milk.

[0026] Bovine BLG is a relatively small protein of approximately 162 amino acid residues, with a molecular weight of approximately 18.3-18.4 kDa. Under physiological conditions, it is predominantly a dimer, but dissociates into monomers below about pH 3, preserving its native state, as determined using NMR. Conversely, BLG also occurs in tetrameric, octameric and other multimeric aggregate forms under a variety of native conditions.

[0027] BLG solutions can form gels under a variety of conditions once the native structure is sufficiently destabilized to allow aggregation: under prolonged heating at low pH and low ionic strength, clear "fine chain" gels are formed, in which the protein molecules assemble into long, rigid fibers.

[0028] In the context of the present invention, the term "BLG" or "β-lactoglobulin" relates to BLG from mammalian species, e.g. in native and / or glycosylated form, including genetic variants of natural origin. The term BLG also encompasses mammalian BLG produced by recombinant microorganisms. The term "BLG" or "β-lactoglobulin" as used herein excludes unfolded, aggregated BLG. The content of BLG is measured according to Example 9.9 of WO2018 / 115520.

[0029] In the context of the present invention, the term "crystal" refers to a solid material whose components (e.g., atoms, molecules, or ions) are arranged in a highly ordered microscopic structure, forming a crystal lattice that extends in all directions. BLG crystals are protein crystals that primarily contain BLGs that are arranged in a highly ordered microscopic structure, forming a crystal lattice that extends in all directions. BLG crystals may be, for example, monolithic or polycrystalline, and may be, for example, intact crystals, crystal fragments, or combinations thereof. Crystal fragments are formed, for example, when intact crystals are subjected to mechanical shear during processing. Crystal fragments also have the highly ordered microscopic structure of crystals, but may lack the flat surfaces and / or uniform edges or corners of intact crystals. See, for example, FIG. 18 for an example of many intact BLG crystals, and FIG. 13 for an example of a BLG crystal fragment. In either case, BLG crystals or crystal fragments can be visually identified as well-defined, small, coherent structures using optical microscopy. BLG crystals or crystal fragments are often at least partially transparent. Protein crystals are further known to be birefringent, and this optical property can be used to identify unknown particles as having a crystalline structure. Amorphous BLG aggregates, on the other hand, appear as poorly defined, opaque, and as open or porous masses of irregular size.

[0030] In the context of the present invention, the term "crystallize" relates to the formation of protein crystals. Crystallization can, for example, occur spontaneously or can be initiated by the addition of crystallization seeds.

[0031] The edible composition comprises BLG in a crystallized and / or isolated form. An edible composition comprising BLG in an isolated form comprises at least 80% w / w BLG based on total solids. An edible composition comprising BLG in a crystallized form comprises at least some BLG crystals, preferably a significant amount of BLG crystals.

[0032] BLG crystals can often be observed by microscopy and can reach a size where they are visible to the eye.

[0033] In the context of the present invention, a liquid that is "supersaturated" or "supersaturated with respect to BLG" contains a concentration of dissolved BLG in the liquid that exceeds the saturation point of BLG at given physical and chemical conditions. The term "supersaturation" is well known in the field of crystallization (see, for example, Gerard Coquerela, "Crystallization of molecular systems from solution: phase diagrams, supersaturation and other basic concepts", Chemical Society Reviews, p. 2286-2300, Issue 7, 2014), and supersaturation can be determined by many different measurement techniques (e.g., by spectroscopy or particle size analysis). In the context of the present invention, supersaturation with respect to BLG is determined by the following procedure.

[0034] Procedure for testing whether a liquid at a particular set of conditions is supersaturated with respect to BLG: a) Transfer a 50 ml sample of the liquid to be tested into a centrifuge tube (VWR Catalog No. 525-0402) with a height of 115 mm, an internal diameter of 25 mm and a capacity of 50 mL. Care must be taken to maintain the sample and its subsequent fractions in the original physical and chemical conditions of the liquid during steps a)-h). b) The sample is immediately centrifuged at 3000 g for 3.0 minutes with a maximum of 30 seconds acceleration and a maximum of 30 seconds deceleration. c) Immediately after centrifugation, transfer as much of the supernatant as possible (without disturbing the pellet if one is formed) to a second centrifuge tube (same type as in step a). d) Take a 0.05 mL subsample from the supernatant (Subsample A). e) 10 mg of BLG crystals (at least 98% pure BLG based on total solids) having a particle size of at most 200 microns is added to a second centrifuge tube and the mixture is vortexed. f) Allow the second tube to stand at the original temperature for 60 minutes. g) Immediately after step f), the second centrifuge tube is centrifuged at 500 g for 10 minutes and another 0.05 mL subsample is then taken from the supernatant (subsample B). h) Collect any centrifugation pellet of step g) and resuspend it in Milli-Q water and immediately inspect the suspension for the presence of crystals visible by microscopy. i) Determine the concentration of BLG in sub-samples A and B using the method outlined in Example 9.9 of WO2018 / 115520 - results are expressed as % BLG w / w relative to the total weight of the sub-sample. The concentration of BLG in sub-sample A is C BLG、A The concentration of BLG in subsample B is called C BLG、B It is called. j) The liquid from which the sample was taken in step a) is BLG、B C BLG、A If lower and crystals are observed in step i), there was (at the particular conditions) supersaturation.

[0035] In the context of the present invention, the terms "liquid" and "solution" encompass compositions that include a combination of liquids and solid or semi-solid particles, such as, for example, protein crystals or other protein particles. Thus, a "liquid" or "solution" may be a suspension or even a slurry. However, a "liquid" and a "solution" are preferably pumpable.

[0036] In the context of the present invention, a dried product, e.g., a powder, comprising "BLG crystals" includes a product obtained by drying a suspension of BLG crystals, where the crystal structure of the wet BLG crystals may have been distorted during the drying process and may have at least partially lost their X-ray diffraction properties. In the same manner, the terms "dried BLG crystals" and "dried BLG crystals" refer to particles obtained from drying wet BLG crystals, which dry particles need not have a crystal structure per se. However, the inventors have observed that when dried BLG crystals are resuspended in cold (4°C) demineralized water in a weight ratio of 2 parts water to 1 part dried BLG crystals, the BLG crystals are rehydrated and regain substantially the same crystal structure (space group type and unit cell dimensions) as before drying.

[0037] The methods of analysis described in the context of PCT application WO2018 / 115520 apply equally to the present invention and should be used to determine the parameters described herein.

[0038] In the context of the present invention, the term "initial protein solution" relates to a protein solution prepared by combining, preferably mixing, one or more type A protein feed(s) and one or more type B protein feed(s). The initial protein solution is supersaturated with respect to BLG and is the BLG-containing solution in which the crystallization of step b) is initiated. If one or more of the protein feed(s) used already contains BLG crystals, the initial protein solution is a first protein solution having a pH in the range of 5.0-6.0 and supersaturated with respect to BLG, which is obtained by combining, preferably mixing, the type A protein feed and the type B protein feed.

[0039] In the context of the present invention, the term "type A protein feed" relates to a protein feed having a pH of at least 5.6 and further comprising BLG. Thus, any BLG-containing protein feed having a pH of at least 5.6 and used to prepare an initial protein solution is considered as a type A protein feed.

[0040] In the context of the present invention, the term "type B protein feed" relates to a protein feed having a pH of at most 5.4 and further comprising BLG. Thus, any BLG-containing protein feed having a pH of at most 5.4 and used to prepare an initial protein solution is considered as a type B protein feed.

[0041] In the context of the present invention, the term "one or more type A protein feed(s)" means that the invention can be carried out with a single type A protein feed or with several type A protein feeds, which are combined with type B protein feed(s) during the preparation of the initial protein solution.

[0042] The term "one or more type B protein feed(s)" in the context of the present invention means that the invention can be carried out either with a single type B protein feed or with several type B protein feeds, which are combined with the type A protein feed(s) during the preparation of the initial protein solution.

[0043] In the context of the present invention, the term "type A protein feed(s), when combined, becomes" describes one or more type A protein feed(s) based on the properties they would have if they were hypothetically mixed into a single type A protein feed in the same amount as used to prepare the initial protein solution. It is important to note that this term also encompasses the use of only a single type A protein feed, in which case the properties relate to the single type A protein feed. It is further important to note that this term does not require that the multiple type A protein feeds are actually combined prior to mixing with the one or more type B protein feed(s).

[0044] In the context of the present invention, the term "type B protein feed(s), when combined, becomes" describes one or more type B protein feed(s) based on the properties they would have if they were hypothetically mixed into a single type B protein feed in the same amounts as used to prepare the initial protein solution. It is important to note that this term also encompasses the use of only a single type B protein feed, in which case the properties relate to the single type B protein feed. It is further important to note that this term does not require that the multiple type B protein feeds are actually combined prior to mixing with the one or more type A protein feed(s).

[0045] The initial protein solution of step a) typically contains non-BLG solids in addition to BLG. In the context of the present invention, the term "non-BLG solids" refers to solids such as carbohydrates, minerals, lipids, peptides and proteins other than BLG. The solids present in the initial protein solution are characteristic of the protein source or sources from which the protein feed is prepared. Protein feeds based on whey protein sources will typically contain other whey proteins, such as, for example, alpha-lactalbumin, caseinomacropeptide and / or bovine serum albumin, in addition to BLG. Protein feeds based on BLG sources prepared by fermentation typically contain impurities from the fermentation broth, such as residual nutrients and metabolic by-products, such as, for example, other proteins, peptides and metabolized nutrients.

[0046] In some preferred embodiments of the invention, the method does not include the separation of step c) and provides an edible composition comprising both BLG crystals and additional protein, e.g., additional whey protein. When this method variant further comprises drying of step f), it provides a dry composition comprising BLG crystals and additional whey protein, i.e., WPC or WPI, in which case at least a portion of the BLG is present in the form of BLG crystals. Preferably, the method comprises steps a), b) and f) in direct sequence.

[0047] When the protein supply is prepared from a whey protein concentrate (WPC), a whey protein isolate (WPI), a serum protein concentrate (SPC) or a serum protein isolate (SPI), the above process variants allow for the preparation of the WPC, WPI, SPC or SPI in liquid or dry form, in which case at least a portion of the BLG is in crystalline form.

[0048] The terms "whey protein concentrate" and "serum protein concentration" refer to a dry or aqueous composition containing a total amount of protein of 20-89% w / w based on total solids.

[0049] The WPC or SPC preferably comprises: 20-89% w / w protein on total solids; BLG, 15-70% w / w of total protein; 8-50% ALA w / w of total protein, and CMP 0-40% w / w on protein.

[0050] Alternatively, and also preferably, the WPC or SPC may comprise: 20-89% w / w protein on total solids; BLG, 15-90% w / w of total protein; 4-50% w / w ALA based on total protein, and CMP 0-40% w / w on protein.

[0051] Preferably, the WPC or SPC comprises: 20-89% w / w protein on total solids; BLG, 15-80% w / w of total protein; 4-50% w / w ALA based on total protein, and CMP 0-40% w / w on protein.

[0052] More preferably the WPC or SPC comprises: 70-89% w / w protein on total solids; BLG, 30-90% w / w of total protein; 4-35% w / w ALA based on total protein, and CMP 0-25% w / w on protein.

[0053] The terms "whey protein isolate" and "serum protein isolate" refer to a dry or aqueous composition containing a total amount of protein of 90-100% w / w relative to total solids.

[0054] The WPI or SPI preferably comprises: 90-100% w / w protein on total solids, BLG, 15-70% w / w of total protein; 8-50% ALA w / w of total protein, and CMP 0-40% w / w of total protein.

[0055] Alternatively, and also preferably, the WPI or SPI may include: 90-100% w / w protein on total solids, BLG, 30-95% w / w of total protein; 4-35% w / w ALA based on total protein, and CMP 0-25% w / w of total protein.

[0056] More preferably, the WPI or SPI may include: 90-100% w / w protein on total solids, BLG, 30-90% w / w of total protein; 4-35% w / w ALA based on total protein, and CMP 0-25% w / w of total protein.

[0057] In some preferred embodiments of the invention, the method further comprises a step d) of washing the BLG crystals, eg the separated BLG crystals obtained from step c).

[0058] In some preferred embodiments of the invention, the method further comprises a step e) of recrystallizing the BLG crystals, eg the BLG crystals obtained from step c) or d).

[0059] The method may, for example, include or even consist of steps a), b), c), d), and e), or the method may include or even consist of steps a), b), c), and e).

[0060] In some particularly preferred embodiments of the invention, the method further comprises a step f) of drying the BLG-containing composition from step b), c), d), or e).

[0061] The method may, for example, comprise or even consist of steps a), b), and f). Alternatively, the method may comprise or even consist of steps a), b), c), and f). Alternatively, the method may comprise or even consist of steps a), b), c), d), and f). Alternatively, the method may comprise or even consist of steps a), b), c), d), e), and f). As mentioned above, step a) of the present invention comprises providing an initial protein solution having a pH in the range of 5-6 and which is supersaturated with respect to BLG.

[0062] The initial protein solution often contains additional proteins in addition to BLG. In the context of the present invention, the term "additional proteins" refers to proteins that are not BLG. The additional proteins present in the initial protein solution typically include one or more non-BLG proteins found in milk serum or whey. Non-limiting examples of such proteins are alpha-lactalbumin, bovine serum albumin, immunoglobulins, caseinomacropeptide (CMP), osteopontin, lactoferrin, and milk fat globule membrane proteins. Alternatively, the additional proteins may be protein impurities or cell debris from the fermentation broth, for example, when BLG is produced by fermentation of a recombinant cell culture.

[0063] In the context of the present invention, the term "whey protein" refers to proteins found in whey or milk serum. The whey proteins of the initial protein solution may be a subset of the protein species found in whey or milk serum, or it may be the complete set of protein species found in whey or / and milk serum. However, the initial protein solution always contains BLG.

[0064] Thus, the initial protein solution may preferably comprise at least one additional whey protein selected from the group consisting of alpha-lactalbumin, bovine serum albumin, immunoglobulins, caseinomacropeptide (CMP), osteopontin, lactoferrin, milk fat globule membrane protein, and combinations thereof.

[0065] Alpha-lactalbumin (ALA) is a protein present in the milk of almost all mammalian species. ALA forms the regulatory subunit of the lactose synthase (LS) heterodimer, and beta-1,4-galactosyltransferase (beta4Gal-T1) forms the catalytic component. Together, these proteins enable LS to generate lactose by transferring a galactose moiety to glucose. As a multimer, alpha-lactalbumin binds strongly to calcium and zinc ions and may have bactericidal or antitumor activity. One of the main structural differences with beta-lactoglobulin is that ALA does not have a free thiol group that can serve as an initiation point for a covalent aggregation reaction. As a result, pure ALA does not form a gel upon denaturation and acidification.

[0066] In the context of the present invention, the term "ALA" or "alpha-lactalbumin" relates to alpha-lactalbumin derived from mammalian species, for example in native and / or glycosylated form, and includes genetic variants of natural origin.

[0067] In some embodiments of the invention the initial protein solution comprises at most 10% w / w casein relative to the total amount of protein, preferably at most 5% w / w, more preferably at most 1% w / w, even more preferably at most 0.5% casein relative to the total amount of protein, hi some preferred embodiments of the invention the initial protein solution does not contain detectable amounts of casein.

[0068] The term "whey" refers to the liquid remaining when casein and milk fat globules are removed from milk, for example by microfiltration or large pore ultrafiltration. Whey may also be called "ideal whey".

[0069] The term "whey protein" or "serum protein" relates to proteins present in whey.

[0070] The term "whey" refers to the liquid supernatant remaining after the casein of milk has been precipitated and removed. Casein precipitation can be accomplished, for example, by acidification of the milk and / or the use of rennet enzymes.

[0071] Several types of whey exist, such as "sweet whey", which is a whey product produced by rennet-based precipitation of casein, and "acid whey" or "sour whey", which is a whey product produced by acid-based precipitation of casein. Acid-based precipitation of casein can be accomplished, for example, by the addition of food acids or by bacterial culture.

[0072] In some preferred embodiments of the invention, the initial protein solution of step a) comprises non-BLG solids in an amount of at least 1% w / w, more preferably at least 2% w / w, even more preferably at least 5% w / w, and most preferably at least 10% w / w, based on total solids.

[0073] In another preferred embodiment of the invention, the initial protein solution of step a) comprises non-BLG solids in an amount of at least 15% w / w, more preferably at least 20% w / w, even more preferably at least 30% w / w, and most preferably at least 40% w / w, based on total solids.

[0074] In some preferred embodiments of the invention, the initial protein solution of step a) comprises non-BLG solids in an amount of 0-70% w / w, more preferably 1-60% w / w, even more preferably 2-50% w / w, and most preferably 3-40% w / w, based on total solids.

[0075] In another preferred embodiment of the invention, the initial protein solution of step a) comprises non-BLG solids in an amount of 0-20% w / w, more preferably 0-15% w / w, even more preferably 0-10% w / w, and most preferably 0-5% w / w based on total solids.

[0076] In a further preferred embodiment of the invention, the initial protein solution of step a) comprises non-BLG solids in an amount of 15-70% w / w, more preferably 20-65% w / w, even more preferably 25-60% w / w, and most preferably 30-50% w / w based on total solids.

[0077] In some preferred embodiments of the invention, the initial protein solution of step a) comprises at least 5% w / w additional whey protein relative to the total amount of protein. Preferably, the initial protein solution of step a) comprises at least 10% w / w additional whey protein relative to the total amount of protein. More preferably, the initial protein solution of step a) comprises at least 15% w / w additional whey protein relative to the total amount of protein. Even more preferably, the initial protein solution of step a) comprises at least 20% w / w additional whey protein relative to the total amount of protein. Most preferably, the initial protein solution of step a) may comprise at least 30% w / w additional whey protein relative to the total amount of protein.

[0078] In another preferred embodiment of the invention, the initial protein solution of step a) comprises at least 1% w / w additional whey protein relative to the total amount of protein. Preferably, the initial protein solution of step a) comprises at least 2% w / w additional whey protein relative to the total amount of protein. Even more preferably, the initial protein solution of step a) comprises at least 3% w / w additional whey protein relative to the total amount of protein. Most preferably, the initial protein solution of step a) may comprise at least 4% w / w additional whey protein relative to the total amount of protein.

[0079] In yet another preferred embodiment of the invention, the initial protein solution of step a) comprises at least 35% w / w additional whey protein relative to the total amount of protein. Preferably, the initial protein solution of step a) may comprise at least 40% w / w additional whey protein relative to the total amount of protein. More preferably, the initial protein solution of step a) may for example comprise at least 45% w / w additional whey protein relative to the total amount of protein. Even more preferably, the initial protein solution of step a) may comprise at least 50% w / w additional whey protein relative to the total amount of protein.

[0080] In some preferred embodiments of the invention, the initial protein solution of step a) comprises additional whey protein in the range of 5-90% w / w based on the total amount of protein. Preferably, the initial protein solution of step a) may comprise additional whey protein in the range of 10-80% w / w based on the total amount of protein. The initial protein solution of step a) may for example comprise additional whey protein in the range of 20-70% w / w based on the total amount of protein. Preferably, the initial protein solution of step a) comprises additional whey protein in the range of 30-70% w / w based on the total amount of protein.

[0081] As mentioned above, the inventors have found that it is possible to crystallize BLG without using organic solvents. This purification approach can also be used to purify preparations containing whey protein (which preparations have already been subjected to some BLG purification), providing a simple way to increase the purity of BLG even further. Thus, in some preferred embodiments of the invention, the initial protein solution of step a) comprises additional whey protein in the range of 1-20% w / w based on the total amount of protein. Preferably, the initial protein solution of step a) may comprise additional whey protein in the range of 2-15% w / w based on the total amount of protein. Even more preferably, the initial protein solution of step a) may comprise additional whey protein in the range of 3-10% w / w based on the total amount of protein, for example.

[0082] In some embodiments of the invention, the initial protein solution of step a) comprises at least 5% w / w ALA based on the total amount of protein. Preferably, the initial protein solution of step a) comprises at least 10% w / w ALA based on the total amount of protein. Even more preferably, the initial protein solution of step a) comprises at least 15% w / w ALA based on the total amount of protein. Alternatively, the initial protein solution of step a) may comprise at least 20% w / w ALA based on the total amount of protein.

[0083] In some preferred embodiments of the invention, the initial protein solution of step a) comprises at least 25% w / w ALA relative to the total amount of protein. Preferably, the initial protein solution of step a) comprises at least 30% w / w ALA relative to the total amount of protein. Preferably, the initial protein solution of step a) comprises at least 35% w / w ALA relative to the total amount of protein. Even more preferably, the initial protein solution of step a) may comprise at least 40% w / w ALA relative to the total amount of protein.

[0084] In some preferred embodiments of the invention, the initial protein solution of step a) comprises ALA in the range of 5-95% w / w based on the total amount of protein. Preferably, the initial protein solution of step a) comprises ALA in the range of 5-70% w / w based on the total amount of protein. Even more preferably, the initial protein solution of step a) may comprise ALA in the range of 10-60% w / w based on the total amount of protein. The initial protein solution of step a) preferably comprises ALA in the range of 12-50% w / w based on the total amount of protein. Even more preferably, the initial protein solution of step a) may comprise ALA in the range of 20-45% w / w based on the total amount of protein.

[0085] In some preferred embodiments of the invention, the initial protein solution of step a) has a weight ratio between BLG and ALA of at least 0.01. Preferably, the initial protein solution of step a) has a weight ratio between BLG and ALA of at least 0.5. Even more preferably, the initial protein solution of step a) has a weight ratio between BLG and ALA of at least 1, such as, for example, at least 2. For example, the initial protein solution of step a) may have a weight ratio between BLG and ALA of at least 3.

[0086] The amounts and concentrations of BLG and other proteins in the initial protein solutions and protein feeds all represent dissolved protein and do not include precipitated or crystallized protein.

[0087] In the context of the present invention, the term "weight ratio" between component X and component Y means the ratio by weight of the calculated X / m Y where m X is the amount (weight) of component X, and m Y is the amount (by weight) of component Y.

[0088] In some preferred embodiments of the invention, the initial protein solution of step a) has a weight ratio between BLG and ALA in the range of 0.01-20. Preferably, the initial protein solution of step a) has a weight ratio between BLG and ALA in the range of 0.2-10. Even more preferably, the initial protein solution of step a) has a weight ratio between BLG and ALA in the range of 0.5-4. For example, the initial protein solution of step a) may have a weight ratio between BLG and ALA in the range of 1-3.

[0089] In some preferred embodiments of the invention, the initial protein solution of step a) comprises at least 1% w / w BLG relative to the total amount of protein. Preferably, the initial protein solution of step a) comprises at least 2% w / w BLG relative to the total amount of protein. Even more preferably, the initial protein solution of step a) comprises at least 5% w / w BLG relative to the total amount of protein. Preferably, the initial protein solution of step a) may comprise at least 10% w / w BLG relative to the total amount of protein.

[0090] In some preferred embodiments of the invention, the initial protein solution of step a) comprises at least 12% w / w BLG relative to the total amount of protein. For example, the initial protein solution of step a) may comprise at least 15% w / w BLG relative to the total amount of protein. The initial protein solution of step a) may comprise, for example, at least 20% w / w BLG relative to the total amount of protein. Alternatively, the initial protein solution of step a) may comprise at least 30% w / w BLG relative to the total amount of protein.

[0091] In another preferred embodiment of the invention, the initial protein solution of step a) comprises at least 40% w / w BLG relative to the total amount of protein. More preferably, the initial protein solution of step a) may comprise at least 45% w / w BLG relative to the total amount of protein. Even more preferably, the initial protein solution of step a) may comprise, for example, at least 50% w / w BLG relative to the total amount of protein. Most preferably, the initial protein solution of step a) may comprise at least 55% w / w BLG relative to the total amount of protein.

[0092] In some particularly preferred embodiments of the invention, the initial protein solution of step a) comprises at most 95% w / w BLG relative to the total amount of protein. Preferably, the initial protein solution of step a) may comprise at most 90% w / w BLG relative to the total amount of protein. More preferably, the initial protein solution of step a) may comprise, for example, at most 85% w / w BLG relative to the total amount of protein. Even more preferably, the initial protein solution of step a) may comprise, for example, at most 80% w / w BLG relative to the total amount of protein. Preferably, the initial protein solution of step a) may comprise at most 78% w / w BLG relative to the total amount of protein. Preferably, the initial protein solution of step a) may comprise at most 75% w / w BLG relative to the total amount of protein.

[0093] In some preferred embodiments of the invention, the initial protein solution of step a) comprises BLG in the range of 1-95% w / w based on the total amount of protein. Preferably, the initial protein solution of step a) may comprise BLG in the range of 5-90% w / w based on the total amount of protein. More preferably, the initial protein solution of step a) comprises BLG in the range of 10-85% w / w based on the total amount of protein. Even more preferably, the initial protein solution of step a) comprises BLG in the range of 10-80% w / w based on the total amount of protein. Most preferably, the initial protein solution of step a) may comprise BLG in the range of 20-70% w / w based on the total amount of protein.

[0094] In another preferred embodiment of the invention, the initial protein solution of step a) comprises BLG in the range of 10-95% w / w relative to the total amount of protein. Preferably, the initial protein solution of step a) may comprise BLG in the range of 12-90% w / w relative to the total amount of protein. More preferably, the initial protein solution of step a) comprises BLG in the range of 15-85% w / w relative to the total amount of protein. Even more preferably, the initial protein solution of step a) comprises BLG in the range of 15-80% w / w relative to the total amount of protein. Most preferably, the initial protein solution of step a) may comprise BLG in the range of 30-70% w / w relative to the total amount of protein.

[0095] In some preferred embodiments of the invention, the initial protein solution of step a) comprises BLG in an amount of at least 0.4% w / w relative to the weight of the initial protein solution. Preferably, the initial protein solution comprises BLG in an amount of at least 1.0% w / w. More preferably, the initial protein solution comprises BLG in an amount of at least 2.0% w / w. Even more preferably, the initial protein solution comprises BLG in an amount of at least 4% w / w.

[0096] Even more preferred is a higher concentration of BLG, preferably the initial protein solution comprises BLG in an amount of at least 6% w / w. More preferably the initial protein solution comprises BLG in an amount of at least 10% w / w. Even more preferred is that the initial protein solution comprises BLG in an amount of at least 15% w / w.

[0097] In some preferred embodiments of the invention, the initial protein solution of step a) comprises an amount of BLG in the range of 0.4-40% w / w by weight of the initial protein solution. Preferably, the initial protein solution comprises an amount of BLG in the range of 1-35% w / w. More preferably, the initial protein solution comprises an amount of BLG in the range of 4-30% w / w. Even more preferably, the initial protein solution comprises an amount of BLG in the range of 10-25% w / w.

[0098] In another preferred embodiment of the invention, the initial protein solution of step a) comprises an amount of BLG in the range of 1-45% w / w relative to the weight of the initial protein solution. Preferably, the initial protein solution comprises an amount of BLG in the range of 3-40% w / w. More preferably, the initial protein solution comprises an amount of BLG in the range of 5-36% w / w. Even more preferably, the initial protein solution comprises an amount of BLG in the range of 7-34% w / w.

[0099] In a further preferred embodiment of the invention, the initial protein solution of step a) comprises an amount of BLG in the range of 6-32% w / w relative to the weight of the initial protein solution. Preferably, the initial protein solution comprises an amount of BLG in the range of 8-30% w / w. More preferably, the initial protein solution comprises an amount of BLG in the range of 10-28% w / w. Even more preferably, the initial protein solution comprises an amount of BLG in the range of 12-26% w / w.

[0100] The initial protein solution is preferably a desalted initial protein solution.

[0101] In this context, the term desalted means that the conductivity of the initial protein solution is at most 15 mS / cm, preferably at most 10 mS / cm, even more preferably at most 8 mS / cm. The UF permeate conductivity of the desalted initial protein solution is preferably at most 7 mS / cm, more preferably at most 4 mS / cm, even more preferably at most 1 mS / cm.

[0102] It is particularly preferred that the initial protein solution is a desalted whey protein concentrate, a desalted whey protein isolate, a desalted whey protein concentrate or a desalted whey protein isolate.

[0103] In some particularly preferred embodiments of the invention, the initial protein solution comprises, or even consists of, demineralized and pH adjusted whey protein concentrate, whey protein concentrate, whey protein isolate, whey protein isolate, or combinations thereof.

[0104] The initial protein solution may, for example, comprise or even consist of a desalted whey protein concentrate. Alternatively, the initial protein solution may comprise or even consist of a desalted whey protein concentrate. Alternatively, the initial protein solution may comprise or even consist of a desalted whey protein isolate. Alternatively, the initial protein solution may comprise or even consist of a desalted whey protein isolate.

[0105] In the context of the present invention, the terms "whey protein concentrate" and "whey protein concentrate" relate to preparations of whey or whey, which preparations contain in the range of approximately 20-89% w / w protein based on total solids.

[0106] In the context of the present invention, the terms "whey protein isolate" and "whey protein isolate" relate to a preparation of whey or whey, which preparation comprises at least 90% w / w protein based on total solids.

[0107] The terms "consisting essentially of" and "consisting essentially of" mean that the claim or feature at issue includes specified materials or steps and those that do not materially affect the basic, novel characteristic(s) of the claimed invention.

[0108] The proteins of the initial protein solution are preferably derived from mammalian milk, preferably from the milk of ruminant animals, such as, for example, cows, sheep, goats, buffalo, camels, llamas, mares and / or deer. Proteins derived from bovine (cow) milk are particularly preferred. Thus, the BLG and any additional proteins are preferably bovine BLG and bovine whey proteins. In another embodiment of the invention, the BLG of the initial protein solution is produced by a recombinant microorganism, but still has the amino acid sequence of BLG derived from mammalian milk.

[0109] The proteins of the initial protein solution are preferably as close as possible to their native state and have preferably been subjected to only mild, non-denaturing heat treatments, if any.

[0110] In some preferred embodiments of the invention, the BLG of the initial protein solution has a lactosylation degree of at most 1. Preferably, the BLG of the initial protein solution has a lactosylation degree of at most 0.6. More preferably, the BLG of the initial protein solution has a lactosylation degree of at most 0.4. Even more preferably, the BLG of the initial protein solution has a lactosylation degree of at most 0.2. Most preferably, the BLG of the initial protein solution has a lactosylation degree of at most 0.1, such as, for example, preferably at most 0.01.

[0111] The degree of lactosylation of BLG is determined according to Czerwenka et al. (J. Agric. Food Chem., Vol. 54, No. 23, 2006, pages 8874-8882).

[0112] In some preferred embodiments of the invention, the initial protein solution has a furosine value of at most 80 mg / 100 g protein. Preferably, the initial protein solution has a furosine value of at most 40 mg / 100 g protein. More preferably, the initial protein solution has a furosine value of at most 20 mg / 100 g protein. Even more preferably, the initial protein solution has a furosine value of at most 10 mg / 100 g protein. Most preferably, the initial protein solution has a furosine value of at most 5 mg / 100 g protein, for example, preferably 0 mg / 100 g protein.

[0113] The initial protein solution typically contains other components in addition to proteins. The initial protein solution may contain other components that are normally found in whey or milk serum, such as, for example, minerals, carbohydrates, and / or lipids. Alternatively or in addition, the initial protein solution may contain components that are not naturally found in whey or milk serum. However, such non-naturally occurring components should preferably be safe for use in food production and preferably also for human consumption.

[0114] The method is particularly advantageous for separating BLG from a crude initial protein solution that contains solids other than BLG.

[0115] The initial protein solution may, for example, comprise carbohydrates, such as, for example, lactose, oligosaccharides and / or hydrolysis products of lactose (i.e., glucose and galactose). The initial protein solution may, for example, comprise carbohydrates in the range of 0-40% w / w, such as, for example, in the range of 1-30% w / w, or in the range of 2-20% w / w.

[0116] In some preferred embodiments of the invention, the initial protein solution comprises at most 20% w / w carbohydrate, preferably at most 10% w / w carbohydrate, more preferably at most 5% w / w carbohydrate, even more preferably at most 2% w / w carbohydrate.

[0117] The initial protein solution may also contain lipids, for example in the form of triglycerides and / or other lipid types such as phospholipids.

[0118] In some embodiments of the invention, the initial protein solution of step a) comprises a total amount of lipids of at most 15% w / w based on total solids. Preferably, the initial protein solution of step a) comprises a total amount of lipids of at most 10% w / w based on total solids. More preferably, the initial protein solution of step a) comprises a total amount of lipids of at most 6% w / w based on total solids. Even more preferably, the initial protein solution of step a) comprises a total amount of lipids of at most 1.0% w / w based on total solids. Most preferably, the initial protein solution of step a) comprises a total amount of lipids of at most 0.5% w / w based on total solids.

[0119] The total amount of protein in the initial protein solution is typically at least 1% w / w based on the weight of the initial protein solution. Preferably, the total amount of protein in the initial protein solution is at least 5% w / w. More preferably, the total amount of protein in the initial protein solution is at least 10% w / w. Even more preferably, the total amount of protein in the initial protein solution is at least 15% w / w.

[0120] In some preferred embodiments of the invention, the total amount of protein in the initial protein solution is in the range of 1-50% w / w. Preferably, the total amount of protein in the initial protein solution is in the range of 5-40% w / w. More preferably, the total amount of protein in the initial protein solution is in the range of 10-30% w / w. Even more preferably, the total amount of protein in the initial protein solution is in the range of 15-25% w / w.

[0121] The total solids of the initial protein solution are typically at least 1% w / w by weight of the initial protein solution. Preferably, the total solids of the initial protein solution are at least 5% w / w. More preferably, the total solids of the initial protein solution are at least 10% w / w. Even more preferably, the total solids of the initial protein solution are at least 15% w / w.

[0122] In some preferred embodiments of the invention, the total solids of the initial protein solution are in the range of 5-50% w / w. Preferably, the total solids of the initial protein solution are in the range of 8-40% w / w. More preferably, the total solids of the initial protein solution are in the range of 10-30% w / w. Even more preferably, the total solids of the initial protein solution are in the range of 15-25% w / w.

[0123] In other preferred embodiments of the invention, the total solids of the initial protein solution are in the range of 8-50% w / w. Preferably, the total solids of the initial protein solution are in the range of 10-45% w / w. More preferably, the total solids of the initial protein solution are in the range of 12-40% w / w. Even more preferably, the total solids of the initial protein solution are in the range of 15-35% w / w.

[0124] The total amount of protein of the initial protein solution is determined according to Example 9.2 of WO2018 / 115520.

[0125] The initial protein solution of step a) is prepared by combining, preferably mixing, one or more type A protein feed(s) with one or more type B protein feed(s). As mentioned above, any BLG-containing protein feed having a pH of at least 5.6 is defined as a type A protein feed, and any BLG-containing protein feed having a pH of at most 5.4 is defined as a type B protein feed.

[0126] In a most preferred embodiment, an initial protein solution is prepared by combining, preferably mixing, a single type A protein supply with a single type B protein supply. Crystallization is preferably initiated by the subsequent addition of a seed composition, preferably in liquid or alternatively powder form.

[0127] The initial protein solution may further comprise protein feed(s) that are neither Type A nor Type B, e.g., protein feed(s) having a pH of approximately 5.5, or protein feed(s) that do not contain BLG, although it is preferred to limit or even avoid the presence of such additional protein feeds unless used to optimize the process, e.g., by providing BLG crystals or other seed material to initiate crystallization.

[0128] Preparation of the initial protein solution may include the addition of other material components in addition to the protein feed, such as the addition of an acid or base, to provide an initial protein solution with a suitable pH. However, it is preferred that the one or more type A protein feed(s) and one or more type B protein feed(s) are already adjusted to provide the desired pH when combined, and thus the addition of other material components is limited or even avoided.

[0129] In the context of the present invention, the terms "one or more type A protein feed(s)" and "one or more type B protein feed(s)" refer to BLG-containing compositions that are combined during the preparation of an initial protein solution that is supersaturated with respect to BLG. As mentioned above, a "type A protein feed" has a pH of at least 5.6 and a "type B protein feed" has a pH of at most 5.4. The protein feed is typically an aqueous liquid and / or powder that contains BLG and impurities. Examples of typical impurities are minerals, carbohydrates, other proteins such as additional proteins, for example.

[0130] Thus, one or more type A protein feed(s), one or more type B protein feed(s), and any additional material components may be combined in any order to provide an initial protein solution.

[0131] The initial protein solution is prepared by combining the protein feeds, preferably mixing one or more type A protein feed(s) with one or more type B protein feed(s), and optionally with any further protein feeds or material components.

[0132] All protein supplies and any additional material components are combined in amounts sufficient to provide an initial protein solution of appropriate chemical composition and as described herein.

[0133] The inventors have found that maximum BLG yields from BLG crystallization are obtained at approximately pH 5.5. Thus, it is preferred that the initial protein solution has a pH closer to 5.5 than: - the pH of the Type A protein feed(s) when they are combined, or - the pH of the B protein feed(s) when they are combined, or - The pH of the Type A protein feed(s) when they are combined and the pH of the Type B protein feed(s) when they are combined.

[0134] It is often preferable to combine the protein feeds to provide an initial protein solution that is in the metastable region with respect to BLG supersaturation, i.e., in the supersaturated region, in which case BLG crystals can grow if seeding is used, but crystallization does not begin spontaneously.

[0135] In some preferred embodiments of the present invention: the -A type protein feed(s), when combined, have a pH of at least 5.6, more preferably at least 5.7, even more preferably at least 5.8, and most preferably at least 5.9; and The -B type protein feed(s), when combined, have a pH of at most 5.4, more preferably at most 5.3, even more preferably 5.2, and most preferably 5.1.

[0136] If only a single type A protein feed is used, the phrase "when combined to result in" refers to the single type A protein feed as such. Similarly, if only a single type B protein feed is used, the phrase "when combined to result in" refers to the single type B protein feed as such.

[0137] Thus, the phrase "the Type A protein feed(s), when mixed, have [particular properties]" means the following: -When only a single type A protein feed is used, it has [specific properties] - When two or more type A protein feeds are used, they have [specific properties] when mixed.

[0138] For example, by stating that the Type A protein feed(s), when mixed, has a pH of at least 5.6, the following is meant: - If only a single type A protein feed is used, it has a pH of at least 5.6 - If two or more type A protein feeds are used, they, when mixed, have a pH of at least 5.6.

[0139] For example, in one embodiment where two type A protein supplies are used, the phrase "when combined results in" means that when the two type A protein supplies are mixed together, a particular property is obtained.

[0140] Thus, in some preferred embodiments of the present invention: -In relation to A-type protein supply: - if only a single type A protein supply is used, it has: - When two or more type A protein feeds are used, they, when mixed, have: a pH of at least 5.6, more preferably at least 5.7, even more preferably at least 5.8, and most preferably at least 5.9; and - In relation to B protein supply: - if only a single B-type protein supply is used, it has: - When two or more B type protein feeds are used, they, when mixed, have: A pH of at most 5.4, more preferably at most 5.3, even more preferably 5.2, most preferably 5.1.

[0141] In another preferred embodiment of the present invention: the -A type protein feed(s), when combined, have a pH of at least 6.0, more preferably at least 6.1, even more preferably at least 6.2, and most preferably at least 6.3; and The -B type protein feed(s), when combined, have a pH of at most 5.0, more preferably at most 4.9, even more preferably at most 4.8, and most preferably at most 4.7.

[0142] Thus, in another preferred embodiment of the present invention: -In relation to A-type protein supply: - if only a single type A protein supply is used, it has: - When two or more type A protein feeds are used, they, when mixed, have: a pH of at least 6.0, more preferably at least 6.1, even more preferably at least 6.2, and most preferably at least 6.3; and - In relation to B protein supply: - if only a single B-type protein supply is used, it has: - When two or more B type protein feeds are used, they, when mixed, have: A pH of at most 5.0, more preferably at most 4.9, even more preferably 4.8, most preferably 4.7.

[0143] In some preferred embodiments of the invention, the Type A protein feed(s), when combined, have a pH in the range of 5.6-10, more preferably 5.8-8, even more preferably 6.0-7, and most preferably 6.1-6.5.

[0144] Thus, in some preferred embodiments of the invention, in the context of a type A protein supply: - if only a single type A protein supply is used, it has: - When two or more type A protein feeds are used, they, when mixed, have: A pH in the range of 5.6-10, more preferably 5.8-8, even more preferably 6.0-7, most preferably 6.1-6.5.

[0145] In other preferred embodiments of the invention, the Type A protein supply(s), when combined, have a pH in the range of 6.0-8, more preferably 6.1-7.5, even more preferably 6.2-7.0, and most preferably 6.2-6.5.

[0146] Thus, in another preferred embodiment of the invention, in the context of an A-type protein supply: - if only a single type A protein supply is used, it has: - When two or more type A protein feeds are used, they, when mixed, have: A pH in the range of 6.0-8, more preferably 6.1-7.5, even more preferably 6.2-7.0, most preferably 6.2-6.5.

[0147] In some preferred embodiments of the invention, the type B protein feed(s), when combined, have a pH in the range of 2-5.4, more preferably 3-5.2, even more preferably 4-5.0, and most preferably 4.5-4.9.

[0148] Thus, in some preferred embodiments of the invention, in the context of a B-type protein supply: - if only a single B-type protein supply is used, it has: - When two or more B type protein feeds are used, they, when mixed, have: A pH in the range of 2-5.4, more preferably 3-5.2, even more preferably 4-5.0, most preferably 4.5-4.9.

[0149] In other preferred embodiments of the invention, the type B protein feed(s), when combined, have a pH in the range of 4.0-5.0, more preferably 4.1-4.9, even more preferably 4.3-4.8, and most preferably 4.5-4.8.

[0150] Thus, in another preferred embodiment of the invention, in the context of the B-type protein supply: - if only a single B-type protein supply is used, it has: - When two or more B type protein feeds are used, they, when mixed, have: A pH in the range of 4.0-5.0, more preferably 4.1-4.9, even more preferably 4.3-4.8, most preferably 4.5-4.8.

[0151] In some preferred embodiments of the invention, at least one of the one or more type A protein feed(s) is not saturated with respect to BLG, and more preferably none of the one or more type A protein feed(s) is saturated with respect to BLG.

[0152] In other preferred embodiments of the invention, at least one of the one or more type B protein feed(s) is not saturated with respect to BLG, and more preferably none of the one or more type B protein feed(s) is saturated with respect to BLG.

[0153] In a further preferred embodiment of the invention, neither the A nor B protein feed(s) are saturated with respect to BLG.

[0154] In some preferred embodiments of the present invention, at least one of the one or more type A protein supply(s) does not contain BLG crystals, and more preferably none of the one or more type A protein supply(s) contains BLG crystals.

[0155] In other preferred embodiments of the invention, at least one of the one or more type B protein supply(s) does not comprise BLG crystals, more preferably none of the one or more type B protein supply(s) comprises BLG crystals.

[0156] It should be noted that even if the feed does not contain BLG crystals, it may still be saturated or even supersaturated with respect to BLG.

[0157] In a further preferred embodiment of the invention, neither the Type A nor Type B protein supply(s) contain BLG crystals.

[0158] In other preferred embodiments of the invention, neither the Type A nor Type B protein supply(s) contain BLG crystals.

[0159] In some preferred embodiments of the invention, at least one of the one or more type A protein feed(s) is not supersaturated with respect to BLG, and more preferably none of the one or more type A protein feed(s) is supersaturated with respect to BLG.

[0160] In other preferred embodiments of the invention, at least one of the one or more type B protein feed(s) is not supersaturated with respect to BLG, and more preferably none of the one or more type B protein feed(s) is supersaturated with respect to BLG.

[0161] In some preferred embodiments of the invention, at least one of the one or more type A protein feed(s) of step a) is saturated with respect to BLG.

[0162] In some preferred embodiments of the invention, at least one of the one or more type B protein feed(s) of step a) is saturated with respect to BLG.

[0163] In some preferred embodiments of the invention, at least one of the one or more type A protein feed(s) is supersaturated with respect to BLG, but is in the metastable zone where spontaneous crystallization does not occur.

[0164] In other preferred embodiments of the invention, at least one of the one or more type B protein feed(s) is supersaturated with respect to BLG, but is in the metastable zone where spontaneous crystallization does not occur.

[0165] In some preferred embodiments of the invention, the type A and type B protein feed(s) that do not contain BLG crystals contribute at least 1% w / w of the BLG content of the initial protein solution, more preferably at least 10% w / w, even more preferably at least 20% w / w, and most preferably at least 40% w / w.

[0166] Preferably, the type A and type B protein feed(s) that do not contain BLG crystals contribute at least 50% w / w of the BLG content of the initial protein solution, more preferably at least 70% w / w, even more preferably at least 80% w / w, and most preferably at least 90% w / w.

[0167] In other preferred embodiments of the invention, the type A and type B protein feed(s) free of BLG crystals contribute at least 92% w / w of the BLG content of the initial protein solution, more preferably at least 94% w / w, even more preferably at least 96% w / w, and most preferably at least 99% w / w.

[0168] For example, it may be preferred for the type A and type B protein feed(s) that do not contain BLG crystals to contribute 100% w / w of the BLG content of the initial protein solution, which is, for example, typically the situation in which seed material is added after preparation of the initial protein solution.

[0169] The inventors have found that it is often desirable for at least some of the protein feed not to be supersaturated with respect to BLG, as this significantly reduces the risk of uncontrolled crystallization during production.

[0170] Thus, in some preferred embodiments of the invention, the type A and type B protein feed that is not supersaturated with respect to BLG contributes at least 1% w / w of the BLG content of the initial protein solution, more preferably at least 10% w / w, even more preferably at least 20% w / w, and most preferably at least 40% w / w.

[0171] Preferably, the type A and type B protein feed, which is not supersaturated with respect to BLG, contributes at least 50% w / w of the BLG content of the initial protein solution, more preferably at least 70% w / w, even more preferably at least 80% w / w, and most preferably at least 90% w / w.

[0172] In a further preferred embodiment of the invention, the type A and type B protein feed that is not supersaturated with respect to BLG contributes at least 92% w / w of the BLG content of the initial protein solution, more preferably at least 94% w / w, even more preferably at least 96% w / w, and most preferably at least 99% w / w.

[0173] It is often preferred that the type A and type B protein feed(s) that are not supersaturated with respect to BLG contribute approximately 100% w / w of the BLG content of the initial protein solution.

[0174] It is preferred that the Type A and Type B protein supplies be prepared, stored, and used at temperatures that avoid protein denaturation and / or degradation.

[0175] In some preferred embodiments of the invention, the type A protein feed(s) when combined have a temperature of at most 60°C, more preferably at most 50°C, even more preferably at most 40°C, and most preferably at most 30°C.

[0176] Thus, in some preferred embodiments of the present invention: -In relation to A-type protein supply: - if only a single type A protein supply is used, it has: - When two or more type A protein feeds are used, they, when mixed, have: A temperature of at most 60°C, more preferably at most 50°C, even more preferably at most 40°C, most preferably at most 30°C.

[0177] In other preferred embodiments of the invention, the type A protein feed(s) when combined have a temperature of at most 20°C, more preferably at most 15°C, even more preferably at most 10°C, and most preferably at most 5°C.

[0178] Thus, in another preferred embodiment of the present invention: -In relation to A-type protein supply: - if only a single type A protein supply is used, it has: - When two or more type A protein feeds are used, they, when mixed, have: A temperature of at most 20°C, more preferably at most 15°C, even more preferably at most 10°C, most preferably at most 5°C.

[0179] In some preferred embodiments of the invention, the type B protein feed(s) when combined have a temperature of at most 60°C, more preferably at most 50°C, even more preferably at most 40°C, and most preferably at most 30°C.

[0180] Thus, in some preferred embodiments of the present invention: - In relation to B protein supply: - if only a single B-type protein supply is used, it has: - When two or more B type protein feeds are used, they, when mixed, have: A temperature of at most 60°C, more preferably at most 50°C, even more preferably at most 40°C, most preferably at most 30°C.

[0181] In other preferred embodiments of the invention, the type B protein feed(s) when combined have a temperature of at most 20°C, more preferably at most 15°C, even more preferably at most 10°C, and most preferably at most 5°C.

[0182] Thus, in another preferred embodiment of the invention, in the context of the B-type protein supply: - if only a single B-type protein supply is used, it has: - When two or more B type protein feeds are used, they, when mixed, have: A temperature of at most 20°C, more preferably at most 15°C, even more preferably at most 10°C, most preferably at most 5°C.

[0183] The inventors have found that initial protein solutions prepared from protein feeds with low conductivity typically provide higher BLG crystallization yields.

[0184] In some preferred embodiments of the invention, the type A protein feed(s), when mixed, has a conductivity of at most 10 mS / cm, more preferably at most 7 mS / cm, even more preferably at most 5 mS / cm, and most preferably at most 4 mS / cm.

[0185] Thus, in some preferred embodiments of the present invention: -In relation to A-type protein supply: - if only a single type A protein supply is used, it has: - When two or more type A protein feeds are used, they, when mixed, have: A conductivity of at most 10 mS / cm, more preferably at most 7 mS / cm, even more preferably at most 5 mS / cm, most preferably at most 4 mS / cm.

[0186] In another preferred embodiment of the invention, the type A protein feed(s), when mixed, has a conductivity of at most 3 mS / cm, more preferably at most 2 mS / cm, even more preferably at most 1 mS / cm, and most preferably at most 0.5 mS / cm.

[0187] Thus, in another preferred embodiment of the invention, in the context of an A-type protein supply: - if only a single type A protein supply is used, it has: - When two or more type A protein feeds are used, they, when mixed, have: A conductivity of at most 3 mS / cm, more preferably at most 2 mS / cm, even more preferably at most 1 mS / cm, most preferably at most 0.5 mS / cm.

[0188] In some preferred embodiments of the invention, the type B protein feed(s), when mixed, has a conductivity of at most 10 mS / cm, more preferably at most 7 mS / cm, even more preferably at most 5 mS / cm, and most preferably at most 4 mS / cm.

[0189] Thus, in some preferred embodiments of the invention, in the context of a B-type protein supply: - if only a single B-type protein supply is used, it has: - When two or more B type protein feeds are used, they, when mixed, have: A conductivity of at most 10 mS / cm, more preferably at most 7 mS / cm, even more preferably at most 5 mS / cm, most preferably at most 4 mS / cm.

[0190] In another preferred embodiment of the invention, the type B protein feed(s), when mixed, has a conductivity of at most 3 mS / cm, more preferably at most 2 mS / cm, even more preferably at most 1 mS / cm, and most preferably at most 0.5 mS / cm.

[0191] Thus, in another preferred embodiment of the invention, in the context of the B-type protein supply: - if only a single B-type protein supply is used, it has: - When two or more B type protein feeds are used, they, when mixed, have: A conductivity of at most 3 mS / cm, more preferably at most 2 mS / cm, even more preferably at most 1 mS / cm, most preferably at most 0.5 mS / cm.

[0192] The inventors further observed that BLG crystallization yields are obtained when the ratio between the conductivity (expressed in mS / cm) of the type A protein feed(s) and type B protein feed(s) and the total amount of protein (expressed in % wt total protein relative to the total weight of the feed(s)) is maintained below a certain threshold value.

[0193] In some preferred embodiments of the invention, the type A protein feed(s), when mixed, has a ratio between conductivity and total amount of protein of at most 0.3, more preferably at most 0.25, more preferably at most 0.20, more preferably at most 0.18, even more preferably at most 0.12, and most preferably at most 0.10.

[0194] Thus, in some preferred embodiments of the present invention: -In relation to A-type protein supply: - if only a single type A protein supply is used, it has: - When two or more type A protein feeds are used, they, when mixed, have: A ratio between the conductivity and the total amount of protein of at most 0.3, more preferably at most 0.25, more preferably at most 0.20, more preferably at most 0.18, even more preferably at most 0.12, most preferably at most 0.10.

[0195] In some preferred embodiments of the invention, the type B protein feed(s), when mixed, has a ratio between conductivity and total amount of protein of at most 0.3, more preferably at most 0.25, more preferably at most 0.20, more preferably at most 0.18, even more preferably at most 0.12, and most preferably at most 0.10.

[0196] Thus, in some preferred embodiments of the invention, in the context of a B-type protein supply: - if only a single B-type protein supply is used, it has: - When two or more B type protein feeds are used, they, when mixed, have: A ratio between the conductivity and the total amount of protein of at most 0.3, more preferably at most 0.25, more preferably at most 0.20, more preferably at most 0.18, even more preferably at most 0.12, most preferably at most 0.10.

[0197] In some preferred embodiments of the invention, the type A protein feed(s), when combined, comprise at least 1% w / w BLG by weight of the combined type A protein feed(s), more preferably at least 3% w / w, even more preferably at least 5% w / w, and most preferably at least 7% w / w BLG by weight of the combined type A protein feed(s).

[0198] Thus, in some preferred embodiments of the invention, in the context of a type A protein supply: - if only a single type A protein supply is used, it contains: - When two or more type A protein feeds are used, they, when mixed, include: At least 1% w / w BLG based on the total weight of the type A protein feed, more preferably at least 3% w / w, even more preferably at least 5% w / w, and most preferably at least 7% w / w BLG based on the total weight of the type A protein feed.

[0199] In other preferred embodiments of the invention, the Type A protein feed(s), when combined, comprise 1-45% w / w BLG by weight of the combined Type A protein feed(s), more preferably 3-40% w / w, even more preferably 5-36% w / w, and most preferably 7-34% w / w BLG by weight of the combined Type A protein feed(s).

[0200] Thus, in another preferred embodiment of the invention, in the context of an A-type protein supply: - if only a single type A protein supply is used, it contains: - When two or more type A protein feeds are used, they, when mixed, include: 1-45% w / w BLG based on the total weight of the Type A protein feed, more preferably 3-40% w / w, even more preferably 5-36% w / w, most preferably 7-34% w / w BLG based on the total weight of the Type A protein feed.

[0201] In a further preferred embodiment of the invention, the Type A protein feed(s) when combined comprise 6-32% w / w BLG by weight of the combined Type A protein feed(s), more preferably 8-30% w / w, even more preferably 10-28% w / w, and most preferably 12-26% w / w BLG by weight of the combined Type A protein feed(s).

[0202] Thus, in a further preferred embodiment of the invention, in the context of an A-type protein supply: - if only a single type A protein supply is used, it contains: - When two or more type A protein feeds are used, they, when mixed, include: 6-32% w / w BLG based on the total weight of the Type A protein feed, more preferably 8-30% w / w, even more preferably 10-28% w / w, and most preferably 12-26% w / w BLG based on the total weight of the Type A protein feed.

[0203] In some preferred embodiments of the invention, the type B protein feed(s) when combined comprise at least 1% w / w BLG by weight of the combined type B protein feed(s), more preferably at least 3% w / w, even more preferably at least 5% w / w, and most preferably at least 7% w / w BLG by weight of the combined type B protein feed(s).

[0204] Thus, in some preferred embodiments of the invention, in the context of a B-type protein supply: - if only a single B-type protein supply is used, it contains: - When two or more B type protein feeds are used, they, when mixed, include: At least 1% w / w BLG based on the total weight of the type B protein feed, more preferably at least 3% w / w, even more preferably at least 5% w / w, and most preferably at least 7% w / w BLG based on the total weight of the type B protein feed.

[0205] In other preferred embodiments of the invention, the type B protein feed(s) when combined comprise 1-45% w / w BLG by weight of the combined type B protein feed(s), more preferably 3-40% w / w, even more preferably 5-36% w / w, and most preferably 7-34% w / w BLG by weight of the combined type B protein feed(s).

[0206] Thus, in another preferred embodiment of the invention, in the context of the B-type protein supply: - if only a single B-type protein supply is used, it contains: - When two or more B type protein feeds are used, they, when mixed, include: 1-45% w / w BLG based on the total weight of the B protein feed, more preferably 3-40% w / w, even more preferably 5-36% w / w, most preferably 7-34% w / w BLG based on the total weight of the B protein feed.

[0207] In a further preferred embodiment of the invention, the type B protein feed(s) when combined comprise 6-32% w / w BLG by weight of the combined type B protein feed(s), more preferably 8-30% w / w, even more preferably 10-28% w / w, and most preferably 12-26% w / w BLG by weight of the combined type B protein feed(s).

[0208] Thus, in a further preferred embodiment of the invention, in the context of the B-type protein supply: - if only a single B-type protein supply is used, it contains: - When two or more B type protein feeds are used, they, when mixed, include: 6-32% w / w BLG based on the total weight of the B protein feed, more preferably 8-30% w / w, even more preferably 10-28% w / w, and most preferably 12-26% w / w BLG based on the total weight of the B protein feed.

[0209] In some preferred embodiments of the invention, the type A protein supply(s), when mixed, comprises BLG in an amount of at least 30% w / w, more preferably at least 40% w / w, even more preferably at least 50% w / w, and most preferably at least 55% w / w, relative to the total amount of protein.

[0210] Thus, in some preferred embodiments of the invention, in the context of a type A protein supply: - if only a single type A protein supply is used, it contains: - When two or more type A protein feeds are used, they, when mixed, include: BLG in an amount of at least 30% w / w, more preferably at least 40% w / w, even more preferably at least 50% w / w, most preferably at least 55% w / w relative to the total amount of protein.

[0211] In some preferred embodiments of the invention, the type B protein supply(s), when mixed, comprises BLG in an amount of at least 30% w / w, more preferably at least 40% w / w, even more preferably at least 50% w / w, and most preferably at least 55% w / w, based on the total amount of protein.

[0212] Thus, in some preferred embodiments of the invention, in the context of a B-type protein supply: - if only a single B-type protein supply is used, it contains: - When two or more B type protein feeds are used, they, when mixed, include: BLG in an amount of at least 30% w / w, more preferably at least 40% w / w, even more preferably at least 50% w / w, most preferably at least 55% w / w relative to the total amount of protein.

[0213] In some preferred embodiments of the invention, the Type A protein feed(s), when combined, comprise non-BLG solids in an amount of at least 1% w / w, more preferably at least 2% w / w, even more preferably at least 5% w / w, and most preferably at least 10% w / w of total solids.

[0214] Thus, in some preferred embodiments of the invention, in the context of a type A protein supply: - if only a single type A protein supply is used, it contains: - When two or more type A protein feeds are used, they, when mixed, include: Non-BLG solids in an amount of at least 1% w / w, more preferably at least 2% w / w, even more preferably at least 5% w / w, and most preferably at least 10% w / w of total solids.

[0215] In other preferred embodiments of the invention, the Type A protein feed(s), when mixed, comprises non-BLG solids in an amount of at least 15% w / w, more preferably at least 20% w / w, even more preferably at least 30% w / w, and most preferably at least 40% w / w, of the total solids.

[0216] Thus, in another preferred embodiment of the invention, in the context of an A-type protein supply: - if only a single type A protein supply is used, it contains: - When two or more type A protein feeds are used, they, when mixed, include: Non-BLG solids in an amount of at least 15% w / w, more preferably at least 20% w / w, even more preferably at least 30% w / w, and most preferably at least 40% w / w of total solids.

[0217] In some preferred embodiments of the invention, the Type A protein feed(s), when combined, contain non-BLG solids in an amount of 0-70% w / w, more preferably 1-60% w / w, even more preferably 2-50% w / w, and most preferably 3-40% w / w of total solids.

[0218] Thus, in some preferred embodiments of the invention, in the context of a type A protein supply: - if only a single type A protein supply is used, it contains: - When two or more type A protein feeds are used, they, when mixed, include: Non-BLG solids in an amount of 0-70% w / w, more preferably 1-60% w / w, even more preferably 2-50% w / w, most preferably 3-40% w / w of the total solids.

[0219] In some preferred embodiments of the invention, the Type A protein feed(s), when mixed, comprises non-BLG solids in an amount of 0-70% w / w, more preferably 0-60% w / w, even more preferably 0-50% w / w, and most preferably 0-40% w / w of total solids.

[0220] Thus, in some preferred embodiments of the invention, in the context of a type A protein supply: - if only a single type A protein supply is used, it contains: - When two or more type A protein feeds are used, they, when mixed, include: Non-BLG solids in an amount of 0-70% w / w, more preferably 1-60% w / w, even more preferably 2-50% w / w, most preferably 3-40% w / w of the total solids.

[0221] In other preferred embodiments of the invention, the Type A protein feed(s), when combined, contain non-BLG solids in an amount of 0-20% w / w, more preferably 0-15% w / w, even more preferably 0-10% w / w, and most preferably 0-5% w / w of total solids.

[0222] Thus, in another preferred embodiment of the invention, in the context of an A-type protein supply: - if only a single type A protein supply is used, it contains: - When two or more type A protein feeds are used, they, when mixed, include: Non-BLG solids in an amount of 0-20% w / w, more preferably 0-15% w / w, even more preferably 0-10% w / w, most preferably 0-5% w / w of total solids.

[0223] In a further preferred embodiment of the invention, the Type A protein feed(s), when combined, comprises non-BLG solids in an amount of 15-70% w / w, more preferably 20-65% w / w, even more preferably 25-60% w / w, and most preferably 30-50% w / w of total solids.

[0224] Thus, in a further preferred embodiment of the invention, in the context of an A-type protein supply: - if only a single type A protein supply is used, it contains: - When two or more type A protein feeds are used, they, when mixed, include: Non-BLG solids in an amount of 15-70% w / w, more preferably 20-65% w / w, even more preferably 25-60% w / w, most preferably 30-50% w / w of the total solids.

[0225] In some preferred embodiments of the invention, the B protein feed(s), when combined, comprises non-BLG solids in an amount of at least 1% w / w, more preferably at least 2% w / w, even more preferably at least 5% w / w, and most preferably at least 10% w / w of total solids.

[0226] Thus, in some preferred embodiments of the invention, in the context of a B-type protein supply: - if only a single B-type protein supply is used, it contains: - When two or more B type protein feeds are used, they, when mixed, include: Non-BLG solids in an amount of at least 1% w / w, more preferably at least 2% w / w, even more preferably at least 5% w / w, and most preferably at least 10% w / w of total solids.

[0227] In other preferred embodiments of the invention, the type B protein feed(s), when combined, comprises non-BLG solids in an amount of at least 15% w / w, more preferably at least 20% w / w, even more preferably at least 30% w / w, and most preferably at least 40% w / w of total solids.

[0228] Thus, in another preferred embodiment of the invention, in the context of the B-type protein supply: - if only a single B-type protein supply is used, it contains: - When two or more B type protein feeds are used, they, when mixed, include: Non-BLG solids in an amount of at least 15% w / w, more preferably at least 20% w / w, even more preferably at least 30% w / w, and most preferably at least 40% w / w of total solids.

[0229] In some preferred embodiments of the invention, the B protein feed(s), when combined, contain non-BLG solids in an amount of 0-70% w / w, more preferably 1-60% w / w, even more preferably 2-50% w / w, and most preferably 3-40% w / w of total solids.

[0230] Thus, in some preferred embodiments of the invention, in the context of a B-type protein supply: - if only a single B-type protein supply is used, it contains: - When two or more B type protein feeds are used, they, when mixed, include: Non-BLG solids in an amount of 0-70% w / w, more preferably 1-60% w / w, even more preferably 2-50% w / w, most preferably 3-40% w / w of the total solids.

[0231] In other preferred embodiments of the invention, the B protein feed(s), when combined, contain non-BLG solids in an amount of 0-20% w / w, more preferably 0-15% w / w, even more preferably 0-10% w / w, and most preferably 0-5% w / w of total solids.

[0232] Thus, in another preferred embodiment of the invention, in the context of the B-type protein supply: - if only a single B-type protein supply is used, it contains: - When two or more B type protein feeds are used, they, when mixed, include: Non-BLG solids in an amount of 0-20% w / w, more preferably 0-15% w / w, even more preferably 0-10% w / w, most preferably 0-5% w / w of total solids.

[0233] In a further preferred embodiment of the invention, the B protein feed(s), when combined, comprises non-BLG solids in an amount of 15-70% w / w, more preferably 20-65% w / w, even more preferably 25-60% w / w, and most preferably 30-50% w / w of total solids.

[0234] Thus, in a further preferred embodiment of the invention, in the context of the B-type protein supply: - if only a single B-type protein supply is used, it contains: - When two or more B type protein feeds are used, they, when mixed, include: Non-BLG solids in an amount of 15-70% w / w, more preferably 20-65% w / w, even more preferably 25-60% w / w, most preferably 30-50% w / w of the total solids.

[0235] In some embodiments of the invention, the protein feed has a low content of denatured protein. Preferably, the protein feeds, when combined, have a degree of protein denaturation of at most 20%, more preferably at most 10%, even more preferably at most 5%, and most preferably at most 2%.

[0236] In some preferred embodiments of the invention, the initial protein solution comprises a total amount of type A protein feed in the range of 1-99% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w, and most preferably 30-60% w / w by weight of the initial protein solution.

[0237] In another preferred embodiment of the invention, the initial protein solution comprises a total amount of type A protein feed in the range of 5-95% w / w, more preferably 15-80% w / w, even more preferably 20-75% w / w, and most preferably 35-70% w / w by weight of the initial protein solution.

[0238] In some preferred embodiments of the invention, the initial protein solution comprises a total amount of type B protein feed(s) in the range of 1-99% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w, and most preferably 30-60% w / w by weight of the initial protein solution.

[0239] In another preferred embodiment of the invention, the initial protein solution comprises a total amount of type B protein feed in the range of 5-95% w / w, more preferably 15-80% w / w, even more preferably 20-75% w / w, and most preferably 35-70% w / w by weight of the initial protein solution.

[0240] In some preferred embodiments of the invention, the initial protein solution comprises: - a total amount of type A protein feed(s) in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w, most preferably 30-60% w / w based on the weight of the initial protein solution, and - a total amount of type B protein feed(s) in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w, most preferably 30-60% w / w based on the weight of the initial protein solution.

[0241] In another preferred embodiment of the invention, the initial protein solution comprises: - a total amount of type A protein feed(s) in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w, most preferably 30-60% w / w based on the weight of the initial protein solution, with a pH of 5.8-9, and - a total amount of type B protein feed(s) having a pH of 2.5-5.2 in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w, most preferably 30-60% w / w based on the weight of the initial protein solution.

[0242] In a further preferred embodiment of the invention, the initial protein solution comprises: - a total amount of type A protein feed(s) in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w, most preferably 30-60% w / w based on the weight of the initial protein solution, with a pH of 6.0-8, and - a total amount of type B protein feed(s) having a pH of 3-5.0 in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w, most preferably 30-60% w / w based on the weight of the initial protein solution.

[0243] In an even more preferred embodiment of the invention, the initial protein solution comprises: - a total amount of type A protein feed(s) in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w, most preferably 30-60% w / w based on the weight of the initial protein solution, with a pH of 6.1-8, and - a total amount of type B protein feed(s) having a pH of 3-4.9 of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w, most preferably 30-60% w / w based on the weight of the initial protein solution.

[0244] In some preferred embodiments of the present invention, all of the protein feed is in liquid form.

[0245] In another preferred embodiment of the invention, at least one of the protein supplies is in a dry form.

[0246] In a further preferred embodiment of the invention, at least one of the protein supplies is in dry form, preferably in powder form, and at least one of the protein supplies is in liquid form.

[0247] The protein feed is preferably prepared from WPC, WPI, SPC, SPI, or combinations thereof.

[0248] The embodiments relating to the chemical composition of the initial protein solution apply equally to type A protein feed(s) and type B protein feed(s), however typically at least one parameter of the protein feed is set to avoid supersaturation, or at least spontaneous crystallization.

[0249] It is often preferred that the BLG supersaturation of the initial protein solution is higher than the following supersaturation: - A type protein feed, if only a single type A protein feed is used, or - Type A protein feeds, when combined, if two or more Type A protein feeds are used.

[0250] In addition, it is often preferred that the supersaturation of the BLG in the initial protein solution is higher than the supersaturation of: - Type B protein feed, if only a single Type B protein feed is used, or - Type B protein feeds, when combined, if more than one Type B protein feed is used.

[0251] It is particularly preferred that the degree of supersaturation of BLG in the initial protein solution is higher than both of the following supersaturations: - A type protein feed, if only a single type A protein feed is used, or - if two or more type A protein feeds are used, the type A protein feeds when combined, and: - Type B protein feed, if only a single Type B protein feed is used, or - Type B protein feeds, when combined, if more than one Type B protein feed is used.

[0252] The degree of supersaturation of a liquid is determined by seeding a sample of the liquid with BLG crystals and quantifying the amount of additional BLG crystal mass produced in the sample when the sample is maintained at the same conditions (e.g., temperature, pressure and humidity) for 24 hours. Quantification of the BLG crystal mass is performed by centrifugation at 15000g for 5 minutes at the temperature of the liquid, followed by quantifying the content of BLG in the resulting pellet using analysis 9.9 of WO2018 / 115520A1. The degree of supersaturation is the percentage of BLG of the liquid sample that can be isolated in the pellet.

[0253] In some preferred embodiments of the invention, in the context of a type A protein supply, if only a single type A protein supply is used, it has the following, or if two or more type A protein supplies are used, they, when mixed, have the following: - a content of BLG of 4-32% w / w, more preferably 6-32% w / w, more preferably 8-30% w / w, even more preferably 10-28% w / w, most preferably 12-26% w / w, based on the total weight of the A-type protein feed, - a pH in the range of 5.6-10, more preferably 5.8-8, even more preferably 6.0-7, most preferably 6.1-6.5; - preferably at a temperature of at most 20°C, more preferably at most 15°C, even more preferably at most 10°C, most preferably at most 5°C, a content of BLG in an amount of preferably at least 30% w / w, more preferably at least 40% w / w, even more preferably at least 50% w / w, most preferably at least 55% w / w, relative to the total amount of protein; -One or more of the following: a conductivity of at most 7 mS / cm, even more preferably at most 5 mS / cm, and most preferably at most 3 mS / cm, and - a ratio between the conductivity and the total amount of protein of at most 0.3, more preferably at most 0.25, most preferably at most 0.20.

[0254] In some preferred embodiments of the invention, in the context of a type B protein feed, if only a single type B protein feed is used, it has the following, or if two or more type B protein feeds are used, they, when mixed, have the following: - a content of BLG of 4-32% w / w relative to the total weight of the B protein feed, more preferably 6-32% w / w, more preferably 8-30% w / w, even more preferably 10-28% w / w, most preferably 12-26% w / w relative to the total weight of the B protein feed, - a pH in the range of 2-5.4, more preferably 3-5.2, even more preferably 4-5.0, most preferably 4.5-4.9; - preferably at a temperature of at most 20°C, more preferably at most 15°C, even more preferably at most 10°C, most preferably at most 5°C, a content of BLG in an amount of preferably at least 30% w / w, more preferably at least 40% w / w, even more preferably at least 50% w / w, most preferably at least 55% w / w, relative to the total amount of protein; -One or more of the following: a conductivity of at most 7 mS / cm, even more preferably at most 5 mS / cm, and most preferably at most 3 mS / cm, and - a ratio between the conductivity and the total amount of protein of at most 0.3, more preferably at most 0.25, most preferably at most 0.20.

[0255] In a particularly preferred embodiment of the invention, - in relation to the A-type protein supply, if only a single A-type protein supply is used, it has: If two or more type A protein feeds are used, they, when mixed, have: - a content of BLG of 4-32% w / w, more preferably 6-32% w / w, more preferably 8-30% w / w, even more preferably 10-28% w / w, most preferably 12-26% w / w, based on the total weight of the A-type protein feed, - a pH in the range of 5.6-10, more preferably 5.8-8, even more preferably 6.0-7, most preferably 6.1-6.5; - preferably at a temperature of at most 20°C, more preferably at most 15°C, even more preferably at most 10°C, most preferably at most 5°C, a content of BLG in an amount of preferably at least 30% w / w, more preferably at least 40% w / w, even more preferably at least 50% w / w, most preferably at least 55% w / w, relative to the total amount of protein; -One or more of the following: a conductivity of at most 7 mS / cm, even more preferably at most 5 mS / cm, and most preferably at most 3 mS / cm, and - a ratio between the conductivity and the total amount of protein of at most 0.3, more preferably at most 0.25, most preferably at most 0.20. And - in relation to the B type protein feed, if only a single B type protein feed is used, it has the following, or if two or more B type protein feeds are used, they, when mixed, have the following: - a content of BLG of 4-32% w / w relative to the total weight of the B protein feed, more preferably 6-32% w / w, more preferably 8-30% w / w, even more preferably 10-28% w / w, most preferably 12-26% w / w relative to the total weight of the B protein feed, - a pH in the range of 2-5.4, more preferably 3-5.2, even more preferably 4-5.0, most preferably 4.5-4.9; - preferably at a temperature of at most 20°C, more preferably at most 15°C, even more preferably at most 10°C, most preferably at most 5°C, a content of BLG in an amount of preferably at least 30% w / w, more preferably at least 40% w / w, even more preferably at least 50% w / w, most preferably at least 55% w / w, relative to the total amount of protein; -One or more of the following: - a conductivity of at most 7 mS / cm, more preferably at most 5 mS / cm, most preferably at most 3 mS / cm, and - a ratio between the conductivity and the total amount of protein of at most 0.3, more preferably at most 0.25, most preferably at most 0.20.

[0256] In some preferred embodiments of the invention, in the context of a type B protein feed, if only a single type B protein feed is used, it has the following, or if two or more type B protein feeds are used, they, when mixed, have the following: - a content of BLG of 4-32% w / w relative to the total weight of the B protein feed, more preferably 6-32% w / w, more preferably 8-30% w / w, even more preferably 10-28% w / w, most preferably 12-26% w / w relative to the total weight of the B protein feed, - a pH in the range of 2-5.4, more preferably 3-5.2, even more preferably 4-5.0, most preferably 4.5-4.9; - preferably at a temperature of at most 20°C, more preferably at most 15°C, even more preferably at most 10°C, most preferably at most 5°C, - preferably a content of non-BLG solids in an amount of 0-70% w / w, more preferably 0-60% w / w, even more preferably 0-50% w / w, most preferably 0-40% w / w, based on the total solids; -One or more of the following: a conductivity of at most 7 mS / cm, even more preferably at most 5 mS / cm, and most preferably at most 3 mS / cm, and - a ratio between the conductivity and the total amount of protein of at most 0.3, more preferably at most 0.25, most preferably at most 0.20.

[0257] In a particularly preferred embodiment of the invention, - in relation to the A-type protein supply, if only a single A-type protein supply is used, it has: If two or more type A protein feeds are used, they, when mixed, have: - a content of BLG of 4-32% w / w, more preferably 6-32% w / w, more preferably 8-30% w / w, even more preferably 10-28% w / w, most preferably 12-26% w / w, based on the total weight of the A-type protein feed, - a pH in the range of 5.6-10, more preferably 5.8-8, even more preferably 6.0-7, most preferably 6.1-6.5; - preferably at a temperature of at most 20°C, more preferably at most 15°C, even more preferably at most 10°C, most preferably at most 5°C, - preferably a content of non-BLG solids in an amount of 0-70% w / w, more preferably 0-60% w / w, even more preferably 0-50% w / w, most preferably 0-40% w / w, based on the total solids; -One or more of the following: a conductivity of at most 7 mS / cm, even more preferably at most 5 mS / cm, and most preferably at most 3 mS / cm, and - a ratio between the conductivity and the total amount of protein of at most 0.3, more preferably at most 0.25, most preferably at most 0.20. And - in relation to the B type protein feed, if only a single B type protein feed is used, it has the following, or if two or more B type protein feeds are used, they, when mixed, have the following: - a content of BLG of 4-32% w / w relative to the total weight of the B protein feed, more preferably 6-32% w / w, more preferably 8-30% w / w, even more preferably 10-28% w / w, most preferably 12-26% w / w relative to the total weight of the B protein feed, - a pH in the range of 2-5.4, more preferably 3-5.2, even more preferably 4-5.0, most preferably 4.5-4.9; - preferably at a temperature of at most 20°C, more preferably at most 15°C, even more preferably at most 10°C, most preferably at most 5°C, - preferably a content of non-BLG solids in an amount of 0-70% w / w, more preferably 0-60% w / w, even more preferably 0-50% w / w, most preferably 0-40% w / w, based on the total solids; -One or more of the following: - a conductivity of at most 7 mS / cm, more preferably at most 5 mS / cm, most preferably at most 3 mS / cm, and - a ratio between the conductivity and the total amount of protein of at most 0.3, more preferably at most 0.25, most preferably at most 0.20.

[0258] In some preferred embodiments of the invention, the protein feed is prepared by one or more of the following process steps: - adjusting the pH, - Reducing the conductivity - Reducing temperature - Increasing the protein concentration - adding an agent that reduces water activity - modifying the ion composition.

[0259] The inventors have found that pressure-driven membrane filtration is well suited for preparing liquid protein feeds.

[0260] In some preferred embodiments of the present invention, at least one of the protein feeds is prepared by a process that includes pressure-driven membrane filtration of protein source, preferably whey protein source.Preferred pressure-driven membrane filtration includes ultrafiltration, microfiltration, nanofiltration and filtration by reverse osmosis.It has been found that ultrafiltration is particularly preferred for preparing protein feeds, and can be used as the only type of pressure-driven membrane filtration during preparation of protein feeds, or can be used in combination with other filtration methods.

[0261] Preparation of protein feeds often includes pH adjustment to provide a protein feed with a desired pH. Preferably, the pH is adjusted to a pH in the range of 6.0-7.0 when preparing a type A protein feed, and to a pH in the range of 4.0-5.0 when preparing a type B protein feed. The pH is preferably adjusted using a food acceptable acid and / or base. Food acceptable acids, such as carboxylic acids, are particularly preferred. Useful examples of such acids are, for example, hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, maleic acid, tartaric acid, lactic acid, citric acid, or gluconic acid, and / or mixtures thereof.

[0262] In some preferred embodiments of the invention, the pH is adjusted using a lactone, such as, for example, D-glucono-δ-lactone, which slowly hydrolyzes and at the same time reduces the pH of the aqueous liquid containing it, and the target pH after hydrolysis of the lactone is complete can be precisely calculated.

[0263] Useful examples of food acceptable bases are, for example, hydroxide sources such as, for example, sodium hydroxide, potassium hydroxide, calcium hydroxide, salts of food acids such as, for example, trisodium citrate, and / or combinations thereof.

[0264] In another preferred embodiment of the invention, the pH is + The H is adjusted by the addition of a cation exchange material in the form of a bead / large particle cation exchange material that is easily removed from the initial protein solution before or even after crystallization. + Adjusting the pH by the addition of a cation exchange material in the form of a cation exchange material is particularly advantageous in the present invention because it reduces the pH without adding negative counter ions that would significantly affect the conductivity of the protein feed.

[0265] In some preferred embodiments of the invention, preparing the initial protein solution involves reducing the conductivity of the protein supply.

[0266] Conductivity values ​​referred to herein are normalized to 25° C. unless otherwise specified.

[0267] The inventors have found that reducing the conductivity of the initial protein solution leads to a higher yield of BLG crystals. The minimum obtainable conductivity of the initial protein solution depends on the composition of the protein and lipid fractions (if any). Some protein species, such as caseinomacropeptide (CMP), contribute more to the conductivity than others. Thus, it is preferred that the conductivity of the protein feed is brought close to a level where the protein and the protein's counterions are the major contributors to the conductivity. Reducing the conductivity often involves removing at least some of the small, free ions that are present in the liquid phase and are not tightly bound to the protein.

[0268] It is often preferred that the protein feed has a conductivity of at most 10 mS / cm. In some preferred embodiments of the invention, the protein feed has a conductivity of at most 5 mS / cm. Preferably, the protein feed has a conductivity of at most 4 mS / cm.

[0269] Lower conductivities are even more preferred and result in higher yields of BLG crystals. Thus, the protein feed preferably has a conductivity of at most 3 mS / cm. In some preferred embodiments of the invention, the protein feed has a conductivity of at most 1 mS / cm. Preferably, the protein feed has a conductivity of at most 0.5 mS / cm.

[0270] The low conductivity of the protein feed is preferably obtained by dialysis or diafiltration. Diafiltration by ultrafiltration is particularly preferred because it allows salts and small charged molecules to be washed away while retaining the protein. In some preferred embodiments of the invention, the same UF unit is used for UF / diafiltration and subsequent concentration of the protein feed.

[0271] The inventors have observed indications that the ratio between the electrical conductivity (expressed in mS / cm) and the total amount of protein in the initial protein solution (expressed in % wt total protein relative to the total weight of the initial protein solution) can be conveniently kept below a certain threshold value, promoting the crystallization of BLG.

[0272] In some preferred embodiments of the invention, the ratio between the conductivity and the total amount of protein in the protein feed is at most 0.3. Preferably, the ratio between the conductivity and the total amount of protein in the protein feed is at most 0.25. Preferably, the ratio between the conductivity and the total amount of protein in the protein feed is at most 0.20. More preferably, the ratio between the conductivity and the total amount of protein in the protein feed is at most 0.18. Even more preferably, the ratio between the conductivity and the total amount of protein in the protein feed is at most 0.12. Most preferably, the ratio between the conductivity and the total amount of protein in the protein feed is at most 0.10.

[0273] For example, it is preferred that the ratio between the conductivity and the total amount of protein of the protein feed is around 0.07, or even lower.

[0274] The inventors have further found that the protein feed can be conveniently adjusted to obtain a UF permeate conductivity of at most 10 mS / cm. The UF permeate conductivity is a measure of the conductivity of the small molecule fraction of a liquid and is measured according to Example 9.10 of WO2018 / 115520. When the term "conductivity" is used as such in this specification, it refers to the conductivity of the liquid in question. When the term "UF permeate conductivity" is used, it refers to the conductivity of the small molecule fraction of a liquid and is measured according to Example 9.10 of WO2018 / 115520.

[0275] Preferably, the UF permeate conductivity of the protein feed is at most 7 mS / cm. More preferably, the UF permeate conductivity of the protein feed may be at most 5 mS / cm. Even more preferably, the UF permeate conductivity of the protein feed may be at most 3 mS / cm.

[0276] Even lower UF permeate conductivities may be used, which is particularly preferred when high yields of BLG must be obtained. Thus, preferably, the UF permeate conductivity of the protein feed is at most 1.0 mS / cm. More preferably, the UF permeate conductivity of the protein feed may be at most 0.4 mS / cm. Even more preferably, the UF permeate conductivity of the protein feed may be at most 0.1 mS / cm. Most preferably, the UF permeate conductivity of the protein feed may be at most 0.04 mS / cm.

[0277] For example, when Milli-Q water is used as a diluent during diafiltration (Milli-Q water has a conductivity of approximately 0.06 μS / cm), even lower UF permeate conductivities can be reached. Thus, the UF permeate conductivity of the protein feed can be at most 0.01 mS / cm. Alternatively, the UF permeate conductivity of the protein feed can be at most 0.001 mS / cm. Alternatively, the UF permeate conductivity of the protein feed can be at most 0.0001 mS / cm.

[0278] In some preferred embodiments of the invention, preparation of the protein feed comprises reducing the temperature.

[0279] The temperature of the protein feed may be at most 30° C., preferably at most 20° C., even more preferably at most 10° C. The inventors have found that lower temperatures provide a higher degree of supersaturation than when the protein feed is mixed, and thus the temperature of the protein feed may be reduced, for example, to at most 5° C., preferably at most 2° C., even more preferably at most 0° C. The temperature may even be lower than 0° C., however, preferably the protein feed should remain pumpable, for example in the form of an ice slurry.

[0280] In some preferred embodiments of the invention, the protein feed is an ice slurry which is then combined to prepare the initial protein solution.

[0281] Preparation of the protein feed preferably includes one or more protein concentration steps, such as ultrafiltration, nanofiltration, reverse osmosis, and / or evaporation.

[0282] Ultrafiltration is particularly preferred because it allows for selective concentration of proteins. As mentioned above, ultrafiltration is preferably used for both diafiltration and concentration during preparation of the protein feed.

[0283] In some preferred embodiments of the invention, the concentration of BLG in the protein feed is below the level at which spontaneous crystallization of BLG occurs.

[0284] In some preferred embodiments of the invention, preparation of the protein feed includes the addition of one or more water activity-reducing agent(s).

[0285] Useful, but non-limiting, examples of such water activity reducing agents are polysaccharides and / or polyethylene glycol (PEG).

[0286] In some preferred embodiments of the invention, preparation of the protein feed involves ion exchange, by dialysis or diafiltration, by addition of new ionic species.

[0287] In yet another preferred embodiment of the invention, preparation of the protein feed comprises reducing the conductivity, for example by diafiltration using a membrane that retains at least BLG. Diafiltration by ultrafiltration is particularly preferred.

[0288] Any suitable protein source may be used to prepare the protein feed. In some preferred embodiments of the invention, the protein source is produced by fermentation of one or more of mammalian cells, recombinant plant cells, recombinant mammalian cells, and / or recombinant microorganisms capable of producing BLG.

[0289] In other preferred embodiments of the invention, the protein source is derived from whey or milk serum from mammalian milk. In particularly preferred embodiments of the invention, the protein source of the protein supply comprises, or even consists of, whey protein concentrate, whey protein concentrate, whey protein isolate, whey protein isolate, or combinations thereof.

[0290] Typically, the protein feed is prepared by combining, preferably mixing, two or more of the above processes.

[0291] In some preferred embodiments of the invention, preparing the protein supply comprises subjecting a protein source, which is preferably a whey protein source, to: - pH adjustment to the desired pH, preferably in the range of 6.0-8 when preparing a type A protein feed and 3-5.0 when preparing a type B protein feed; and -Concentration by ultrafiltration, nanofiltration and / or reverse osmosis.

[0292] In another preferred embodiment of the invention, the preparation of the protein supply comprises subjecting a protein source, preferably a whey protein source, to: - pH adjustment to the desired pH, preferably in the range of 6.0-8 when preparing a type A protein feed and 3-5.0 when preparing a type B protein feed; - concentration by ultrafiltration, nanofiltration and / or reverse osmosis, - Ultrafiltration / diafiltration to provide a protein feed with low conductivity, and - Temperature regulation to maintain the temperature of the liquid stream during processing at a maximum of 20°C, more preferably at a maximum of 15°C, even more preferably at a maximum of 10°C, and most preferably at a maximum of 5°C.

[0293] The inventors have further found that the BLG yield of the present process can be improved by controlling the molar ratio between the sum of sodium plus potassium to the sum of calcium and magnesium. Higher relative amounts of calcium and magnesium surprisingly appear to increase the yield of BLG and thus increase the efficiency of BLG recovery of the present process.

[0294] In some preferred embodiments of the invention, the type A protein feed(s), when combined, have a molar ratio between Na+K and Ca+Mg of at most 4. More preferably, the type A protein feed(s), when combined, have a molar ratio between Na+K and Ca+Mg of at most 2. Even more preferably, the type A protein feed(s), when combined, have a molar ratio between Na+K and Ca+Mg of at most 1.5, even more preferably at most 1.0. Most preferably, the type A protein feed(s), when combined, have a molar ratio between Na+K and Ca+Mg of at most 0.5, such as for example at most 0.2.

[0295] The molar ratio between Na+K and Ca+Mg is (m Na +m K ) / (m Ca +mMg ) where m Na is the content of element Na in moles, m K is the content of element K in moles, and m Ca is the content of element Ca in moles, and m Mg is the content of elemental Mg expressed in moles

[0296] In some preferred embodiments of the invention, the type B protein feed(s), when combined, have a molar ratio between Na+K and Ca+Mg of at most 4. More preferably, the type B protein feed(s), when combined, have a molar ratio between Na+K and Ca+Mg of at most 2. Even more preferably, the type B protein feed(s), when combined, have a molar ratio between Na+K and Ca+Mg of at most 1.5, even more preferably at most 1.0. Most preferably, the type A protein feed(s), when combined, have a molar ratio between Na+K and Ca+Mg of at most 0.5, such as for example at most 0.2.

[0297] In some embodiments of the invention, the protein feed has a low content of denatured protein. Preferably, the protein feed has a degree of protein denaturation of at most 20%, preferably at most 10%, more preferably at most 5%, and most preferably at most 2%.

[0298] In some preferred embodiments of the invention, the initial protein solution preparation has a pH in the range of 5-6.

[0299] All pH values ​​are measured using a pH glass electrode and normalized to 25°C.

[0300] The initial protein solution may have a pH in the range of, for example, 4.9-6.1. The pH of the initial protein solution may be in the range of, for example, 5.0-6.1. Alternatively, the pH of the initial protein solution may be in the range of 5.1-6.1. Preferably, the pH of the initial protein solution is in the range of 5.1-6.0.

[0301] In some preferred embodiments of the invention, the pH of the initial protein solution is in the range of 5.0-6.0. Preferably, the pH of the initial protein solution is in the range of 5.1-6.0. More preferably, the pH of the initial protein solution is in the range of 5.1-5.9. Even more preferably, the pH of the initial protein solution may be in the range of 5.2-5.8. Most preferably, the pH of the initial protein solution is in the range of 5.4-5.6.

[0302] The inventors have observed that maximum BLG crystallization yields are obtained at approximately pH 5.5, and that the pH of the initial protein solution can be conveniently selected outside pH 5.5, but can be brought closer to the optimum pH during crystallization, for example, by the addition of food acid or additional type B or type A protein feed. Thus, in some preferred embodiments of the invention, the pH of the initial protein solution is in the range of 5.0-5.3 or 5.7-6.0.

[0303] The inventors have found that it is advantageous to avoid spontaneous crystallization during crystallization, and that BLG crystals formed by controlled crystallization in the metastable zone have a more uniform size distribution and it is easier to more efficiently separate the BLG crystals resulting from spontaneous BLG crystallization from the mother liquor.

[0304] Thus, in some preferred embodiments of the present invention, the initial protein solution, and preferably also the crystallizing protein solution, is controlled to avoid spontaneous BLG crystallization, for example, the initial protein solution is supersaturated with respect to BLG, but preferably in the metastable zone, meaning that spontaneous BLG crystallization cannot occur.

[0305] The inventors have found that a lower conductivity of the initial protein solution leads to a higher yield of BLG crystals. The minimum obtainable conductivity of the initial protein solution depends on the composition of the protein and lipid fractions (if any). Some protein species, such as caseinomacropeptide (CMP), contribute more to the conductivity than others. Thus, it is preferred that the conductivity of the protein feed is brought close to a level where the protein and protein counterions are the major contributors to the conductivity. Reducing the conductivity often involves removing at least some of the small, free ions that are present in the liquid phase and are not tightly bound to the protein.

[0306] It is often preferred that the initial protein solution has a conductivity of at most 10 mS / cm. In some preferred embodiments of the invention, the initial protein solution has a conductivity of at most 5 mS / cm. Preferably, the initial protein solution has a conductivity of at most 4 mS / cm.

[0307] Lower conductivities are even more preferred, resulting in higher yields of BLG crystals. Thus, the initial protein solution preferably has a conductivity of at most 3 mS / cm. In some preferred embodiments of the invention, the initial protein solution has a conductivity of at most 1 mS / cm. Preferably, the initial protein solution has a conductivity of at most 0.5 mS / cm.

[0308] The inventors have observed indications that the ratio between the electrical conductivity (expressed in mS / cm) and the total amount of protein of the initial protein solution (expressed in % wt total protein relative to the total weight of the initial protein solution) can be conveniently kept below a certain threshold value, promoting the crystallization of BLG.

[0309] In some preferred embodiments of the invention, the ratio between the conductivity of the initial protein solution and the total amount of protein is at most 0.3. Preferably, the ratio between the conductivity of the initial protein solution and the total amount of protein is at most 0.25. Preferably, the ratio between the conductivity of the initial protein solution and the total amount of protein is at most 0.20. More preferably, the ratio between the conductivity of the initial protein solution and the total amount of protein is at most 0.18. Even more preferably, the ratio between the conductivity of the initial protein solution and the total amount of protein is at most 0.12. Most preferably, the ratio between the conductivity of the initial protein solution and the total amount of protein is at most 0.10.

[0310] For example, it is preferred that the ratio between the conductivity of the initial protein solution and the total amount of protein is approximately 0.07 or less.

[0311] The inventors have further found that the initial protein solution may conveniently have a UF permeate conductivity of at most 10 mS / cm. The UF permeate conductivity is a measure of the conductivity of the small molecule fraction of the liquid and is measured according to Example 9.10 of WO2018 / 115520. When the term "conductivity" is used as such in this specification, it refers to the conductivity of the liquid in question. When the term "UF permeate conductivity" is used, it refers to the conductivity of the small molecule fraction of the liquid and is measured according to Example 9.10 of WO2018 / 115520.

[0312] Preferably, the UF permeability conductivity of the initial protein solution is at most 7 mS / cm. More preferably, the UF permeability conductivity of the initial protein solution may be at most 5 mS / cm. Even more preferably, the UF permeability conductivity of the initial protein solution may be at most 3 mS / cm.

[0313] Even lower UF permeate conductivities may be used, which is particularly preferred when high yields of BLG must be obtained. Thus, preferably, the UF permeate conductivity of the initial protein solution is at most 1.0 mS / cm. More preferably, the UF permeate conductivity of the initial protein solution may be at most 0.4 mS / cm. Even more preferably, the UF permeate conductivity of the initial protein solution may be at most 0.1 mS / cm. Most preferably, the UF permeate conductivity of the initial protein solution may be at most 0.04 mS / cm.

[0314] For example, when Milli-Q water is used as a diluent during diafiltration (Milli-Q water has a conductivity of approximately 0.06 μS / cm), an even lower UF permeate conductivity can be reached. Thus, the UF permeate conductivity of the initial protein solution can be at most 0.01 mS / cm. Alternatively, the UF permeate conductivity of the initial protein solution can be at most 0.001 mS / cm. Alternatively, the UF permeate conductivity of the initial protein solution can be at most 0.0001 mS / cm.

[0315] The inventors have found that lower temperatures increase the BLG crystallization yield. In some preferred embodiments of the present invention, the temperature of the initial protein solution is at most 30° C., more preferably at most 20° C., and most preferably at most 10° C. Lower temperatures provide higher supersaturation, and thus the temperature of the initial protein solution is preferably at most 5° C., more preferably at most 2° C., and even more preferably at most 0° C. The temperature may even be lower than 0° C., however, preferably the initial protein solution should remain pumpable, e.g., in the form of an ice slurry.

[0316] In some preferred embodiments of the invention, the initial protein solution is an ice slurry at the beginning of the BLG crystallization. Alternatively, or in addition, the crystallization initial protein solution can be converted to or maintained as an ice slurry during the BLG crystallization in step b).

[0317] The inventors have further found that the BLG yield of the present process can be improved by controlling the molar ratio between the sum of sodium plus potassium to the sum of calcium and magnesium. Higher relative amounts of calcium and magnesium surprisingly appear to increase the yield of BLG and thus increase the efficiency of BLG recovery of the present process.

[0318] In some preferred embodiments of the present invention, the initial protein solution of step a) has a molar ratio between Na+K and Ca+Mg of at most 4. More preferably, the initial protein solution of step a) has a molar ratio between Na+K and Ca+Mg of at most 2. Even more preferably, the initial protein solution of step a) has a molar ratio between Na+K and Ca+Mg of at most 1.5, even more preferably of at most 1.0. Most preferably, the initial protein solution of step a) has a molar ratio between Na+K and Ca+Mg of at most 0.5, such as for example at most 0.2.

[0319] The molar ratio between Na+K and Ca+Mg is (m Na +m K ) / (m Ca +m Mg ) where m Na is the content of element Na in moles, m K is the content of element K in moles, and m Ca is the content of element Ca in moles, and m Mg is the content of elemental Mg expressed in moles

[0320] It is particularly preferred that the initial protein solution is supersaturated with respect to BLG by salting-in, and thus BLG can be crystallized from the initial protein solution in salting-in mode.

[0321] In some embodiments of the invention, the initial protein solution has a low content of denatured protein, especially when the edible BLG product of the invention has a degree of protein denaturation. Preferably, the initial protein solution has a degree of protein denaturation of at most 20%, preferably at most 10%, more preferably at most 5%, most preferably at most 2%.

[0322] Step b) of the method involves crystallizing at least some of the BLG of the supersaturated initial protein solution.

[0323] It is particularly preferred that the crystallization in step b) is carried out in salting-in mode, i.e. in a liquid with low ionic strength and conductivity, as opposed to the salting-out mode, in which a significant amount of salt is added to the solution to induce crystallization.

[0324] The crystallization of BLG in step b) may for example include one or more of the following: - waiting for crystallization to occur, - addition of crystallization seeds, -Increasing the supersaturation of BLG even further, and / or -Mechanical stimulation.

[0325] In some preferred embodiments of the invention, step b) comprises adding crystallization seeds to the initial protein solution. The inventors have found that the addition of crystallization seeds allows control of when and where BLG crystallization occurs to avoid sudden clogging and unintended shutdown of process equipment during production. For example, it is often desirable to avoid the onset of crystallization while concentrating the protein feed.

[0326] In principle, any seed material that initiates the crystallization of BLG can be used, however, it is preferred that hydrated or dried BLG crystals are used for seeding, to avoid adding additional impurities to the initial protein solution.

[0327] The crystallization seeds may be in dry form or may form part of a suspension when added to the initial protein solution. Adding a suspension containing crystallization seeds, e.g., BLG crystals, is currently preferred as it is believed to provide a faster initiation of crystallization. Such a suspension preferably contains crystallization seeds and has a pH in the range of 5-6 and a conductivity of at most 10 mS / cm.

[0328] In some embodiments of the invention, at least some of the crystallization seeds are located on a solid phase that is contacted with the initial protein solution.

[0329] The crystallization seeds preferably have a particle size smaller than the desired size of the BLG crystals.The size of the crystallization seeds can be modified by removing the largest seeds by sieving or other size fractionation processes.For example, particle size reduction by grinding can also be employed before particle size fractionation.

[0330] In some embodiments of the invention, at least 90% w / w of the crystallization seeds have a particle size (measured by sieving analysis) in the range of 0.1-600 microns. For example, at least 90% w / w of the crystallization seeds may have a particle size in the range of 1-400 microns. Preferably, at least 90% w / w of the crystallization seeds may have a particle size in the range of 5-200 microns. More preferably, at least 90% w / w of the crystallization seeds may have a particle size in the range of 5-100 microns.

[0331] The particle size and dosage of the crystallization seeds can be adjusted to provide optimal crystallization of the BLG.

[0332] In some preferred embodiments of the invention, at least one of the one or more type A or type B protein feed(s) already contains BLG crystals, in which case no further seeding is necessary, although it is often preferable to ensure a sufficient amount of crystallization seeds.

[0333] In some preferred embodiments of the invention, step b) involves increasing the supersaturation of the BLG even further, preferably to a supersaturation at which crystallization of the BLG begins immediately, i.e., within at most 20 minutes, preferably within at most 5 minutes. This is also called the nucleation zone, where crystallites form spontaneously and initiate the crystallization process.

[0334] The degree of supersaturation can be increased, for example, by one or more of the following: Further increasing the protein concentration of the initial protein solution Further cooling of the initial protein solution - To bring the initial protein solution closer to the optimal pH for BLG crystallization - Reducing the electrical conductivity even further.

[0335] It is particularly preferred to further increase the protein concentration of the initial protein solution and / or to bring the initial protein solution closer to the optimal pH for BLG crystallization (approximately pH 5.5) by addition of additional type A or B protein feed.

[0336] In some preferred embodiments of the invention, step b) involves waiting for BLG crystals to form, which can take several hours and is typically due to the initial protein solution being only slightly supersaturated with respect to BLG and to no crystallization seeds being added.

[0337] In some preferred embodiments of the invention, providing the initial protein solution (step a) and crystallizing BLG (step b) are performed as two separate steps. However, if the initial protein solution already contains at least some BLG crystals, step b) starts as soon as the initial protein solution is prepared.

[0338] In some preferred embodiments of the invention, step b) preferably comprises further adjustment of the crystallizing protein solution to increase or at least maintain the degree of supersaturation of BLG, which further adjustment preferably results in an increased yield of BLG crystals or provides better quality BLG crystals, e.g. in the form of larger and more uniform crystal size.

[0339] Such additional adjustments may include one or more of the following: -Increasing even further the protein concentration of the protein solution to be crystallized -Cooling the protein solution to be crystallized to a lower temperature - To bring the protein solution to be crystallized closer to the optimal pH for BLG crystallization. - Reducing even further the conductivity of the protein solution to be crystallized.

[0340] In some preferred embodiments of the invention, the crystallizing protein solution is maintained in the metastable zone during step b) to avoid the spontaneous formation of new crystallites.

[0341] It is particularly preferred to further increase the protein concentration of the crystallizing protein solution and / or bring the crystallizing protein solution closer to the optimum pH for BLG crystallization (approximately pH 5.5) by addition of additional type A or type B protein feed. The pH adjustment is preferably performed gradually to avoid spontaneous crystallization or large local pH differences. A pH adjustment rate in the range of 0.05-0.5 pH-units / hour has been found to be advantageous for controlling BLG crystallization.

[0342] It is further particularly preferred that the temperature of the protein solution to be crystallized is reduced during step b) to a temperature at least 2° C. below the temperature of the initial protein solution, more preferably at least 4° C. below, most preferably at least 6° C. below the temperature of the initial protein solution. However, it is preferred that the protein solution to be crystallized is not completely frozen.

[0343] The inventors have observed that maximum BLG crystallization yields are obtained at approximately pH 5.5, and that the pH of the initial protein solution can be conveniently selected outside the optimum, but can be brought closer to the optimum pH during crystallization, for example by the addition of food acid, or more preferably by additional type B or type A protein feed.

[0344] Thus, in some preferred embodiments of the present invention, the pH of the initial protein solution is in the range of 5.0-5.3 or 5.7-6.0. In such preferred embodiments of the present invention, it is further preferred to adjust the pH of the crystallizing protein solution to bring the pH of the crystallizing protein solution closer to pH 5.5 than the pH of the initial protein solution, preferably by adding an additional type A protein feed or an additional type B protein feed. It is particularly preferred that the crystallizing protein solution is adjusted to approximately pH 5.5 during crystallization. The additional type A protein feed or the additional type B protein feed used to adjust the crystallizing protein solution preferably has a conductivity that is equal to or lower than that of the crystallizing protein solution. It is further preferred to combine said pH adjustment during crystallization with a reduction in the temperature of the crystallizing protein solution, so that the crystallizing protein solution is cooled, for example simultaneously or sequentially, to a temperature lower than that of the initial protein solution.

[0345] In some preferred embodiments of the present invention: - the pH of the initial protein solution of step a) is in the range of 5.0-5.3 or 5.7-6.0 and the temperature of the initial protein solution is in the range of 5-20°C; - during step b) the pH of the protein solution to be crystallized is adjusted to a pH in the range of 5.4-5.6, preferably by adding an additional type A protein feed if the pH of the initial protein solution is in the range of 5.0-5.3, or by adding an additional type B protein feed if the pH of the initial protein solution is in the range of 5.7-6.0, and - During step b), the temperature of the protein solution to be crystallized is reduced to a temperature that is at least 2°C lower than the temperature of the initial protein solution.

[0346] The terms "additional type A protein supply" and "additional type B protein supply" refer to BLG-containing compositions that are added to an initial protein solution to cause crystallization or that are added to a protein solution to be crystallized to modify at least its pH and BLG content. The characteristics and preferences described in connection with "type A protein supply" and "type B protein supply" apply equally to "additional type A protein supply" and "additional type B protein supply", respectively.

[0347] The inventors have determined the crystal lattice structure of the isolated BLG crystals by X-ray crystallography and have not found similar crystals in the prior art.

[0348] In some preferred embodiments of the invention, at least some of the BLG crystals obtained in step b) are in the orthorhombic space group P2 1 2 1 2 1 has.

[0349] Preferably, at least some of the obtained BLG crystals are in the orthorhombic space group P2. 1 2 1 2 1 , and unit cell dimensions a = 68.68 (± 5%) A, b = 68.68 (± 5%) A, and c = 156.65 (± 5%) A; and unit cell integration angles α = 90 degrees, β = 90 degrees, and γ = 90 degrees.

[0350] In some preferred embodiments of the invention, at least some of the resulting BLG crystals are in the orthorhombic space group P2 1 2 1 2 1 , and unit cell dimensions a=68.68(±2%) A, b=68.68(±2%) A, and c=156.65(±2%) A; and unit cell integration angles α=90 degrees, β=90 degrees, and γ=90 degrees.

[0351] Even more preferably, at least some of the obtained BLG crystals are in the orthorhombic space group P2 1 2 1 2 1 , and unit cell dimensions a=68.68(±1%) A, b=68.68(±1%) A, and c=156.65(±1%) A; and unit cell integration angles α=90 degrees, β=90 degrees, and γ=90 degrees.

[0352] Most preferably, at least some of the resulting BLG crystals are in the orthorhombic space group P2. 1 2 1 2 1 , and unit cell dimensions a=68.68A, b=68.68A, and c=156.65A; and unit cell integration angles α=90 degrees, β=90 degrees, and γ=90 degrees.

[0353] In some particularly preferred embodiments of the invention, the method includes a step c) of separating at least some of the BLG crystals from the remaining liquid of the BLG crystal-containing solution, which is particularly preferred when purification of BLG is desired.

[0354] Step c) may for example comprise separating the BLG crystals to a solids content of at least 30% w / w. Preferably, step c) comprises separating the BLG crystals to a solids content of at least 40% w / w. Even more preferably, step c) comprises separating the BLG crystals to a solids content of at least 50% w / w.

[0355] The inventors have found that a high solids content is advantageous for the purification of BLG because the aqueous portion adhering to the separated BLG crystals typically contains impurities that should be avoided. In addition, a high solids content reduces the energy consumption for converting the separated BLG crystals to a dry product, such as a powder, which increases the BLG yield obtained from a drying unit with a fixed capacity.

[0356] In some preferred embodiments of the invention, step c) comprises separating the BLG crystals to a solids content of at least 60%. Preferably, step c) comprises separating the BLG crystals to a solids content of at least 70%. Even more preferably, step c) comprises separating the BLG crystals to a solids content of at least 80%.

[0357] In some preferred embodiments of the invention, the separation in step c) comprises one or more of the following operations: - Centrifugation, -Decantation, -filtration, - Sedimentation, -A combination of the above.

[0358] These unit operations are well known to those of skill in the art and are readily performed. Separation by filtration can include, for example, the use of vacuum filtration, dynamic cross-flow filtration (DCF), a filtrate press, or a filter centrifuge.

[0359] Different pore sizes for filtration can be used based on the desired results. Preferably, the filter allows native whey proteins and small aggregates to pass, but retains BLG crystals. The filter preferably has a nominal pore size of at least 0.1 microns. The filter may, for example, have a nominal pore size of at least 0.5 microns. Even more preferably, the filter may have a nominal pore size of at least 2 microns.

[0360] Filters with larger pore sizes can also be used, and are in fact preferred when primarily large crystals must be separated from a liquid containing BLG crystals. In some embodiments of the invention, the filter has a nominal pore size of at least 5 microns. Preferably, the filter has a nominal pore size of at least 20 microns. Even more preferably, the filter may have a pore size of at least 40 microns.

[0361] The filter may have a pore size in the range of, for example, 0.03-5000 microns, such as, for example, 0.1-5000 microns. Preferably, the filter may have a pore size in the range of 0.5-1000 microns. Even more preferably, the filter may have a pore size in the range of 5-800 microns, such as, for example, in the range of 10-500 microns or in the range of 50-500 microns.

[0362] In some preferred embodiments of the invention, the filter has a pore size in the range of 0.03-100 microns. Preferably, the filter may have a pore size in the range of 0.1-50 microns. More preferably, the filter may have a pore size in the range of 4-40 microns. Even more preferably, the filter may have a pore size in the range of 5-30 microns, for example, in the range of 10-20 microns.

[0363] The advantage of using a filter with a pore size larger than 1 micron is that bacteria and other microorganisms are also at least partially removed during separation, and optionally also during washing and / or recrystallization, thus making it possible to produce highly pure BLG with both a very low bacterial load, while also avoiding heat damage to the protein.

[0364] Another advantage of using filters with pore sizes larger than 1 micron is that removal of water and subsequent drying is easier and requires less energy.

[0365] The remaining liquid that is separated from the BLG crystals can be recycled into one or more protein feeds during preparation of the initial protein solution.

[0366] In some preferred embodiments of the invention, step c) uses a filter centrifuge. In other preferred embodiments of the invention, step c) uses a decanter centrifuge. Results (see Example 13 of WO2018 / 115520) show that the use of a filter centrifuge and / or a decanter centrifuge to separate the BLG crystals from the mother liquor provides a more robust operation of the method than, for example, vacuum filtration.

[0367] It is often preferred to dry the formed filter cake using a drying gas to reduce the moisture content of the filter cake, preferably to enable it to be stripped from the filter. The use of a drying gas can form part of the separation step, or alternatively, the final drying step if the filter cake is converted directly into a dry edible BLG composition.

[0368] In some preferred embodiments of the invention, step c) uses a DCF unit.

[0369] In some preferred embodiments of the invention, step c) is carried out using a DCF unit fitted with a membrane capable of retaining BLG crystals, the DCF permeate is recycled and forms part of the initial protein solution or protein feed, and the DCF retentate can be recovered or returned to the crystallization tank. Preferably, the DCF permeate is treated, for example by ultrafiltration / diafiltration, to make it supersaturated with respect to BLG before mixing with the initial protein solution or protein feed.

[0370] Advantageously, these embodiments do not require the temperature of the liquid stream to be raised above 15°C, and are therefore less prone to microbial contamination than process variants that require higher temperatures. Another industrial advantage of these embodiments is that the level of supersaturation can be easily controlled and maintained at a level where unwanted, spontaneous crystallization does not occur. Thus, the temperature of the liquid stream in these embodiments of the process is preferably at most 15°C, more preferably at most 12°C, even more preferably at most 10°C, and most preferably at most 5°C.

[0371] These embodiments are exemplified in WO2018 / 115520 in Example 10. These embodiments may be carried out as a batch process or a continuous process.

[0372] In some preferred embodiments of the invention, the method comprises a step d) of washing the BLG crystals, e.g. the isolated BLG crystals of c). The washing can consist of a single washing step or multiple washing steps.

[0373] The washing in step d) preferably involves contacting the BLG crystals with a washing solution without completely dissolving the BLG crystals, and then separating the remaining BLG crystals from the washing solution.

[0374] The wash liquid is preferably selected to avoid complete dissolution of the BLG crystals and may, for example, comprise, or even consist essentially of, cold demineralized water, cold tap water, or cold reverse osmosis permeate.

[0375] The wash solution may have a pH in the range of 5-6, preferably in the range of 5.0-6.0, even more preferably in the range of 5.1-6.0, such as, for example, in the range of 5.1-5.9.

[0376] The cleaning liquid may have a conductivity of at most 0.1 mS / cm, preferably at most 0.02 mS / cm, even more preferably at most 0.005 mS / cm.

[0377] A cleaning liquid having even lower conductivity may be used, for example the cleaning liquid may have a conductivity of at most 1 microS / cm, or alternatively the cleaning liquid may have a conductivity of at most 0.1 microS / cm, such as for example around 0.05 microS / cm.

[0378] The washing step is preferably carried out at low temperature to limit dissolution of the crystallized BLG. The temperature of the washing solution is preferably at most 30°C, more preferably at most 20°C, even more preferably at most 10°C.

[0379] The washing step may, for example, be carried out at at most 5° C., more preferably at most 2° C., such as, for example, at approximately 0° C. Temperatures below 0° C. may be used as long as the washing solution does not freeze at that temperature, for example due to the presence of one or more freezing point depressant(s).

[0380] In some embodiments of the invention the washing solution comprises BLG, for example in an amount of at least 1% w / w, preferably in an amount of at least 3% w / w, for example in an amount of 4% w / w.

[0381] The washing in step d) typically dissolves at most 80% w / w, preferably at most 50% w / w, even more preferably at most 20% w / w of the initial amount of BLG crystals. Preferably, the washing in step d) dissolves at most 15% w / w, more preferably at most 10% w / w, even more preferably at most 5% w / w of the initial amount of BLG crystals.

[0382] The weight ratio between the total amount of washing liquid and the initial amount of separated BLG crystals is often at least 1, preferably at least 2, more preferably at least 5. For example, the weight ratio between the amount of washing liquid and the initial amount of separated BLG crystals may be at least 10. Alternatively, the weight ratio between the total amount of washing liquid and the initial amount of separated BLG crystals may be at least 20, such as at least 50 or at least 100, for example.

[0383] The term "total amount of washing liquid" relates to the total amount of washing liquid used during the entire process.

[0384] In some preferred embodiments of the invention, the one or more wash sequences occur in the same or a similar filter array as the BLG crystal separation: the filter cake containing primarily BLG crystals is added to one or more wash sequences, and the washes are removed through the filters while the remaining portion of the BLG crystals resides within the filter cake.

[0385] In a particularly preferred embodiment of the invention, the separation of step c) is carried out using a filter that retains BLG crystals. The filter cake is then contacted with one or more volumes of washing liquid that move through the filter cake and the filter. Each volume of washing liquid is often preferred to be at most 10 times the volume of the filter cake, preferably at most 5 times the volume of the filter cake, more preferably at most 1 time the volume of the filter cake, even more preferably at most 0.5 times the volume of the filter cake, for example at most 0.2 times the volume of the filter cake. The volume of the filter cake includes both the solids and the fluids (liquid and gas) of the filter cake. The filter cake is preferably washed in this way at least twice, preferably at least 4 times, even more preferably at least 6 times.

[0386] The used wash solution from step d) can for example be recycled to the protein feed or initial protein solution, in which case the washed-out BLG can be isolated again.

[0387] The method may further comprise step e), which comprises a recrystallization step. The recrystallization step may, for example, comprise: - dissolving the separated BLG crystals in a recrystallization solution; - adjusting the recrystallization solution to obtain a supersaturation with respect to BLG; - crystallizing BLG in a supersaturated adjusted recrystallization solution; and - Separating the BLG crystals from the remaining conditioned recrystallization solution.

[0388] Alternatively, and often more preferably, the separated BLG crystals are converted into one or more type A protein feed(s) and one or more type B protein feed(s), which are combined to form a new supersaturated protein solution, e.g., a whey protein solution, as in step a), in which case the new protein solution BLG is recrystallized.

[0389] Step e) may involve either a single recrystallization sequence or multiple recrystallization sequences.

[0390] In some embodiments of the invention, the BLG crystals of step c) or d) are recrystallized at least twice. For example, the BLG crystals may be recrystallized at least three times, such as, for example, at least four times.

[0391] The washing and recrystallization steps can be combined in any order and performed multiple times if necessary.

[0392] The separated BLG crystals of step c) can for example be subjected to the following process sequence: - one or more steps of washing (step d), followed by - one or more steps of recrystallization (step e).

[0393] Alternatively, the isolated BLG crystals of step c) can be subjected to the following process sequence: - one or more steps of recrystallization (step e), followed by - one or more steps of washing (step d).

[0394] Several washing and recrystallization steps may be carried out, for example in the following order: - one or more steps of washing (step d), - one or more steps of recrystallization (step e), - one or more steps of washing (step d), and - one or more steps of recrystallization (step e). Or, for example, the following order is also possible: - one or more steps of recrystallization (step e), - one or more steps of washing (step d), - one or more steps of recrystallization (step e). - one or more steps of washing (step d).

[0395] In some embodiments of the invention, the method further comprises subjecting the separated BLG to an additional BLG enrichment step, e.g. based on chromatography or selective filtration. However, in other preferred embodiments of the invention, the method does not comprise an additional BLG enrichment step after step b). By the term "additional BLG enrichment step" is meant a process step that enriches BLG relative to the total amount of protein, which step is not related to crystallization of BLG or handling of BLG crystals. One example of such an additional BLG enrichment step is ion exchange chromatography. Washing of BLG crystals and / or recrystallization of BLG are not considered as "additional BLG enrichment steps".

[0396] In some particularly preferred embodiments of the invention, the method comprises a drying step f), in which the BLG-containing composition from steps b), c), d), or e) is converted into a dry composition.

[0397] In the context of the present invention, the term "dry" means that the composition or product in question contains at most 6% w / w water, preferably less.

[0398] In the context of the present invention, the term "BLG-containing composition" is used to describe the composition that is subjected to drying in step f).

[0399] In the context of the present invention, a "BLG-containing composition derived from step b), c), d), or e)" means a composition comprising at least some of the BLG from step b), c), d), or e). In some preferred embodiments of the invention, the "BLG-containing composition derived from step b), c), d), or e)" is obtained directly from step b), c), d), or e). However, in other preferred embodiments of the invention, the "BLG-containing composition derived from step b), c), d), or e)" is the result of further processing of the composition obtained directly from step b), c), d), or e).

[0400] It is often preferred that the BLG-containing composition comprises a significant amount of the BLG present in the composition directly obtained from steps b), c), d), or e). In some preferred embodiments of the invention, the BLG-containing composition from steps b), c), d), or e) comprises at least 50% w / w of the BLG obtained from steps b), c), d), or e), preferably at least 70%, and even more preferably at least 80%.

[0401] Preferably, the BLG-containing composition from step b), c), d), or e) comprises at least 85% w / w of BLG obtained from step b), c), d), or e). More preferably, the BLG-containing composition from step b), c), d), or e) comprises at least 90% w / w of BLG obtained from step b), c), d), or e). Even more preferably, the BLG-containing composition from step b), c), d), or e) comprises at least 95% w / w of BLG obtained from step b), c), d), or e). Most preferably, the BLG-containing composition from step b), c), d), or e) comprises 100% w / w of BLG obtained from step b), c), d), or e).

[0402] In some preferred embodiments of the invention, the drying step comprises one or more of spray drying, freeze drying, spin flash drying, rotary drying, and / or fluidized bed drying.

[0403] In some particularly preferred embodiments of the invention, the drying step comprises a BLG-containing composition in which the BLG crystals are dissolved and the resulting powder does not contain BLG crystals formed by step b) or by recrystallization prior to the drying step. These embodiments are preferred if the edible BLG composition is similar to, for example, a conventional, dried whey protein powder.

[0404] BLG crystals can be dissolved, for example, by: -Increase in temperature, -Increasing the electrical conductivity, for example by adding one or more salts -Changes in pH, e.g. outside the range of 5-6, - Decreasing the concentration of BLG, e.g. by dilution -or any combination of the above.

[0405] Spray drying is the currently preferred method of drying BLG-containing compositions that are free of BLG crystals.

[0406] In other particularly preferred embodiments of the invention, the drying step includes a BLG-containing composition that still contains BLG crystals, and the resulting powder contains BLG crystals. These embodiments are preferred when the edible BLG composition has a higher density than conventional, dried whey protein powders.

[0407] In some particularly preferred embodiments of the invention, the drying step results in a BLG-containing composition that still contains BLG crystals, and the resulting powder contains BLG crystals. These embodiments are preferred when the edible BLG-composition has a higher density than conventional, dried whey protein powders.

[0408] As shown in Example 7 of WO2018 / 115520, the present inventors have previously spray-dried a slurry of BLG crystals and discovered that the dried BLG crystals can retain at least some of their crystalline structure when resuspended in cold demineralized water. It is particularly advantageous to avoid exposing the BLG-containing composition containing BLG crystals to a heat treatment regime that dissolves a significant amount of the BLG crystals before spraying. Thus, if pre-heating of the BLG-containing composition containing BLG crystals is used before spraying, it is preferable to carefully control the heat load.

[0409] In some embodiments of the invention, the BLG-containing composition comprising BLG crystals has a temperature at most 70° C., preferably at most 60° C., and more preferably at most 50° C., when it reaches the outlet of the spray-device (e.g., nozzle or atomizer). In some preferred embodiments of the invention, the BLG-containing composition comprising BLG crystals has a temperature at most 40° C., preferably at most 30° C., more preferably at most 20° C., even more preferably at most 10° C., and most preferably at most 5° C., when it reaches the outlet of the spray-device.

[0410] The spray-device of a spray dryer is a device, such as a nozzle or atomizer, which converts the solution or suspension to be dried into small droplets which enter the drying chamber of the spray dryer.

[0411] It is particularly preferred that the BLG-containing composition comprising BLG crystals has a temperature in the range of 0-50°C when it reaches the outlet of the spray-apparatus, preferably in the range of 2-40°C, more preferably in the range of 4-35°C, and most preferably in the range of 5-10°C when it reaches the outlet of the spray-apparatus.

[0412] In some preferred embodiments of the invention, the BLG-containing composition upon reaching the outlet of the spray-device has a crystallinity of BLG of at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, and most preferably at least 90%, e.g., for example, preferably 97-100%. The BLG-containing composition may be a BLG isolate, e.g., comprising more than 90% w / w BLG relative to total protein, or it may contain significant amounts of other proteins and thus may comprise at most 90% w / w BLG relative to total protein.

[0413] In some preferred embodiments of the invention, the BLG-containing composition may have the protein composition of a conventional liquid WPC or WPI or a conventional liquid SPC or SPI as described herein, but upon reaching the outlet of the spray-device, may have a crystallinity of BLG of at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, most preferably at least 90%, e.g., for example preferably 97-100%.

[0414] The inlet temperature of the gas into the spray dryer is preferably in the range of 140-220° C., more preferably in the range of 160-200° C., even more preferably in the range of 170-190° C., for example preferably around 180° C. The outlet temperature of the gas from the spray dryer is preferably in the range of 50-95° C., more preferably in the range of 70-90° C., even more preferably in the range of 80-88° C., for example preferably around 85° C. As a rule of thumb it is said that the solids to be subjected to spray drying are heated to a temperature 10-15° C. below the gas outlet temperature.

[0415] In some preferred embodiments of the invention, the spray dryer is preferably at a temperature in the range of 50-85°C, more preferably in the range of 60-80°C, even more preferably in the range of 65-75°C, such as, for example, preferably around 70°C.

[0416] In some preferred embodiments of the invention, the BLG-containing composition to be dried is mixed with dried BLG isolate to raise the solids content to a level where the mixture can be dried by fluidized bed drying. This is also called backmixing and allows for very cost-effective drying of the BLG product. These embodiments are particularly preferred for BLG-containing compositions that contain BLG crystals.

[0417] An advantage of this method is that the BLG-containing composition being dried can have a very high solids content prior to the drying step, thus less water must be removed and less energy is consumed in the drying operation.

[0418] In some preferred embodiments of the invention, the BLG-containing composition from step b), c), d), or e) has a solids content of at least 20% w / w. Preferably, the BLG-containing composition from step b), c), d), or e) has a solids content of at least 30% w / w. More preferably, the BLG-containing composition from step b), c), d), or e) has a solids content of at least 40% w / w. Even more preferably, the BLG-containing composition from step b), c), d), or e) has a solids content of at least 50% w / w, such as for example at least 60% w / w.

[0419] In another preferred embodiment of the invention, the BLG-containing composition from step b), c), d), or e) has a solids content in the range of 20-80% w / w. Preferably, the BLG-containing composition from step b), c), d), or e) has a solids content in the range of 30-70% w / w. More preferably, the BLG-containing composition from step b), c), d), or e) has a solids content in the range of 40-65% w / w. Even more preferably, the BLG-containing composition from step b), c), d), or e) has a solids content in the range of 50-65% w / w, e.g., approximately 60% w / w.

[0420] The inventors have found that the higher the crystallinity of the BLG-containing composition, the less water is bound to the BLG-containing composition and a higher total solids content of the BLG-containing composition can be achieved prior to the drying step. Thus, in some preferred embodiments of the invention, the BLG-containing composition has a crystallinity of at least 10% w / w of BLG. Preferably, the BLG of the BLG-containing composition has a crystallinity of at least 20% w / w. More preferably, the BLG of the BLG-containing composition has a crystallinity of at least 30% w / w. Even more preferably, the BLG of the BLG-containing composition has a crystallinity of at least 40% w / w.

[0421] Even higher degrees of crystallinity are often preferred. Thus, in some preferred embodiments of the invention, the BLG of the BLG-containing composition has a degree of crystallinity of at least 50% w / w. Preferably, the BLG of the BLG-containing composition has a degree of crystallinity of at least 60% w / w. More preferably, the BLG of the edible BLG composition has a degree of crystallinity of at least 70% w / w. Even more preferably, the BLG of the BLG-containing composition has a degree of crystallinity of at least 80% w / w. Most preferably, the BLG of the BLG-containing composition has a degree of crystallinity of at least 90% w / w, preferably at least 95% w / w, more preferably at least 97% w / w, and even more preferably at least 99% w / w.

[0422] The inventors have found that a reduced content of water tends to increase the crystallinity of BLG in a composition, and thus a composition having a high water:BLG ratio (e.g., a suspension in water containing 4% BLG crystals) tends to have a lower crystallinity of BLG than a composition having a lower water:BLG ratio under the same conditions (e.g., a filter cake or wet isolated crystals).

[0423] The process of the present invention can be operated using mild temperatures that do not damage the nutritional value of any of the BLG and other whey proteins of the initial protein solution.

[0424] In some preferred embodiments of the invention, the BLG is not subjected to temperatures in excess of 90° C. during the process. Preferably, the BLG is not subjected to temperatures in excess of 80° C. during the process. Even more preferably, the BLG is not subjected to temperatures in excess of 75° C. during the process. It should be noted that although spray drying often employs temperatures in excess of 150° C., the short exposure times and the concomitant evaporation of water mean that the spray dried protein does not experience temperatures in excess of 50-70° C.

[0425] The inventors have seen indications that extended heating during the drying step reduces the amount of BLG that is in crystalline form. In some preferred embodiments of the invention, the heat exposure during the drying step is kept low enough to provide a degree of denaturation of BLG of at most 10%, preferably at most 4%, more preferably at most 1%, even more preferably at most 0.4%, and even more preferably at most 0.1%. Most preferably, the drying step does not lead to any detectable denaturation of BLG.

[0426] The degree of denaturation caused by the drying step is determined by the BLG content (relative to the total solids) in the BLG-containing composition dried in step f) (c ステップf前 ) and the BLG content (based on total solids) in the reconstituted, dried composition is determined and calculated by using the following formula:

number

[0427] Some preferred embodiments of the invention relate to a method for preparing an edible composition comprising a crystalline form of beta-lactoglobulin (BLG), the method comprising the steps of: a) providing an initial protein solution comprising BLG and at least one additional whey protein, said initial protein solution being supersaturated with respect to BLG and having a pH in the range of 5-6, said initial protein solution comprising: 70-100% w / w protein on total solids, - 30-90% w / w BLG based on total protein, preferably 30-70% w / w BLG - 4-50% w / w ALA, and preferably 8-35% w / w ALA, based on total protein; - 0-25% w / w CMP on protein, at least 10% w / w protein, based on the total weight of the initial protein solution; b) crystallizing the BLG in the supersaturated initial protein solution, preferably by adding crystallization seeds, thereby obtaining a BLG crystal-containing solution; and f) drying the BLG crystal-containing solution or concentrate of step b), said BLG crystal-containing solution preferably having a crystallinity of at least 30% BLG; This method does not include steps c), d) or e).

[0428] The initial protein solution is preferably a desalted protein solution, preferably having a ratio between conductivity and total amount of protein of at most 0.3 and / or a UF permeate conductivity of at most 7 mS / cm.

[0429] In these embodiments, the BLG crystals are not separated from the BLG crystal-containing solution, but are dried to obtain a high density edible protein composition in powder form.

[0430] Another preferred embodiment of the invention relates to a method for preparing an edible composition comprising a crystalline form of beta-lactoglobulin (BLG), the method comprising the steps of: a) providing an initial protein solution comprising BLG and at least one additional whey protein, said initial protein solution being supersaturated with respect to BLG and having a pH in the range of 5-6, said initial protein solution comprising: 70-100% w / w protein on total solids, - 30-90% w / w BLG based on total protein, preferably 30-70% - 4-50% w / w ALA based on total protein, and preferably 8-35% - 0-25% w / w CMP based on total protein, at least 10% w / w protein, based on the total weight of the initial protein solution; b) crystallizing the BLG in the supersaturated initial protein solution, preferably by addition of crystallization seeds, thus obtaining a BLG crystal-containing solution; c) separating the BLG crystals from the remaining liquid of the BLG crystal-containing solution; d) optionally washing the separated BLG crystals obtained from step c); e) optionally recrystallizing the BLG crystals obtained from step c) or d); and f) drying the BLG-containing composition derived from, preferably directly obtained from, step c), d), or e), wherein the BLG-containing composition comprises BLG crystals and preferably has a crystallinity of at least 30% BLG.

[0431] The initial protein solution is preferably a desalted protein solution, preferably having a ratio between conductivity and total amount of protein of at most 0.3 and / or a UF permeate conductivity of at most 7 mS / cm.

[0432] These embodiments are particularly useful for producing low mineral and low phosphorus edible BLG compositions in the form of a dense powder.

[0433] Yet another preferred embodiment of the invention relates to a method for preparing an edible composition comprising β-lactoglobulin in isolated form, the method comprising the steps of: a) providing an initial protein solution comprising BLG and at least one additional whey protein, said initial protein solution being supersaturated with respect to BLG and having a pH in the range of 5-6, said initial protein solution comprising: 70-100% w / w protein on total solids, - 30-90% w / w of BLG, preferably 30-70% w / w of BLG relative to the total protein; - 5-50% w / w ALA, and preferably 8-35% w / w ALA, based on total protein; - 0-25% w / w CMP based on total protein, at least 10% w / w protein, based on the total weight of the initial protein solution; b) crystallizing the BLG in the supersaturated initial protein solution, preferably by addition of crystallization seeds, thus obtaining a BLG crystal-containing solution; c) separating the BLG crystals from the remaining liquid of the BLG crystal-containing solution; d) optionally washing the separated BLG crystals obtained from step c); e) optionally recrystallizing the BLG crystals obtained from step c) or d); and f) drying the BLG-containing composition from step c), d), or e), wherein the BLG-containing composition is free of BLG crystals.

[0434] The initial protein solution is preferably a desalted protein solution, preferably having a ratio between conductivity and total amount of protein of at most 0.3 and / or a UF permeate conductivity of at most 7 mS / cm.

[0435] In these embodiments, the BLG crystals are dissolved prior to drying.

[0436] In some preferred embodiments, the method is carried out as a batch process. Alternatively, and sometimes preferred, the method may be carried out as a semi-batch process. In other preferred embodiments, the method is carried out as a continuous process.

[0437] An advantage of the present method is that it is much faster than comparable methods for BLG crystallization in the prior art: the period from initial preparation of the protein feed to completion of separation in step c can be at most 10 hours, preferably at most 4 hours, more preferably at most 2 hours, and even more preferably at most 1 hour.

[0438] In some preferred embodiments of the invention, the BLG crystal-containing solution remaining after the BLG crystals are separated in step c) is recovered and used directly as an ingredient in food production or subjected to further processing according to PCT application WO2020 / 002,422 (hereby incorporated by reference for all purposes). If so, the method provides an edible, alpha-lactalbumin-enriched whey protein composition in addition to an edible BLG composition.

[0439] An edible BLG composition preferably comprises BLG in an amount of at least 30% w / w based on total protein, and total protein based on total solids in an amount of at least 30% w / w.

[0440] In some preferred embodiments of the invention, the BLG of the edible BLG composition has a lactosylation degree of at most 1. Preferably, the BLG of the edible BLG composition has a lactosylation degree of at most 0.6. More preferably, the BLG of the edible BLG composition has a lactosylation degree of at most 0.4. Even more preferably, the BLG of the edible BLG composition has a lactosylation degree of at most 0.2. Most preferably, the BLG of the edible BLG composition has a lactosylation degree of at most 0.1, such as, for example, preferably at most 0.01.

[0441] In some preferred embodiments of the invention, the BLG of the edible BLG composition comprises at least 90% w / w non-lactosylated BLG, preferably at least 95% w / w non-lactosylated BLG, and even more preferably at least 98% w / w non-lactosylated BLG.

[0442] The percentage of non-lactosylated BLG is determined according to Example 9.1 of WO2018 / 115520.

[0443] In some preferred embodiments of the invention, the BLG of the edible BLG composition has a crystallinity of at least 10% w / w. Preferably, the BLG of the edible BLG composition has a crystallinity of at least 20% w / w. More preferably, the BLG of the edible BLG composition has a crystallinity of at least 30% w / w. Even more preferably, the BLG of the edible BLG composition has a crystallinity of at least 40% w / w.

[0444] Even higher degrees of crystallinity are often preferred. Thus, in some preferred embodiments of the invention, the BLG of the edible BLG composition has a degree of crystallinity of at least 50% w / w. Preferably, the BLG of the edible BLG composition has a degree of crystallinity of at least 60% w / w. More preferably, the BLG of the edible BLG composition has a degree of crystallinity of at least 70% w / w. Even more preferably, the BLG of the edible BLG composition has a degree of crystallinity of at least 80% w / w. Most preferably, the BLG of the edible BLG composition has a degree of crystallinity of at least 90% w / w, preferably at least 95% w / w.

[0445] The crystallinity of BLG in a liquid having a pH in the range of 5-6 is measured according to Example 9.7 of WO2018 / 115520. The crystallinity of BLG in a powder material is measured according to Example 9.8 of WO2018 / 115520. If the edible composition is a dry product but not in powder form, it must be converted to a powder, for example by grinding or milling, before being subjected to the method of Example 9.8 of WO2018 / 115520.

[0446] In some preferred embodiments of the invention, the edible BLG composition is a WPC, WPI, SPC, or SPI, in which case at least some of the BLG is in crystalline form. The edible BLG composition can, for example, comprise at most 90% w / w BLG based on the total amount of protein, and have a crystallinity of at least 10% BLG. For example, the edible BLG composition can comprise at most 80% w / w BLG based on the total amount of protein, and have a crystallinity of at least 10% BLG. The edible BLG composition can, for example, comprise 30-70% w / w BLG based on the total amount of protein, and have a crystallinity of at least 10% BLG.

[0447] In another preferred embodiment of the invention, the edible BLG composition comprises at most 90% w / w BLG relative to the total amount of protein and has a crystallinity of at least 30% BLG. Preferably, the edible BLG composition comprises at most 80% w / w BLG relative to the total amount of protein and has a crystallinity of at least 30% BLG. Even more preferably, the edible BLG composition comprises 30-70% w / w BLG relative to the total amount of protein and has a crystallinity of at least 30% BLG.

[0448] The inventors have found that the present invention makes it possible to prepare an edible whey protein product having a very low content of phosphorus and other minerals, which is advantageous for patients suffering from kidney disease or who otherwise have reduced kidney function.

[0449] The edible BLG composition is preferably a low phosphorus composition.

[0450] In the context of the present invention, the term "low phosphorus" relates to a composition, such as a liquid, powder or another food product, having a total phosphorus content of at most 100 mg phosphorus / 100 g protein. Preferably, the low phosphorus composition has a total phosphorus content of at most 80 mg phosphorus / 100 g protein. More preferably, the low phosphorus composition may have a total phosphorus content of at most 50 mg phosphorus / 100 g protein. Even more preferably, the low phosphorus composition may have a total phosphorus content of at most 20 mg phosphorus / 100 g protein. Even more preferably, the low phosphorus composition may have a total phosphorus content of at most 5 mg phosphorus / 100 g protein. The low phosphorus composition according to the present invention may be used as a food ingredient for the production of food products for patient groups with reduced renal function.

[0451] Thus, in some particularly preferred embodiments of the invention, the edible BLG composition comprises at most 80 mg phosphorus / 100 g protein. Preferably, the edible BLG composition comprises at most 30 mg phosphorus / 100 g protein. More preferably, the edible BLG composition comprises at most 20 mg phosphorus / 100 g protein. Even more preferably, the edible BLG composition comprises at most 10 mg phosphorus / 100 g protein. Most preferably, the edible BLG composition comprises at most 5 mg phosphorus / 100 g protein.

[0452] The phosphorus content relates to the total amount of elemental phosphorus in the composition in question and is determined according to Example 9.5 of WO 2018 / 115520.

[0453] In another preferred embodiment of the invention, the edible BLG composition is a low-mineral composition.

[0454] The term "low-mineral" in the context of the present invention relates to a composition, such as a liquid, powder or another food product, that has at least one, preferably two and even more preferably all of the following: - maximum ash content of 1.2% w / w based on total solids; - a total calcium and magnesium content of at most 0.3% w / w based on total solids; - A total content of sodium and potassium of at most 0.10% w / w based on total solids; -Total phosphorus content of at most 100 mg phosphorus / 100 g protein.

[0455] Preferably, the low mineral composition has at least one, preferably two or more, and even more preferably all of the following: - maximum ash content of 0.7% w / w based on total solids; - A total calcium and magnesium content of at most 0.2% w / w based on total solids, - A total content of sodium and potassium of at most 0.08% w / w based on total solids; -Total phosphorus content of at most 80 mg phosphorus / 100 g protein.

[0456] Even more preferably, the reduced mineral composition has at least one, preferably two or more, and even more preferably all of the following: - maximum ash content of 0.5% w / w based on total solids; - A total calcium and magnesium content of at most 0.15% w / w based on total solids; - A total content of sodium and potassium of at most 0.06% w / w based on total solids; -Total phosphorus content of at most 50 mg phosphorus / 100 g protein.

[0457] It is particularly preferred that the low mineral composition comprises: - maximum ash content of 0.5% w / w based on total solids; - A total calcium and magnesium content of at most 0.15% w / w based on total solids; - A total content of sodium and potassium of at most 0.06% w / w based on total solids; -Total phosphorus content of at most 50 mg phosphorus / 100 g protein.

[0458] In some preferred embodiments of the invention, the edible BLG composition comprises a total amount of protein of at least 25% w / w based on the total solids of the edible BLG composition. Preferably, the edible BLG composition comprises a total amount of protein of at least 50% w / w based on the total solids of the edible BLG composition. More preferably, the edible BLG composition comprises a total amount of protein of at least 75% w / w based on the total solids of the edible BLG composition. Even more preferably, the edible BLG composition comprises a total amount of protein of at least 90% w / w based on the total solids of the edible BLG composition.

[0459] In some preferred embodiments of the invention, the total amount of protein in the edible BLG composition is in the range of 25-100% w / w of total solids. Preferably, the total amount of protein in the edible BLG composition is in the range of 50-100% w / w of total solids. More preferably, the total amount of protein in the edible BLG composition is in the range of 75-100% w / w of total solids. Even more preferably, the total amount of protein in the edible BLG composition is in the range of 90-100% w / w of total solids.

[0460] In some preferred embodiments of the invention, the edible BLG composition comprises at least 75% w / w BLG based on the total amount of protein. Preferably, the edible BLG composition may comprise at least 90% w / w BLG based on the total amount of protein. More preferably, the edible BLG composition may comprise at least 95% w / w BLG based on the total amount of protein. Even more preferably, the edible BLG composition may comprise at least 97% w / w BLG based on the total amount of protein. Most preferably, the edible BLG composition comprises approximately 100% w / w BLG based on the total amount of protein.

[0461] In some preferred embodiments of the invention, the edible BLG composition comprises at most 10% w / w carbohydrates, preferably at most 5% w / w carbohydrates, more preferably at most 1% w / w carbohydrates, and even more preferably at most 0.1% w / w carbohydrates.

[0462] The edible BLG composition may also include lipids, for example in the form of other lipid types such as triglycerides and / or phospholipids.

[0463] In some embodiments of the invention, the edible BLG composition comprises a total amount of lipid of at most 1% w / w based on total solids. Preferably, the edible BLG composition comprises a total amount of lipid of at most 0.5% w / w based on total solids. More preferably, the edible BLG composition comprises a total amount of lipid of at most 0.1% w / w based on total solids. Even more preferably, the edible BLG composition comprises a total amount of lipid of at most 0.05% w / w based on total solids. Most preferably, the edible BLG composition comprises a total amount of lipid of at most 0.01% w / w based on total solids.

[0464] In some preferred embodiments of the invention, the edible BLG composition is a dry composition, such as a powder. It is particularly preferred that the edible BLG composition is a spray-dried powder.

[0465] The inventors have observed that edible BLG compositions in powder form, in which at least some of the BLG is in crystalline form when dried, have a higher density than comparable BLG compositions without BLG crystals (see Example 7). This high density effect is also quite surprisingly observed for edible BLG compositions in powder form obtained from a spray-dried BLG crystal slurry.

[0466] Thus, in some preferred embodiments of the invention, the edible BLG composition in powder form has a bulk density of at least 0.40 g / mL. Preferably, the edible BLG composition in powder form has a bulk density of at least 0.45 g / mL. More preferably, the edible BLG composition in powder form has a bulk density of at least 0.50 g / mL. Even more preferably, the edible BLG composition in powder form has a bulk density of at least 0.6 g / mL. The edible BLG composition in powder form may, for example, have a bulk density of at least 0.7 g / mL.

[0467] The bulk density advantage applies to both powders of edible BLG compositions in which BLG is nearly the only protein present and powders of edible BLG compositions in which the concentration of BLG is not fortified relative to other proteins present in the initial protein solution. Thus, the invention provides high density powders of both isolated BLG and crude whey protein (containing significant amounts of ALA and other whey proteins in addition to BLG).

[0468] In some preferred embodiments of the invention, the edible BLG composition in powder form has a bulk density of at least 0.45 g / mL and comprises at least 70% w / w protein relative to the total weight of the composition. More preferably, the edible BLG composition in powder form has a bulk density of at least 0.50 g / mL and comprises at least 70% w / w protein relative to the total weight of the composition. Even more preferably, the edible BLG composition in powder form has a bulk density of at least 0.6 g / mL and comprises at least 70% w / w protein relative to the total weight of the composition. The edible BLG composition in powder form can, for example, have a bulk density of at least 0.7 g / mL and comprises at least 70% w / w protein relative to the total weight of the composition.

[0469] In another preferred embodiment of the invention, the edible BLG composition in powder form has a bulk density of at least 0.45 g / mL and comprises at least 80% w / w protein relative to the total weight of the composition. More preferably, the edible BLG composition in powder form has a bulk density of at least 0.50 g / mL and comprises at least 80% w / w protein relative to the total weight of the composition. Even more preferably, the edible BLG composition in powder form has a bulk density of at least 0.6 g / mL and comprises at least 80% w / w protein relative to the total weight of the composition. The edible BLG composition in powder form can, for example, have a bulk density of at least 0.7 g / mL and comprises at least 80% w / w protein relative to the total weight of the composition.

[0470] The edible BLG composition in powder form may have, for example, a bulk density in the range of 0.40-1.5 g / mL and comprises at least 80% w / w protein relative to the total weight of the composition. Preferably, the powdered edible BLG composition may have a bulk density in the range of 0.45-1.0 g / mL and comprises at least 80% w / w protein relative to the total weight of the composition. More preferably, the powdered edible BLG composition may have a bulk density in the range of 0.50-0.9 g / mL and comprises at least 80% w / w protein relative to the total weight of the composition. Even more preferably, the powdered edible BLG composition may have a bulk density in the range of 0.6-0.9 g / mL and comprises at least 80% w / w protein relative to the total weight of the composition. The powdered edible BLG composition may have, for example, a bulk density in the range of 0.6-0.8 g / mL and comprises at least 80% w / w protein relative to the total weight of the composition.

[0471] The inventors have found that the high density powders of the invention advantageously allow for more cost-effective packaging and logistics of the powders, as less packaging material is required per kg of powder and more powder (mass) can be transported in a given container or truck.

[0472] The edible BLG composition in powder form may have, for example, a bulk density in the range of 0.40-1.5 g / mL. Preferably, the edible BLG composition has a bulk density in the range of 0.45-1.0 g / mL. More preferably, the edible BLG composition may have a bulk density in the range of 0.50-0.9 g / mL. Even more preferably, the edible BLG composition may have a bulk density in the range of 0.6-0.9 g / mL. The edible BLG composition may have, for example, a bulk density in the range of 0.6-0.8 g / mL.

[0473] In another preferred embodiment of the invention, the edible BLG composition in powder form has a bulk density in the range of 0.50-1.5 g / mL. Preferably, the powdered edible BLG composition has a bulk density in the range of 0.55-1.0 g / mL. More preferably, the powdered edible BLG composition may have a bulk density in the range of 0.60-1.0 g / mL. Even more preferably, the powdered edible BLG composition has a bulk density in the range of 0.65-1.0 g / mL. The powdered edible BLG composition may preferably have a bulk density in the range of 0.70-1.0 g / mL.

[0474] The edible BLG composition in powder form may have, for example, a bulk density in the range of 0.40-1.5 g / mL and comprises at least 70% w / w protein relative to the total weight of the composition. Preferably, the powdered edible BLG composition may have a bulk density in the range of 0.45-1.0 g / mL and comprises at least 70% w / w protein relative to the total weight of the composition. More preferably, the powdered edible BLG composition may have a bulk density in the range of 0.50-0.9 g / mL and comprises at least 70% w / w protein relative to the total weight of the composition. Even more preferably, the powdered edible BLG composition may have a bulk density in the range of 0.6-0.9 g / mL and comprises at least 70% w / w protein relative to the total weight of the composition. The powdered edible BLG composition may have, for example, a bulk density in the range of 0.6-0.8 g / mL and comprises at least 70% w / w protein relative to the total weight of the composition.

[0475] The edible BLG composition in powder form may have, for example, a bulk density in the range of 0.40-1.5 g / mL and comprises at least 80% w / w protein relative to the total weight of the composition. Preferably, the powdered edible BLG composition may have a bulk density in the range of 0.45-1.0 g / mL and comprises at least 80% w / w protein relative to the total weight of the composition. More preferably, the powdered edible BLG composition may have a bulk density in the range of 0.50-0.9 g / mL and comprises at least 80% w / w protein relative to the total weight of the composition. Even more preferably, the powdered edible BLG composition may have a bulk density in the range of 0.6-0.9 g / mL and comprises at least 80% w / w protein relative to the total weight of the composition. The powdered edible BLG composition may have, for example, a bulk density in the range of 0.6-0.8 g / mL and comprises at least 80% w / w protein relative to the total weight of the composition.

[0476] In another preferred embodiment of the invention, the edible BLG composition in powder form has a bulk density in the range of 0.50-1.5 g / mL and comprises at least 70% w / w protein relative to the total weight of the composition. Preferably, the powdered edible BLG composition has a bulk density in the range of 0.55-1.0 g / mL and comprises at least 70% w / w protein relative to the total weight of the composition. More preferably, the powdered edible BLG composition may have a bulk density in the range of 0.60-1.0 g / mL and comprises at least 70% w / w protein relative to the total weight of the composition. Even more preferably, the powdered edible BLG composition has a bulk density in the range of 0.65-1.0 g / mL and comprises at least 70% w / w protein relative to the total weight of the composition. The powdered edible BLG composition may preferably have a bulk density in the range of 0.70-1.0 g / mL and comprises at least 70% w / w protein relative to the total weight of the composition.

[0477] In another preferred embodiment of the invention, the edible BLG composition in powder form has a bulk density in the range of 0.50-1.5 g / mL and comprises at least 80% w / w protein relative to the total weight of the composition. Preferably, the powdered edible BLG composition has a bulk density in the range of 0.55-1.0 g / mL and comprises at least 80% w / w protein relative to the total weight of the composition. More preferably, the powdered edible BLG composition may have a bulk density in the range of 0.60-1.0 g / mL and comprises at least 80% w / w protein relative to the total weight of the composition. Even more preferably, the powdered edible BLG composition has a bulk density in the range of 0.65-1.0 g / mL and comprises at least 80% w / w protein relative to the total weight of the composition. The powdered edible BLG composition may preferably have a bulk density in the range of 0.70-1.0 g / mL and comprises at least 80% w / w protein relative to the total weight of the composition.

[0478] The bulk density of the powder is measured according to Example 9.3 of WO2018 / 115520.

[0479] The inventors have observed indications that BLG compositions according to the present invention have better long-term stability than similar BLG compositions, particularly when at least some of the BLG is present in the form of BLG crystals, which is believed to provide better storage stability of the BLG molecule.

[0480] In some preferred embodiments of the invention, the dried BLG composition has a furosine value of at most 80 mg / 100 g protein, preferably at most 60 mg / 100 g protein, more preferably at most 40 mg / 100 g protein, even more preferably at most 20 mg / 100 g protein after 60 days at 30° C. Most preferably, the dried BLG composition has a furosine value of at most 10 mg / 100 g protein after 60 days at 30° C.

[0481] In some preferred embodiments of the invention, the dry BLG composition has a furosine value of at most 80 mg / 100 g protein, preferably at most 60 mg / 100 g protein, more preferably at most 40 mg / 100 g protein, even more preferably at most 20 mg / 100 g protein. Most preferably, the dry BLG composition has a furosine value of at most 10 mg / 100 g protein. Preferably, the dry BLG composition has a furosine value of 0 mg / 100 g protein.

[0482] In some preferred embodiments of the invention, the BLG of the dry BLG composition has a lactosylation degree of at most 1, preferably at most 0.6, more preferably 0.2, even more preferably at most 0.1, and most preferably at most 0.01 after 60 days at 30°C.

[0483] In some preferred embodiments of the invention, the edible BLG composition is a liquid composition. The liquid edible BLG composition preferably comprises at least 20% w / w water, more preferably at least 30% w / w water, and even more preferably at least 40% w / w water.

[0484] A liquid edible BLG composition may, for example, comprise water in the range of 20-90% w / w, more preferably in the range of 30-80% w / w, even more preferably at least 40% w / w.

[0485] The inventors have surprisingly found that the edible BLG composition according to the present invention has a low degree of protein denaturation, even when spray drying is used to prepare the edible BLG powder composition (see Example 11 of WO2018 / 115520).

[0486] Thus, in some preferred embodiments of the invention, the edible BLG composition has a degree of protein denaturation of at most 10%. Preferably, the edible BLG composition has a degree of protein denaturation of at most 5%. More preferably, the edible BLG composition has a degree of protein denaturation of at most 2%. Even more preferably, the edible BLG composition has a degree of protein denaturation of at most 1%. Even more preferably, the edible BLG composition has a degree of protein denaturation of at most 0.5%.

[0487] In some preferred embodiments of the invention, the edible BLG composition is a dry powder, preferably a spray-dried powder, and has a degree of protein denaturation of at most 2%, preferably at most 1.5%. More preferably, the dry edible BLG composition, for example in the form of a spray-dried powder, has a degree of protein denaturation of at most 1.0%. Even more preferably, the dry edible BLG composition, for example in the form of a spray-dried powder, has a degree of protein denaturation of at most 0.8%. Even more preferably, the dry edible BLG composition, for example in the form of a spray-dried powder, has a degree of protein denaturation of at most 0.5%.

[0488] In some preferred embodiments of the invention, the edible BLG composition comprises: - Maximum 6% w / w water -At least 80% total protein to total solids - at least 95% BLG based on total protein, and The edible BLG composition comprises: -It is a dry powder, - has a bulk density of at least 0.50 g / mL, preferably at least 0.60 g / mL.

[0489] In another preferred embodiment of the invention, the edible BLG composition comprises: - Maximum 6% w / w water -At least 80% total protein to total solids - at least 95% BLG based on total protein, and The edible BLG composition -It is a dry powder, - has a bulk density of at least 0.50 g / mL, preferably at least 0.60 g / mL, and - has a crystallinity of at least 20%, preferably at least 40% of BLG.

[0490] In a further preferred embodiment of the invention, the edible BLG composition comprises: - Maximum 6% w / w water -At least 80% total protein to total solids - at least 95% BLG based on total protein, and The edible BLG composition comprises: -It is a dry powder, - has a bulk density of at least 0.50 g / mL, preferably at least 0.60 g / mL, and - has a degree of protein denaturation of at most 2%, preferably at most 1.0%.

[0491] In a further preferred embodiment of the invention, the edible BLG composition comprises: - Maximum 6% w / w water At least 80% total protein based on total solids; - at least 95% BLG based on total protein; - Maximum 80mg phosphorus / 100g protein The edible BLG composition comprises: -It is a dry powder.

[0492] In a further preferred embodiment of the invention, the edible BLG composition comprises: - Maximum 6% w / w water At least 90% total protein based on total solids; - at least 97% BLG based on total protein; - Maximum 50mg phosphorus / 100g protein The edible BLG composition comprises: -It is a dry powder.

[0493] In another preferred embodiment of the invention, the edible BLG composition comprises: - Maximum 6% w / w water at least 80% total protein by total solids, preferably at least 90% total protein by total solids; - 30-90% BLG based on total protein, - 8-25% w / w ALA based on total protein; The edible BLG composition comprises: is a dry powder, and - has a crystallinity of at least 20%, preferably at least 40% of BLG.

[0494] In some preferred embodiments of the invention, the edible BLG composition comprises: - 20-80% w / w of water, preferably 20-60% w / w of water, At least 80% total protein, preferably at least 90% total protein based on total solids - at least 95% BLG based on total protein; - Maximum 80mg phosphorus / 100g protein The edible BLG composition comprises: - has a crystallinity of at least 20%, preferably at least 40, of BLG, and Optionally, it has a degree of protein denaturation of at most 2%, preferably at most 1.0%.

[0495] The edible compositions according to these embodiments are particularly useful for preparing edible BLG compositions in dry form, and are particularly suitable for spray drying and preparation of high density whey protein powders having a standard concentration profile of whey protein species whey protein but containing at least some of the BLG in the form of dried BLG crystals.

[0496] In another preferred embodiment of the invention, the edible BLG composition comprises: - 20-80% w / w of water, preferably 20-60% w / w of water, at least 80% total protein by total solids, preferably at least 90% total protein by total solids; - 30-79% BLG based on total protein, - 8-25% w / w ALA based on total protein; The edible BLG composition comprises: - has a crystallinity of at least 20%, preferably at least 40% of BLG.

[0497] The edible compositions according to these embodiments are particularly useful for preparing edible BLG compositions in dry form, and are particularly suitable for spray drying and preparation of high density whey protein powders having a standard concentration profile of whey protein species whey protein but containing at least some of the BLG in the form of dried BLG crystals.

[0498] The process is preferably carried out at a temperature in the range of 1-65°C, preferably 2-50°C, more preferably 3-20°C, even more preferably 4-15°C.

[0499] Particularly preferred embodiments of the invention are described in the following numbered embodiments: Numbered embodiment 1. A method for preparing an edible composition comprising beta-lactoglobulin (BLG), preferably in crystallized and / or isolated form, the method comprising the steps of: a) preparing an initial protein solution comprising BLG, said initial protein solution being supersaturated with respect to BLG and having a pH in the range of 5-6; b) crystallizing BLG in a supersaturated, initial protein solution, preferably in salting-out mode, thereby obtaining a BLG crystal-containing solution; and c) optionally separating the BLG crystals from the remaining liquid of the BLG crystal-containing solution; Step a) comprises preparing an initial protein solution by combining, preferably mixing, one or more type A protein feed(s) with one or more type B protein feed(s), - "Type A protein feed" is defined as a protein feed that contains BLG and has a pH of at least 5.6, and - A method wherein a "type B protein feed" is defined as a protein feed that contains BLG and has a pH of at most 5.4.

[0500] Numbered Embodiment 2. A method according to any of the preceding numbered embodiments, comprising: the -A type protein feed(s), when combined, have a pH of at least 5.6, more preferably at least 5.7, even more preferably at least 5.8, and most preferably at least 5.9; and - the type B protein feed(s) when combined have a pH of at most 5.4, more preferably at most 5.3, even more preferably 5.2, and most preferably 5.1.

[0501] Numbered Embodiment 3. A method according to any of the preceding numbered embodiments, comprising: the -A type protein feed(s), when combined, have a pH of at least 6.0, more preferably at least 6.1, even more preferably at least 6.2, and most preferably at least 6.3; and - the type B protein feed(s) when combined have a pH of at most 5.0, more preferably at most 4.9, even more preferably 4.8, and most preferably 4.7.

[0502] Numbered embodiment 4. The method according to any of the preceding numbered embodiments, wherein the type A protein feed(s), when combined, have a pH in the range of 5.6-10, more preferably 5.8-8, even more preferably 6.0-7, and most preferably 6.1-6.5.

[0503] Numbered Embodiment 5. A method according to any of the preceding numbered embodiments, wherein the type A protein feed, when combined, has a pH in the range of 6.0-8, more preferably 6.1-7.5, even more preferably 6.2-7.0, and most preferably 6.2-6.5.

[0504] Numbered Embodiment 6. The method according to any of the preceding numbered embodiments, wherein the type B protein feed, when combined, has a pH in the range of 2-5.4, more preferably 3-5.2, even more preferably 4-5.0, and most preferably 4.5-4.9.

[0505] Numbered Embodiment 7. A method according to any of the preceding numbered embodiments, wherein the type B protein feed, when combined, has a pH in the range of 4.0-5.0, more preferably 4.1-4.9, even more preferably 4.3-4.8, and most preferably 4.5-4.8.

[0506] Numbered embodiment 8. A method according to any of the preceding numbered embodiments, wherein at least one of the one or more type A protein feeds is not saturated with respect to BLG, and more preferably none of the one or more type A protein feeds is saturated with respect to BLG.

[0507] Numbered embodiment 9. A method according to any of the preceding numbered embodiments, wherein at least one of the one or more type B protein feeds is not saturated with respect to BLG, and more preferably none of the one or more type B protein feeds is saturated with respect to BLG.

[0508] Numbered embodiment 10. A method according to any of the preceding numbered embodiments, wherein neither the Type A nor Type B protein feed is saturated with respect to BLG.

[0509] Numbered embodiment 11. A method according to any of the preceding numbered embodiments, wherein at least one of the one or more type A protein supplies does not contain BLG crystals, more preferably none of the one or more type A protein supplies contains BLG crystals.

[0510] Numbered embodiment 12. A method according to any of the preceding numbered embodiments, wherein at least one of the one or more type B protein supplies does not contain BLG crystals, more preferably none of the one or more type B protein supplies contains BLG crystals.

[0511] Numbered embodiment 13. A method according to any of the preceding numbered embodiments, wherein neither the type A nor type B protein supply comprises BLG crystals.

[0512] Numbered embodiment 14. A method according to any of the preceding numbered embodiments, wherein at least one of the one or more type A protein feeds is not supersaturated with respect to BLG, and more preferably none of the one or more type A protein feeds is supersaturated with respect to BLG.

[0513] Numbered Embodiment 15. A method according to any of the preceding numbered embodiments, wherein neither the type A nor type B protein supply comprises BLG crystals.

[0514] Numbered embodiment 16. A method according to any of the preceding numbered embodiments, wherein at least one of the one or more type A protein feeds is not supersaturated with respect to BLG, and more preferably none of the one or more type A protein feeds is supersaturated with respect to BLG.

[0515] Numbered embodiment 17. A method according to any of the preceding numbered embodiments, wherein at least one of the one or more type B protein supplies is not supersaturated with respect to BLG, and more preferably none of the one or more types is supersaturated with respect to BLG.

[0516] Numbered embodiment 18. A method according to any of the preceding numbered embodiments, wherein at least one of the one or more type A protein supplies of step a) is saturated with respect to BLG.

[0517] Numbered embodiment 19. A method according to any of the preceding numbered embodiments, wherein at least one of the one or more type B protein supplies of step a) is saturated with respect to BLG.

[0518] Numbered embodiment 20. A method according to any of the preceding numbered embodiments, wherein the type A and type B protein feed(s) that do not comprise BLG crystals contribute at least 1% w / w of the BLG content of the initial protein solution, more preferably at least 10% w / w, even more preferably at least 20% w / w, and most preferably at least 40% w / w.

[0519] Numbered embodiment 21. A method according to any of the preceding numbered embodiments, wherein the type A and type B protein feed(s) that do not comprise BLG crystals contribute at least 50% w / w of the BLG content of the initial protein solution, more preferably at least 70% w / w, even more preferably at least 80% w / w, and most preferably at least 90% w / w.

[0520] Numbered embodiment 22. A method according to any of the preceding numbered embodiments, wherein the type A and type B protein feed(s) that do not comprise BLG crystals contribute at least 92% w / w of the BLG content of the initial protein solution, more preferably at least 94% w / w, even more preferably at least 96% w / w, and most preferably at least 99% w / w.

[0521] Numbered embodiment 23. A method according to any of the preceding numbered embodiments, wherein the type A and type B protein feed(s) that are not supersaturated with respect to BLG contribute at least 1% w / w of the BLG content of the initial protein solution, more preferably at least 10% w / w, even more preferably at least 20% w / w, and most preferably at least 40% w / w.

[0522] Numbered embodiment 24. A method according to any of the preceding numbered embodiments, wherein the type A and type B protein feed(s) that are not supersaturated with respect to BLG contribute at least 50% w / w of the BLG content of the initial protein solution, more preferably at least 70% w / w, even more preferably at least 80% w / w, and most preferably at least 90% w / w.

[0523] Numbered embodiment 25. A method according to any of the preceding numbered embodiments, wherein the type A and type B protein feed(s) that are not supersaturated with respect to BLG contribute at least 92% w / w of the BLG content of the initial protein solution, more preferably at least 94% w / w, even more preferably at least 96% w / w, and most preferably at least 99% w / w.

[0524] Numbered embodiment 26. A method according to any of the preceding numbered embodiments, wherein the type A protein feed(s) when combined have a temperature of at most 60°C, more preferably at most 50°C, even more preferably at most 40°C, and most preferably at most 30°C.

[0525] Numbered embodiment 27. A method according to any of the preceding numbered embodiments, wherein the type A protein feed(s) when combined have a temperature of at most 20°C, more preferably at most 15°C, even more preferably at most 10°C, and most preferably at most 5°C.

[0526] Numbered embodiment 28. A method according to any of the preceding numbered embodiments, wherein the type B protein feed(s) when combined have a temperature of at most 60°C, more preferably at most 50°C, even more preferably at most 40°C, and most preferably at most 30°C.

[0527] Numbered embodiment 29. A method according to any of the preceding numbered embodiments, wherein the type B protein feed(s) when combined have a temperature of at most 20°C, more preferably at most 15°C, even more preferably at most 10°C, and most preferably at most 5°C.

[0528] Numbered embodiment 30. The method according to any of the preceding numbered embodiments, wherein the type A protein feed(s) when combined comprise at least 1% w / w BLG by weight of the combined type A protein feed(s), more preferably at least 3% w / w, even more preferably at least 5% w / w, and most preferably at least 7% w / w BLG by weight of the combined type A protein feed(s).

[0529] Numbered Embodiment 31. The method according to any of the preceding numbered embodiments, wherein the Type A protein feed(s) when combined comprise 1-45% w / w BLG by weight of the combined Type A protein feed(s), more preferably 3-40% w / w, even more preferably 5-36% w / w, and most preferably 7-34% w / w BLG by weight of the combined Type A protein feed(s).

[0530] Numbered embodiment 32. The method according to any of the preceding numbered embodiments, wherein the type B protein feed(s) when combined comprise at least 1% w / w BLG by weight of the combined type B protein feed(s), more preferably at least 3% w / w, even more preferably at least 5% w / w, and most preferably at least 7% w / w BLG by weight of the combined type B protein feed(s).

[0531] Numbered embodiment 33. The method according to any of the preceding numbered embodiments, wherein the type B protein feed(s) when combined comprise 1-45% w / w BLG by weight of the combined type B protein feed(s), more preferably 3-40% w / w, even more preferably 5-36% w / w, and most preferably 7-34% w / w BLG by weight of the combined type B protein feed(s).

[0532] Numbered Embodiment 34. A method according to any of the preceding numbered embodiments, wherein the initial protein solution comprises a total amount of type A protein feed in the range of 1-99% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w, and most preferably 30-60% w / w by weight of the initial protein solution.

[0533] Numbered Embodiment 35. A method according to any of the preceding numbered embodiments, wherein the initial protein solution comprises a total amount of type B protein feed(s) in the range of 1-99% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w, and most preferably 30-60% w / w by weight of the initial protein solution.

[0534] Numbered embodiment 36. The method according to any of the preceding numbered embodiments, wherein the initial protein solution comprises: - a total amount of type A protein feed(s) in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w, most preferably 30-60% w / w based on the weight of the initial protein solution, and - a total amount of type B protein feed(s) in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w, most preferably 30-60% w / w based on the weight of the initial protein solution.

[0535] Numbered embodiment 37. The method according to any of the preceding numbered embodiments, wherein the initial protein solution comprises: - a total amount of type A protein feed(s) in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w, most preferably 30-60% w / w based on the weight of the initial protein solution, with a pH of 5.8-8; and - a total amount of type B protein feed(s) having a pH of 3-5.2 in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w, most preferably 30-60% w / w based on the weight of the initial protein solution.

[0536] Numbered embodiment 38. The method according to any of the preceding numbered embodiments, wherein the initial protein solution comprises: - a total amount of type A protein feed(s) in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w, most preferably 30-60% w / w based on the weight of the initial protein solution, with a pH of 6.0-8, and - a total amount of type B protein feed(s) having a pH of 3-5.0 in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w, most preferably 30-60% w / w based on the weight of the initial protein solution.

[0537] Numbered embodiment 39. The method according to any of the preceding numbered embodiments, wherein the initial protein solution comprises: - a total amount of type A protein feed(s) in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w, most preferably 30-60% w / w based on the weight of the initial protein solution, with a pH of 6.1-8, and - a total amount of type B protein feed(s) having a pH of 3-4.9 of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w, most preferably 30-60% w / w based on the weight of the initial protein solution.

[0538] Numbered Embodiment 40. A method according to any of the preceding numbered embodiments, wherein at least one of the protein supplies used to prepare the initial protein solution is in liquid form.

[0539] Numbered Embodiment 41. The method according to any of the preceding numbered embodiments, wherein all of the protein feed is in liquid form.

[0540] Numbered Embodiment 42. The method according to any of the previous numbered embodiments, wherein at least one of the protein supplies is in a dry form, preferably a powder form, and at least one of the protein supplies is in a liquid form.

[0541] Numbered Embodiment 43. The method according to any of the preceding numbered embodiments, wherein all of the protein supplies are in dry form.

[0542] Numbered embodiment 44. A method according to any of the preceding numbered embodiments, further comprising a step d) of washing the BLG crystals, e.g., the separated crystals obtained from step c).

[0543] Numbered Embodiment 45. The method according to any of the preceding numbered embodiments, further comprising a step e) of recrystallizing the BLG crystals, e.g., the BLG crystals obtained from step c) or d).

[0544] Numbered Embodiment 46. The method according to any of the preceding numbered embodiments, further comprising a step f) of drying the BLG-containing composition from step b), c), d), or e).

[0545] Numbered Embodiment 47. The method according to any of the previous numbered embodiments, wherein the initial protein solution of step a) comprises at least 1% w / w non-BLG solids relative to total solids.

[0546] Numbered Embodiment 48. The method according to any of the preceding numbered embodiments, wherein the initial protein solution of step a) comprises at least 5% w / w ALA relative to the total amount of protein.

[0547] Numbered embodiment 49. The method according to any of the preceding numbered embodiments, wherein the initial protein solution of step a) comprises at least 15% w / w additional whey protein based on the total amount of protein.

[0548] Numbered Embodiment 50. The method according to any of the preceding numbered embodiments, wherein the initial protein solution of step a) comprises at most 40% w / w BLG relative to the total amount of protein.

[0549] Numbered Embodiment 51. The method according to any of the previous numbered embodiments, wherein the initial protein solution of step a) comprises at least 5% w / w BLG by weight of the initial protein solution.

[0550] Numbered Embodiment 52. The method according to any of the preceding numbered embodiments, wherein the protein supply is prepared from whey protein concentrate, whey protein concentrate, whey protein isolate, and / or whey protein isolate.

[0551] Numbered Embodiment 53. A method according to any of the preceding numbered embodiments, wherein the ratio between the conductivity of the initial protein solution and the total amount of protein is at most 0.3.

[0552] Numbered Embodiment 54. A method according to any of the preceding numbered embodiments, wherein the UF permeate conductivity of the initial protein solution is at most 7 mS / cm.

[0553] Numbered embodiment 55. The method according to any of the preceding numbered embodiments, wherein one or more of the protein supply(s) is prepared by subjecting one or more whey protein materials to one or more of the following adjustments: -adjusting the pH, -Reducing electrical conductivity - Reducing temperature - increasing protein concentration, and -Adding agents that reduce water activity.

[0554] Numbered Embodiment 56. The method according to any of the preceding numbered embodiments, wherein preparing the one or more protein supplies comprises adjusting the pH of the whey protein material.

[0555] Numbered Embodiment 57. A method according to any of the preceding numbered embodiments, wherein preparing the one or more protein supplies comprises reducing the conductivity of the whey protein material.

[0556] Numbered Embodiment 58. The method according to any of the preceding numbered embodiments, wherein preparing the one or more protein supplies comprises reducing the temperature of the whey protein material.

[0557] Numbered Embodiment 59. The method according to any of the preceding numbered embodiments, wherein preparing the one or more protein supplies comprises increasing the total protein concentration of the whey protein material.

[0558] Numbered Embodiment 60. The method according to any of the preceding numbered embodiments, wherein one or more of the protein supplies are derived from whey protein concentrate, whey protein concentrate, whey protein isolate, and / or whey protein isolate.

[0559] Numbered Embodiment 61. The method according to any of the preceding numbered embodiments, wherein one or more of the protein supplies comprises whey protein concentrate, whey protein concentrate, whey protein isolate, and / or whey protein isolate protein.

[0560] Numbered embodiment 62. The method according to any of the preceding numbered embodiments, wherein the crystallization of BLG in step b) comprises one or more of the following: - waiting for crystallization to occur, - addition of crystallization seeds, -Increasing the supersaturation of BLG even further, and / or -Mechanical stimulation.

[0561] Numbered embodiment 63. A method according to any of the preceding numbered embodiments, wherein the crystallization of BLG in step b) comprises the addition of one or more additional protein supplies comprising BLG while the crystallization of BLG is progressing.

[0562] Numbered embodiment 64. A method according to any of the preceding numbered embodiments, wherein step c) comprises separating the BLG crystals from the remainder of the initial protein solution.

[0563] Numbered embodiment 65. The method according to numbered embodiment 64, wherein step c) comprises separating the BLG crystals to a solids content of at least 30% w / w, preferably at least 40% w / w, and even more preferably at least 50% w / w.

[0564] Numbered embodiment 66. A method according to any of numbered embodiments 44-65, wherein the washing in step d) comprises contacting the separated BLG crystals with a washing solution without completely dissolving the BLG crystals, and then separating the remaining BLG crystals from the washing solution.

[0565] Numbered embodiment 67. A method according to numbered embodiment 66, wherein the washing in step d) dissolves at most 80% w / w of the initial amount of BLG crystals, preferably at most 50% w / w, and even more preferably at most 20% w / w of the initial amount of BLG crystals.

[0566] Numbered embodiment 68. The method according to any of numbered embodiments 45-67, wherein the recrystallization step comprises: - dissolving the separated BLG crystals in a recrystallization solution; - adjusting the recrystallization solution to obtain a supersaturation with respect to BLG; - crystallizing BLG in a supersaturated adjusted recrystallization solution; and - Separating the BLG crystals from the remaining conditioned recrystallization solution.

[0567] Numbered Embodiment 69. The method according to any of numbered embodiments 45-68, wherein the BLG crystals of step d) are recrystallized at least twice.

[0568] Numbered Embodiment 70. The method according to any of numbered embodiments 46-69, wherein the drying step includes one or more of spray drying, freeze drying, spin flash dryer, rotary drying, and / or fluidized bed drying.

[0569] Numbered embodiment 71. A method according to any of the preceding numbered embodiments, comprising: -In relation to A-type protein supply: - if only a single type A protein supply is used, it has: - When two or more type A protein feeds are used, they, when mixed, have: a pH of at least 5.6, more preferably at least 5.7, even more preferably at least 5.8, and most preferably at least 5.9; and - In relation to B protein supply: - if only a single B-type protein supply is used, it has: - if two or more B type protein feeds are used, they, when mixed, have the following: A pH of at most 5.4, more preferably at most 5.3, even more preferably 5.2, most preferably 5.1.

[0570] Numbered embodiment 72. A method according to any of the preceding numbered embodiments, comprising: -In relation to A-type protein supply: - if only a single type A protein supply is used, it has: - When two or more type A protein feeds are used, they, when mixed, have: a pH of at least 6.0, more preferably at least 6.1, even more preferably at least 6.2, and most preferably at least 6.3; and - In relation to B protein supply: - if only a single B-type protein supply is used, it has: - if two or more B type protein feeds are used, they, when mixed, have the following: A pH of at most 5.0, more preferably at most 4.9, even more preferably 4.8, most preferably 4.7.

[0571] Numbered embodiment 73. A method according to any of the preceding numbered embodiments, comprising, in connection with the type A protein supply: - if only a single type A protein supply is used, it has: - if two or more type A protein feeds are used, they, when mixed, have the following: A pH in the range of 5.6-10, more preferably 5.8-8, even more preferably 6.0-7, most preferably 6.1-6.5.

[0572] Numbered embodiment 74. A method according to any of the preceding numbered embodiments, comprising, in connection with the type A protein supply: - if only a single type A protein supply is used, it has: - if two or more type A protein feeds are used, they, when mixed, have the following: A pH in the range of 6.0-8, more preferably 6.1-7.5, even more preferably 6.2-7.0, most preferably 6.2-6.5.

[0573] Numbered embodiment 75. A method according to any of the preceding numbered embodiments, comprising, in relation to the B-type protein supply: - if only a single B-type protein supply is used, it has: - if two or more B type protein feeds are used, they, when mixed, have the following: A pH in the range of 2-5.4, more preferably 3-5.2, even more preferably 4-5.0, most preferably 4.5-4.9.

[0574] Numbered embodiment 76. A method according to any of the preceding numbered embodiments, comprising, in relation to the B-type protein supply: - if only a single B-type protein supply is used, it has: - if two or more B type protein feeds are used, they, when mixed, have the following: A pH in the range of 4.0-5.0, more preferably 4.1-4.9, even more preferably 4.3-4.8, most preferably 4.5-4.8.

[0575] Numbered embodiment 77. A method according to any of the preceding numbered embodiments, comprising: at least one of the one or more type A protein supplies does not contain BLG crystals, more preferably none of the one or more type A protein supplies contains BLG crystals; and / or A method wherein at least one of the one or more type B protein supplies does not comprise BLG crystals, more preferably none of the one or more type B protein supplies comprises BLG crystals.

[0576] Numbered embodiment 78. A method according to any of the preceding numbered embodiments, wherein at least one of the one or more type A protein feeds is not supersaturated with respect to BLG, more preferably none of the one or more type A protein feeds is supersaturated with respect to BLG, and / or A method wherein at least one of the one or more type B protein supplies is not supersaturated with respect to BLG, and more preferably none of the one or more types is supersaturated with respect to BLG.

[0577] Numbered embodiment 79. A method according to any of the preceding numbered embodiments, wherein the type A and type B protein feed(s) that do not comprise BLG crystals contribute at least 1% w / w of the BLG content of the initial protein solution, more preferably at least 10% w / w, even more preferably at least 20% w / w, and most preferably at least 40% w / w.

[0578] Numbered embodiment 80. A method according to any of the preceding numbered embodiments, wherein the type A and type B protein feed(s) that do not comprise BLG crystals contribute at least 50% w / w of the BLG content of the initial protein solution, more preferably at least 70% w / w, even more preferably at least 80% w / w, and most preferably at least 90% w / w.

[0579] Numbered embodiment 81. A method according to any of the preceding numbered embodiments, comprising, in connection with the type A protein supply: - if only a single type A protein supply is used, it has: - if two or more type A protein feeds are used, they, when mixed, have the following: A temperature of at most 60°C, more preferably at most 50°C, even more preferably at most 40°C, most preferably at most 30°C.

[0580] Numbered embodiment 82. A method according to any of the preceding numbered embodiments, comprising, in relation to the B-type protein supply: - if only a single B-type protein supply is used, it has: - if two or more B type protein feeds are used, they, when mixed, have the following: A temperature of at most 60°C, more preferably at most 50°C, even more preferably at most 40°C, most preferably at most 30°C.

[0581] Numbered embodiment 83. A method according to any of the preceding numbered embodiments, comprising, in connection with the type A protein supply: - if only a single type A protein supply is used, it contains: - if two or more type A protein feeds are used, they, when mixed, may comprise the following process: At least 1% w / w BLG based on the total weight of the type A protein feed, more preferably at least 3% w / w, even more preferably at least 5% w / w, and most preferably at least 7% w / w BLG based on the total weight of the type A protein feed.

[0582] Numbered embodiment 84. A method according to any of the preceding numbered embodiments, comprising, in relation to the B-type protein supply: - if only a single B-type protein supply is used, it contains: - if two or more B type protein supplies are used, they, when mixed, may comprise the following process: At least 1% w / w BLG based on the total weight of the type B protein feed, more preferably at least 3% w / w, even more preferably at least 5% w / w, and most preferably at least 7% w / w BLG based on the total weight of the type B protein feed.

[0583] Numbered embodiment 85. The method according to any of the preceding numbered embodiments, wherein the initial protein solution comprises: - a total amount of type A protein feed(s) in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w, most preferably 30-60% w / w based on the weight of the initial protein solution, with a pH of 6.0-8, and - a total amount of type B protein feed(s) having a pH of 3-5.0 in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w, most preferably 30-60% w / w based on the weight of the initial protein solution.

[0584] Numbered Embodiment 86. A method according to any of the preceding numbered embodiments, comprising: - in relation to the A-type protein supply, if only a single A-type protein supply is used, it has: If two or more type A protein feeds are used, they, when mixed, have: - a content of BLG of 4-32% w / w, more preferably 6-32% w / w, more preferably 8-30% w / w, even more preferably 10-28% w / w, most preferably 12-26% w / w, based on the total weight of the A-type protein feed, - a pH in the range of 5.6-10, more preferably 5.8-8, even more preferably 6.0-7, most preferably 6.1-6.5; - preferably at a temperature of at most 20°C, more preferably at most 15°C, even more preferably at most 10°C, most preferably at most 5°C, a content of BLG in an amount of preferably at least 30% w / w, more preferably at least 40% w / w, even more preferably at least 50% w / w, most preferably at least 55% w / w, relative to the total amount of protein; -One or more of the following: a conductivity of at most 7 mS / cm, even more preferably at most 5 mS / cm, and most preferably at most 3 mS / cm, and a ratio between the conductivity and the total amount of protein of at most 0.3, more preferably at most 0.25, most preferably at most 0.20; And, - in relation to the B type protein feed, if only a single B type protein feed is used, it has the following, or if two or more B type protein feeds are used, they, when mixed, have the following: - a content of BLG of 4-32% w / w relative to the total weight of the B protein feed, more preferably 6-32% w / w, more preferably 8-30% w / w, even more preferably 10-28% w / w, most preferably 12-26% w / w relative to the total weight of the B protein feed, - a pH in the range of 2-5.4, more preferably 3-5.2, even more preferably 4-5.0, most preferably 4.5-4.9; - preferably at a temperature of at most 20°C, more preferably at most 15°C, even more preferably at most 10°C, most preferably at most 5°C, a content of BLG in an amount of preferably at least 30% w / w, more preferably at least 40% w / w, even more preferably at least 50% w / w, most preferably at least 55% w / w, relative to the total amount of protein; -One or more of the following: - a conductivity of at most 7 mS / cm, more preferably at most 5 mS / cm, most preferably at most 3 mS / cm, and - a ratio between the conductivity and the total amount of protein of at most 0.3, more preferably at most 0.25, most preferably at most 0.20.

[0585] Numbered Embodiment 87. A method according to any of the preceding numbered embodiments, comprising: - in relation to the A-type protein supply, if only a single A-type protein supply is used, it has: If two or more type A protein feeds are used, they, when mixed, have: - a content of BLG of 4-32% w / w, more preferably 6-32% w / w, more preferably 8-30% w / w, even more preferably 10-28% w / w, most preferably 12-26% w / w, based on the total weight of the A-type protein feed, - a pH in the range of 5.6-10, more preferably 5.8-8, even more preferably 6.0-7, most preferably 6.1-6.5; - preferably at a temperature of at most 20°C, more preferably at most 15°C, even more preferably at most 10°C, most preferably at most 5°C, a content of BLG in an amount of preferably at least 30% w / w, more preferably at least 40% w / w, even more preferably at least 50% w / w, most preferably at least 55% w / w, relative to the total amount of protein; -One or more of the following: a conductivity of at most 7 mS / cm, even more preferably at most 5 mS / cm, and most preferably at most 3 mS / cm, and a ratio between the conductivity and the total amount of protein of at most 0.3, more preferably at most 0.25, most preferably at most 0.20; And, - in relation to the B type protein feed, if only a single B type protein feed is used, it has the following, or if two or more B type protein feeds are used, they, when mixed, have the following: - a content of BLG of 4-32% w / w relative to the total weight of the B protein feed, more preferably 6-32% w / w, more preferably 8-30% w / w, even more preferably 10-28% w / w, most preferably 12-26% w / w relative to the total weight of the B protein feed, - a pH in the range of 2-5.4, more preferably 3-5.2, even more preferably 4-5.0, most preferably 4.5-4.9; - preferably at a temperature of at most 20°C, more preferably at most 15°C, even more preferably at most 10°C, most preferably at most 5°C, a content of BLG in an amount of preferably at least 30% w / w, more preferably at least 40% w / w, even more preferably at least 50% w / w, most preferably at least 55% w / w, relative to the total amount of protein; -One or more of the following: - a conductivity of at most 7 mS / cm, more preferably at most 5 mS / cm, most preferably at most 3 mS / cm, and - a ratio between the conductivity and the total amount of protein of at most 0.3, more preferably at most 0.25, most preferably at most 0.20.

[0586] Numbered embodiment 88. The method according to any of the preceding numbered embodiments, wherein the type A protein supply(s) and the type B protein supply(s) contribute at least 50% w / w of the protein content of the initial protein solution, more preferably at least 70% w / w, even more preferably at least 80% w / w, and most preferably at least 90% w / w.

[0587] Numbered embodiment 89. The method according to any of the preceding numbered embodiments, wherein the type A protein supply(s) and the type B protein supply(s) contribute at least 92% w / w of the protein content of the initial protein solution, more preferably at least 94% w / w, even more preferably at least 96% w / w, and most preferably at least 99% w / w.

[0588] Numbered embodiment 90. The method according to any of the preceding numbered embodiments, wherein the type A protein supply(s) and the type B protein supply(s) contribute at least 50% w / w of the solids content of the initial protein solution, more preferably at least 70% w / w, even more preferably at least 80% w / w, and most preferably at least 90% w / w.

[0589] Numbered embodiment 91. The method according to any of the preceding numbered embodiments, wherein the type A protein feed(s) and type B protein feed(s) contribute at least 92% w / w of the solids content of the initial protein solution, more preferably at least 94% w / w, even more preferably at least 96% w / w, and most preferably at least 99% w / w.

[0590] Numbered embodiment 92. The method according to any of the preceding numbered embodiments, wherein the type A protein supply(s) and type B protein supply(s) contribute at least 50% w / w of the weight of the initial protein solution, more preferably at least 70% w / w, even more preferably at least 80% w / w, and most preferably at least 90% w / w.

[0591] Numbered embodiment 93. The method according to any of the preceding numbered embodiments, wherein the type A protein supply(s) and type B protein supply(s) contribute at least 92% w / w of the weight of the initial protein solution, more preferably at least 94% w / w, even more preferably at least 96% w / w, and most preferably at least 99% w / w.

[0592] Numbered embodiment 94. The method according to any of the preceding numbered embodiments, wherein the degree of supersaturation of the BLG in the initial protein solution is higher than both of the following supersaturations: - A type protein feed, if only a single type A protein feed is used, or - if two or more type A protein feeds are used, the type A protein feeds when combined, and - Type B protein feed, if only a single Type B protein feed is used, or - Type B protein feeds, when combined, if more than one Type B protein feed is used.

[0593] The present invention has been described above with reference to specific embodiments. However, other embodiments than those described above are equally possible within the scope of the invention. The different features and steps of the various embodiments and aspects of the invention can be combined in other ways than those described herein, unless otherwise specified.

[0594] Working Example Example 1: Crystallization based on mixing of two WPI streams introduction: During initial trials of large-scale implementation of the method of WO2018 / 115520, frequent spontaneous BLG crystallization events were observed in an ultrafiltration unit used to prepare whey protein solutions, leading to production interruptions and time-consuming cleaning of process units affected by the crystallization.

[0595] The inventors then had the idea that instead of producing a supersaturated protein solution of WO2018 / 115520 from a single feed, which may become supersaturated with respect to BLG under unfavourable conditions during preparation (e.g. the occurrence of high local protein concentrations and / or sudden cooling), it would be more prudent to produce a supersaturated whey protein solution from two (or more) feeds, each having a pH that is clearly not supersaturated with respect to BLG (or is only supersaturated to a limited extent), and then mixing the two (or more) feeds to obtain a suitable pH for supersaturation of BLG, i.e. a pH close to pH 5.5.

[0596] This new approach allows for the preparation of feeds with very high protein content and without the risk of uncontrolled BLG crystallization. In addition, this approach allows for the preparation of the feeds, and subsequently the initial protein solutions, at temperatures significantly lower than the optimal growth of the microorganisms, which is advantageous since less heat treatment is required to reduce the microbial load of the final product to an acceptable level. The inventors have further found that the pH adjustments made with the present type A and / or type B protein feeds contribute only to a very limited change in the conductivity of the initial protein solution, in contrast to the same pH changes made with conventional food acids or food bases. This is beneficial since the reduced conductivity of the initial protein solution typically results in higher BLG crystallization yields.

[0597] The feasibility of this new approach was demonstrated in this example, where the first protein feed (Batch 1, Type A protein feed) had a pH of 6.2 and the second protein feed (Batch 2, Type B protein feed) had a pH of 4.9. Despite the high protein concentrations, neither of the feeds was supersaturated. However, by simply mixing the two feeds in appropriate amounts (to obtain a pH close to 5.5), a supersaturated protein mixture was obtained from which BLG could be crystallized.

[0598] process: The whey feedstock for this example was a lactose-depleted UF retentate derived from sweet whey from a standard cheese production process, the whey was fat-reduced through a Synder FR membrane prior to use. Two batches were produced from this feedstock and conditioned in an ultrafiltration setup using an Alfa Laval GR82PE membrane with a 30 mill spacer and 1.5-3.0 bar feed pressure. The product was desalted using polish water as the diafiltration medium, the feed having a concentration of 18 percent TS (total solids) ±5 percent. Diafiltration was continued at least until the drop in conductivity in the retentate was less than 0.02 mS / cm over a 20 minute period during which the TS in the retentate was stable (±0.5 percent TS). The retentate was then concentrated to approximately 22 percent TS. The feed compositions for batches 1 and 2 can be seen in Tables 1.1 and 1.2.

[0599] The first batch (Batch 1) was pH adjusted to pH 6.1 with food grade hydrochloric acid (HCl) before UF treatment at 10-12°C. The other batch (Batch 2) was pH adjusted to pH 4.9 with food grade hydrochloric acid (HCl) before UF treatment at 10-12°C. [Table 1] [Table 2]

[0600] After UF treatment, the temperature of the two batches was adjusted to 10° C., and then portions of the batches were combined in five different ratios to generate protein solutions in which crystallization was carried out. See Table 1.3 for details. Each mixture was seeded with 0.1% w / w seed material at 10° C., cooled to 5° C. after seeding, and left with stirring overnight.

[0601] To determine whether batches 1 and 2 were already supersaturated, samples of each of the two batches were taken before mixing as above, similarly sheeted, and stored overnight at 10° C. Visual inspection by microscopy the following morning showed that no crystals were present after incubation, therefore it can be concluded that batches 1 and 2 were not supersaturated.

[0602] Seed material was generated by combining small portions of batches 1 and 2, allowing them to reach a final pH of 5.5, and then adding a small amount of dried BLG crystal material. The seed solution was then placed on ice with stirring for 1 hour.

[0603] Samples of the protein solution were taken before seeding and after crystallization and centrifuged at 3000 g for 5 min, and samples of the supernatant were analyzed by RP-HPLC as described in Example 1 of WO2018 / 115520, and the crystallization yield was calculated by the following formula:

number

[0604] result: As can be seen from the table below, it is possible to adjust the whey protein feed to a pH region with low (or even no) supersaturation with respect to BLG, thus reducing the risk of spontaneous crystallization in the UF plant and increasing the maximum protein concentration that can be used without spontaneous BLG crystallization. The two feeds were then mixed to provide an initial protein solution with high BLG supersaturation. [Table 3]

[0605] Example 2: Crystallization based on mixing of two WPI streams using commercial WPI The whey materials for this example were commercially available Lacprodan DI-9213 and another homemade pH 6.2 WPI.

[0606] A pH 6.2 WPI was made from lactose-depleted UF retentate derived from sweet whey from a standard cheese production process, the whey was fat-reduced prior to use through a Synder FR membrane. The feed was pH adjusted to pH 6.2 prior to UF processing at 10-12°C using food grade hydrochloric acid. Batches were produced from this feed and conditioned using an ultrafiltration setup using an Alfa Laval GR82PE membrane with a 30 mil spacer and 1.5-3.0 bar feed pressure. The product was desalted using polish water as the diafiltration medium, the feed had a concentration of 18 percent TS ± 5 percent. Diafiltration was continued at least until the drop in conductivity in the retentate was less than 0.02 mS / cm over a 20 minute period during which the TS in the retentate was stable (± 0.5 percent TS). The retentate was then concentrated to approximately 22 percent TS, the feed compositions for batches 1 and 2 can be seen in Tables 2.1 and 2.2.

[0607] Batch 2 was made from Lacprodan DI-9213 in powder form. The powder was conditioned by rehydrating it to a TS of 16.3 and leaving it at 10° C. with stirring until all the powder was dissolved. [Table 4] [Table 5]

[0608] After conditioning, the two batches were processed and analyzed as in Example 1 (except that only two solutions were made).

[0609] The properties of the mixtures can be seen in Table 2.3.

[0610] result: As can be seen from the table below, it is possible to adjust the whey protein feed to a pH region that is not supersaturated with respect to BLG, thus reducing the risk of spontaneous crystallization and increasing the protein concentration to a level that is possible without risk of crystallization in the UF plant, and then the two are subsequently combined to obtain a high degree of supersaturation. As expected, it can be seen that higher conductivity affects the yield. [Table 6]

[0611] Example 3: Crystallization based on mixing of two WPI streams, pH 4.0 and 6.1 The whey feedstock for this example was a lactose-depleted UF retentate derived from sweet whey from a standard cheese production process, and the whey was fat-reduced prior to use through a Synder FR membrane. Two batches were produced from this feedstock and conditioned by an ultrafiltration setup using an Alfa Laval GR82PE membrane with a 30 mil spacer and 1.5-3.0 bar feed pressure. The product was desalted using polish water as the diafiltration medium, and the feed had a concentration of 18 percent TS ± 5 percent. Diafiltration was continued at least until the drop in conductivity in the retentate was less than 0.02 mS / cm over a 20 minute period, during which the TS in the retentate was stable (± 0.5 percent TS). The retentate was then concentrated to approximately 22 percent TS, and the feed composition for batches 1 and 2 can be seen in Tables 3.1 and 3.2.

[0612] One batch (Batch 1) was pH adjusted with food grade hydrochloric acid (HCl) to pH 6.1 before UF processing at 10-12°C. The other batch (Batch 2) was pH adjusted with food grade hydrochloric acid (HCl) to pH 4.0 before UF processing at 10-12°C. [Table 7] [Table 8]

[0613] After conditioning, the two batches were processed and analyzed as in Example 1 of WO2018 / 115520 (except that only four solutions were made).

[0614] The properties of the mixtures can be seen in Table 3.3.

[0615] result: As can be seen from the table below, it is possible to prepare a whey protein feed in a region with low supersaturation with respect to BLG, thus reducing the risk of spontaneous crystallization and increasing the protein concentration to a level that is possible without risk of crystallization in the UF plant, and then the two are subsequently combined to obtain a high supersaturation. [Table 9]

[0616] Example 4: Crystal Size Distribution The whey feedstock for this example was lactose-depleted UF retentate derived from sweet whey from a standard cheese production process, and the whey was fat-reduced before use through a Synder FR membrane. From this feedstock, two batches for each crystallization were produced and conditioned by ultrafiltration setup using an Alfa Laval GR82PE membrane with a 30 mil spacer and 1.5-3.0 bar feed pressure. The product was desalted using polish water as the diafiltration medium, and the feed had a concentration of 18 percent TS ± 5 percent. Diafiltration was continued at least until the drop in conductivity in the retentate was less than 0.02 mS / cm over a 20 minute period, during which the TS in the retentate was stable (± 0.5 percent TS). The retentate was then concentrated to approximately 22 percent TS, and the feed composition for batches 1 and 2 can be seen in the table below.

[0617] One batch (Batch 1) was pH adjusted with food grade hydrochloric acid (HCl) to approximately pH 6.1 prior to UF processing at 10-12° C. The other batch (Batch 2) was pH adjusted with food grade hydrochloric acid (HCl) to pH 4.8 prior to UF processing at 10-12° C.

[0618] Crystallization was carried out in a 300 L crystallization tank using 0.1% w / w seed material, which was generated as in Example 1.

[0619] First Crystallization [Table 10] [Table 11] After conditioning, the two batches were combined to obtain a final pH of 5.50, the mixture was seeded, immediately cooled to 5° C., and left overnight to crystallize. A sample of the slurry was analyzed as described in Example 1, and the yield was calculated. The particle size distribution of the crystal slurry was analyzed by Malvern particle size characterization.

[0620] Second Crystallization [Table 12] [Table 13] After conditioning, the two batches were combined to obtain a final pH of 5.45, the mixture was seeded, immediately cooled to 5° C., and left to crystallize overnight. A sample of the slurry was analyzed as described in Example 1, and the yield was calculated. The particle size distribution of the crystal slurry was analyzed by Malvern particle size characterization.

[0621] Third crystallization: [Table 14] [Table 15] After conditioning, the two batches were combined to obtain a pH of 5.80, the mixture was seeded and incubated for 1 hour, then adjusted to a final pH of 5.45 by further addition of batch 2 over 2 hours, then immediately cooled to 5° C. and left overnight to crystallize. A sample of the slurry was analyzed as described in Example 1 and the yield was calculated. The particle size distribution of the crystal slurry was analyzed by Malvern particle size characterization.

[0622] result: In Table 4.7, the crystallization results are shown and as can be seen, the crystal size distribution was similar even though different raw materials were used and the crystallization process was different. [Table 16]

Claims

1. A method for preparing an edible composition comprising beta-lactoglobulin (BLG), preferably in crystallized and / or isolated form, comprising the steps of: a) preparing an initial protein solution comprising BLG, said initial protein solution being supersaturated with respect to BLG and having a pH in the range of 5-6; b) crystallizing BLG in said supersaturated, initial protein solution, preferably in salting mode, thereby obtaining a BLG crystal-containing solution; and c) Optionally, separating the BLG crystals from the remaining liquid of the BLG crystal-containing solution. Including, Step a) comprises preparing said initial protein solution by mixing one or more type A protein feed(s) with one or more type B protein feed(s), - "Type A protein feed" is defined as a protein feed that contains BLG and has a pH of at least 5.6, and - A method wherein a "type B protein feed" is defined as a protein feed that contains BLG and has a pH of at most 5.

4.

2. - In relation to A-type protein supply: - if only a single type A protein feed is used, it has: - If two or more type A protein feeds are used, they, when mixed, have: a pH of at least 5.6, more preferably at least 5.7, even more preferably at least 5.8, and most preferably at least 5.9; and - In relation to B-type protein supply: - if only a single B-type protein supply is used, it has: - if two or more type B protein feeds are used, they, when mixed, have: A pH of at most 5.4, more preferably at most 5.3, even more preferably 5.2, most preferably 5.

1.

3. - In relation to A-type protein supply: - if only a single type A protein feed is used, it has: - If two or more type A protein feeds are used, they, when mixed, have: a pH of at least 6.0, more preferably at least 6.1, even more preferably at least 6.2, and most preferably at least 6.3; and - In relation to B-type protein supply: - if only a single B-type protein supply is used, it has: - if two or more type B protein feeds are used, they, when mixed, have: A pH of at most 5.0, more preferably at most 4.9, even more preferably 4.8, most preferably 4.

7.

4. In relation to Type A protein supply: - if only a single type A protein feed is used, it has: - if two or more type A protein feeds are used, they, when mixed, have: A pH in the range of 5.6-10, more preferably 5.8-8, even more preferably 6.0-7, most preferably 6.1-6.

5.

5. In relation to Type A protein supply: - if only a single type A protein feed is used, it has: - if two or more type A protein feeds are used, they, when mixed, have: A pH in the range of 6.0-8, more preferably 6.1-7.5, even more preferably 6.2-7.0, most preferably 6.2-6.

5.

6. In relation to B-type protein supplies: - if only a single B-type protein supply is used, it has: - if two or more type B protein feeds are used, they, when mixed, have: A pH in the range of 2-5.4, more preferably 3-5.2, even more preferably 4-5.0, most preferably 4.5-4.

9.

7. In relation to B-type protein supplies: - if only a single B-type protein supply is used, it has: - if two or more type B protein feeds are used, they, when mixed, have: A pH in the range of 4.0-5.0, more preferably 4.1-4.9, even more preferably 4.3-4.8, most preferably 4.5-4.

8.

8. at least one of said one or more type A protein supplies does not contain BLG crystals, more preferably none of said one or more type A protein supplies contains BLG crystals; and / or 8. The method of any of claims 1 to 7, wherein at least one of said one or more type B protein supplies does not comprise BLG crystals, more preferably none of said one or more type B protein supplies comprises BLG crystals.

9. at least one of said one or more type A protein feeds is not supersaturated with respect to BLG, more preferably none of said one or more type A protein feeds is supersaturated with respect to BLG; and / or A method according to any preceding claim, wherein at least one of said one or more type B protein supplies is not supersaturated with respect to BLG, more preferably none of said one or more types is supersaturated with respect to BLG.

10. 10. The method according to any of claims 1 to 9, wherein the type A and type B protein feed(s) free of BLG crystals contribute at least 1% w / w of the BLG content of said initial protein solution, more preferably at least 10% w / w, even more preferably at least 20% w / w, most preferably at least 40% w / w.

11. 11. The method according to any of claims 1 to 10, wherein the type A and type B protein feed(s) free of BLG crystals contribute at least 50% w / w of the BLG content of said initial protein solution, more preferably at least 70% w / w, even more preferably at least 80% w / w, most preferably at least 90% w / w.

12. In relation to Type A protein supply: - if only a single type A protein feed is used, it has: - if two or more type A protein feeds are used, they, when mixed, have: A temperature of at most 60°C, more preferably at most 50°C, even more preferably at most 40°C, most preferably at most 30°C.

13. In relation to B-type protein supplies: - if only a single B-type protein supply is used, it has: - if two or more type B protein feeds are used, they, when mixed, have: A temperature of at most 60°C, more preferably at most 50°C, even more preferably at most 40°C, most preferably at most 30°C.

14. In relation to Type A protein supply: - if only a single type A protein supply is used, it comprises: - if two or more type A protein supplies are used, they, when mixed, comprise: At least 1% w / w BLG based on the total weight of the Type A protein feed, more preferably at least 3% w / w, even more preferably at least 5% w / w, and most preferably at least 7% w / w BLG based on the total weight of the Type A protein feed.

15. In relation to B-type protein supplies: - if only a single B-type protein supply is used, it contains: - if two or more B-type protein supplies are used, they, when mixed, comprise: At least 1% w / w BLG based on the total weight of the B protein feed, more preferably at least 3% w / w, even more preferably at least 5% w / w, and most preferably at least 7% w / w BLG based on the total weight of the B protein feed.

16. A method according to any preceding claim, wherein the initial protein solution comprises a total amount of type A protein feed in the range of 1-99% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w, and most preferably 30-60% w / w, based on the weight of the initial protein solution.

17. A method according to any preceding claim, wherein the initial protein solution comprises a total amount of type B protein feed(s) in the range of 1-99% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w, and most preferably 30-60% w / w by weight of the initial protein solution.

18. The method of any one of claims 1 to 17, wherein the initial protein solution comprises: - a total amount of type A protein feed(s) in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w, most preferably 30-60% w / w based on the weight of said initial protein solution, having a pH of 5.8-8, and - a total amount of type B protein feed(s) in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w, most preferably 30-60% w / w based on the weight of said initial protein solution, having a pH of 3-5.

2.

19. The method according to any of claims 1 to 18, wherein the initial protein solution comprises: - a total amount of type A protein feed(s) in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w, most preferably 30-60% w / w based on the weight of said initial protein solution, having a pH of 6.0-8, and - a total amount of type B protein feed(s) in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w, most preferably 30-60% w / w based on the weight of said initial protein solution, having a pH of 3-5.

0.

20. 20. The method of any of claims 1 to 19, wherein the type A protein supply(s) and the type B protein supply(s) contribute at least 50% w / w of the protein content of the initial protein solution, more preferably at least 70% w / w, even more preferably at least 80% w / w, most preferably at least 90% w / w.

21. 21. The method of any of claims 1 to 20, wherein the type A protein supply(s) and the type B protein supply(s) contribute at least 92% w / w of the protein content of said initial protein solution, more preferably at least 94% w / w, even more preferably at least 96% w / w, most preferably at least 99% w / w.

22. 22. The method of any of claims 1 to 21, wherein the type A protein supply(s) and the type B protein supply(s) contribute at least 50% w / w of the solids content of the initial protein solution, more preferably at least 70% w / w, even more preferably at least 80% w / w, most preferably at least 90% w / w.

23. 23. The method of any of claims 1 to 22, wherein the type A protein supply(s) and the type B protein supply(s) contribute at least 92% w / w of the solids content of the initial protein solution, more preferably at least 94% w / w, even more preferably at least 96% w / w, most preferably at least 99% w / w.

24. 24. The method of any of claims 1 to 23, wherein the type A protein supply(s) and type B protein supply(s) contribute at least 50% w / w of the weight of said initial protein solution, more preferably at least 70% w / w, even more preferably at least 80% w / w, most preferably at least 90% w / w.

25. 25. The method of any of claims 1 to 24, wherein the type A protein supply(s) and the type B protein supply(s) contribute at least 92% w / w of the weight of said initial protein solution, more preferably at least 94% w / w, even more preferably at least 96% w / w, most preferably at least 99% w / w.

26. The method according to any of claims 1 to 25, wherein the degree of supersaturation of the BLG of the initial protein solution is higher than both of the following supersaturations: - A type protein feed, if only a single A type protein feed is used, or - the type A protein feeds, when combined, if more than one type A protein feed is used; and - B type protein feed, if only a single B type protein feed is used, or - Type B protein feeds, when combined, if more than one Type B protein feed is used.

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