Production of novel beta-lactoglobulin preparations and related methods, uses and food products

Abstract

This application relates to the production of novel β-lactoglobulin preparations and related methods, uses, and food products. The present invention relates to novel methods for producing isolated β-lactoglobulin compositions and / or compositions containing crystallized β-lactoglobulin. The present invention also relates to novel β-lactoglobulin compositions, uses of these compositions, and food products containing these compositions.

Description

[0001] This application is a divisional application of an application filed on December 22, 2017, with application number 201780084095.1, and invention name “Production of novel β-lactoglobulin preparations and related methods, uses and food products”. Technical Field

[0002] The present invention relates to novel methods for producing isolated β-lactoglobulin compositions and / or compositions containing crystallized β-lactoglobulin. The present invention also relates to novel β-lactoglobulin compositions, uses of these compositions and food products comprising these compositions. Background Art

[0003] The concept of milk protein fractionation is well known in the art and has developed over the past few decades into a range of techniques for preparing compositions enriched in various milk protein species, each with specific properties and characteristics.

[0004] The isolation of beta-lactoglobulin (BLG) from milk serum or whey is the subject of numerous publications and typically involves multiple separation steps and often chromatographic techniques to obtain a purified beta-lactoglobulin product.

[0005] For example, 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) describe the purification of BLG from fresh milk by low-temperature acid coagulation of casein and by subjecting the resulting acid whey to a combination of affinity chromatography (DEAE agarose) and gel permeation chromatography. The BLG composition obtained is said to contain 0.985 g of β-lactoglobulin per 1 g of protein.

[0006] Slack et al. (Journal of Food Processing and Preservation, Vol. 10, 1986, pp. 19-30) explored a different approach and prepared a BLG-rich precipitate by adjusting the pH of demineralized acid whey and sweet whey to pH 4.65 and separating the formed precipitate by centrifugation and decantation. The resulting precipitate particles were described as relatively insoluble and contained significant amounts of protein impurities in the otherwise BLG. No crystal formation was observed. It should be noted that the BLG precipitate that can form at pH 4.65 is not a BLG crystal.

[0007] Palmer (Crystalline Globulin from Cow's Milk, J. Biol. Chem., Vol. 104, 1934, pp. 359-372) reported a laborious and time-consuming process for producing acid whey-based protein crystals using several sequences of salt precipitation of unwanted proteins, pH adjustment, and dialysis (to remove other unwanted proteins). Finally, when a highly purified BLG solution was obtained, BLG crystallized. The process lasted for more than 12 days and required the addition of toluene. Therefore, the procedure disclosed in Palmer is incompatible with safe food production and provides a clearly inedible food product.

[0008] 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) disclosed an improvement over the Palmer method that allowed the preparation of beta-lactoglobulin crystals in days rather than weeks. However, the improved method still required the removal of unwanted proteins prior to crystallization and also used toluene for crystallization, which made it incompatible with safe food production.

[0009] JP H10 218755 A discloses the production of a cosmetic composition containing a melanin production inhibitor, which includes BLG as an active ingredient. The document also indicates that BLG can be isolated, for example, by adding hydrochloric acid to milk to precipitate casein, followed by filtration to obtain whey. The pH of the whey is adjusted to 6.0, and ammonium sulfate is added in a semi-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 and dialyzed at pH 5.2 to separate crystals, and β-lactoglobulin is prepared at a rate of approximately 1.8 g per liter of whey. However, the general process steps of the proposed process described in JP H10 218755 A are insufficient to result in the formation of BLG crystals. Therefore, the document does not disclose the realization of BLG or BLG crystal crystallization.

[0010] US Pat. No. 2,790,790 discloses a process for precipitating proteins from solution, and more specifically, for fractionally precipitating relatively unconjugated proteins from aqueous solutions using sodium chloride as a precipitant. The process is shown to be useful for isolating BLG by NaCl-induced precipitation at pH 3.6-3.8. In Example II of the document, it is shown that the NaCl precipitate can be dialyzed in the usual manner to form crystalline B-lactoglobulin. However, US Pat. No. 2,790,790 does not demonstrate that BLG crystals can actually be formed at pH 3.6-3.8, and does not mention the meaning of dialyzing the BLG precipitate in the "usual manner." Therefore, the document does not disclose the realization of BLG or BLG crystals. Summary of the Invention

[0011] Serendipitously, the inventors surprisingly discovered that high-purity BLG crystals can be prepared directly from a crude whey protein solution containing significant amounts of other whey proteins in addition to BLG, and without the use of organic solvents such as toluene. This is contrary to common wisdom in the art, which teaches that proteins must be highly purified before one wishes to crystallize them, and that not all proteins can be crystallized.

[0012] This discovery has the potential to transform the way whey proteins are processed and fractionated in the dairy industry and pioneer the efficient and gentle production of highly purified BLG that can be safely used as a food ingredient.

[0013] Thus, one aspect of the present invention relates to a method for preparing an edible composition comprising beta-lactoglobulin (BLG) in crystallized and / or isolated form, the method comprising the steps of:

[0014] a) providing a whey protein solution comprising BLG and at least one further whey protein, said whey protein solution being supersaturated with respect to BLG and having a pH in the range of 5-6,

[0015] b) crystallizing BLG in the supersaturated whey protein solution, and

[0016] c) Optionally, separating the BLG crystals from the remaining whey protein solution.

[0017] The inventors have also found that an edible whey protein composition in powder form containing BLG crystals has a significantly higher bulk density than comparable compositions of the prior art. This is advantageous because it simplifies the handling of the powder and results in less dust.

[0018] Thus, another aspect of the present invention relates to an edible composition comprising β-lactoglobulin in crystalline and / or isolated form, such as obtainable by one or more of the methods described herein. The edible composition may, for example, be a powder comprising β-lactoglobulin crystals and having a bulk density of at least 0.40 g / mL. Alternatively, the edible composition may be a liquid suspension or slurry comprising β-lactoglobulin crystals.

[0019] In the context of the present invention, a dried product, such as a powder, comprising "BLG crystals" comprises a product obtained by drying a suspension of BLG crystals, whereas the crystal structure of the wet BLG crystals may have been deformed during the drying process and may have at least partially lost its X-ray diffraction characteristics. Likewise, the terms "dried BLG crystals" and "dried BLG crystals" refer to particles obtained by drying wet BLG crystals, and such dried particles themselves do not necessarily have a crystalline structure. However, the inventors observed that when the dried BLG crystals were resuspended in cold (4°C) demineralized water at a weight ratio of 2 parts water to 1 part dried BLG crystals, the BLG crystals rehydrated and recovered substantially the same crystal structure (space group type and unit cell size) as before drying.

[0020] BLG is well known to be an important source of essential amino acids, including, for example, leucine, and therefore the edible BLG compositions provided herein have several interesting nutritional uses.

[0021] Another aspect of the present invention relates to an isolated BLG crystal having an orthorhombic space group P 21 21 21 and a unit cell size as well as And wherein the crystal has unit cell integration angles α=90°, β=90°, and γ=90°.

[0022] Yet another aspect of the present invention relates to the use of an edible composition as defined herein as a food ingredient.

[0023] Another aspect of the present invention relates to a food product comprising an edible composition as defined herein and a fat source and / or a carbohydrate source.

[0024] This application provides the following:

[0025] 1) A method for preparing an edible composition comprising beta-lactoglobulin (BLG) in crystallized and / or isolated form, the method comprising the following steps:

[0026] a) providing a whey protein solution comprising BLG and at least one additional whey protein, said whey protein solution:

[0027] - is supersaturated with respect to BLG and has a pH in the range of 5-6,

[0028] - contains BLG in an amount of at most 90% (w / w),

[0029] b) crystallizing BLG in said supersaturated whey protein solution, preferably in salting mode, and

[0030] c) Optionally, separating the BLG crystals from the remaining whey protein solution.

[0031] 2) The method according to 1), further comprising the step d) washing the BLG crystals, for example, the separated crystals obtained from step c).

[0032] 3) The method according to 1) or 2), further comprising the step e) recrystallizing the BLG crystals, such as the BLG crystals obtained from step c) or d).

[0033] 4) The method according to any one of the preceding claims, further comprising the step f) drying the BLG-containing composition from step b), c), d) or e).

[0034] 5) The method according to any one of the preceding claims, wherein the whey protein solution of step a) contains at least 5% (w / w) ALA relative to the total amount of protein.

[0035] 6) The method according to any one of the preceding claims, wherein the whey protein solution of step a) comprises at least 15% (w / w) additional whey protein relative to the total amount of protein.

[0036] 7) The method according to any one of the preceding claims, wherein the whey protein solution of step a) contains at least 1% (w / w) BLG relative to the total amount of protein.

[0037] 8) The method according to any one of the preceding claims, wherein the whey protein solution of step a) comprises at least 0.4% (w / w) BLG relative to the weight of the whey protein solution.

[0038] 9) The method according to any one of the preceding claims, wherein the whey protein solution comprises whey protein concentrate, whey protein concentrate, whey protein isolate and / or whey protein isolate.

[0039] 10) The method according to any one of the preceding claims, wherein the ratio of the electrical conductivity of the whey protein solution to the total amount of protein is at most 0.3.

[0040] 11) The method according to any of the preceding claims, wherein the UF permeate conductivity of the whey protein solution is at most 7 mS / cm.

[0041] 12) The method according to any of the preceding claims, wherein the supersaturated whey protein solution is prepared by adjusting the whey protein feed by one or more of the following:

[0042] - Adjust pH,

[0043] - Reduce electrical conductivity

[0044] - Lower the temperature

[0045] - Increase protein concentration, and

[0046] - Add agents that reduce water activity.

[0047] 13) A method according to any preceding claim, wherein the preparation of the whey protein solution involves adjusting the pH of the whey protein feed.

[0048] 14) A method according to any preceding claim, wherein the preparation of the whey protein solution involves reducing the conductivity of the whey protein feed.

[0049] 15) A method according to any preceding claim, wherein the preparation of the whey protein solution involves reducing the temperature of the whey protein feed.

[0050] 16) A method according to any preceding claim, wherein the preparation of the whey protein solution involves increasing the total protein concentration of the whey protein feed.

[0051] 17) The method according to any of the preceding claims, wherein the BLG crystallization in step b) involves one or more of the following:

[0052] - Wait for crystallization to occur,

[0053] - Add seed crystals,

[0054] - further increase the supersaturation of BLG, and / or

[0055] - Mechanical stimulation.

[0056] 18) The method according to any one of 1) to 17), wherein step c) comprises separating the BLG crystals to a solid content of at least 30% (w / w), preferably at least 40% (w / w), and even more preferably at least 50% (w / w).

[0057] 19) The method according to any one of 2) to 19), wherein the washing in step d) involves contacting the separated BLG crystals with a washing liquid without completely dissolving the BLG crystals, and then separating the remaining BLG crystals from the washing liquid.

[0058] 20) The method according to 19), wherein the washing of 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.

[0059] 21) The method according to any one of 3) to 20), wherein the recrystallization step involves:

[0060] - Dissolve the separated BLG crystals in the recrystallization liquid,

[0061] - adjusting the recrystallization liquid to obtain supersaturation with respect to BLG,

[0062] - crystallizing BLG in a supersaturated conditioned recrystallization solution, and

[0063] - Separating the BLG crystals from the remaining conditioned recrystallization solution.

[0064] 22) The method according to any one of 3) to 21), wherein the BLG crystals of step d) are recrystallized at least twice.

[0065] 23) The method according to any one of 4) to 22), wherein the drying step involves one or more of spray drying, freeze drying, spin flash dryer, rotary drying and / or fluidized bed drying.

[0066] 24) An edible BLG composition obtainable by one or more methods according to any one of 1) to 23).

[0067] 25) An edible BLG composition comprising at least 90% (w / w) BLG relative to total solids.

[0068] 26) An edible BLG composition according to 25) having a BLG crystallinity of at least 10%.

[0069] 27) An edible BLG composition according to any one of 24) to 26), comprising at most 90% (w / w) BLG relative to the total amount of protein and having a BLG crystallinity of at least 10%.

[0070] 28) The edible BLG composition according to any one of 24) to 27), wherein the composition is a dry composition.

[0071] 29) The dry BLG composition according to 28), which is in the form of a powder having a bulk density of at least 0.4 g / mL, preferably in the form of a spray-dried powder.

[0072] 30) The dry BLG composition according to 28) or 29), comprising

[0073] - at least 20% (w / w) BLG relative to the total amount of protein, and

[0074] - BLG crystallinity of at least 10%.

[0075] 31) The edible BLG composition according to any one of 24) to 27), wherein the composition is a liquid composition.

[0076] 32) The edible BLG composition according to any one of 24) to 31), wherein the composition is a low mineral composition.

[0077] 33) The edible BLG composition according to any one of 24) to 32), wherein the composition is a low-phosphorus composition.

[0078] 34) Use of the edible BLG composition according to any one of 24) to 33) as a food ingredient.

[0079] 35) Use of the low-phosphorus edible BLG composition according to any one of 24) to 33) as a food ingredient in the production of a low-phosphorus food product.

[0080] 36) A food product comprising an edible BLG composition according to any one of 24) to 33) and at least one additional ingredient such as, for example, a fat source and / or a carbohydrate source.

[0081] 37) The food product according to 36), which is a dry food product comprising carbohydrates and protein, comprising at least 1% (w / w) BLG, wherein:

[0082] i) the BLG has a crystallinity of at least 10%, and / or

[0083] ii) BLG accounts for at least 90% (w / w) of the total protein.

[0084] 38) The food product according to any one of 36) to 37), which is a low-phosphorus food product comprising at most 80 mg phosphorus per 100 g protein.

[0085] 39) The food product according to any one of 36) to 38), which is a dairy product, a candy, a beverage, a protein bar or an enteral nutrition composition.

[0086] 40) The food product according to any one of 36) to 39) in the form of a beverage:

[0087] - comprising an edible product according to any one of 24) to 33) so as to provide a total amount of BLG of at least 1% (w / w),

[0088] - sweeteners,

[0089] - at least one edible acid,

[0090] - have a pH in the range of 2.5-4.0, and

[0091] - Up to 80mg phosphorus per 100g protein.

[0092] 41) An isolated BLG crystal having an orthorhombic space group P 21 21 21 and a unit cell size as well as and having unit cell integration angles α=90°, β=90°, and γ=90°.

[0093] 42) The isolated BLG crystals according to 41), comprising at least 20% (w / w) BLG and at most about 80% (w / w) water. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 Two superimposed chromatograms of a sweet whey based crude whey protein solution (solid line) and the mother liquor obtained after crystallization (dashed line) are shown. The difference between the solid and dashed lines is due to the removal of BLG crystals.

[0095] Figure 2 is a micrograph of the recovered BLG crystals from Example 1.

[0096] Figure 3 is a chromatogram of the recovered BLG crystals from Example 1.

[0097] Figure 4 is a graph showing the relationship between the electrical conductivity of the whey protein solution and the yield of recovered BLG crystals obtained.

[0098] Figure 5 is a graph showing the relationship between the temperature and conductivity of the whey protein solution and the yield of recovered BLG crystals obtained.

[0099] Figure 6 The relationship between the total protein content of a whey protein solution (indirectly indicated by the Brix which is proportional to the protein content) and the yield of recovered BLG crystals obtained is shown.

[0100] Figure 7 Shown are chromatograms of feed 1 of Example 3 (solid line) and the mother liquor obtained after crystallization and removal of BLG crystals (dashed line).

[0101] Figure 8 is a microscope photograph of a sample taken during the early stages of crystallization of Charge 1 of Example 3.

[0102] Figure 9 is a microscope photograph of a sample taken after the crystallization of Charge 1 of Example 3 was completed.

[0103] Figure 10 A chromatogram of washed BLG crystals obtained from Feed 1 of Example 3 is shown.

[0104] Figure 11 Shown are chromatograms of feed 2 of Example 3 (solid line) and the mother liquor obtained after crystallization and removal of BLG crystals (dashed line).

[0105] Figure 12 Shown are pictures of feed 2 of Example 3 before (left picture) and after (right picture) crystallization.

[0106] Figure 13 Micrographs of whole and fragments of BLG crystals obtained from Feed 2 of Example 3 are shown.

[0107] Figure 14 and Figure 15 It was shown that increasing the conductivity or changing the pH of the BLG crystal slurry resulted in the dissolution of the BLG crystals.

[0108] Figure 16 The chromatogram of the BLG crystal precipitate isolated after washing with 2 volumes of MilliQ water is shown in Figure 1. The chromatogram clearly shows that the crystals contain BLG of very high purity.

[0109] Figure 17 Chromatograms showing the protein composition of feed 3 (solid line) and the resulting mother liquor (dashed line).

[0110] Figure 18 is a micrograph of BLG crystals recovered from Feed 3 of Example 3.

[0111] Figure 19 Shown is the chromatogram of recovered BLG crystals of Feed 3 of Example 3 (without any washing step).

[0112] Figure 20 The effect of increasing conductivity on the yield of recovered BLG crystals is shown.

[0113] Figure 21 This is a micrograph of a BLG crystal formed with a conductivity of 4.20 mS / cm.

[0114] Figure 22 Shown is a micrograph of BLG crystals at the early stages of crystallization from an SPC-based whey protein solution.

[0115] Figure 23 The difference in bulk density between a standard whey protein isolate (WPI) and a high purity BLG composition of the present invention, which contains BLG crystals, is demonstrated.

[0116] Figure 24 is a photograph of a rotary filter where the BLG crystals of Example 3, Feed 1, have been separated from the mother liquor.

[0117] Figure 25 are photographs of subsamples of the six low phosphorus beverage samples of Example 8. From left to right, the subsamples are Samples A, B, C, D, E, and F.

[0118] Figure 26 is a schematic illustration of a crystallization process variant of Example 10 using DCF to separate BLG crystals from the mother liquor.

[0119] Figure 27 Three photographs of the filter cake obtained by separating BLG crystals and mother liquor using a filter centrifuge are shown. DETAILED DESCRIPTION

[0120] As mentioned above, one aspect of the present invention relates to a method for preparing an edible composition comprising beta-lactoglobulin (BLG) in crystallized and / or isolated form, the method comprising the steps of:

[0121] a) providing a whey protein solution comprising BLG and at least one further whey protein, said whey protein solution being supersaturated with respect to BLG and having a pH in the range of 5-6,

[0122] b) crystallizing BLG in the supersaturated whey protein solution, and

[0123] c) Optionally, separating the BLG crystals from the remaining whey protein solution.

[0124] In the context of the present invention, the term "edible composition" relates to a composition that is safe for human consumption and use as a food ingredient and does not contain problematic amounts of toxic components (such as toluene) or other unwanted organic solvents. BLG is the most predominant protein in bovine whey and milk serum and is present in several genetic variants, with the major proteins in bovine milk being labeled A and B. BLG is a lipocalin and can bind to many hydrophobic molecules, suggesting that BLG plays a role in the transport of such hydrophobic molecules. BLG has also been shown to be able to bind iron via siderophores and may have a role in combating pathogens. Homologues of BLG are absent in human breast milk.

[0125] Bovine BLG is a relatively small protein with approximately 162 amino acid residues and a molecular weight of approximately 18.3-18.4 kDa. Under physiological conditions, it is primarily a dimer, but dissociates into monomers below approximately pH 3, retaining its native state, as determined using NMR. Conversely, BLG also exists as tetramers, octamers, and other multimeric aggregates under various natural conditions.

[0126] When the native structure is unstable enough to allow aggregation, BLG solutions can form gels under various conditions. Upon prolonged heating at low pH and low ionic strength, transparent 'thin-strand' gels are formed in which the protein molecules assemble into long, stiff fibers.

[0127] In the context of the present invention, the term "BLG" or "β-lactoglobulin" relates to BLG from mammalian species, eg in native and / or glycosylated form, and includes naturally occurring genetic variants.

[0128] In the context of the present invention, the term "crystal" relates to a solid material whose constituents (e.g. atoms, molecules or ions) are arranged in a highly ordered microstructure, forming a lattice extending in all directions. BLG crystals are protein crystals which primarily contain BLG arranged in a highly ordered microstructure, forming a lattice extending in all directions. BLG crystals may be, for example, monolithic or polycrystalline and may be, for example, intact crystals, crystal fragments or a combination thereof. Crystal fragments are formed, for example, when intact crystals are subjected to mechanical shearing during processing. Crystal fragments also have a highly ordered crystal microstructure, but may lack the uniform surface and / or uniform edges or corners of the intact crystal. See, for example Figure 18 , for many examples of complete BLG crystals, and Figure 13 are examples of BLG crystal fragments. In both cases, BLG crystals or crystal fragments can be visually identified using an optical microscope as well-defined, compact, and coherent structures. BLG crystals or crystal fragments are typically at least partially transparent. In addition, protein crystals are known to be birefringent, and this optical property can be used to identify unknown particles as having a crystalline structure. On the other hand, non-crystalline BLG aggregates appear to be undefined, opaque, and appear as open or porous masses of irregular size.

[0129] In the context of the present invention, the term "crystallization" relates to the formation of protein crystals. Crystallization may, for example, occur spontaneously or be initiated by the addition of seed crystals.

[0130] The edible composition comprises BLG in crystalline and / or isolated form. An edible composition comprising BLG in isolated form comprises at least 80% (w / w) BLG relative to total solids. An edible composition comprising BLG in crystalline form comprises at least some BLG crystals, and preferably a significant amount of BLG crystals.

[0131] BLG crystals can often be viewed through a microscope and may even reach a size that makes them visible to the naked eye.

[0132] In the context of the present invention, a liquid that is "supersaturated" or "supersaturated with respect to BLG" contains a concentration of dissolved BLG that is above the saturation point of BLG in the liquid under given physical and chemical conditions. The term "supersaturation" is well known in the art of crystallization (see, for example, Gérard Coquerela, "Crystallization of molecular systems from solution: phase diagrams, supersaturation and other basic concepts", Chemical Society Reviews, pp. 2286-2300, No. 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.

[0133] Procedure for testing whether a liquid is supersaturated with respect to BLG under a specific set of conditions:

[0134] a) Transfer 50 ml of the liquid sample to be tested into a centrifuge tube (VWR catalog number 525-0402) with a height of 115 mm, an inner diameter of 25 mm, and a capacity of 50 mL. During steps a) to h), care should be taken to maintain the sample and its subsequent fractions in the original physical and chemical conditions of the liquid.

[0135] b) Immediately centrifuge the sample at 3000 g for 3.0 minutes with a maximum acceleration of 30 seconds and a maximum deceleration of 30 seconds.

[0136] c) Immediately after centrifugation, transfer as much of the supernatant as possible (without disturbing the pellet if one has formed) to a second centrifuge tube (of the same type as in step a)

[0137] d) Take a 0.05 mL subsample of the supernatant (subsample A)

[0138] e) 10 mg of BLG crystals having a particle size of at most 200 microns (at least 98% pure BLG relative to total solids) are added to a second centrifuge tube and the mixture is stirred.

[0139] f) The second centrifuge tube was left to stand at the original temperature for 60 minutes.

[0140] g) Immediately after step f), the second centrifuge tube was centrifuged at 500 g for 10 minutes, and then another 0.05 mL subsample of the supernatant was taken (subsample B).

[0141] h) If present, recover the pellet from step g), resuspend it in milliQ water and immediately examine the suspension for the presence of crystals visible under a microscope.

[0142] i) The concentration of BLG in subsamples A and B was determined using the method outlined in Example 9.9 - the results are expressed as % BLG w / w relative to the total weight of the subsample. The BLG concentration of subsample A is referred to as C BLG,A , and the BLG concentration of subsample B is called C BLG,B .

[0143] j) If C BLG,B Lower than C BLG,A And if crystals are observed in step i), the liquid from which the sample of step a) was taken is supersaturated (under the specific conditions).

[0144] In the context of the present invention, the terms "liquid" and "solution" encompass compositions containing a combination of a liquid and solid or semi-solid particles (e.g., protein crystals or other protein particles). Thus, a "liquid" or "solution" may be a suspension or even a slurry. However, "liquids" and "solutions" are preferably pumpable.

[0145] In some preferred embodiments of the present invention, the method does not include the separation step c) and provides an edible composition comprising both BLG crystals and additional whey protein. If this method variant further includes the drying step f), it provides a dried composition comprising BLG crystals and additional whey protein (i.e., WPC or WPI), wherein at least a portion of the BLG is in the form of BLG crystals. Preferably, the method comprises steps a), b) and f) in direct order.

[0146] If the whey protein feed is whey protein concentrate (WPC), whey protein isolate (WPI), serum protein concentrate (SPC) or serum protein isolate (SPI), the above-described process variants can produce WPC, WPI, SPC or SPI in liquid or dry form, wherein at least a portion of the BLG is in crystalline form.

[0147] The terms "whey protein concentrate" and "serum protein concentrate" relate to dry or aqueous compositions containing a total amount of protein of 20% to 89% (w / w) relative to total solids.

[0148] WPC or SPC preferably contains:

[0149] 20%-89% (w / w) protein relative to total solids,

[0150] 15%-70% (w / w) BLG relative to total protein,

[0151] 8%-50% (w / w) ALA relative to total protein, and

[0152] 0-40% (w / w) CMP relative to protein.

[0153] Alternatively, and preferably, the WPC or SPC may contain:

[0154] 20%-89% (w / w) protein relative to total solids,

[0155] 15%-90% (w / w) BLG relative to total protein,

[0156] 4%-50% (w / w) ALA relative to total protein, and

[0157] 0-40% (w / w) CMP relative to protein.

[0158] Preferably, the WPC or SPC contains:

[0159] 20%-89% (w / w) protein relative to total solids,

[0160] 15%-80% (w / w) BLG relative to total protein,

[0161] 4%-50% (w / w) ALA relative to total protein, and

[0162] 0-40% (w / w) CMP relative to protein.

[0163] More preferably, the WPC or SPC contains:

[0164] 70%-89% (w / w) protein relative to total solids,

[0165] 30%-90% (w / w) BLG relative to total protein,

[0166] 4%-35% (w / w) ALA relative to total protein, and

[0167] 0-25% (w / w) CMP relative to protein.

[0168] The terms "whey protein isolate" and "serum protein isolate" relate to dry or aqueous compositions containing a total amount of protein of 90-100% (w / w) relative to total solids.

[0169] WPI or SPI preferably contains:

[0170] 90%-100% (w / w) protein relative to total solids,

[0171] 15%-70% (w / w) BLG relative to total protein,

[0172] 8%-50% (w / w) ALA relative to total protein, and

[0173] 0-40% (w / w) CMP relative to total protein.

[0174] Alternatively, and preferably, the WPI or SPI may contain:

[0175] 90%-100% (w / w) protein relative to total solids,

[0176] 30%-95% (w / w) BLG relative to total protein,

[0177] 4%-35% (w / w) ALA relative to total protein, and

[0178] 0-25% (w / w) CMP relative to total protein.

[0179] More preferably, the WPI or SPI may contain:

[0180] 90%-100% (w / w) protein relative to total solids,

[0181] 30%-90% (w / w) BLG relative to total protein,

[0182] 4%-35% (w / w) ALA relative to total protein, and

[0183] 0-25% (w / w) CMP relative to total protein.

[0184] In some preferred embodiments of the present invention, the method further comprises the step d) washing the BLG crystals, for example, the separated BLG crystals obtained from step c).

[0185] In some preferred embodiments of the present invention, the method further comprises the step e) recrystallizing the BLG crystals, such as the BLG crystals obtained from step c) or d).

[0186] The method may for example comprise or even consist of steps a), b), c), d) and e). Alternatively, the method may comprise or even consist of steps a), b), c) and e).

[0187] In some particularly preferred embodiments of the present invention, the method further comprises the step f) drying the BLG-containing composition from step b), c), d) or e).

[0188] The method may, for example, comprise or even consist of steps a), b), and f).

[0189] Alternatively, the method may comprise or even consist of steps a), b), c) and f).

[0190] Alternatively, the method may comprise or even consist of steps a), b), c), d) and f).

[0191] Alternatively, the method may comprise or even consist of steps a), b), c), d), e) and f).

[0192] As stated, step a) of the present invention involves providing a whey protein solution comprising BLG and at least one additional whey protein.

[0193] In the context of the present invention, the term "whey protein" refers to proteins found in whey or milk whey. The whey protein of a whey protein solution may be a subset of the protein species found in whey or milk whey, or it may be the complete set of protein species found in whey and / or milk whey. However, a whey protein solution always contains BLG.

[0194] In the context of the present invention, the term "additional proteins" refers to proteins that are not BLG. Additional proteins present in a whey protein solution typically comprise one or more non-BLG proteins found in milk serum or whey. Non-limiting examples of such proteins are α-lactalbumin, bovine serum albumin, immunoglobulins, casein macropeptide (CMP), osteopontin, lactoferrin, and milk fat globule membrane protein.

[0195] Therefore, the whey protein solution may preferably contain at least one additional whey protein selected from the group consisting of α-lactalbumin, bovine serum albumin, immunoglobulins, casein macropeptide (CMP), osteopontin, lactoferrin, milk fat globule membrane protein and combinations thereof.

[0196] α-Lactalbumin (ALA) is a protein present in the milk of nearly all mammalian species. ALA forms the regulatory subunit of the lactose synthase (LS) heterodimer, and β-1,4-galactosyltransferase (β4Gal-T1) forms the catalytic component. Together, these proteins enable LS to produce lactose by transferring the galactose moiety to glucose. As a polymer, α-lactalbumin strongly binds calcium and zinc ions and may possess bactericidal or antitumor activity. One of its major structural differences from β-lactoglobulin is that ALA lacks any free thiol groups that could serve as initiation points for covalent aggregation reactions. As a result, pure ALA does not form gels upon denaturation and acidification.

[0197] In the context of the present invention, the term "ALA" or "alpha-lactalbumin" relates to alpha-lactalbumin from mammalian species, eg in native and / or glycosylated form, and includes naturally occurring genetic variants.

[0198] In some embodiments of the present invention, the whey 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), and even more preferably at most 0.5% casein relative to the total amount of protein. In some preferred embodiments of the present invention, the whey protein solution does not contain any detectable amount of casein.

[0199] The term "serum" refers to the liquid that remains when casein and milk fat globules are removed from milk, for example by microfiltration or macroporous ultrafiltration. Serum may also be referred to as "ideal whey."

[0200] The term "whey protein" or "serum protein" relates to proteins present in whey.

[0201] The term "whey" relates to the liquid supernatant remaining after the casein of milk has been precipitated and removed. Casein precipitation may be accomplished, for example, by acidifying the milk and / or using rennet.

[0202] There are several types of whey: for example, "sweet whey," which is a whey product produced by rennet-based casein precipitation, and "acid whey" or "sour whey," which is a whey product produced by acid-based casein precipitation. Acid-based casein precipitation can be accomplished, for example, by adding food acids or by bacterial culture.

[0203] In some preferred embodiments of the present invention, the whey protein solution of step a) comprises at least 5% (w / w) additional whey protein relative to the total amount of protein. Preferably, the whey protein solution of step a) comprises at least 10% (w / w) additional whey protein relative to the total amount of protein. More preferably, the whey 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 whey protein solution of step a) comprises at least 20% (w / w) additional whey protein relative to the total amount of protein. Most preferably, the whey protein solution of step a) may comprise at least 30% (w / w) additional whey protein relative to the total amount of protein.

[0204] In other preferred embodiments of the present invention, the whey protein solution of step a) comprises at least 1% (w / w) additional whey protein relative to the total amount of protein. Preferably, the whey 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 whey protein solution of step a) comprises at least 3% (w / w) additional whey protein relative to the total amount of protein. Most preferably, the whey protein solution of step a) may comprise at least 4% (w / w) additional whey protein relative to the total amount of protein.

[0205] In yet other preferred embodiments of the present invention, the whey protein solution of step a) comprises at least 35% (w / w) additional whey protein relative to the total amount of protein. Preferably, the whey 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 whey 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 whey protein solution of step a) may comprise at least 50% (w / w) additional whey protein relative to the total amount of protein.

[0206] In some preferred embodiments of the present invention, the whey protein solution of step a) comprises additional whey protein in the range of 5%-90% (w / w) relative to the total amount of protein. Preferably, the whey protein solution of step a) may comprise additional whey protein in the range of 10%-80% (w / w) relative to the total amount of protein. The whey protein solution of step a) may, for example, comprise additional whey protein in the range of 20%-70% (w / w) relative to the total amount of protein. Preferably, the whey protein solution of step a) comprises additional whey protein in the range of 30%-70% (w / w) relative to the total amount of protein.

[0207] As described, the inventors have discovered that BLG can be crystallized without the use of organic solvents. This purification approach can also be used to refine preparations containing whey protein that have undergone some BLG purification; and provides a simple method for further improving the purity of BLG. Therefore, in some preferred embodiments of the present invention, the whey protein solution of step a) contains additional whey protein in the range of 1%-20% (w / w) relative to the total amount of protein. Preferably, the whey protein solution of step a) may contain additional whey protein in the range of 2%-15% (w / w) relative to the total amount of protein. Even more preferably, the whey protein solution of step a) may, for example, contain additional whey protein in the range of 3%-10% (w / w) relative to the total amount of protein.

[0208] In some embodiments of the present invention, the whey protein solution of step a) comprises at least 5% (w / w) ALA relative to the total amount of protein. Preferably, the whey protein solution of step a) comprises at least 10% (w / w) ALA relative to the total amount of protein. Even more preferably, the whey protein solution of step a) comprises at least 15% (w / w) ALA relative to the total amount of protein. Alternatively, the whey protein solution of step a) may comprise at least 20% (w / w) ALA relative to the total amount of protein.

[0209] In some preferred embodiments of the present invention, the whey protein solution of step a) comprises at least 25% (w / w) ALA relative to the total amount of protein. Preferably, the whey protein solution of step a) comprises at least 30% (w / w) ALA relative to the total amount of protein. The whey protein solution of step a) preferably comprises at least 35% (w / w) ALA relative to the total amount of protein. Even more preferably, the whey protein solution of step a) may comprise at least 40% (w / w) ALA relative to the total amount of protein.

[0210] In some preferred embodiments of the present invention, the whey protein solution of step a) contains ALA in the range of 5%-95% (w / w) relative to the total amount of protein. Preferably, the whey protein solution of step a) contains ALA in the range of 5%-70% (w / w) relative to the total amount of protein. Even more preferably, the whey protein solution of step a) may contain ALA in the range of 10%-60% (w / w) relative to the total amount of protein. The whey protein solution of step a) preferably contains ALA in the range of 12%-50% (w / w) relative to the total amount of protein. Even more preferably, the whey protein solution of step a) may contain ALA in the range of 20%-45% (w / w) relative to the total amount of protein.

[0211] In some preferred embodiments of the present invention, the weight ratio between BLG and ALA in the whey protein solution of step a) is at least 0.01. Preferably, the weight ratio between BLG and ALA in the whey protein solution of step a) is at least 0.5. Even more preferably, the weight ratio between BLG and ALA in the whey protein solution of step a) is at least 1, such as at least 2. For example, the weight ratio between BLG and ALA in the whey protein solution of step a) may be at least 3.

[0212] The amounts and concentrations of BLG and other proteins in the whey protein solution and whey protein feed refer to dissolved protein and do not include precipitated or crystallized protein.

[0213] In the context of the present invention, the term "weight ratio" between component X and component Y means the weight ratio calculated by m X / m Y The value obtained, where m X is the amount (by weight) of component X, and m Y is the amount of component Y (by weight).

[0214] In some preferred embodiments of the present invention, the weight ratio between BLG and ALA in the whey protein solution of step a) is in the range of 0.01-20. Preferably, the weight ratio between BLG and ALA in the whey protein solution of step a) is in the range of 0.2-10. Even more preferably, the weight ratio between BLG and ALA in the whey protein solution of step a) is in the range of 0.5-4. For example, the weight ratio between BLG and ALA in the whey protein solution of step a) is in the range of 1-3.

[0215] In some preferred embodiments of the present invention, the whey protein solution of step a) contains at least 1% (w / w) BLG relative to the total amount of protein. Preferably, the whey protein solution of step a) contains at least 2% (w / w) BLG relative to the total amount of protein. Even more preferably, the whey protein solution of step a) contains at least 5% (w / w) BLG relative to the total amount of protein. Preferably, the whey protein solution of step a) may contain at least 10% (w / w) BLG relative to the total amount of protein.

[0216] In some preferred embodiments of the present invention, the whey protein solution of step a) contains at least 12% (w / w) BLG relative to the total amount of protein. For example, the whey protein solution of step a) may contain at least 15% (w / w) BLG relative to the total amount of protein. The whey protein solution of step a) may, for example, contain at least 20% (w / w) BLG relative to the total amount of protein. Alternatively, the whey protein solution of step a) may contain at least 30% (w / w) BLG relative to the total amount of protein.

[0217] In some particularly preferred embodiments of the present invention, the whey protein solution of step a) contains up to 95% (w / w) BLG relative to the total amount of protein. Preferably, the whey protein solution of step a) may contain up to 90% (w / w) BLG relative to the total amount of protein. More preferably, the whey protein solution of step a) may, for example, contain up to 85% (w / w) BLG relative to the total amount of protein. Even more preferably, the whey protein solution of step a) may, for example, contain up to 80% (w / w) BLG relative to the total amount of protein. Preferably, the whey protein solution of step a) may contain up to 78% (w / w) BLG relative to the total amount of protein. Preferably, the whey protein solution of step a) may contain up to 75% (w / w) BLG relative to the total amount of protein.

[0218] In some preferred embodiments of the present invention, the whey protein solution of step a) contains BLG in the range of 1%-95% (w / w) relative to the total amount of protein. Preferably, the whey protein solution of step a) may contain BLG in the range of 5%-90% (w / w) relative to the total amount of protein. More preferably, the whey protein solution of step a) contains BLG in the range of 10%-85% (w / w) relative to the total amount of protein. Even more preferably, the whey protein solution of step a) contains BLG in the range of 10%-80% (w / w) relative to the total amount of protein. Most preferably, the whey protein solution of step a) may contain BLG in the range of 20%-70% (w / w) relative to the total amount of protein.

[0219] In other preferred embodiments of the present invention, the whey protein solution of step a) contains BLG in the range of 10%-95% (w / w) relative to the total amount of protein. Preferably, the whey protein solution of step a) may contain BLG in the range of 12%-90% (w / w) relative to the total amount of protein. More preferably, the whey protein solution of step a) contains BLG in the range of 15%-85% (w / w) relative to the total amount of protein. Even more preferably, the whey protein solution of step a) contains BLG in the range of 15%-80% (w / w) relative to the total amount of protein. Most preferably, the whey protein solution of step a) may contain BLG in the range of 30%-70% (w / w) relative to the total amount of protein.

[0220] In some preferred embodiments of the present invention, the whey protein solution of step a) comprises at least 0.4% (w / w) BLG relative to the weight of the whey protein solution. Preferably, the whey protein solution comprises at least 1.0% (w / w) BLG. More preferably, the whey protein solution comprises at least 2.0% (w / w) BLG. Even more preferably, the whey protein solution comprises at least 4% (w / w) BLG.

[0221] Higher concentrations of BLG are even more preferred, and preferably the whey protein solution comprises at least 6% (w / w) BLG. More preferably, the whey protein solution comprises at least 10% (w / w) BLG. Even more preferably, the whey protein solution comprises at least 15% (w / w) BLG.

[0222] In some preferred embodiments of the present invention, the whey protein solution of step a) comprises BLG in the range of 0.4%-40% (w / w) relative to the weight of the whey protein solution. Preferably, the whey protein solution comprises BLG in the range of 1%-35% (w / w). More preferably, the whey protein solution comprises BLG in the range of 4%-30% (w / w). Even more preferably, the whey protein solution comprises BLG in the range of 10%-25% (w / w).

[0223] Any suitable whey protein source can be used to prepare the whey protein solution.In some preferred embodiments of the present invention, the whey protein solution comprises or even consists of whey protein concentrate, whey protein concentrate, whey protein isolate, whey protein isolate or a combination thereof.

[0224] Preferably, the whey protein solution is a demineralized whey protein solution.

[0225] In the present context, the term demineralized means that the electrical conductivity of the whey protein solution is at most 15 mS / cm, and preferably at most 10 mS / cm, and even more preferably at most 8 mS / cm. The UF permeate conductivity of the demineralized whey protein solution is preferably at most 7 mS / cm, more preferably at most 4 mS / cm, and even more preferably at most 1 mS / cm.

[0226] It is particularly preferred that the whey protein solution is a demineralized whey protein concentrate, a demineralized whey protein isolate, a demineralized whey protein concentrate or a demineralized whey protein isolate.

[0227] In some particularly preferred embodiments of the present invention, the whey protein solution comprises or even consists of demineralized and pH-adjusted whey protein concentrate, whey protein concentrate, whey protein isolate, whey protein isolate or a combination thereof.

[0228] The whey protein solution may, for example, comprise or even consist of a demineralized whey protein concentrate. Alternatively, the whey protein solution may comprise or even consist of a demineralized whey protein concentrate. Alternatively, the whey protein solution may comprise or even consist of a demineralized whey protein isolate. Alternatively, the whey protein solution may comprise or even consist of a demineralized whey protein isolate.

[0229] In the context of the present invention, the terms "whey protein concentrate" and "milk albumen concentrate" relate to preparations of whey or milk serum containing in the range of about 20% to 89% (w / w) protein relative to total solids.

[0230] In the context of the present invention, the terms "whey protein isolate" and "milk albumen isolate" relate to whey or a preparation of milk serum containing at least 90% (w / w) protein relative to total solids.

[0231] The terms “consists essentially of” and “consisting essentially of” mean that the claim or feature in question includes the specified materials or steps and those that do not materially affect the basic novel characteristic(s) of the claimed invention.

[0232] The protein of the whey protein solution is preferably derived from mammalian milk, and preferably from ruminant milk, such as cows, sheep, goats, buffaloes, camels, llamas, mares and / or deer. Protein from bovine (cow) milk is particularly preferred. Therefore, BLG and the additional whey protein are preferably bovine BLG and bovine whey protein.

[0233] The proteins of the whey protein solution are preferably as close to their native state as possible and, if possible, have preferably only undergone mild heat treatment.

[0234] In some preferred embodiments of the present invention, the BLG of the whey protein solution has a degree of lactosylation of at most 1. Preferably, the BLG of the whey protein solution has a degree of lactosylation of at most 0.6. More preferably, the BLG of the whey protein solution has a degree of lactosylation of at most 0.4. Even more preferably, the BLG of the whey protein solution has a degree of lactosylation of at most 0.2. Most preferably, the BLG of the whey protein solution has a degree of lactosylation of at most 0.1, such as preferably at most 0.01.

[0235] The degree of lactosylation of BLG was determined according to Czerwenka et al. (J. Agric. Food Chem., Vol. 54, No. 23, 2006, pp. 8874-8882).

[0236] In some preferred embodiments of the present invention, the whey protein solution has a furosine value of at most 80 mg / 100 g protein. Preferably, the whey protein solution has a furosine value of at most 40 mg / 100 g protein. More preferably, the whey protein solution has a furosine value of at most 20 mg / 100 g protein. Even more preferably, the whey protein solution has a furosine value of at most 10 mg / 100 g protein. Most preferably, the whey protein solution has a furosine value of at most 5 mg / 100 g protein, for example, preferably a furosine value of 0 mg / 100 g protein.

[0237] In addition to protein, whey protein solutions typically also contain other components. Whey protein solutions can contain other components commonly found in whey or milk whey, such as minerals, carbohydrates and / or lipids. Alternatively or additionally, whey protein solutions can contain non-natural components of whey or milk whey. However, such non-natural components should preferably be safe for food production purposes, and preferably also safe for human consumption.

[0238] The present method is particularly advantageous for separating BLG from a crude whey protein solution containing other solids in addition to BLG.

[0239] The whey protein solution may for example contain carbohydrates, such as lactose, oligosaccharides and / or hydrolysates of lactose (i.e. glucose and galactose). The whey protein solution may for example contain carbohydrates in the range of 0-40% (w / w), such as in the range of 1%-30% (w / w) or in the range of 2%-20% (w / w).

[0240] In some preferred embodiments of the present invention, the whey protein solution contains at most 20% (w / w) carbohydrates, preferably at most 10% (w / w) carbohydrates, more preferably at most 5% (w / w) carbohydrates, and even more preferably at most 2% (w / w) carbohydrates.

[0241] The whey protein solution may also include lipids, for example in the form of triglycerides and / or other lipid types such as phospholipids.

[0242] In some embodiments of the invention, the whey protein solution of step a) comprises a total amount of lipids of at most 15% (w / w) relative to the total solids. Preferably, the whey protein solution of step a) comprises a total amount of lipids of at most 10% (w / w) relative to the total solids. More preferably, the whey protein solution of step a) comprises a total amount of lipids of at most 6% (w / w) relative to the total solids. Even more preferably, the whey protein solution of step a) comprises a total amount of lipids of at most 1.0% (w / w) relative to the total solids. Most preferably, the whey protein solution of step a) comprises a total amount of lipids of at most 0.5% (w / w) relative to the total solids.

[0243] The total amount of protein in the whey protein solution is typically at least 1% (w / w) relative to the weight of the whey protein solution. Preferably, the total amount of protein in the whey protein solution is at least 5% (w / w). More preferably, the total amount of protein in the whey protein solution is at least 10% (w / w). Even more preferably, the total amount of protein in the whey protein solution is at least 15% (w / w).

[0244] In some preferred embodiments of the present invention, the total protein content of the whey protein solution is in the range of 1%-50% (w / w). Preferably, the total protein content of the whey protein solution is in the range of 5%-40% (w / w). More preferably, the total protein content of the whey protein solution is in the range of 10%-30% (w / w). Even more preferably, the total protein content of the whey protein solution is in the range of 15%-25% (w / w).

[0245] The total protein content of the whey protein solution was determined according to Example 9.2.

[0246] The whey protein solution is typically prepared by making one or more adjustments to the whey protein feed that result in a whey protein solution that is supersaturated with respect to BLG.

[0247] The feed is preferably WPC, WPI, SPC, SPI or a combination thereof.

[0248] In the context of the present invention, the term "whey protein feed" relates to a composition that is converted into a whey protein solution supersaturated with respect to BLG. A whey protein feed is typically an aqueous liquid comprising BLG and at least one additional whey protein, but is usually not supersaturated with respect to BLG.

[0249] The embodiments relating to the chemical composition of the whey protein solution apply equally to the whey protein feed, however typically at least one parameter of the whey protein feed is set to avoid supersaturation or at least spontaneous crystallization.

[0250] In some preferred embodiments of the present invention, the supersaturated whey protein solution is prepared by making one or more of the following adjustments to the whey protein feed:

[0251] - Adjust pH,

[0252] - Reduce electrical conductivity

[0253] - Lower the temperature

[0254] - Increase protein concentration

[0255] - Adding agents to reduce water activity

[0256] - Modified ion composition

[0257] In some preferred embodiments of the present invention, the preparation of the whey protein solution involves adjusting the pH of the whey protein feed to a pH in the range of 5-6.

[0258] All pH values ​​were measured using a pH glass electrode and normalized to 25 °C.

[0259] The whey protein solution may, for example, have a pH in the range of 4.9-6.1. The pH of the whey protein solution may, for example, be in the range of 5.0-6.1. Alternatively, the pH of the whey protein solution may be in the range of 5.1-6.1. Preferably, the pH of the whey protein solution is in the range of 5.1-6.0.

[0260] In some preferred embodiments of the present invention, the pH of the whey protein solution is in the range of 5.0-6.0. Preferably, the pH of the whey protein solution is in the range of 5.1-6.0. More preferably, the pH of the whey protein solution is in the range of 5.1-5.9. Even more preferably, the pH of the whey protein solution may be in the range of 5.2-5.9. Most preferably, the pH of the whey protein solution is in the range of 5.2-5.8.

[0261] The pH is preferably adjusted using a food-acceptable acid and / or base. Food-acceptable acids are particularly preferred, such as carboxylic acids. 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 present invention, a lactone is used to adjust the pH, such as D-glucono-δ-lactone, which slowly hydrolyzes and simultaneously lowers the pH of the aqueous liquid containing it. The target pH after the lactone hydrolysis is completed can be accurately calculated.

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

[0264] In other preferred embodiments of the present invention, by adding H + The pH is adjusted by adding a cation exchange material in the form of beads / large particles. The beads / large particles cation exchange material can be easily removed from the whey protein solution before or even after crystallization. + Adjusting the pH using a cation exchange material in the form of a cation exchange material is particularly advantageous in the present invention as it lowers the pH without adding negative counterions that would significantly affect the conductivity of the whey protein feed.

[0265] In some preferred embodiments of the present invention, the preparation of the whey protein solution involves reducing the conductivity of the whey protein feed.

[0266] Unless otherwise stated, conductivity values ​​reported herein have been normalized to 25°C.

[0267] The inventors have found that reducing the conductivity of the whey protein solution leads to a higher yield of BLG crystals. The minimum achievable conductivity of the whey protein solution depends on the composition of the protein fraction and the lipid fraction (if any). Some protein species, such as casein macropeptide (CMP), contribute more to conductivity than other protein species. Therefore, it is preferred to make the conductivity of the whey protein feed close to a level where protein and protein counterions are the main contributors to conductivity. The reduction in conductivity generally involves removing at least some small free ions that are present in the liquid phase and are not tightly bound to the protein.

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

[0269] Lower conductivity is even more preferred and results in a higher yield of BLG crystals. Therefore, the whey protein solution preferably has a conductivity of at most 3 mS / cm. In some preferred embodiments of the present invention, the whey protein solution has a conductivity of at most 1 mS / cm. Preferably, the whey protein solution has a conductivity of at most 0.5 mS / cm.

[0270] The electrical conductivity of the whey protein feed is preferably reduced by dialysis or diafiltration. Diafiltration by ultrafiltration is particularly preferred because it allows washing out salts and small charged molecules while retaining protein. In some preferred embodiments of the present invention, identical UF units are used for UF / diafiltration and subsequently for concentrating the whey protein feed.

[0271] The inventors have found indications that the ratio between the electrical conductivity (expressed in mS / cm) and the total amount of protein (expressed as % wt. total protein relative to the total weight of the whey protein solution) in the whey protein solution can advantageously be kept at or below a certain threshold value in order to promote crystallization of BLG.

[0272] In some preferred embodiments of the present invention, the ratio of the electrical conductivity of the whey protein solution to the total amount of protein is at most 0.3. Preferably, the ratio of the electrical conductivity of the whey protein solution to the total amount of protein is at most 0.25. Preferably, the ratio of the electrical conductivity of the whey protein solution to the total amount of protein is at most 0.20. More preferably, the ratio of the electrical conductivity of the whey protein solution to the total amount of protein is at most 0.18. Even more preferably, the ratio of the electrical conductivity of the whey protein solution to the total amount of protein is at most 0.12. Most preferably, the ratio of the electrical conductivity of the whey protein solution to the total amount of protein is at most 0.10.

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

[0274] The inventors have also discovered that the whey protein feed can advantageously be adjusted to provide a whey protein solution having a UF permeate conductivity of at most 10 mS / cm. 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. When the term "conductivity" is used herein, 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.

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

[0276] If a high yield of BLG is to be obtained, even lower UF permeate conductivity can be used and is particularly preferred. Therefore, preferably, the UF permeate conductivity of the whey protein solution is at most 1.0 mS / cm. More preferably, the UF permeate conductivity of the whey protein solution may be at most 0.4 mS / cm. Even more preferably, the UF permeate conductivity of the whey protein solution may be at most 0.1 mS / cm. Most preferably, the UF permeate conductivity of the whey protein solution may be at most 0.04 mS / cm.

[0277] Even lower UF permeate conductivity can be achieved, for example, by using MilliQ water as a diluent during diafiltration (MilliQ water has a conductivity of approximately 0.06 μS / cm). Thus, the UF permeate conductivity of the whey protein solution may be at most 0.01 mS / cm. Alternatively, the UF permeate conductivity of the whey protein solution may be at most 0.001 mS / cm. Alternatively, the UF permeate conductivity of the whey protein solution may be at most 0.0001 mS / cm.

[0278] In some preferred embodiments of the present invention, the preparation of the whey protein solution involves reducing the temperature of the whey protein feed.

[0279] For example, preparation of a whey protein solution may involve reducing the temperature of the whey protein feed to at least 5° C., preferably at least 10° C., and even more preferably at least 15° C. For example, preparation of a whey protein solution may involve reducing the temperature of the whey protein feed to at least 20° C.

[0280] The temperature of the whey protein feed can, for example, be lowered to at most 30° C., preferably at most 20° C., and even more preferably to at most 10° C. The inventors have found that even lower temperatures provide higher supersaturation, therefore, the temperature of the whey protein feed can, for example, be lowered to at most 5° C., preferably at most 2° C., and even more preferably to at most 0° C. The temperature can even be below 0° C., but preferably the whey protein solution should remain pumpable, for example in the form of an ice slurry.

[0281] In some preferred embodiments of the present invention, the whey protein solution is an ice slurry before BLG crystallization is initiated. Alternatively or additionally, the whey protein solution may be converted to an ice slurry or maintained as an ice slurry during the BLG crystallization of step b).

[0282] In some particularly preferred embodiments of the present invention, the preparation of the whey protein solution involves increasing the total protein concentration of the whey protein feed. The whey protein feed can, for example, undergo one or more protein concentration steps, such as ultrafiltration, nanofiltration, reverse osmosis and / or evaporation, thereby concentrating to obtain the whey protein solution.

[0283] Ultrafiltration is particularly preferred as it allows selective concentration of protein, while the concentrations of salts and carbohydrates are barely affected.As mentioned above, ultrafiltration is preferably used for both diafiltration and concentration of whey protein feed.

[0284] In some preferred embodiments of the present invention, the BLG concentration of the whey protein solution is below a level at which spontaneous crystallization of BLG occurs. It is therefore generally preferred to terminate the modification of the whey protein feed when the whey protein solution is in the metastable region, i.e., in the supersaturated region, where BLG crystals can grow but crystallization does not initiate spontaneously when seeding is used.

[0285] In some preferred embodiments of the present invention, the preparation of the whey protein solution involves adding one or more water activity reducing agents to the whey protein feed.

[0286] Useful but non-limiting examples of such water activity reducing agents are polysaccharides and / or polyethylene glycol (PEG).

[0287] In some preferred embodiments of the invention, the preparation of the whey protein solution involves modifying the ionic composition of the whey protein feed, for example, by ion exchange, by addition of new ionic species, by dialysis or diafiltration.

[0288] Typically, the whey protein solution is prepared by combining two or more of the above process steps to produce supersaturation.

[0289] In some preferred embodiments of the present invention, preparation of the whey protein solution involves subjecting a whey protein feed to at least:

[0290] - concentration at a temperature above 10°C, for example using ultrafiltration, nanofiltration or reverse osmosis, and

[0291] - Subsequently cooled to a temperature below 10°C.

[0292] In other preferred embodiments of the present invention, the preparation of the whey protein solution involves subjecting the whey protein feed to at least

[0293] - concentrated at a pH above 6.0, and

[0294] - followed by addition of an acid (e.g. GDL or H + In yet other preferred embodiments of the present invention, the preparation of the whey protein solution involves subjecting the whey protein feed to at least:

[0295] - Reduction of conductivity by diafiltration, for example using a membrane that retains at least the BLG.

[0296] In a further preferred embodiment of the invention, the preparation of the whey protein solution involves subjecting the whey protein feed to a combination of at least:

[0297] - Adjust pH to 5-6,

[0298] - Reduction of conductivity by diafiltration using a membrane that retains at least BLG,

[0299] - concentrating the protein at a temperature above 10°C, for example using ultrafiltration, nanofiltration or reverse osmosis, and

[0300] - Finally, cool to a temperature below 10°C.

[0301] The inventors have also discovered that the BLG yield of the present process can be improved by controlling the molar ratio between the sum of sodium + potassium and the sum of calcium and magnesium. Surprisingly, higher relative amounts of calcium and magnesium appear to increase the BLG yield and, therefore, the BLG recovery efficiency of the present process.

[0302] In some preferred embodiments of the present invention, the molar ratio between Na+K and Ca+Mg in the whey protein solution of step a) is at most 4. More preferably, the molar ratio between Na+K and Ca+Mg in the whey protein solution of step a) is at most 2. Even more preferably, the molar ratio between Na+K and Ca+Mg in the whey protein solution of step a) is at most 1.5, and even more preferably at most 1.0. Most preferably, the molar ratio between Na+K and Ca+Mg in the whey protein solution of step a) is at most 0.5, for example at most 0.2.

[0303] The molar ratio of Na+K to Ca+Mg is calculated as (m Na +m K) / (m Ca +m Mg ), where m Na is the content of element Na in mol, m K is the content of element K in mol, m Ca is the content of element Ca in mol, and m Mg is the content of element Mg in mol.

[0304] It is particularly preferred that the whey protein solution is supersaturated with respect to BLG by salting-in so that BLG can be crystallized from the whey protein solution in salting-in mode.

[0305] In some embodiments of the present invention, particularly if the edible BLG product of the present invention also has a degree of protein denaturation, the whey protein solution has a low content of denatured protein. Preferably, the whey protein solution has a degree of protein denaturation of at most 2%, preferably at most 1.5%, more preferably at most 1.0%, and most preferably at most 0.8%.

[0306] Step b) of the process involves crystallizing at least some of the BLG of the supersaturated whey protein solution.

[0307] Particularly preferably, the crystallization of step b) is carried out in a salting-in mode, i.e. in a liquid having low ionic strength and low electrical conductivity. This is in contrast to the salting-out mode, in which a significant amount of salt is added to the solution to induce crystallization.

[0308] The BLG crystallization of step b) may, for example, involve one or more of the following:

[0309] - Wait for crystallization to occur,

[0310] - Add seed crystals,

[0311] - further increase the supersaturation of BLG, and / or

[0312] - Mechanical stimulation.

[0313] In some preferred embodiments of the present invention, step b) involves adding seed crystals to the whey protein solution. The inventors have discovered that adding seed crystals can control the time and location of BLG crystallization to avoid sudden blockages of process equipment and unexpected stops in the production process. For example, it is generally desirable to avoid the onset of crystallization when feeding whey protein concentrate.

[0314] In principle, any seed material that initiates BLG crystallization can be used. However, it is preferred to use hydrated BLG crystals or dried BLG crystals for seeding to avoid adding further impurities to the whey protein solution.

[0315] The seed crystals may be in dry form or may form part of a suspension when added to the whey protein solution. Adding a suspension containing seed crystals, such as BLG crystals, is currently preferred as it appears to provide a faster initiation of crystallization. Preferably, such a suspension containing seed crystals has a pH in the range of 5-6 and a conductivity of at most 10 mS / cm.

[0316] In some embodiments of the invention, at least some of the seeds are located on a solid phase that is in contact with the whey protein solution.

[0317] The seed crystals preferably have a particle size smaller than the desired size of the BLG crystals. The size of the seed crystals can be changed by removing the largest seeds through screening or other size classification processes. The particle size can also be reduced (e.g., by grinding) before size classification.

[0318] In some embodiments of the present invention, at least 90% (w / w) of the seed crystals have a particle size in the range of 0.1-600 microns (as measured by sieve analysis). For example, at least 90% (w / w) of the seed crystals may have a particle size in the range of 1-400 microns. Preferably, at least 90% (w / w) of the seed crystals may have a particle size in the range of 5-200 microns. More preferably, at least 90% (w / w) of the seed crystals may have a particle size in the range of 5-100 microns.

[0319] The particle size and dosage of the seed crystals can be adjusted to provide optimal crystallization of BLG.

[0320] In some preferred embodiments of the present invention, seeds are added to the whey protein feed before supersaturation with respect to BLG is achieved, but preferably in such a way that at least some seeds are still present when supersaturation is achieved. This can be accomplished, for example, by adding seeds when the whey protein feed is close to supersaturation (e.g., during cooling, concentration, and / or pH adjustment), with supersaturation being achieved before the seeds are completely dissolved.

[0321] In some preferred embodiments of the present invention, step b) involves further increasing the supersaturation of BLG, preferably to a point where crystallization of BLG begins immediately (i.e., within 20 minutes at most, and preferably within 5 minutes at most). This is also referred to as the nucleation zone, where crystallites spontaneously form and the crystallization process begins.

[0322] Supersaturation can be increased, for example, by one or more of the following:

[0323] - Further increase the protein concentration of whey protein solution

[0324] - Further cooling of the whey protein solution

[0325] - Make the whey protein solution closer to the optimal pH for BLG crystallization

[0326] -Further reduce the conductivity.

[0327] In some preferred embodiments of the invention, step b) involves waiting for BLG crystals to form. This may take several hours and is typically for a whey protein solution that is only slightly supersaturated with respect to BLG and to which no seeds have yet been added.

[0328] In some preferred embodiments of the present invention, providing the whey protein solution (step a) and crystallization of BLG (step b) are performed as two separate steps.

[0329] However, in other preferred embodiments of the present invention, step b) involves additional adjustments to the whey protein solution to crystallize in order to increase the supersaturation of BLG or at least maintain supersaturation. The additional adjustments result in an increased yield of BLG crystals.

[0330] Such additional adjustments may involve one or more of the following:

[0331] -Further increase the protein concentration of crystallized whey protein solution

[0332] - Cooling the crystallized whey protein solution to an even lower temperature

[0333] - Brings crystallized whey protein solution even closer to the optimal pH for BLG crystallization

[0334] -Further reduce the conductivity of crystallized whey protein solution.

[0335] In some preferred embodiments of the present invention, during step b), the crystallized whey protein solution is kept in a metastable zone to avoid spontaneous formation of new crystallites.

[0336] The inventors have determined the lattice structure of isolated BLG crystals by X-ray crystallography, and similar crystals have not been found in the prior art.

[0337] In some preferred embodiments of the present invention, at least some of the BLG crystals obtained during step b) have orthorhombic space group P 21 21 21.

[0338] Preferably, at least some of the obtained BLG crystals have an orthorhombic space group P 21 21 21, and a unit cell size as well as And the unit cell integration angles α = 90°, β = 90°, and γ = 90°.

[0339] In some preferred embodiments of the present invention, at least some of the obtained BLG crystals have an orthorhombic space group P 21 21 21, and a unit cell size as well as And the unit cell integration angles α = 90°, β = 90°, and γ = 90°.

[0340] Even more preferably, at least some of the obtained BLG crystals may have an orthorhombic space group P 21 21 21, and a unit cell size as well as And the unit cell integration angles α = 90°, β = 90°, and γ = 90°.

[0341] Most preferably, at least some of the BLG crystals obtained have an orthorhombic space group P 21 21 21, a unit cell size as well as And the unit cell integration angles α = 90°, β = 90°, and γ = 90°.

[0342] In some particularly preferred embodiments of the present invention, the method comprises step c): separating at least some of the BLG crystals from the remaining whey protein solution. This is particularly preferred when purification of BLG is desired.

[0343] Step c) may, for example, comprise separating the BLG crystals to a solid content of at least 30% (w / w). Preferably, step c) comprises separating the BLG crystals to a solid content of at least 40% (w / w). Even more preferably, step c) comprises separating the BLG crystals to a solid content of at least 50% (w / w).

[0344] The inventors have found that a high solids content is beneficial for the purification of BLG because the aqueous fraction adhering to the isolated BLG crystals typically contains impurities that should be avoided. In addition, a high solids content reduces the energy consumption of converting the isolated BLG crystals into a dry product (e.g., powder) and increases the BLG yield obtained from a drying unit with a given capacity.

[0345] In some preferred embodiments of the present invention, step c) comprises separating the BLG crystals to a solid content of at least 60%. Preferably, step c) comprises separating the BLG crystals to a solid content of at least 70%. Even more preferably, step c) comprises separating the BLG crystals to a solid content of at least 80%.

[0346] In some preferred embodiments of the present invention, the separation in step c) involves one or more of the following operations:

[0347] - centrifugation,

[0348] - decantation,

[0349] -filter,

[0350] -settlement,

[0351] -A combination of the above.

[0352] These unit operations are well known and readily accomplished to those skilled in the art.Separation by filtration may, for example, involve the use of vacuum filtration, dynamic cross-flow filtration (DCF), a filter press or a filter centrifuge.

[0353] Depending on the desired result, different pore sizes can be used for filtration. Preferably, the filter allows native whey protein 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 can, for example, have a nominal pore size of at least 0.5 microns. Even more preferably, the filter can have a nominal pore size of at least 2 microns.

[0354] Filters with larger pore sizes can also be used, and are in fact preferred if the primary purpose is to separate large crystals from a liquid containing BLG crystals. In some embodiments of the present 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.

[0355] The filter may, for example, have a pore size in the range of 0.03-5000 microns (e.g., 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, for example, in the range of 10-500 microns or in the range of 50-500 microns.

[0356] In some preferred embodiments of the present 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, a pore size in the range of 10-20 microns.

[0357] The advantage of using a filter with a pore size greater than 1 micron is that bacteria and other microorganisms are also at least partially removed during the separation process and optionally during washing and / or recrystallization. Thus, the present method can produce high-purity BLG at very low bacterial loads while avoiding thermal damage to the protein.

[0358] Another advantage of using filters with a pore size greater than 1 micron is that water removal and subsequent drying becomes easier and less energy intensive.

[0359] During the preparation of the whey protein solution, the remaining whey protein solution separated from the BLG crystals may be recycled to the whey protein feed.

[0360] In some preferred embodiments of the present invention, step c) uses a filter centrifuge. In other preferred embodiments of the present invention, step c) uses a decanter centrifuge. Preliminary results (see Example 13) have shown that using a filter centrifuge and / or a decanter centrifuge to separate BLG crystals from the mother liquor provides a more robust process than, for example, vacuum filtration.

[0361] It is generally preferred to dry the formed filter cake with a drying gas to reduce the moisture content of the filter cake and preferably to allow the filter cake to be stripped from the filter. The use of a drying gas may form part of the separation step, or alternatively may form a final drying step if the filter cake is to be converted directly to a dry edible BLG composition.

[0362] In some preferred embodiments of the present invention, step c) employs a DCF unit.

[0363] Preliminary tests (see Example 12) have shown that the use of a DCF unit having a membrane pore size in the range of 0.03-5 microns and preferably in the range of 0.3-1.0 microns provides efficient separation of BLG crystals, and the inventors have observed that the DCF unit can be operated for a sufficient time to separate crystals from even large batches of whey protein solutions containing BLG crystals.

[0364] In some preferred embodiments of the present invention, step c) is performed using a DCF unit equipped with a membrane capable of retaining BLG crystals, the DCF permeate is recycled to form part of the whey protein solution or whey 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 render it supersaturated with BLG before mixing with the whey protein solution or whey protein feed.

[0365] Advantageously, these embodiments do not require the temperature of the liquid stream to be raised above 15°C and are therefore less susceptible to microbial contamination than process variants requiring higher temperatures. Another industrial advantage of these embodiments is that the level of supersaturation is easily controlled and can be maintained at a level where unwanted spontaneous crystallization does not occur. Therefore, during these embodiments of the process, the temperature of the liquid stream is preferably at most 15°C, more preferably at most 12°C, and even more preferably at most 10°C, and most preferably at most 5°C.

[0366] These embodiments are illustrated in Example 10 and are Figure 26 These embodiments can be implemented as a batch process or a continuous process.

[0367] In some preferred embodiments of the present invention, the method includes step d): washing the BLG crystals, such as the separated BLG crystals of c). Washing may include a single washing step or multiple washing steps.

[0368] The washing of step d) preferably involves contacting the BLG crystals with a washing liquid without completely dissolving the BLG crystals, and subsequently separating the remaining BLG crystals from the washing liquid.

[0369] The wash liquid is preferably chosen to avoid complete dissolution of the BLG crystals and may, for example, comprise or even consist essentially of cold demineralised water, cold tap water or cold reverse osmosis permeate.

[0370] The pH of the wash liquor may be in the range of 5-6, preferably in the range of 5.0-6.0, and even more preferably in the range of 5.1-6.0, such as in the range of 5.1-5.9.

[0371] The conductivity of the wash liquid may be at most 0.1 mS / cm, preferably at most 0.02 mS / cm, and even more preferably at most 0.005 mS / cm.

[0372] A washing liquid with an even lower conductivity may be used. For example, the washing liquid may have a conductivity of at most 1 μS / cm. Alternatively, the conductivity of the washing liquid may be at most 0.1 μS / cm, for example about 0.05 μS / cm.

[0373] The washing step is preferably performed at low temperature to limit the dissolution of the crystalline BLG. The temperature of the washing liquid is preferably at most 30°C, more preferably at most 20°C, and even more preferably at most 10°C.

[0374] The washing step may be performed, for example, at a temperature of at most 5° C., more preferably at a temperature of at most 2° C., for example at about 0° C. Temperatures below 0° C. may be used so far that the washing liquid does not freeze at this temperature, for example due to the presence of one or more freezing point depressants.

[0375] In some embodiments of the present invention, the wash liquor contains BLG in an amount of at least 1% (w / w), and preferably in an amount of at least 3% (w / w), such as 4% (w / w).

[0376] The washing of step d) typically dissolves at most 80% (w / w) of the initial amount of BLG crystals, preferably at most 50% (w / w) of the initial amount of BLG crystals, and even more preferably at most 20% (w / w). Preferably, the washing of step d) dissolves at most 15% (w / w) of the initial amount of BLG crystals, more preferably at most 10% (w / w) of the initial amount of BLG crystals, and even more preferably at most 5% (w / w).

[0377] The weight ratio between the total amount of wash liquid and the initial amount of separated BLG crystals is typically at least 1, preferably at least 2, and more preferably at least 5. For example, the weight ratio between the amount of wash liquid and the initial amount of separated BLG crystals can be at least 10. Alternatively, the weight ratio between the total amount of wash liquid and the initial amount of separated BLG crystals can be at least 20, e.g., at least 50 or at least 100.

[0378] The term "total amount of washing liquid" relates to the total amount of washing liquid used in the entire process.

[0379] In some preferred embodiments of the present invention, one or more wash sequences are performed using the same filter arrangement as used for BLG crystal separation or a similar filter arrangement. The filter cake containing primarily BLG crystals is added to one or more wash liquid sequences, the wash liquid is removed by the filter, and the remaining BLG crystal portion remains in the filter cake.

[0380] In a particularly preferred embodiment of the present invention, the separation of step c) is performed using a filter that retains BLG crystals. Subsequently, the filter cake is contacted with one or more amounts of washing liquid that move through the filter cake and the filter. It is generally preferred that each amount of washing liquid is 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 times 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 the solids and fluids (liquid and gas) of the filter cake. The filter cake is preferably washed in this manner at least 2 times, preferably at least 4 times, and even more preferably at least 6 times.

[0381] The spent washing liquid from step d) can, for example, be recycled to the whey protein feed or the whey protein solution, where the washed-out BLG can be separated again.

[0382] The method may further comprise step e), which involves a recrystallization step comprising:

[0383] - Dissolve the separated BLG crystals in the recrystallization liquid,

[0384] - adjusting the recrystallization liquid to obtain supersaturation with respect to BLG,

[0385] - crystallizing BLG in a supersaturated conditioned recrystallization solution, and

[0386] - Separating the BLG crystals from the remaining conditioned recrystallization solution.

[0387] Step e) may comprise a single recrystallization sequence or multiple recrystallization sequences.

[0388] In some embodiments of the present invention, the BLG crystals of step c) or d) are recrystallized at least 2 times. For example, the BLG crystals can be recrystallized at least 3 times, such as at least 4 times.

[0389] The washing and recrystallization steps can be combined in any order and performed multiple times if desired.

[0390] The isolated BLG crystals of step c) may for example undergo a processing sequence:

[0391] - one or more washing steps (step d), followed by

[0392] - one or more recrystallization steps (step e).

[0393] Alternatively, the isolated BLG crystals of step c) may be subjected to the following processing sequence:

[0394] - one or more recrystallization steps (step e), followed by

[0395] - one or more washing steps (step d).

[0396] It is also possible to combine multiple steps of washing and recrystallization, for example, in the following order:

[0397] - one or more washing steps (step d),

[0398] - one or more recrystallization steps (step e),

[0399] - one or more washing steps (step d), and

[0400] - one or more recrystallization steps (step e).

[0401] Or for example in sequence:

[0402] - one or more recrystallization steps (step e),

[0403] - one or more washing steps (step d),

[0404] - one or more recrystallization steps (step e).

[0405] - one or more washing steps (step d)

[0406] In some embodiments of the present invention, the method further involves subjecting the isolated BLG to an additional BLG enrichment step, for example based on chromatography or selective filtration. However, in other preferred embodiments of the present invention, the method does not comprise an additional BLG enrichment step after step b). The term "additional BLG enrichment step" means a process step that enriches BLG relative to the total amount of protein, which step is independent of the crystallization of BLG or the processing of BLG crystals. An example of such an additional BLG enrichment step is ion exchange chromatography. Washing of BLG crystals and / or recrystallization of BLG is not considered an "additional BLG enrichment step".

[0407] In some particularly preferred embodiments of the present invention, the method involves a drying step f) wherein the BLG-containing composition from step b), c), d), or e) is converted into a dry composition.

[0408] In the context of the present invention, the term "dry" means that the composition or product in question comprises at most 6% (w / w) water and preferably even less.

[0409] In the context of the present invention, the term "BLG-containing composition" is used to describe the composition subjected to the drying step f).

[0410] In the context of the present invention, a "BLG-containing composition from step b), c), d), or e)" means a composition from step b), c), d), or e) that contains at least some BLG. In some preferred embodiments of the present invention, the "BLG-containing composition from step b), c), d), or e)" is obtained directly from step b), c), d), or e). However, in other preferred embodiments of the present invention, the "BLG-containing composition 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).

[0411] It is generally preferred that the BLG-containing composition contains a significant amount of BLG present in the composition obtained directly from step b), c), d), or e). In some preferred embodiments of the present invention, the BLG-containing composition from step b), c), d), or e) comprises at least 50% (w / w), preferably at least 70%, and even more preferably at least 80% of the BLG obtained from step b), c), d), or e).

[0412] 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).

[0413] In some preferred embodiments of the present invention, the drying step involves one or more of spray drying, freeze drying, spin flash drying, spin drying and / or fluidized bed drying.

[0414] In some particularly preferred embodiments of the present invention, the drying step involves a BLG-containing composition in which the BLG crystals have dissolved and in which 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 to be similar to an edible BLG composition having, for example, conventional dried whey protein powder.

[0415] BLG crystals can be dissolved, for example, by:

[0416] - Warming up,

[0417] - increasing the conductivity, for example by adding one or more salts,

[0418] - changes in pH, e.g. outside the range of 5-6,

[0419] - reducing the concentration of BLG, for example by dilution,

[0420] - or a combination of the above.

[0421] Spray drying is the currently preferred method for drying BLG-containing compositions free of BLG crystals.

[0422] In other particularly preferred embodiments of the present invention, the drying step involves a BLG-containing composition that still contains BLG crystals, and wherein the resulting powder contains BLG crystals. These embodiments are preferred if the edible BLG composition is to have a higher density than conventional dried whey protein powders.

[0423] In some particularly preferred embodiments of the present invention, the drying step involves a BLG-containing composition that still contains BLG crystals, and wherein the resulting powder contains BLG crystals. These embodiments are preferred if the edible BLG composition is to have a higher density than conventional dried whey protein powders.

[0424] As described in Example 7, the inventors discovered that a slurry of BLG crystals can be spray-dried while retaining at least some crystalline structure when the dried BLG crystals are resuspended in cold, demineralized water. It is particularly advantageous to avoid exposing a BLG-containing composition containing BLG crystals to a heat treatment regimen that dissolves a significant amount of the BLG crystals prior to spraying. Therefore, if preheating of a BLG-containing composition containing BLG crystals prior to spraying is employed, it is preferred that the heat load be carefully controlled.

[0425] In some embodiments of the present invention, the BLG-containing composition containing BLG crystals has a temperature of at most 70° C., preferably at most 60° C., more preferably at most 50° C., upon reaching the outlet of the spray device (e.g., a nozzle or an atomizer). In some preferred embodiments of the present invention, the BLG-containing composition containing BLG crystals has a temperature of 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. upon reaching the outlet of the spray device.

[0426] The spraying device of a spray dryer is, for example, a device such as a nozzle or an atomizer, which converts the solution or suspension to be dried into droplets which enter the drying chamber of the spray dryer.

[0427] It is particularly preferred that the BLG-containing composition containing BLG crystals has a temperature in the range of 0-50°C when it reaches the outlet of the spray device, preferably has a temperature in the range of 2°C-40°C, more preferably in the range of 4°C-35°C, and most preferably in the range of 5°C-10°C when it reaches the outlet of the spray device.

[0428] In some preferred embodiments of the present invention, the composition containing BLG has a BLG crystallinity 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%, such as preferably 97%-100%, when it reaches the outlet of the spray device. The composition containing BLG may be a BLG isolate, for example containing BLG in an amount exceeding 90% (w / w) relative to the total protein, or it may contain significant amounts of other proteins and thus contain BLG in an amount of up to 90% (w / w) relative to the total protein.

[0429] In some preferred embodiments of the present invention, the composition containing BLG can have a protein composition as described herein as a traditional liquid WPC or WPI or a traditional liquid SPC or SPI, but has a BLG crystallinity 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., preferably 97%-100%) when reaching the outlet of the spray device.

[0430] The inlet temperature of the gas to the spray dryer is preferably in the range of 140° C. to 220° C., more preferably in the range of 160° C. to 200° C., and even more preferably in the range of 170° C. to 190° C., such as preferably about 180° C. The outlet temperature of the gas from the spray dryer is preferably in the range of 50° C. to 95° C., more preferably in the range of 70° C. to 90° C., and even more preferably in the range of 80° C. to 88° C., such as preferably about 85° C. According to experience, the solid subjected to spray drying is said to be heated to a temperature 10° C. to 15° C. lower than the gas outlet temperature.

[0431] In some preferred embodiments of the present invention, the spray dryer is preferably in the range of 50-85°C, more preferably in the range of 60-80°C, and even more preferably in the range of 65-75°C, such as preferably around 70°C.

[0432] The concept of spray drying a suspension of BLG crystals has not been disclosed in the prior art and is itself an independent aspect of the present invention.

[0433] Thus, one aspect of the present invention relates to a method of producing a spray-dried edible powder composition comprising BLG, said composition comprising dried BLG crystals, said method comprising the steps of:

[0434] - providing a liquid BLG-containing composition comprising BLG crystals and preferably having a BLG crystallinity of at least 20%, said liquid BLG-containing composition preferably comprising at least 10% (w / w) total solids, and preferably comprising at least 5% (w / w) BLG, and

[0435] - Atomizing the liquid BLG-containing composition into a drying chamber of an operating spray dryer to convert the liquid BLG-containing composition comprising BLG crystals into a powder.

[0436] In some preferred embodiments of the present invention, the BLG-containing composition to be dried is mixed with dried BLG isolate to raise the solids content to a level that allows the mixture to be dried by fluidized bed drying. This is also known as backmixing and allows for very cost-effective drying of the BLG product. These embodiments are particularly preferred for BLG-containing compositions containing BLG crystals.

[0437] One advantage of the present process is that the BLG-containing composition to be dried can have a very high solids content before the drying step, so that less water has to be removed and less energy is consumed in the drying operation.

[0438] In some preferred embodiments of the present 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), for example, at least 60% (w / w).

[0439] In other preferred embodiments of the present invention, the composition containing BLG from step b), c), d), or e) has a solid content in the range of 20%-80% (w / w). Preferably, the composition containing BLG from step b), c), d), or e) has a solid content in the range of 30%-70% (w / w). More preferably, the composition containing BLG from step b), c), d), or e) has a solid content in the range of 40%-65% (w / w). Even more preferably, the composition containing BLG from step b), c), d), or e) has a solid content in the range of 50%-65% (w / w), for example, about 60% (w / w).

[0440] The inventors discovered 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 before the drying step.

[0441] Thus, in some preferred embodiments of the present invention, the composition comprising BLG has a BLG crystallinity of at least 10% (w / w). Preferably, the BLG of the composition comprising BLG has a crystallinity of at least 20% (w / w). More preferably, the BLG of the composition comprising BLG has a crystallinity of at least 30% (w / w). Even more preferably, the BLG of the composition comprising BLG has a crystallinity of at least 40% (w / w).

[0442] Even higher crystallinity is generally preferred. Thus, in some preferred embodiments of the present invention, the BLG of the BLG-containing composition has a crystallinity of at least 50% (w / w). Preferably, the BLG of the BLG-containing composition has a crystallinity of at least 60% (w / w). More preferably, the BLG of the edible BLG composition has a crystallinity of at least 70% (w / w). Even more preferably, the BLG of the BLG-containing composition has a crystallinity of at least 80% (w / w). Most preferably, the BLG of the BLG-containing composition has a 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).

[0443] The inventors have discovered that decreasing water content tends to increase the BLG crystallinity of a composition. Thus, under the same conditions, a composition with a high water:BLG ratio (e.g., a suspension of 4% BLG crystals in water) tends to have a lower BLG crystallinity than a composition with a lower water:BLG ratio (e.g., a filter cake or wet isolated crystals).

[0444] The method of the present invention can be operated using mild temperatures that do not compromise the nutritional value of the BLG and other whey proteins of the whey protein solution.

[0445] In some preferred embodiments of the present invention, the BLG is not subjected to temperatures above 90° C. during the process. Preferably, the BLG is not subjected to temperatures above 80° C. during the process. Even more preferably, the BLG is not subjected to temperatures above 75° C. during the process. It should be noted that even though spray drying typically employs temperatures exceeding 150° C., the short exposure time and simultaneous evaporation of water means that the spray-dried protein does not experience temperatures above 50° C.-70° C.

[0446] The inventors have seen evidence that prolonged heating during the drying step reduces the amount of BLG in crystalline form. In some preferred embodiments of the invention, the heat exposure during the drying step is kept low enough so that the degree of denaturation of the BLG is 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 result in any detectable denaturation of the BLG.

[0447] The degree of denaturation caused by the drying step is determined by determining the BLG content (c 步骤f前 ) (relative to total solids) and the BLG content in the reconstituted dry composition (relative to total solids) and were calculated using the following formula:

[0448] Degree of denaturation = ((c步骤f前 -c 步骤f后 ) / c 步骤f前 )*100%

[0449] Some preferred embodiments of the present invention relate to a method of preparing an edible composition comprising beta-lactoglobulin (BLG) in crystalline form, the method comprising the steps of:

[0450] a) providing a whey protein solution comprising BLG and at least one further whey protein, said whey protein solution being supersaturated with respect to BLG and having a pH in the range of 5-6, said whey protein solution comprising:

[0451] - 70%-100% (w / w) protein relative to total solids,

[0452] - 30%-90% (w / w) BLG relative to total protein, and preferably 30%-70% (w / w) BLG

[0453] - 4% to 50% (w / w) ALA relative to total protein, and preferably 8% to 35% (w / w) ALA,

[0454] - 0-25% (w / w) CMP relative to protein,

[0455] - at least 10% (w / w) protein relative to the total weight of the whey protein solution,

[0456] b) crystallizing BLG in a supersaturated whey protein solution, preferably by adding seeds, and

[0457] f) drying the BLG-containing composition obtained directly from step b), said BLG-containing composition preferably having a BLG crystallinity of at least 30%,

[0458] The method does not comprise steps c), d) or e).

[0459] The whey protein solution is preferably a demineralised whey protein solution and preferably has a ratio between its conductivity and the total amount of protein of at most 0.3 and / or a UF permeate conductivity of at most 7 mS / cm.

[0460] In these examples, the BLG crystals were not separated from the whey protein solution, but were dried and produced a high density edible BLG composition in powder form.

[0461] The present invention also relates to edible compositions obtainable by these embodiments.

[0462] Other preferred embodiments of the present invention relate to a method for preparing an edible composition comprising beta-lactoglobulin (BLG) in crystalline form, said method comprising the steps of:

[0463] a) providing a whey protein solution comprising BLG and at least one further whey protein, said whey protein solution being supersaturated with respect to BLG and having a pH in the range of 5-6, said whey protein solution comprising:

[0464] - 70%-100% (w / w) protein relative to total solids,

[0465] - 30%-90%, and preferably 30%-70% (w / w) BLG relative to total protein

[0466] - 4%-50%, and preferably 8%-35% (w / w) ALA relative to total protein

[0467] - 0-25% (w / w) CMP relative to total protein.

[0468] - at least 10% (w / w) protein relative to the total weight of the whey protein solution,

[0469] b) crystallizing BLG in a supersaturated whey protein solution, preferably by adding seed crystals,

[0470] c) separating the BLG crystals from the remaining whey protein solution,

[0471] d) optionally, washing the separated BLG crystals obtained from step c),

[0472] e) optionally, recrystallizing the BLG crystals obtained from step c) or d), and

[0473] f) drying the BLG-containing composition originating from and preferably directly obtained from step c), d) or e), said BLG-containing composition comprising BLG crystals and preferably having a BLG crystallinity of at least 30%.

[0474] The whey protein solution is preferably a demineralised whey protein solution and preferably has a ratio between its conductivity and the total amount of protein of at most 0.3 and / or a UF permeate conductivity of at most 7 mS / cm.

[0475] These embodiments are particularly suitable for preparing low mineral and low phosphorus edible BLG compositions in high density powder form.

[0476] The present invention also relates to edible compositions obtainable by these embodiments.

[0477] Yet other preferred embodiments of the present invention relate to a method for preparing an edible composition comprising β-lactoglobulin in isolated form, said method comprising the steps of:

[0478] a) providing a whey protein solution comprising BLG and at least one further whey protein, said whey protein solution being supersaturated with respect to BLG and having a pH in the range of 5-6, said whey protein solution comprising:

[0479] - 70%-100% (w / w) protein relative to total solids,

[0480] - 30%-90% (w / w) BLG relative to total protein, and preferably 30%-70% (w / w) BLG,

[0481] - 5% to 50% (w / w) ALA relative to total protein, and preferably 8% to 35% (w / w) ALA,

[0482] - 0-25% (w / w) CMP relative to total protein.

[0483] - at least 10% (w / w) protein relative to the total weight of the whey protein solution,

[0484] b) crystallizing BLG in a supersaturated whey protein solution, preferably by adding seed crystals,

[0485] c) separating the BLG crystals from the remaining whey protein solution,

[0486] d) optionally, washing the separated BLG crystals obtained from step c),

[0487] e) optionally, recrystallizing the BLG crystals obtained from step c) or d), and

[0488] f) drying the BLG-containing composition from step c), d), or e), said BLG-containing composition not comprising BLG crystals.

[0489] The whey protein solution is preferably a demineralised whey protein solution and preferably has a ratio between its conductivity and the total amount of protein of at most 0.3 and / or a UF permeate conductivity of at most 7 mS / cm.

[0490] In these examples, the BLG crystals were dissolved prior to drying.

[0491] The present invention also relates to edible compositions obtainable by these embodiments.

[0492] In some preferred embodiments, the method is implemented in a batch process. Alternatively, and sometimes preferably, the method can be implemented as a semi-batch process. In other preferred embodiments, the method is implemented as a continuous process.

[0493] An advantage of the present method is that it is much faster than comparable methods of BLG crystallization of the prior art. The duration from initial conditioning of the whey protein feed to completion of the separation of step c may be at most 10 hours, preferably at most 4 hours, more preferably at most 2 hours, and even more preferably at most 1 hour.

[0494] Further aspects of the invention relate to isolated BLG crystals obtainable from the methods described herein.

[0495] In the context of the present invention, the term "isolated BLG crystals" relates to BLG crystals which have been separated from the solution from which they were formed but which may still contain internal water, ie hydrated BLG molecules of the crystals.

[0496] The isolated crystals preferably have an orthorhombic space group P 21 21 21.

[0497] Preferably, the isolated BLG crystals have an orthorhombic space group P 21 21 21, and a unit cell size as well as And it has unit cell integration angles α=90° (±2%), β=90° (±2%), and γ=90° (±2%).

[0498] In some preferred embodiments of the present invention, the isolated BLG crystals have an orthorhombic space group P 21 21 21, and a unit cell size as well as And it has unit cell integration angles α=90° (±1%), β=90° (±1%), and γ=90° (±1%).

[0499] Even more preferably, the isolated BLG crystals may have an orthorhombic space group P 21 21 21, and a unit cell size as well as And it has unit cell integration angles α=90° (±0.5%), β=90° (±0.5%), and γ=90° (±0.5%).

[0500] Most preferably, the isolated BLG crystals have an orthorhombic space group P 21 21 21, and a unit cell size as well as And it has unit cell integration angles α=90°, β=90°, and γ=90°.

[0501] The isolated BLG crystals may, for example, comprise at least 20% (w / w) BLG and up to 80% (w / w) water. Preferably, the isolated BLG crystals may comprise at least 40% (w / w) BLG and water in the range of 0-60% (w / w). Even more preferably, the isolated BLG crystals comprise BLG in the range of 40%-60% (w / w) and about 40% to about 60% (w / w) water.

[0502] The inventors have discovered that the BLG crystals of the present invention unexpectedly have the ability to recover their original crystal structure after drying and rehydration. This is particularly advantageous in applications that benefit from the BLG crystal structure.

[0503] A further aspect of the present invention relates to an edible composition comprising beta-lactoglobulin, such as an edible composition obtainable by a process as defined herein.

[0504] Another aspect of the present invention relates to an edible BLG composition comprising at least 90% (w / w) BLG relative to total solids.Such an edible BLG composition is obtainable by a process as defined herein.

[0505] A further aspect of the present invention relates to an edible BLG composition comprising dried BLG crystals, at least 20% (w / w) BLG relative to total solids, and preferably having a crystallinity of at least 20% with respect to BLG. Such an edible BLG composition comprising dried BLG crystals is obtainable by a process as defined herein.

[0506] In some preferred embodiments of the present invention, the BLG of the edible BLG composition has a degree of lactosylation of at most 1. Preferably, the BLG of the edible BLG composition has a degree of lactosylation of at most 0.6. More preferably, the BLG of the edible BLG composition has a degree of lactosylation of at most 0.4. Even more preferably, the BLG of the edible BLG composition has a degree of lactosylation of at most 0.2. Most preferably, the BLG of the edible BLG composition has a degree of lactosylation of at most 0.1, such as preferably at most 0.01.

[0507] In some preferred embodiments of the present 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.

[0508] The percentage of non-lactosylated BLG was determined according to Example 9.1.

[0509] In some preferred embodiments of the present 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).

[0510] Even higher crystallinity is generally preferred. Thus, in some preferred embodiments of the present invention, the BLG of the edible BLG composition has a crystallinity of at least 50% (w / w). Preferably, the BLG of the edible BLG composition has a crystallinity of at least 60% (w / w). More preferably, the BLG of the edible BLG composition has a crystallinity of at least 70% (w / w). Even more preferably, the BLG of the edible BLG composition has a crystallinity of at least 80% (w / w). Most preferably, the BLG of the edible BLG composition has a crystallinity of at least 90% (w / w), and preferably at least 95% (w / w).

[0511] The crystallinity of BLG in liquids with a pH range of 5-6 was measured according to Example 9.7. The crystallinity of BLG in powdered materials was measured according to Example 9.8. If the edible composition is a dry product rather than a powder, it must be converted to a powder, for example by grinding or milling, before the method of Example 9.8 is performed.

[0512] In some preferred embodiments of the present invention, the edible BLG composition is WPC, WPI, SPC or SPI, wherein at least some of the BLG is in crystalline form. The edible BLG composition can be, for example, a composition comprising up to 90% (w / w) BLG relative to the total amount of protein and having a BLG crystallinity of at least 10%. For example, the edible BLG composition can be a composition comprising up to 80% (w / w) BLG relative to the total amount of protein and having a BLG crystallinity of at least 10%. The edible BLG composition can be a composition comprising 30%-70% (w / w) BLG relative to the total amount of protein and having a BLG crystallinity of at least 10%.

[0513] In other preferred embodiments of the present invention, the edible BLG composition comprises up to 90% (w / w) BLG relative to the total amount of protein and has a BLG crystallinity of at least 30%. Preferably, the edible BLG composition may comprise up to 80% (w / w) BLG relative to the total amount of protein and have a BLG crystallinity of at least 30%. Even more preferably, the edible BLG composition may comprise 30%-70% (w / w) BLG relative to the total amount of protein and have a BLG crystallinity of at least 30%.

[0514] The inventors have found that the present invention enables the preparation of edible whey protein products having very low levels of phosphorus and other minerals, which is beneficial for patients suffering from kidney disease or otherwise having reduced kidney function.

[0515] The edible BLG composition is preferably a low phosphorus composition.

[0516] In the context of the present invention, term " low-phosphorus " relates to total phosphorus content and is the composition of 100mg phosphorus / 100g protein at the most, for example liquid, powder or other food products.Preferably, low-phosphorus composition has the total phosphorus content / 100g protein of 80mg at the most.More preferably, low-phosphorus composition can have the total phosphorus content / 100g protein of 50mg at the most.Even more preferably, low-phosphorus composition can have the total phosphorus content / 100g protein of 20mg phosphorus at the most.Even more preferably, low-phosphorus composition can have the total phosphorus content / 100g protein of 5mg phosphorus at the most.Low-phosphorus composition according to the present invention can be used as the food product ingredient for the production of renal function reduction patient group food.

[0517] Thus, in some particularly preferred embodiments of the present 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.

[0518] The phosphorus content relates to the total amount of elemental phosphorus of the composition in question and is determined according to Example 9.5.

[0519] In other preferred embodiments of the present invention, the edible BLG composition is a low mineral composition.

[0520] In the context of the present invention, the term "low in minerals" relates to a composition, such as a liquid, powder or other food product, having at least one, preferably two, and even more preferably all of the following:

[0521] - an ash content of at most 1.2% (w / w) relative to the total solids,

[0522] - a total calcium and magnesium content of up to 0.3% (w / w) relative to the total solids,

[0523] - a total sodium and potassium content of up to 0.10% (w / w) relative to the total solids,

[0524] - A total phosphorus content of at most 100 mg phosphorus per 100 g protein.

[0525] Preferably, the low mineral composition has at least one, preferably two or more, and even more preferably all of the following:

[0526] - an ash content of at most 0.7% (w / w) relative to the total solids,

[0527] - a total calcium and magnesium content of up to 0.2% (w / w) relative to the total solids,

[0528] - a total sodium and potassium content of up to 0.08% (w / w) relative to the total solids,

[0529] - A total phosphorus content of up to 80 mg phosphorus per 100 g protein.

[0530] Even more preferably, the low mineral composition has at least one, preferably two or more, and even more preferably all of the following:

[0531] - an ash content of at most 0.5% (w / w) relative to the total solids,

[0532] - a total calcium and magnesium content of up to 0.15% (w / w) relative to the total solids,

[0533] - a total sodium and potassium content of up to 0.06% (w / w) relative to the total solids,

[0534] - A total phosphorus content of up to 50 mg phosphorus per 100 g protein.

[0535] It is particularly preferred that the low mineral composition has the following:

[0536] - an ash content of at most 0.5% (w / w) relative to the total solids,

[0537] - a total calcium and magnesium content of up to 0.15% (w / w) relative to the total solids,

[0538] - a total sodium and potassium content of up to 0.06% (w / w) relative to the total solids,

[0539] - A total phosphorus content of up to 50 mg phosphorus per 100 g protein.

[0540] In some preferred embodiments of the present invention, the edible BLG composition comprises a total amount of protein of at least 25% (w / w) relative to 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) relative to 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) relative to 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) relative to the total solids of the edible BLG composition.

[0541] In some preferred embodiments of the present invention, the total amount of protein in the edible BLG composition is in the range of 25%-100% (w / w) relative to the total solids. Preferably, the total amount of protein in the edible BLG composition is in the range of 50%-100% (w / w). More preferably, the total amount of protein in the edible BLG composition is in the range of 75%-100% (w / w) relative to the total solids. Even more preferably, the total amount of protein in the edible BLG composition is in the range of 90%-100% (w / w) relative to the total solids.

[0542] In some preferred embodiments of the present invention, the edible BLG composition comprises at least 75% (w / w) BLG relative to the total amount of protein. Preferably, the edible BLG composition may comprise at least 90% (w / w) BLG relative to the total amount of protein. More preferably, the edible BLG composition may comprise at least 95% (w / w) BLG relative to the total amount of protein. Even more preferably, the edible BLG composition may comprise at least 97% (w / w) BLG relative to the total amount of protein. Most preferably, the edible BLG composition comprises about 100% (w / w) BLG relative to the total amount of protein.

[0543] In some preferred embodiments of the present invention, the edible BLG composition contains 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.

[0544] Edible BLG compositions may also include lipids, for example, in the form of triglycerides and / or other lipid types such as phospholipids.

[0545] In some embodiments of the present invention, the edible BLG composition comprises a total amount of lipids of at most 1% (w / w) relative to the total solids. Preferably, the edible BLG composition comprises a total amount of lipids of at most 0.5% (w / w) relative to the total solids. More preferably, the edible BLG composition comprises a total amount of lipids of at most 0.1% (w / w) relative to the total solids. Even more preferably, the edible BLG composition comprises a total amount of lipids of at most 0.05% (w / w) relative to the total solids. Most preferably, the edible BLG composition comprises a total amount of lipids of at most 0.01% (w / w) relative to the total solids.

[0546] In some preferred embodiments of the present invention, the edible BLG composition is a dry composition, and for example a powder. It is particularly preferred that the edible BLG composition is a spray-dried powder.

[0547] The inventors observed that the density of edible BLG compositions in powder form (wherein at least some BLG is in crystalline form upon drying) is higher than the density of comparable BLG compositions without BLG crystals (see Example 7). This high density effect was also quite unexpectedly observed for edible BLG compositions in powder form obtained from a spray-dried BLG crystal slurry.

[0548] Thus, in some preferred embodiments of the present 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 can, for example, have a bulk density of at least 0.7 g / mL.

[0549] The advantages of bulk density apply both to edible BLG composition powders in which BLG is virtually the only protein present and to edible BLG composition powders in which the concentration of BLG is not enriched relative to other proteins present in a whey protein solution. Thus, the present invention provides high density powders of both isolated BLG and crude whey protein that, in addition to BLG, also contains significant amounts of ALA and other whey proteins.

[0550] In some preferred embodiments of the present 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 may, for example, have a bulk density of at least 0.7 g / mL and comprise at least 70% (w / w) protein relative to the total weight of the composition.

[0551] In other preferred embodiments of the present 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 may, for example, have a bulk density of at least 0.7 g / mL and comprise at least 80% (w / w) protein relative to the total weight of the composition.

[0552] The edible BLG composition in powder form may, for example, have a bulk density in the range of 0.40-1.5 g / mL and comprise 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 0.45-1.0 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 comprise 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 0.6-0.9 g / mL and comprises at least 80% (w / w) protein relative to the total weight of the composition. The powdered edible BLG composition may, for example, have a bulk density in the range of 0.6-0.8 g / mL and comprise at least 80% (w / w) protein relative to the total weight of the composition.

[0553] The inventors have found that the high density powder of the present invention advantageously allows for more cost-effective packaging and logistics of the powder, as less packaging material is required per kg of powder and more powder (mass) can be transported in a given container or truck.

[0554] The edible BLG composition in powder form may, for example, have a bulk density in the range of 0.40-1.5 g / mL. Preferably, the powdered edible BLG composition has a bulk density in the range of 0.45-1.0 g / mL. More preferably, the powdered edible BLG composition may have a bulk density in the range of 0.50-0.9 g / mL. Even more preferably, the powdered edible BLG composition has a bulk density in the range of 0.6-0.9 g / mL. The powdered edible BLG composition may, for example, have a bulk density in the range of 0.6-0.8 g / mL.

[0555] In other preferred embodiments of the present 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.

[0556] The edible BLG composition in powder form may, for example, have a bulk density in the range of 0.40-1.5 g / mL and comprise 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 0.45-1.0 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 comprise 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 0.6-0.9 g / mL and comprises at least 70% (w / w) protein relative to the total weight of the composition. The powdered edible BLG composition may, for example, have a bulk density in the range of 0.6-0.8 g / mL and comprise at least 70% (w / w) protein relative to the total weight of the composition.

[0557] The edible BLG composition in powder form may, for example, have a bulk density in the range of 0.40-1.5 g / mL and comprise 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 0.45-1.0 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 comprise 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 0.6-0.9 g / mL and comprises at least 80% (w / w) protein relative to the total weight of the composition. The powdered edible BLG composition may, for example, have a bulk density in the range of 0.6-0.8 g / mL and comprise at least 80% (w / w) protein relative to the total weight of the composition.

[0558] In other preferred embodiments of the present 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 comprise 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 comprise at least 70% (w / w) protein relative to the total weight of the composition.

[0559] In other preferred embodiments of the present 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 comprise 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 comprise at least 80% (w / w) protein relative to the total weight of the composition.

[0560] The bulk density of the powders was measured according to Example 9.3.

[0561] The inventors have seen evidence that BLG compositions according to the present invention have better long-term stability than similar BLG compositions. This is particularly true when at least some of the BLG is present in the form of BLG crystals, which appear to provide better storage stability of the BLG molecules.

[0562] In some preferred embodiments of the present 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, and even more preferably at most 20 mg / 100 g protein after 60 days at 30° C. Most preferably, the dry BLG composition has a furosine value of at most 10 mg / 100 g protein after 60 days at 30° C.

[0563] In some preferred embodiments of the present 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, and 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.

[0564] In some preferred embodiments of the present invention, the BLG of the dried BLG composition has a degree of lactosylation 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.

[0565] In some preferred embodiments of the present 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.

[0566] The liquid edible BLG composition may, for example, comprise in the range of 20-90% (w / w) water, more preferably in the range of 30-80% (w / w) water, even more preferably at least 40% (w / w) water.

[0567] The inventors have found that edible BLG compositions according to the present invention have unexpectedly low levels of protein denaturation, even when spray drying has been used to prepare the edible BLG powder compositions (see Example 11).

[0568] Thus, in some preferred embodiments of the present invention, the edible BLG composition has a protein denaturation degree of at most 2%. Preferably, the edible BLG composition has a protein denaturation degree of at most 1.5%. More preferably, the edible BLG composition has a protein denaturation degree of at most 1.0%. Even more preferably, the edible BLG composition has a protein denaturation degree of at most 0.8%. Even more preferably, the edible BLG composition has a protein denaturation degree of at most 0.5%.

[0569] In some preferred embodiments of the present invention, the edible BLG composition is a dry powder, and preferably a spray-dried powder, and has a protein denaturation level of at most 2%, and preferably at most 1.5%. More preferably, the dry edible BLG composition, for example in the form of a spray-dried powder, has a protein denaturation level 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 protein denaturation level 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 protein denaturation level of at most 0.5%.

[0570] In some preferred embodiments of the present invention, the edible BLG composition comprises:

[0571] - Up to 6% (w / w) water

[0572] - At least 80% total protein relative to total solids

[0573] - at least 95% BLG relative to total protein, and

[0574] The edible BLG composition:

[0575] - is a dry powder, and

[0576] - having a bulk density of at least 0.50 g / mL, and preferably at least 0.60 g / mL.

[0577] In other preferred embodiments of the present invention, the edible BLG composition comprises:

[0578] - Up to 6% (w / w) water

[0579] - At least 80% total protein relative to total solids

[0580] - at least 95% BLG relative to total protein, and

[0581] The edible BLG composition:

[0582] - is a dry powder,

[0583] - has a bulk density of at least 0.50 g / mL, and preferably at least 0.60 g / mL, and

[0584] - having a BLG crystallinity of at least 20%, and preferably at least 40%.

[0585] In a further preferred embodiment of the present invention, the edible BLG composition comprises:

[0586] - Up to 6% (w / w) water

[0587] - At least 80% total protein relative to total solids

[0588] - at least 95% BLG relative to total protein, and

[0589] The edible BLG composition:

[0590] - is a dry powder,

[0591] - has a bulk density of at least 0.50 g / mL, and preferably at least 0.60 g / mL, and - has a degree of protein denaturation of at most 2%, and preferably at most 1.0%.

[0592] In a further preferred embodiment of the present invention, the edible BLG composition comprises:

[0593] - Up to 6% (w / w) water

[0594] - at least 80% total protein relative to total solids,

[0595] - at least 95% BLG relative to total protein,

[0596] - Up to 80 mg phosphorus / 100 g protein.

[0597] The edible BLG composition:

[0598] -It is a dry powder.

[0599] In yet another preferred embodiment of the present invention, the edible BLG composition comprises:

[0600] - Up to 6% (w / w) water

[0601] - at least 90% total protein relative to total solids,

[0602] - at least 97% BLG relative to total protein,

[0603] - Up to 50 mg phosphorus / 100 g protein.

[0604] The edible BLG composition:

[0605] -It is a dry powder.

[0606] In other preferred embodiments of the present invention, the edible BLG composition comprises:

[0607] - Up to 6% (w / w) water

[0608] at least 80% total protein relative to the total solids, and preferably at least 90% total protein relative to the total solids,

[0609] - 30%-70% BLG relative to total protein,

[0610] - 8%-25% (w / w) ALA relative to total protein,

[0611] The edible BLG composition:

[0612] - is a dry powder, and

[0613] - having a BLG crystallinity of at least 20%, and preferably at least 40%.

[0614] In some preferred embodiments of the present invention, the edible BLG composition comprises:

[0615] - 20% to 80% (w / w) of water, and preferably 20% to 60% (w / w) of water,

[0616] - At least 80% total protein relative to total solids, and preferably at least 90% total protein

[0617] - at least 95% BLG relative to total protein,

[0618] - Up to 80 mg phosphorus / 100 g protein.

[0619] The edible BLG composition:

[0620] - has a BLG crystallinity of at least 20%, preferably at least 40%, and

[0621] - optionally having a degree of protein denaturation of at most 2%, and preferably at most 1.0%.

[0622] 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 preparing a high density whey protein powder having a normal concentration profile of the whey protein species whey protein but containing at least some BLG in the form of dried BLG crystals.

[0623] In other preferred embodiments of the present invention, the edible BLG composition comprises:

[0624] - 20% to 80% (w / w) of water, and preferably 20% to 60% (w / w) of water,

[0625] at least 80% total protein relative to the total solids, and preferably at least 90% total protein relative to the total solids,

[0626] - 30%-70% BLG relative to total protein,

[0627] - 8%-25% (w / w) ALA relative to total protein,

[0628] The edible BLG composition:

[0629] - having a BLG crystallinity of at least 20%, and preferably at least 40%.

[0630] 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 preparing a high density whey protein powder having a normal concentration profile of the whey protein species whey protein but containing at least some BLG in the form of dried BLG crystals.

[0631] Yet another aspect of the present invention relates to the use of an edible BLG composition as defined herein as a food ingredient.

[0632] For example, it is preferred to use the low phosphorus edible BLG composition as defined herein as a food product ingredient in the production of low phosphorus food products.

[0633] Another aspect of the present invention relates to a food product comprising an edible BLG composition as defined herein and at least one additional ingredient, such as a fat source and / or a carbohydrate source.

[0634] In some preferred embodiments of the present invention, the food product is a dry food comprising carbohydrates and protein, such as a bar, said dry food comprising at least 1% (w / w), preferably at least 5% BLG, wherein:

[0635] i) the BLG crystallinity is at least 20%, preferably at least 40%, and / or

[0636] ii) BLG comprises at least 90% (w / w) of total protein.

[0637] In some particularly preferred embodiments of the present invention, the food product is a low-phosphorus food comprising at most 100 mg phosphorus / 100 g protein, preferably at most 80 mg phosphorus / 100 g protein, more preferably at most 40 mg phosphorus / 100 g protein, and even more preferably at most 20 mg phosphorus / 100 g protein.

[0638] BLG has a favorable amino acid profile and preferably contributes a significant portion of the protein in a food product. This is particularly interesting if the food product is a low-mineral or low-phosphorus food. In some preferred embodiments of the present invention, the edible BLG composition comprises at least 25% (w / w), or at least 50% (w / w), more preferably at least 80% (w / w), and even more preferably at least 90% (w / w) of the total protein in the food product. Perhaps even most preferably, the edible BLG composition contributes all of the protein in the food product.

[0639] In some preferred embodiments of the present invention, the low-phosphorus edible BLG composition comprises at least 25% (w / w), or at least 50% (w / w), more preferably at least 80% (w / w), and even more preferably at least 90% (w / w) of the total protein in the low-phosphorus food product. It may even be most preferred that the low-phosphorus edible BLG composition contributes to all of the protein in the low-phosphorus food product.

[0640] Non-limiting examples of food products are eg dairy products, confectionery, beverages, protein bars, enteral nutrition compositions, bakery products.

[0641] In some preferred embodiments of the present invention, the food product is a beverage. The beverage preferably comprises:

[0642] - an edible BLG composition as defined herein, so as to provide a total amount of BLG of at least 1% (w / w), preferably at least 5% (w / w), more preferably at least 8% (w / w), and even more preferably at least 12% (w / w),

[0643] - sweeteners, such as sugar sweeteners and / or non-sugar sweeteners,

[0644] - at least one edible acid, such as citric acid or other suitable edible acid,

[0645] -optionally, flavoring, and

[0646] - Up to 80mg phosphorus / 100g protein

[0647] The pH range is 2.5-4.0.

[0648] The inventors have recognized that it is not trivial to prepare an acidic, high protein, low mineral beverage or liquid from a dry, edible BLG composition comprising BLG crystals. Dry, edible BLG compositions comprising BLG crystals typically produce a pH in the range of 5-6 when resuspended in water, and the addition of acid or salt to alter the pH or increase conductivity also increases the mineral load of the resulting liquid / beverage.

[0649] However, the inventors have discovered that if carboxylic acids, lactones, carboxylic anhydrides or combinations thereof are used to lower the pH, unnecessary minerals are not added and the mineral composition of the beverage / liquid can be better controlled.

[0650] Thus, one aspect of the present invention relates to a process for producing an acidified low-mineral liquid using an edible BLG composition comprising BLG crystals as an ingredient, the process comprising the steps of:

[0651] - providing one or more acidifying agents selected from the group consisting of carboxylic acids, lactones, carboxylic anhydrides or combinations thereof,

[0652] - contacting an edible BLG composition comprising BLG crystals with one or more acidulants and optionally other ingredients (e.g., water, a fat source, and / or a carbohydrate source) in an amount sufficient to adjust the pH to 2-4.5, and preferably 2.5-4.0, and to dissolve the BLG crystals,

[0653] Thus forming a liquid.

[0654] The liquid can, for example, be used as a beverage, or it can be used as an ingredient in the production of other food products.

[0655] If the edible BLG composition used in the process is provided in dry form, for example as a powder, it is generally preferred to rehydrate it in water before adding the acidulant.

[0656] The edible BLG composition used in the process is preferably present in the liquid in an amount sufficient to provide 1%-30% (w / w) protein, preferably 2%-25% (w / w) protein, more preferably 4%-20% (w / w) protein, and even more preferably 5%-16% (w / w) protein.

[0657] The edible BLG composition used in the process preferably has a BLG crystallinity of at least 30%, preferably at least 50%, and even more preferably at least 70%.

[0658] Examples of suitable acidifying agents are:

[0659] - carboxylic acids, such as acetic acid, maleic acid, tartaric acid, lactic acid, citric acid, gluconic acid or mixtures thereof,

[0660] - lactones, such as D-glucono-delta-lactone,

[0661] -Carboxylic anhydride.

[0662] In some preferred embodiments, the edible BLG composition comprising BLG crystals used in the process is preferably a low-phosphorus composition, and any other ingredients used in the process are preferably selected so that the final liquid is also a low-phosphorus composition.

[0663] In other preferred embodiments, the edible BLG composition comprising BLG crystals used in the process is preferably a low mineral composition, and any other ingredients used in the process are preferably selected such that the final liquid is also a low mineral composition.

[0664] The process is preferably carried out at a temperature in the range of 1-65°C, preferably 2-50°C, more preferably in the range of 3-20°C, even more preferably in the range of 4-15°C.

[0665] The present invention has been described above with reference to a number of specific embodiments. However, other embodiments than those described above are also possible within the scope of the present invention. Unless otherwise indicated, the different features and steps of the various embodiments and aspects of the present invention may be combined in other ways than those described herein.

[0666] Examples

[0667] Example 1: Crystallization of β-lactoglobulin from crude whey protein concentrate

[0668] plan:

[0669] The BLG crystallization process was fed with a lactose-depleted UF retentate derived from sweet whey from a standard cheese production process and filtered through a 1.2-micron filter. The sweet whey feed was adjusted to a feed concentration of 21% TS (total solids) ± 5 using an ultrafiltration setup using a Koch HFK-328 membrane with a 46-mil spacer and a feed pressure of 1.5-3.0 bar and polishing water (water filtered by reverse osmosis to obtain a conductivity of no more than 0.05 mS / cm) as the diafiltration medium. The temperature of the feed and retentate was approximately 12° C. during ultrafiltration. The pH was then adjusted to approximately 5.40 by adding HCl. The diafiltration continued until the retentate conductivity dropped below 0.03 mS / cm over a 20-minute period. The retentate was then concentrated to approximately 30% TS (approximately 23.1% total protein relative to the total weight of the concentrated retentate). A sample of the concentrated retentate was centrifuged at 3000 g for 5 minutes, but no visible precipitate was formed. The supernatant was subjected to HPLC analysis. The composition of the feed is shown in Table 1.

[0670] The concentrated retentate was seeded with 0.5 g / L of pure BLG crystal material obtained from spontaneous BLG crystallization (as described in Example 3 in the context of Feed 2). The seeding material was prepared by washing the BLG crystal slurry five times in milliQ water, collecting the BLG crystals after each wash. After washing, the BLG crystals were freeze-dried, ground with a pestle and mortar, and then passed through a 200-micron sieve. Thus, the particle size of the seed crystals was less than 200 microns.

[0671] The concentrated retentate was transferred to a 300 L crystallization tank, where it was cooled to approximately 4°C and maintained at this temperature overnight with gentle stirring. The next morning, a sample of the cooled concentrated retentate was transferred to a test tube and examined visually and microscopically. Rapidly settling crystals formed significantly overnight. A laboratory sample of the mixture containing the crystals and mother liquor was further cooled to 0°C in an ice-water bath. The mother liquor and crystals were separated by centrifugation at 3000 g for 5 minutes, and samples of the supernatant and precipitate were removed for HPLC analysis. The crystals were washed once in cold polishing water, then centrifuged again, and the precipitate was then freeze-dried.

[0672] Table 1 Concentrations of selected feed components normalized to 95% (w / w) total solids

[0673]

[0674] Quantification of BLG relative yield by HPLC:

[0675] All samples were diluted to the same extent by adding polished water. The samples were filtered through a 0.22 micron filter. For each sample, the same volume was loaded onto a 5μm C4 The sample was loaded on an HPLC system equipped with an LC column 250 x 4.6 mm (Part No. 00G-4167-E0) and detected at 214 nm.

[0676] The samples were run using the following conditions:

[0677] Buffer A: MilliQ water, 0.1% w / w TFA

[0678] Buffer B: HPLC grade acetonitrile, 0.085% w / w TFA

[0679] Flow rate: 1ml / min

[0680] Gradient: 0-30 min 82%-55% A and 18%-45% B; 30-32 min 55%-10% A and 45%-90% B; 32.5-37.5 min 10% A and 90% B; 38-48 min 10%-82% A and 90%-18% B.

[0681] Data processing:

[0682] Since all samples were processed in the same way, we could directly compare the areas of the BLG peak to obtain relative yields. Since the crystals contained only BLG and the samples were processed in the same way, the concentration of α-lactalbumin (ALA), and therefore the area of ​​ALA, should be the same in all samples. Therefore, the area of ​​ALA before and after crystallization was used as a correction factor (cf) when calculating relative yields.

[0683]

[0684] The relative yield was calculated by the following equation:

[0685]

[0686] result:

[0687] Figure 1 Overlaid chromatograms are shown before and after crystallization of BLG from sweet whey. The "before crystallization" sample is represented by a solid black line, while the "after crystallization" sample is represented by a dashed line. Clearly, a substantial decrease in BLG concentration has occurred, and using the yield calculation described above, the yield of removed BLG was determined to be 64.5% (w / w).

[0688] Study the crystal slurry by microscopy; Figure 2As can be seen in the figure, the sample contains hexagonal crystals, many of which are much larger than 200 microns in size, indicating that the observed crystals are not just seeding crystals. The crystals easily break when pressed with a needle, confirming that they are protein crystals.

[0689] Figure 3 The chromatogram of the washed crystalline product is shown, and in this case BLG accounts for 98.9% of the total area of ​​the chromatogram.The purity of the BLG product can be increased even further by further washing.

[0690] in conclusion:

[0691] This example unexpectedly demonstrates that BLG can be selectively crystallized from crude whey protein concentrate (which contains more than 48% non-BLG proteins relative to total protein) and that the resulting BLG crystal isolate has extremely high purity. This discovery opens up a new approach to industrial milk protein separation, in which BLG is separated from other protein components in a gentle manner that preferably avoids prolonged exposure to high temperatures and problematic chemicals.

[0692] Example 2: Effects of conductivity and temperature on BLG yield

[0693] plan:

[0694] Using the same feed, experimental and analytical setup as in Example 1, retentate samples (approximately 13.9% (w / w) total protein) were taken at different conductivity levels during UF diafiltration to investigate the effect of conductivity on BLG crystal yield. The samples were cooled to 4°C and held at this temperature overnight (however, the inventors have observed that 30 minutes or even less may be sufficient to reach equilibrium), and then the three samples were cooled to 0°C in ice water and held at this temperature for at least 1 hour to demonstrate the effect of temperature on yield. The results for the 4°C sample can be found in the table below. Figure 4 Seen in.

[0695] After diafiltration was complete, samples were taken at Brix 21, 24, and 32.5 during concentration. These samples were first cooled to 4°C and held at this temperature overnight. The yield of BLG crystals was measured as described in Example 1. The samples were then cooled to 0°C in ice water and held at this temperature for at least 1 hour. The yield of BLG crystals was then measured again.

[0696] result:

[0697] When the relative yield of BLG is plotted against the conductivity in the sample (e.g. Figure 4 There is a clear correlation between lower conductivity and higher relative yield of BLG.

[0698] exist Figure 5In the figure, the yields of three samples with different conductivity at two temperatures (4°C and 0°C) are shown. It can be seen that the lower the temperature, the higher the yield of BLG. Further lowering the temperature is expected to increase the yield.

[0699] Figure 6 Shown is the effect of protein concentration on the relative yield of BLG at 4° C. and 0° C. The graph shows a clear correlation between protein concentration (here measured by Brix) and the relative yield of BLG, indicating that relative yield continues to increase as protein concentration increases.

[0700] in conclusion:

[0701] The inventors have observed that many parameters affect the efficiency of the crystallization process. At a given pH value, the yield of BLG can be increased by reducing the conductivity, increasing the concentration of BLG and lowering the temperature.

[0702] Example 3: Crystallization of BLG in three whey protein solutions

[0703] plan:

[0704] Three different types of whey protein-containing raw materials were tested as crystallization feeds using the same experimental and analytical setup as in Example 1. However, no inoculation was used in the experiments with Feed 2. Feeds 1 and 2 were based on sweet whey and were fat-reduced through a Synder FR membrane prior to processing as described in Example 1. Feed 3 was derived from acid whey.

[0705] The compositions of the three feeds can be seen below in Tables 2, 3, and 4. Feed 3 crystallized at 21% TS (13.3% w / w total protein relative to the total weight of the feed), which is a significantly lower concentration than the other two (26.3% (w / w) total protein in Feed 1 and 25.0% (w / w) in Feed 2).

[0706] The crystallized slurry of feed 1 was centrifuged at 1500 g for 5 minutes on a Maxi-spin filter with a 0.45 μm CA membrane, and then 2 volumes of MilliQ water were added to the filter cake before centrifugation again. The resulting filter cake was analyzed by HPLC. A photograph of the Maxi-spin filter holding the precipitate (filter cake) of crystallized feed 1 is shown in Figure 24 The precipitate from feed 2 was washed with 2 volumes of MilliQ water and centrifuged again under standard conditions before analysis by HPLC. The precipitate from feed 3 was analyzed without washing.

[0707] The crystals produced from feed 2 were diluted to 10% TS and the pH was adjusted to pH 7 using 1 M NaOH to reverse the crystallization. NaCl was added to the crystal slurry from feed 2 (36% TS) to reverse the crystallization.

[0708] Table 2 Concentrations of selected components of feed 1 (whey protein concentrate based on sweet whey). BDL in wet sample = below detection limit

[0709]

[0710] Table 3 Concentrations of selected components of feed 2 (ALA-reduced whey protein concentrate based on sweet whey). BDL = below detection limit in wet non-standard samples.

[0711]

[0712]

[0713] Table 4 Concentrations of selected components of feed 3 (acid whey based whey protein concentrate).

[0714]

[0715] result:

[0716] Feed 1:

[0717] exist Figure 7 In the chromatograms of the protein composition of the feed (solid line) and the mother liquor (dashed line) can be seen. Clearly, most of the BLG was recovered as crystals by the process. The yield of isolated BLG (calculated as described in Example 1) was approximately 65% ​​relative to the total amount of BLG in the feed.

[0718] Figure 8 Here is a microscope picture of a sample taken during the early stages of the crystallization period. Figure 9 These are microscope images of samples taken at the end of crystallization. It's clear from both images that BLG crystals are relatively fragile. Some crystals appear to have broken during stirring, transforming from hexagonal or rhombohedral shapes into crystal fragments that still appear very compact and well-defined, but with more irregular shapes.

[0719] Figure 10 The chromatogram shows BLG crystals separated and washed on a spin filter. As shown, the purity is very high and the removal efficiency of other whey proteins is very high.

[0720] Feed 2:

[0721] exist Figure 11In the graph, the protein composition of feed 2 (solid line) and the resulting mother liquor (dashed line) can be seen. Clearly, most of the BLG has been removed and the yield calculated relative to the total amount of BLG in feed 2 is 82%.

[0722] Figure 12 Feed 2 is shown before (left) and after (right) crystallization. During crystallization, the feed changes from a clear liquid (with the stirring magnet visible) to a milky white opaque liquid.

[0723] Figure 13 A micrograph of a BLG crystal is shown. Although most of the crystal is fractured, a hexagonal shape can be seen.

[0724] Figure 16 The chromatogram of the BLG crystal precipitate isolated after washing with 2 volumes of MilliQ water is shown in Figure 1. The chromatogram clearly shows that the crystals contain BLG of very high purity.

[0725] Figure 14 and Figure 15 The results of increasing the conductivity (by adding NaCl) or changing the pH (adjusting the pH to 7 by adding NaOH) so that the environment is no longer favorable for crystal structure are shown. In both cases, the milky white suspension turns into a clear liquid as the BLG crystals dissolve.

[0726] The mineral composition of the crystal preparation obtained from Feed 2 is provided in Table 5. We note that the phosphorus to protein ratio is very low, which makes the crystal preparation suitable as a protein source for patients with kidney disease.

[0727]

[0728] Feed 3:

[0729] exist Figure 17 In Figure 3, the chromatograms of the protein composition of feed 3 (solid line) and the resulting mother liquor (dashed line) are shown. Clearly, a large portion of BLG was isolated (the calculated yield was 70.3% relative to the total amount of BLG in the feed). A higher yield would have been achieved if the protein content had been higher before crystallization.

[0730] Figure 18 This is a micrograph of BLG crystals separated from feed 3 (substantially free of CMP). The crystals have a rectangular shape, as opposed to a hexagonal shape. The rectangular crystals appear to be more stable than the hexagonal crystals. Figure 19 The chromatogram shows the crystalline precipitate isolated without washing; the chromatogram clearly shows that the crystals are BLG crystals, but with a rectangular shape rather than a hexagonal shape (compare e.g. Figure 18 The rectangular crystal shape and Figure 2 hexagonal crystal shape).

[0731] Table 6 Concentrations of selected components in the crystal formulation obtained from feed 3.

[0732]

[0733]

[0734] The crystal formulation from feed 3 contained 45 mg P / 100 g protein. We note that the phosphorus to protein ratio is very low, which makes the crystal formulation suitable as a protein source for patients with kidney disease.

[0735] in conclusion:

[0736] All three feeds are suitable for BLG crystallization. BLG crystals are readily soluble by adding salt or increasing pH or temperature. The novel method makes it possible to prepare BLG preparations with very low phosphorus content, making them suitable as a protein source for patients with kidney disease. Example 4 Effect of pH on BLG Crystal Yield

[0737] plan:

[0738] The same protocol and experimental setup as in Example 1 (using reduced fat sweet whey protein concentrate) was used, except that the pH was adjusted for each experiment to the levels described in Table 8. The protein concentration at the start of the crystallization step was approximately 24% (w / w).

[0739] The pH was adjusted with dilute NaOH solution (>4%) or dilute HCl (>3.6%) solution to study the effect of pH on the crystallization process and the yield obtained.After crystallization, BLG crystals were isolated by centrifugation as described in Example 1.

[0740] Table 7 Concentration ranges for selected components of the feed used in Example 4.

[0741]

[0742]

[0743] Table 8 Target pH of samples

[0744] sample Target pH 1 4.80 2 5.20 3 5.50 4 5.80 5 6.00 6 6.20

[0745] result:

[0746] The yield was calculated as described in Example 1. Note that the starting sample was taken before the addition of the seeding material. Therefore, if the sample is not supersaturated with respect to BLG, the seeding material will dissolve and contribute to the total BLG concentration, in which case the BLG yield will appear negative.

[0747] Table 9 Calculated sample yields based on HPLC measurements.

[0748]

[0749]

[0750] in conclusion:

[0751] This experiment confirmed that BLG crystallization can be achieved in the salting mode within the pH range of 5-6.

[0752] Example 5: Studying the impact of increasing conductivity levels

[0753] plan:

[0754] The same protocol and experimental setup as in Example 1 were used, except that samples were taken at different conductivities. The feedstock shown in Table 10 was adjusted and used as feed for the crystallization process. Prior to UF, feedstock samples were taken and NaCl was added to increase conductivity to investigate the conductivity level at which BLG crystals could grow. The protein content during crystallization was approximately 16.7% (w / w).

[0755] Table 10 Composition ranges of feed used in Example 5.

[0756]

[0757]

[0758] result:

[0759] Samples were processed as described in Example 1. Figure 20 The yields calculated at different conductivities in the retentate are shown. After pH adjustment, the point at 3.53 mS / cm is the raw material. All points greater than 3.53 are the result of adding NaCl to increase conductivity. Points less than 3.53 are the result of diafiltration on the UF system. The yield at 4.93 mS / cm is close to zero and is not considered significant.

[0760] The UF permeate conductivity of the retentate sample with a conductivity of 4.93 mS / cm was about 5.7 mS / cm. The UF permeate conductivity of the retentate sample with a conductivity of 3.53 mS / cm was about 4.35 mS / cm.

[0761] from Figure 20 As can be seen in the graph, BLG crystals form in the feed at a conductivity below 4.93 mS / cm (total protein content of about 16.7% (w / w) at 4° C.). A BLG yield of about 75% is achieved at a conductivity of about 2 mS / cm in the retentate and about 1.6 mS / cm in the UF permeate.

[0762] Figure 21 is a micrograph of crystals formed in the retentate at 4.20 mS / cm, showing the expected BLG crystal features.

[0763] in conclusion:

[0764] The specific feed of Example 5 allowed the formation of BLG crystals at less than 4.93 mS / cm (corresponding to a UF permeate conductivity of 5.75 mS / cm and a conductivity to total protein ratio of 0.057). The upper limit of conductivity is expected to depend on protein concentration and protein composition. For example, higher protein concentrations and / or increased levels of highly charged proteins or other macromolecules (e.g., CMP) are expected to increase the upper limit of conductivity at which BLG crystals can be formed.

[0765] Example 6: Crystallization of BLG in serum protein concentrate

[0766] Serum protein concentrate (SPC) was prepared by microfiltration of skim milk using a Synder FR membrane and subjected to a processing temperature of approximately 50°C. The retentate obtained contained essentially all casein and residual fat, and also contained some serum proteins, lactose, and minerals. The permeate contained molecules that could permeate the membrane, including serum proteins, lactose, and minerals, but essentially no casein or fat. The permeate was then prepared for crystallization as described in Example 1 (see Table 11 for the composition of the feed), and the resulting BLG crystals were characterized as described in Example 1. However, instead of performing all UF operations at 12°C, the temperature of the retentate was increased from 12°C to 25°C when the conductivity of the retentate approached 1 mS / cm. The temperature was increased to avoid spontaneous crystallization of BLG during UF concentration.

[0767] Table 11 Concentrations of selected components of the feed (serum protein concentrate). BDL = below detection limit in wet non-standard samples.

[0768]

[0769]

[0770] Similar to the crystallization of Examples 1-5, the BLG of the SPC feed formed crystals that could be isolated in very high purity (confirmed by chromatography as in the previous examples) and provided a BLG yield of 70% relative to the total amount of BLG of the SPC feed. Figure 22 , a BLG crystal from an early stage of crystallization is shown. As shown previously, the crystals have a rectangular or square shape, as opposed to the hexagonal shape observed, for example, in Example 2.

[0771] Example 7: Preparation of spray-dried BLG crystals and determination of bulk density

[0772] A portion of the BLG crystals produced in Example 3 (using Feed 2) was separated on a decanter centrifuge at 1200 g, 5180 RPM, 110 RPM Diff. (with a 64 mil spacer (mil means 1 / 1000 inch)) and a flow rate of 25-30 L / h. The BLG crystalline phase was then mixed with finishing water in a 1:1 ratio and then separated again on a decanter centrifuge using the same settings. The BLG crystalline phase was then mixed with finishing water to form a slurry containing about 25% dry matter and having a BLG crystallinity of about 80, which was then dried on a pilot plant spray dryer at an inlet temperature of 180°C and an outlet temperature of 85°C without any preheating. The temperature of the liquid stream was 10°C-12°C until spray drying. The moisture content of the resulting powder sampled at the outlet was 4.37% (w / w).

[0773] The crystallinity of BLG in the slurry was about 90%.

[0774] The inventors also successfully separated a slurry of BLG crystals and mother liquor on a decanter centrifuge at 350 g, 2750 RPM, 150 RPM Diff. (with a 64 mil spacer) and a flow rate of 75 L / h. The BLG crystal phase was then mixed with polishing water in a ratio of 1:2. The BLG crystal phase was then mixed with polishing water to form a thinner slurry, which was then dried on a pilot plant spray dryer using the same parameters as above.

[0775] The bulk density of the spray-dried powder was then measured according to Example 9.3 and compared to the bulk density of standard WPI dried on the same equipment. The standard WPI was found to have a bulk density of 0.39 g / mL (based on 625 punches), which is at the high end of the normal range for WPI powders. However, the bulk density of the spray-dried BLG crystal formulation was 0.68 g / mL, which is more than 75% higher than the bulk density of the standard WPI (see, e.g., Figure 23 ). This was indeed unexpected and offers many advantages related to logistics and applications.

[0776] Table 12 Concentrations of selected components of the spray-dried BLG crystal formulation of Example 7. BDL = Below Detection Limit

[0777]

[0778] A sample of the spray-dried BLG crystal preparation was then resuspended in cold demineralized water, and the BLG crystals were still clearly visible under a microscope. Addition of citric acid or NaCl dissolved the BLG crystals and transformed the opaque crystal suspension into a clear liquid.

[0779] The inventors have seen evidence that prolonged heating during the drying step reduces the amount of BLG in crystalline form. It is therefore preferred that the heat exposure of the BLG crystal preparation is as low as possible.

[0780] in conclusion:

[0781] This example demonstrates that a slurry containing BLG crystals can be spray dried and that the BLG crystals are still present in the resuspended spray-dried powder if the heating during the drying step is controlled.

[0782] The inventors also found that the bulk density of whey protein powder containing BLG crystals is much higher than that obtained by normal spray drying of a dissolved protein stream. High-density powders allow for more cost-effective packaging and logistics of the powder, as less packaging material is required per kg of powder, and more powder (mass) can be shipped in a given container or truck.

[0783] High density powders also appear to be easier to handle and less bulky and dusty during manufacturing and use.

[0784] Example 8: Low-phosphorus protein beverage

[0785] Six low phosphorus beverage samples were prepared using the purified BLG product from Example 3 (crystalline preparation obtained from Feed 3). All dry ingredients were mixed with demineralized water to obtain 10 kg of each sample and hydrated at 10°C for 1 hour.

[0786] Table 13 Composition of six beverage samples.

[0787]

[0788] Subsamples of six samples were obtained to Turbidity was measured on a 3000IR turbidimeter and viscosity was measured on a Gilson vicoman. The results are shown in the table below.

[0789] Table 14 Measured viscosity and turbidity of six beverage samples.

[0790] sample Viscosity (Cp) NTU A 1.42 36.2 B 2.37 46.3 C 2.69 4.9 D 2.70 5.0 E 1.45 63.1 F 2.25 82.1

[0791] Photographs of test tubes containing subsamples of the six low-phosphorus beverage samples are shown in Figure 25 From left to right, the subsamples are samples A, B, C, D, E, and F. Visual inspection of the test tubes verified the turbidity measurements and noted that all beverage samples were transparent, with samples C and D (pH 3.0) being particularly clear. The low viscosity confirmed that the beverage samples were easy to drink.

[0792] All ingredients used to prepare the beverages are low in phosphorus and free of unnecessary minerals. Consequently, the resulting beverages have a phosphorus content of approximately 45 mg P / 100 g protein and typically have a very low mineral content. Therefore, these six beverages are suitable for use as protein beverages for patients with kidney disease.

[0793] Example 9 - Analytical Method

[0794] Example 9.1 Determination of lactosylated BLG and non-lactosylated BLG:

[0795] The amounts of lactosylated BLG and native BLG were quantified using LC-MS.

[0796] The analysis was performed on a 6410 Triple Quad MS also from Agilent Technologies coupled to a HP1200 series HPLC from Agilent Technologies. For separation prior to ionization, Symmetry 300 was used. TM A C18 column (WAT106172: 5 μm solid phase particles, column size 2.1 x 150 mm) was used, and protein detection was performed at 214 nm. Prior to analysis, samples were filtered through a 0.22 μm filter. All samples were run in duplicate.

[0797] The analysis was performed using the following conditions:

[0798] HPLC

[0799] Buffer A: 99.9% MilliQ-vand with 0.1% TFA

[0800] Buffer B: 9.9% MilliQ-vand, 90% acetonitrile, 0.1% TFA

[0801] Flow rate: 0.3mL / min

[0802] gradient:

[0803] 0-20min: 85%-60% A and 15%-40% B

[0804] 20-45 min: 60%-50% A and 40%-50% B

[0805] 45-55min: 0% A and 100% B

[0806] 55-70 min 85% A and 15% B

[0807] Sample loading: 40 μL

[0808] The column temperature was set at 60°C.

[0809] Mass Spectrometry:

[0810] Ions with m / z values ​​of 100-2000 were detected and the resulting data were evaluated in MassHunter Workstation software (version B.04.00). Deconvolution was used to group all forms of the same species (mass). Further inquiries were made to the mass between 18 kDa and 20 kDa. The intact mass of BLG-A was 18.361 kDa, and that of BLG-B was 18.276 kDa. Lactosylation increased the protein mass by 324 Da, and by examining this mass region, up to 5 lactosylated proteins could be detected. Relative quantification was performed by comparing the signal intensity of each mass, ignoring the ionization differences of the different species.

[0811] Example 9.2: Determination of total protein

[0812] The total protein content (true protein) of the samples was determined by:

[0813] 1) The total nitrogen of the sample was determined according to ISO 8968-1 / 2 | IDF 020-1 / 2 - Milk - Determination of nitrogen content - Part 1 / 2: Nitrogen content by Kjeldahl.

[0814] 2) The non-protein nitrogen of the sample was determined according to ISO 8968-4 | IDF 020-4 - Milk - Determination of nitrogen content - Part 4: Determination of non-protein nitrogen content.

[0815] 3) Calculate the total amount of protein as (m 总氮 –m 非蛋白氮 )*6.38.

[0816] Example 9.3: Determination of loose density and bulk density

[0817] The density of a dry powder is defined as the relationship between the weight and volume of the powder, which is analyzed under specific conditions using a special Stampf volumeter (i.e. a graduated cylinder). Density is typically expressed in g / ml or kg / L.

[0818] In this method, a sample of dry powder is tapped in a graduated cylinder. After a specified number of taps, the volume of the product is read and the density is calculated.

[0819] This method can define three types of densities:

[0820] Pour density, which is the mass divided by the volume of the powder after transfer to a specified graduated cylinder.

[0821] Loose density is the mass of the powder divided by the volume of the powder after 100 taps according to the conditions specified in this standard.

[0822] Bulk density is the mass of the powder divided by the volume of the powder after 625 taps according to the conditions specified in this standard.

[0823] The method uses a special graduated cylinder of 250 ml, graduated from 0 to 250 ml, a weight of 190±15 g (J. Engelsmann A.G. 67059 Ludwigshafen / Rh) and a Stampf volumeter, for example from J. Engelsmann AG.

[0824] The loose density and bulk density of the dry product are determined by the following procedure.

[0825] Preprocessing:

[0826] The samples to be measured were stored at room temperature.

[0827] Then mix the sample thoroughly by repeatedly rotating and turning the container (to avoid breaking up the particles). The container should not be filled more than 2 / 3 full.

[0828] program:

[0829] Weigh 100.0 ± 0.1 g of powder and transfer it into a graduated cylinder. The volume V0 is read in ml.

[0830] If all 100g of powder does not fit into the graduated cylinder, reduce the amount to 50 or 25g.

[0831] Fix the cylinder to the Stampf volumeter and tap 100 times. Use a spatula to level the surface and read the volume V. 100 (ml).

[0832] Change the number of taps to 625 (including 100 taps). After tapping the surface, read the volume V 625 (ml).

[0833] Density calculation:

[0834] Calculate the loose density and bulk density in g / ml according to the following formula:

[0835] Bulk density = M / V

[0836] Wherein M represents the weighed sample in grams and V represents the volume in ml after 625 taps.

[0837] Example 9.4: Determination of water content of powders

[0838] Determination of the moisture content of food products according to ISO 5537:2004 (Milk powder - Determination of moisture content (Reference method)). NMKL is the Nordic Metodikkomité for Food Analysis. )”.

[0839] Example 9.5: Determination of the total amount of calcium, magnesium, sodium, potassium and phosphorus

[0840] The total amount of calcium, magnesium, sodium, potassium, and phosphorus was determined using the following procedure: the sample was first decomposed using microwave digestion, and then the total amount of one or more minerals was determined using an ICP device.

[0841] equipment:

[0842] The microwave oven was from Anton Paar and the ICP was an Optima 2000 DV from PerkinElmer Inc.

[0843] Material:

[0844] 1M HNO3

[0845] Yttrium in 2% HNO3

[0846] Appropriate standards for calcium, magnesium, sodium, potassium, and phosphorus in 5% HNO3

[0847] Preprocessing:

[0848] Weigh out a desired amount of powder and transfer it to a microwave digestion tube. Add 5 mL of 1 M HNO₃. Digest the sample in the microwave according to the microwave instructions. Place the digestion tube in a fume hood, remove the cap, and allow the volatile fumes to evaporate.

[0849] Measurement procedure:

[0850] The pretreated samples were transferred to digestion tubes using a known amount of Milli-Q water. A solution of yttrium in 2% HNO3 was added to the digestion tubes (approximately 0.25 mL per 50 mL of diluted sample) and diluted to a known volume using Milli-Q water. The samples were analyzed on an ICP using the manufacturer's protocol.

[0851] Blind samples were prepared by diluting a mixture of 10 mL of 1 M HNO 3 and 0.5 mL of a solution of yttrium in 2% HNO 3 with Milli-Q water to a final volume of 100 mL.

[0852] Prepare at least three standards with concentrations covering the expected sample concentrations.

[0853] Example 9.6: Determination of furosine value:

[0854] Furosine values ​​were determined as described in "Maillard Reaction Evaluation by Furosine Determination During Infant Cereal Processing", Guerra-Hernandez et al., Journal of Cereal Science 29 (1999) 171–176, and the total amount of protein was determined according to Example 9.2. Furosine values ​​are reported in the unit mg furosine per 100 g protein.

[0855] Example 9.7: Determination of BLG crystallinity in liquid

[0856] The following method was used to determine the crystallinity of BLG in liquids with a pH in the range of 5-6.

[0857] a) A 10 mL sample of the liquid in question was transferred to a Maxi-spin filter with a 0.45 micron pore size CA membrane.

[0858] b) Immediately spin the filter at 1500 g for 5 min. Keep the centrifuge at 2°C.

[0859] c) Add 2 mL of cold milliQ water (2°C) to the retentate side of the spin filter and immediately spin the filter at 1500 g for 5 min while maintaining centrifugal cooling at 2°C. Collect the permeate (Permeate A), measure the volume, and determine the BLG concentration by HPLC using the method outlined in Example 9.9.

[0860] d) Add 4 mL of 2M NaCl to the retentate side of the filter, stir rapidly and allow the mixture to stand at 25°C for 15 minutes.

[0861] e) Immediately spin the filter at 1500 g for 5 min and collect the permeate (Permeate B)

[0862] f) Determine the total weight of BLG in Permeate A and Permeate B using the method outlined in Example 9.9 and convert the results to total weight of BLG rather than weight percentage. The weight of BLG in Permeate A is referred to as m 渗透物A The weight of BLG in permeate B is called m 渗透物B .

[0863] g) The crystallinity of the liquid with respect to BLG is determined as:

[0864] Crystallinity = m 渗透物B / (m 渗透物A +m渗透物B )*100%

[0865] Example 9.8: Determination of BLG crystallinity in dry powder

[0866] This method was used to determine the crystallinity of BLG in dry powder.

[0867] a) 5.0 g of powder sample was mixed with 20.0 g of cold milliQ water (2°C) and allowed to stand at 2°C for 5 minutes.

[0868] b) A sample of the liquid in question was transferred to a Maxi-spin filter with a 0.45 micron CA membrane.

[0869] c) Immediately spin the filter at 1500 g for 5 min. Keep the centrifuge at 2°C.

[0870] d) Add 2 mL of cold milli-Q water (2° C.) to the retentate side of the spin filter and immediately spin the filter at 1500 g for 5 min. Collect the permeate (Permeate A), measure the volume, and determine the BLG concentration by HPLC using the method outlined in Example 9.9. The result is converted to total weight of BLG rather than weight percentage. The weight of BLG in Permeate A is referred to as m 渗透物A

[0871] f) The BLG crystallinity in the powder was then calculated using the following formula:

[0872]

[0873] where m 总BLG is the total amount of BLG in the powder sample of step a).

[0874] If the total amount of BLG of a powder sample is unknown, this can be determined by suspending another 5 g of powder sample (from the same powder source) in 20.0 grams of milliQ water, adjusting the pH to 7.0 by adding aqueous NaOH, leaving the mixture at 25° C. under stirring for 1 hour, and finally determining the total amount of BLG of the powder sample using Example 9.9.

[0875] Example 9.9: Determination of the total amount of BLG, ALA and CMP in an aqueous liquid

[0876] The contents of α-lactalbumin, β-lactoglobulin and CMP were analyzed by HPLC analysis at 0.4 mL / min. 25 microliters of the filtered sample were injected onto two TSKgel3000PWxl (7.8 mm 30 cm, Tosohass, Japan) columns connected in series with an attached precolumn PWxl (6 mm × 4 cm, Tosohass, Japan) equilibrated in an eluent consisting of 465 g MilliQ water, 417.3 g acetonitrile and 1 mL trifluoroacetic acid, and a UV detector at 210 nm was used.

[0877] The native α-lactalbumin (C α ), β-lactoglobulin (C β ) and casein macropeptide (C CMP ) content.

[0878] The total amount of additional protein (non-BLG protein) was determined by subtracting the amount of BLG from the total amount of protein (determined according to Example 9.2).

[0879] Example 9.10: Determination of UF Permeate Conductivity

[0880] Transfer 15 mL of sample to an Amicon Ultra-15 centrifugal filter unit with a 3 kDa cutoff (3000 NMWL) and centrifuge at 4000 g for 20-30 minutes, or until a sufficient volume of UF permeate for conductivity measurement accumulates in the bottom of the filter unit. Conductivity is measured immediately after centrifugation. Sample handling and centrifugation are performed at the temperature of the sample source.

[0881] Example 9.11: Determination of the degree of protein denaturation of a whey protein composition

[0882] Denatured whey protein is known to have lower solubility at pH 4.6 than at pH 7.0, and the degree of denaturation of a whey protein composition is determined by measuring the amount of soluble protein at pH 4.6 relative to the total amount of protein at pH 7.0.

[0883] More specifically, the whey protein composition to be analyzed (e.g., powder or aqueous solution) is converted into:

[0884] - a first aqueous solution containing 5.0% (w / w) total protein and having a pH of 7.0, and

[0885] - A second aqueous solution containing 5.0% (w / w) total protein and having a pH of 4.6.

[0886] pH adjustment was performed using 3% (w / w) NaOH (aq) or 5% (w / w) HCl (aq).

[0887] The total protein content (P) of the first aqueous solution was determined according to Example 9.2. pH 7.0 ).

[0888] The second aqueous solution was stored at room temperature for 2 h and then centrifuged at 3000 g for 5 minutes. A sample of the supernatant was recovered and analyzed according to Example 9.2 to determine the total protein (S pH 4.6 ).

[0889] The degree of protein denaturation D of the whey protein composition is calculated as follows:

[0890] D=((P pH 7.0 -S pH 4.6 ) / P pH 7.0 )*100%

[0891] Example 9.12: Detection of Dried BLG Crystals in Powder

[0892] The presence of dried BLG crystals in the powder can be identified by:

[0893] The powder sample to be analyzed was resuspended and gently mixed in demineralized water at a temperature of 4°C in a weight ratio of 2 parts water to 1 part powder and allowed to rehydrate at 4°C for 1 hour.

[0894] The rehydrated sample is examined microscopically to identify the presence of crystals, preferably using plane polarized light to detect birefringence.

[0895] The crystalline material is isolated and subjected to X-ray crystallography to verify the presence of the crystal structure and preferably also to verify that the crystal lattice (space group and unit cell size) corresponds to that of BLG crystals.

[0896] The chemical composition of the isolated crystalline material was analyzed to verify that the solid consisted primarily of BLG. Example 10: Crystallization by UF-based dynamic cross-flow filtration

[0897] The feed to the crystallization tank was prepared as described in Example 1, except that the diafiltration was performed at pH 5.92 and the final TS was 20%.

[0898] After the feed was conditioned (the feed composition can be seen in Table 15), it was transferred to a 300 L crystallization tank and the pH was initially adjusted to pH 5.80 and the temperature was maintained at 10°C-12°C. After the pH was adjusted, seeding material was added, which was produced in the same manner as described in Example 1, but derived from a non-spontaneous crystallization process. The feed was inoculated with seeding material to a concentration of 0.5 g seeding material per liter of feed. After inoculation, the temperature on the cooling hood was set to 5°C, the pH was slowly adjusted to 5.50, and the mixture was allowed to crystallize for about one hour, after which a DCF (dynamic cross-flow filtration) unit was connected to the crystallization tank, as shown in FIG. Figure 26 The DCF unit was equipped with a Kerafol ceramic membrane with a pore size of 500 nm, the TMP (transmembrane pressure) was set to 0.4 bar, and the rotation speed of the membrane was 32 Hz.

[0899] The retentate from the DCF was returned to the crystallization tank, while the permeate was used as feed in a UF (ultrafiltration) unit equipped with a Koch HFK-328 membrane with a 46 mil spacer. In the UF unit, the temperature was raised to but not above 12° C. The amount of diafiltration water added was adjusted so that the retentate from the UF returned to the crystallization tank was approximately 21% TS when demineralizing the mother liquor (ML).

[0900] Diafiltration on the ML was continued until the conductivity difference between the permeate and the diafiltration water was less than 50 μS / cm. At this point, the amount of diafiltration water was adjusted so that the retentate was approximately 30% TS. As BLG was removed as crystals, the amount of TS in the ML decreased; this continuous removal of excess water and minerals made it possible to increase the overall yield, as it appeared that the concentration of other proteins during BLG crystallization had limited, if any, effect on BLG solubility within the range explored.

[0901] The composition of the ML permeate from the DCF can be seen in Table 16. The initial 300 L of feed has been reduced to approximately 100 L of ML. Based on mass conservation, the relative yield of BLG is calculated to be 92%.

[0902] Table 15 Selected components of the feed used in Example 10.

[0903]

[0904] Table 16 Protein composition of the final mother liquor obtained in Example 10.

[0905]

[0906]

[0907] in conclusion:

[0908] By continuously removing excess minerals and water from the matrix in which BLG crystallization occurs, BLG yields can be significantly increased, and the process can be performed at low temperatures.

[0909] Example 11: Degree of protein denaturation of different whey protein products

[0910] The degree of protein denaturation of commercial products and four edible BLG compositions of the present invention was compared. The samples are described below.

[0911] sample A: BiPro (commercially available as WPI; Davisco, USA) B: BLG crystal slurry as is - no drying (present invention) C: Freeze-dried BLG crystal slurry (present invention) D: BLG crystals were re-dissolved (pH 7) and freeze-dried E: Spray-dried BLG crystal slurry (present invention)

[0912] Sample BE was prepared as follows:

[0913] A crystal slurry was prepared as described in Example 12 and isolated as described in Example 7. Some of the isolated BLG slurry was removed and divided into four portions.

[0914] Sample B: The first portion of the isolated BLG crystal slurry was re-dissolved without any drying by adjusting the pH of the BLG crystal slurry to 7.01 using 3% NaOH; the sample was then diluted to B x 6 to make an approximately 5% protein solution.

[0915] Sample C: A second portion of the isolated BLG crystal slurry was freeze-dried. The powder was then resuspended in polish water, the pH was adjusted to 7.09 using 3% NaOH, and the sample was then diluted to 6 Brix to prepare an approximately 5% protein solution.

[0916] Sample D: A third portion of the isolated BLG crystal slurry was re-dissolved by adjusting the pH to 7.0 using 3% NaOH and then freeze-dried. The freeze-dried powder was then re-suspended in polishing water and the pH was measured to be 7.07. The sample was then diluted to 6 Brix to make an approximately 5% protein solution.

[0917] Sample E: A fourth portion of the isolated BLG crystal slurry was processed and spray dried as described in Example 7. The powder was then resuspended in polishing water and the pH was adjusted to 7.04 using 3% NaOH. The sample was then diluted to 6 Brix to make an approximately 5% protein solution.

[0918] The degree of protein denaturation of each sample was determined according to Example 9.11 and the results are presented in Table 17.

[0919] Table 17 compares the degree of protein denaturation of a commercially available WPI product (Bipro) and four BLG products of the present invention.

[0920]

[0921] in conclusion:

[0922] Regardless of the drying method, the edible BLG compositions of the present invention have surprisingly low levels of denatured protein; only one-tenth the level found in commercially available WPI for comparison. Particularly surprising is that the spray-dried BLG crystal slurry product still has the lowest level of denaturation of all the products.

[0923] Example 12: Isolation of Crystals by Dynamic Cross-Flow Filtration

[0924] The crystallization process was fed with a lactose-depleted UF retentate derived from sweet whey from a standard cheese production process and filtered through a 1.2-micron filter. The sweet whey feed was adjusted to a feed concentration of 10% TS (total solids) ± 5 and polishing water (water filtered by reverse osmosis to a conductivity of at most 0.05 mS / cm) using an ultrafiltration setup using a Koch HFK-328 membrane with a feed pressure of 1.5-3.0 bar. The temperature of the feed and retentate was approximately 12°C during ultrafiltration. The pH was then adjusted to approximately 5.60 by adding HCl. The diafiltration continued until the retentate conductivity was below 1.30 mS / cm. The feed was then heated to 25°C, after which the retentate was concentrated to approximately 27% TS (approximately 21% total protein relative to the total weight of the concentrated retentate). At the end of the concentration, the permeate conductivity was 0.33 mS / cm. A sample of the concentrated retentate was centrifuged at 3000 g for 5 minutes, but no visible precipitate formed.

[0925] The concentrated retentate was transferred to a 300 L crystallization tank, where it was cooled to approximately 6° C. and maintained at this temperature overnight with gentle stirring. The next morning, the retentate had crystallized. The mother liquor and crystals were separated by centrifugation at 3000 g for 5 minutes, and samples of the supernatant and precipitate were removed for HPLC analysis. The BLG yield for this process was calculated to be 67%.

[0926] Crystal slurry from a 300 L tank was used to feed an Andritz DCF 152S system using a disk membrane with a pore size of 500 nm. Filtration was performed at 8°C, a rotation speed of 32 Hz, and a transmembrane pressure of 0.4 bar. The system operated as a dead-end filtration system, where the retentate accumulated in the filter chamber, unlike in larger units where the retentate is continuously removed. Filtration ran steadily for just over 40 minutes, at which point solids accumulated in the filter chamber began to affect filtration.

[0927] During DFC operation, the amount of crystal quality increases significantly.

[0928] in conclusion.

[0929] DCF provides a stable and efficient means for separating crystals from ML. If necessary, a wash solution can be added to the DCF.

[0930] Example 13: Crystal separation using a filter centrifuge

[0931] Using the same feed and the same crystallization process as in Example 12, the separation was tested on a filter centrifuge HZ 25 / 0.1 equipped with a filter cloth having a pore size of approximately 20 μm.

[0932] Test 1: 4 L of feed was fed to a filter centrifuge operating at 60 g. After all feed was added, the centrifuge was accelerated to 250 g to dry the filter cake. The filter cake contained 47.6% TS; the composition of the filter cake is shown in Table 18.

[0933] After cleaning, 7 L of the same feed as above was fed into a centrifuge (60 g). The centrifuge was then accelerated to 250 g for dehydration for about 5 minutes, after which it was decelerated again to 60 g and 0.25 L of finishing water was added for washing. After the washing water was added, the centrifuge was accelerated again to 250 g for dehydration. The TS of the filter cake was measured to be 47%. The filter cake is shown in FIG. Figure 27 A. The compositions of the filter cake, ML fraction, and wash liquor after washing are shown in Table 18. After the filter cake was dewatered, an attempt was made to peel it from the sides of the centrifuge; Figure 27 As shown in .C, the top layer did accumulate and fall through the intended tube, but the lower layer was too wet and sticky to peel properly, as shown in Figure 27 .B.

[0934] Table 18 Concentrations of selected components of the composition provided in Example 13.

[0935]

[0936]

[0937] 1) Protein composition % (w / w) relative to the weight of the solution

[0938] 2) Concentrations of other selected components (% w / w normalized to 95% total solids relative to the total weight of the composition)

[0939] in conclusion:

[0940] A filter centrifuge offers an interesting option for obtaining a BLG filter cake that is so pure that even without washing, ALA and CMP are below the levels required for quantification. By applying even a small amount of wash medium to the filter cake, the mineral content of the filter cake can be further reduced, as shown by the protein composition of the wash water in Table 18. Washing also reduces the non-BLG protein content of the filter cake, as shown by the wash water used. The wash water used contained a greater ALA:BLG ratio than that in the filter cake. This suggests that the washing step has a greater tendency to remove ALA (and possibly other non-BLG proteins) than BLG.

[0941] The filter cake produced in this way is non-peelable, yet still permeable. This allows the option of adding a drying gas at a given temperature to reduce the moisture content of the filter cake to a level that makes the top layer peelable. Alternatively, the filter cake can be redissolved in the centrifuge by adding an appropriate amount of acid, base, or salt to an aqueous solution in a siphon centrifuge-type setup.

[0942] Example 14: Effect of Mineral Composition of Whey Protein Solutions

[0943] In this example the effect of the molar ratio between monovalent and divalent metal cations on BLG yield was investigated.

[0944] Two samples were compared:

[0945] Sample A: with overweight Na + (Source: Na2SO4)

[0946] Sample B: with overweight Ca 2+ (Source: CaSO4)

[0947] The same type of raw material as used in Example 1 was adjusted with 2.5% sulfuric acid. The pH of the samples was adjusted to approximately pH 5.4; the exact pH is reported in Table 20. The original volume of each sample was 250 mL. Both samples were dialyzed against approximately 24 L of cold polishing water in another 24 L container. For all dialysis processes, dialysis tubing OrDial D-Clean MWCO3500 (Item No. 63034405) was used. The container was continuously stirred during the dialysis process, and the dialysis was performed in a cooler at 4°C. The first dialysis was performed overnight.

[0948] To remove excess ions after the first dialysis, transfer the dialysis bag to a container containing 2 L of saline solution. The concentrations are as follows:

[0949] Sample A: Na2SO4 (sodium sulfate) 0.059M,

[0950] Sample B: CaSO4 (calcium sulfate) 0.059M.

[0951] The first salt dialysis was performed overnight. The conductivity, pH, and Brix after the first salt dialysis are recorded in Table 20. The salt solution was changed to fresh salt solution and the dialysis was continued over the weekend.

[0952] After the second salt dialysis, the tube was transferred to a 24 L container filled with approximately 24 L of cold polish water and dialyzed overnight to remove excess ions prior to crystallization.

[0953] After the last dialysis step, the protein concentration was slightly below the preferred concentration. Therefore, the sample was concentrated on a Pellicon XL UF laboratory setup using a 10 kDa cut-off membrane and a peristaltic pump running at 75 mL / h. The mineral content of the samples is shown in Table 19 along with the raw materials.

[0954] The samples were then inoculated with 0.5 g / L of the aforementioned inoculum material and allowed to crystallize overnight at 4°C. The next day, crystal precipitation was visible in all samples. HPLC samples of each sample were prepared by centrifuging them at 3000 g for 5 minutes, and then a sample of the supernatant was analyzed. The results are shown in Table 21.

[0955] Table 19 Concentrations of selected mineral components in the feedstock of Example 14 and in Samples A and B.

[0956]

[0957]

[0958] 1) The variation is related to the concentration of a given component in the feedstock

[0959] Table 20 pH, conductivity and Brix at various stages in the preparation of samples A and B.

[0960]

[0961] Table 21 Concentration of BLG in samples with different ratios between monovalent and divalent cations.

[0962]

[0963]

[0964] in conclusion:

[0965] Table 21 documents that if a high molar ratio between monovalent and divalent cations is avoided, less residual amount of BLG is left in the mother liquor (and a higher yield of isolated BLG crystals is obtained). The molar ratio between monovalent and divalent cations, and indeed Na+K to Ca+Mg, can be controlled to increase the BLG yield of the process of the present invention.

Claims

1. A method for preparing an edible composition comprising beta-lactoglobulin (BLG) in isolated form, the method comprising the steps of: a) providing a whey protein solution comprising native BLG and at least one additional whey protein, said whey protein solution being supersaturated with respect to BLG, having a pH in the range of 5-6, and having: - a conductivity of at most 5 mS / cm, and / or - a ratio between the electrical conductivity expressed in mS / cm and the total amount of protein of the whey protein solution expressed in % wt total protein relative to the total weight of the whey protein solution of at most 0.3, b) crystallizing BLG in the supersaturated whey protein solution in a salting-out mode, and c) Separating the BLG crystals from the remaining whey protein solution.

2. The method according to claim 1, further comprising the step d) washing the separated crystals obtained from step c).

3. The method according to claim 1, further comprising the step e) recrystallizing the BLG crystals obtained from step c).

4. The method according to claim 1, further comprising the step of drying the BLG-containing composition from step c).

5. The method according to claim 1, wherein The whey protein solution of step a) comprises at least 5% w / w α-lactalbumin relative to the total amount of protein.

6. The method according to claim 1, wherein The whey protein solution of step a) comprises at least 15% w / w of additional whey protein relative to the total amount of protein.

7. The method according to claim 1, wherein The whey protein solution of step a) comprises at least 1% w / w native BLG relative to the total amount of protein.

8. The method according to claim 1, wherein The whey protein solution of step a) comprises at least 4% w / w native BLG relative to the weight of said whey protein solution.

9. The method according to claim 1, wherein The whey protein solution comprises whey protein concentrate, whey protein concentrate, whey protein isolate and / or whey protein isolate.

10. The method according to claim 1, wherein The ratio of the electrical conductivity of the whey protein solution to the total amount of protein is at most 0.

25.

11. The method according to claim 1, wherein The UF permeate conductivity of the whey protein solution is at most 7 mS / cm.

12. The method according to claim 1, wherein The supersaturated whey protein solution is prepared by making one or more of the following adjustments to the whey protein feed: - Adjust pH, - Reduce electrical conductivity - Lower the temperature - Increase protein concentration, and - Add agents that reduce water activity.

13. The method according to claim 12, wherein: Preparation of the whey protein solution involves adjusting the pH of the whey protein feed.

14. The method according to claim 12, wherein: Preparation of the whey protein solution involves reducing the conductivity of the whey protein feed.

15. The method according to claim 12, wherein: Preparation of the whey protein solution involves reducing the temperature of the whey protein feed.

16. The method according to claim 12, wherein: Preparation of the whey protein solution involves increasing the total protein concentration of the whey protein feed.

17. The method according to claim 1, wherein The BLG crystallization of step b) involves one or more of the following: - Wait for crystallization to occur, - Add seed crystals, - further increase the supersaturation of BLG, and / or - Mechanical stimulation.

18. The method according to claim 1, wherein Step c) comprises isolating the BLG crystals to a solids content of at least 30% w / w.

19. The method according to claim 2, wherein: Washing in step d) involves contacting the separated BLG crystals with a washing liquid without completely dissolving the BLG crystals, and subsequently separating the remaining BLG crystals from the washing liquid.

20. The method according to claim 19, wherein The washing of step d) dissolves at most 80% w / w of the initial amount of BLG crystals.

21. The method according to claim 3, wherein The recrystallization step involves: - Dissolve the separated BLG crystals in the recrystallization liquid, - adjusting the recrystallization liquid to obtain supersaturation with respect to BLG, - crystallizing BLG in a supersaturated conditioned recrystallization solution, and - Separating the BLG crystals from the remaining conditioned recrystallization solution.

22. The method according to claim 3, wherein The BLG crystals of step d) are recrystallized at least twice.

23. The method according to claim 4, wherein The drying step involves one or more of spray drying, freeze drying, spin drying and / or fluidized bed drying.

24. The method according to claim 4, wherein The drying step is performed by spray drying.

25. The method according to claim 4, wherein The drying step involves a BLG-containing composition in which the BLG crystals have dissolved and wherein the resulting powder does not contain BLG crystals formed by step b) or by recrystallization prior to the drying step.

26. The method according to claim 25, wherein The BLG crystals have been dissolved by: - Warming up, - Increase electrical conductivity, - change the pH, - Reduce the concentration of BLG, - or a combination of the above.

27. The method according to claim 25, wherein The drying step is performed by spray drying.

28. The method according to claim 4, wherein The BLG-containing composition from step c), d) or e) has a solids content of at least 20% w / w.

29. The method according to claim 4, wherein The heat exposure during the drying step is kept low enough so that the degree of denaturation of the BLG is at most 10%.

30. An edible BLG composition obtainable by the method according to any one of claims 1 to 29, in the form of a powder prepared by spray drying, and comprising: - up to 6% w / w water, - a total amount of proteins of at least 80% w / w relative to the total solids of said edible BLG composition, - at least 90% w / w of native BLG relative to the total amount of said protein, The edible BLG composition may also include one or more of the following: - has a bulk density of at least 0.45 g / mL, - contain at most 80 mg phosphorus per 100 g protein, - the BLG of the edible BLG composition has a degree of lactosylation of at most 1, - Has a protein denaturation degree of up to 2%.

31. An edible BLG composition according to claim 30 comprising at least 95% w / w native BLG relative to the total amount of protein.

32. An edible BLG composition according to claim 30, comprising at most 10% w / w carbohydrates.

33. The edible BLG composition of claim 30 comprising a total amount of lipids of at most 0.5% w / w relative to total solids.

34. The edible BLG composition of claim 30, having a bulk density of at least 0.5 g / mL.

35. The edible BLG composition of claim 30, wherein The BLG of the edible BLG composition has a degree of lactosylation of at most 0.

2.

36. The edible BLG composition of claim 30, wherein The edible BLG composition comprises at most 50 mg phosphorus per 100 g protein.

37. The edible BLG composition of claim 30, which is in the form of a dry BLG composition that is a powder and has a furosine value of at most 80 mg / 100 g protein after 60 days at 30°C.

38. The edible BLG composition of claim 30, having a degree of protein denaturation of at most 1.5%.

39. The edible BLG composition of claim 30, comprising: - up to 6% w / w water, - at least 80% w / w total protein relative to total solids, - at least 95% w / w native BLG relative to total protein, and The edible BLG composition has a bulk density of at least 0.45 g / mL.

40. The edible BLG composition of claim 30, comprising: - up to 6% w / w water, - at least 80% w / w total protein relative to total solids, - at least 95% w / w native BLG relative to total protein, and The edible BLG composition has a degree of protein denaturation of at most 2%.

41. The edible BLG composition of claim 30, comprising: - up to 6% w / w water, - at least 80% w / w total protein relative to total solids, - at least 95% w / w native BLG relative to total protein, and - Up to 80 mg phosphorus / 100 g protein.

42. The edible BLG composition of claim 30, comprising: - up to 6% w / w water, - at least 80% w / w total protein relative to total solids, - at least 95% w / w native BLG relative to total protein, and The edible BLG composition has a furosine value of at most 80 mg / 100 g protein after 60 days at 30°C.

43. The edible BLG composition of claim 30, comprising: - up to 6% w / w water, - at least 80% w / w total protein relative to total solids, - at least 95% w / w native BLG relative to total protein, and wherein the BLG of the edible BLG composition has a degree of lactosylation of at most 0.

2.

44. The edible BLG composition of claim 30, comprising: - up to 6% w / w water, - at least 80% w / w total protein relative to total solids, - at least 97% w / w native BLG relative to total protein, and The edible BLG composition has a degree of protein denaturation of at most 2%.

45. The edible BLG composition of claim 30, comprising: - up to 6% w / w water, - at least 80% w / w total protein relative to total solids, - at least 95% w / w native BLG relative to total protein, - Up to 50 mg phosphorus / 100 g protein.

46. ​​The edible BLG composition of claim 30, comprising: - up to 6% w / w water, - at least 80% w / w total protein relative to total solids, - at least 95% w / w native BLG relative to total protein, and The edible BLG composition has a furosine value of at most 40 mg / 100 g protein after 60 days at 30°C.

47. The edible BLG composition of claim 30, comprising: - up to 6% w / w water, - at least 80% w / w total protein relative to total solids, - at least 97% w / w native BLG relative to total protein, and wherein the BLG of the edible BLG composition has a degree of lactosylation of at most 0.2.

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