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

Abstract

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

Description

[0001] This application is a divisional application of the application with the application date of 22 December 2017, the application number 201780084095.1, and the invention title "Production of novel beta-lactoglobulin preparations and related methods, uses and food products". TECHNICAL FIELD

[0002] The present invention relates to a new method of producing an isolated beta-lactoglobulin composition and / or a composition containing crystalline beta-lactoglobulin. The present invention further relates to new beta-lactoglobulin compositions, uses of these compositions and food products comprising these compositions. BACKGROUND

[0003] The concept of fractionation of milk proteins is well known in the art and has developed over the past decades into a range of techniques to prepare 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 many 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 beta-Lactoglobulin, J Dairy Sci., Vol. 84 (3), 2001, pp. 562-571) describe the purification of BLG from freshly expressed milk by low temperature acid coagulation of casein and by subjecting the obtained acid whey to a combination of affinity chromatography (DEAE Sepharose) and gel permeation chromatography. The obtained BLG composition is said to contain 0.985 g beta-lactoglobulin per 1 g protein.

[0006] Slack et al. (Journal of Food Processing and Preservation, Vol. 10, 1986, pp. 19-30) explore a different approach and prepare a BLG-enriched precipitate by adjusting the pH of demineralised acid whey and sweet whey to pH 4.65 and isolating the formed precipitate by centrifugation and decantation. The obtained precipitate particles are described as relatively insoluble and contain significant amounts of protein impurities in addition to BLG. No crystal formation is observed. It is 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) reports a laborious and time-consuming process for producing protein crystals based on acid whey using salt precipitation of unwanted proteins, pH adjustment and dialysis to remove other unwanted proteins of several sequences. Finally, when a highly purified BLG solution is obtained, BLG crystallizes. The process lasts more than 12 days and requires the addition of toluene. Thus, 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) discloses an improved process over the Palmer method which allows the preparation of beta-lactoglobulin crystals in the order of days rather than weeks. However, the improved method still requires the removal of unwanted proteins prior to crystallization and also uses toluene for crystallization which makes it incompatible with safe food production.

[0009] JP H10 218755 A discloses the production of a cosmetic composition containing a melanin production inhibitor comprising BLG as an active ingredient. The document further 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-saturation amount; precipitated proteins are removed by salting-out and the filtrate is recovered. The filtrate is saturated with ammonium sulfate and precipitated proteins are recovered. The recovered proteins are again dissolved in water and dialyzed at pH 5.2 to isolate crystals, and beta-lactoglobulin is prepared at a ratio of about 1.8 g from 1 L of whey. However, the general process steps of the proposed process described in JP H10 218755 A are not sufficient to result in the formation of BLG crystals. Thus, the document does not contain a disclosure of the achievement of BLG or BLG crystal crystallization.

[0010] US 2 790 790 discloses a process for precipitating proteins from solution, and more specifically for fractionally precipitating relatively unconjugated proteins from aqueous solutions by using sodium chloride as a precipitant. It is indicated that the process can be used for the isolation of BLG by NaCI-induced precipitation at pH 3.6-3.8. In Example II of the document, it is indicated that the NaCI-precipitate can be dialysed in the usual way to form crystalline B-lactoglobulin. However, US 2 790 790 does not demonstrate that it is actually possible to form BLG crystals at pH 3.6-3.8, and does not refer to the meaning of the "usual way" of dialysing BLG precipitates. Thus, the document does not contain a disclosure of the realisation of BLG or BLG crystal crystallisation. SUMMARY

[0011] By chance, the inventors surprisingly found that it is possible to directly prepare high purity BLG crystals in a crude lactoferrin solution containing significant amounts of other whey proteins in addition to BLG, and without using organic solvents such as toluene. This is contrary to the common wisdom in the art that proteins must be highly purified before one wishes to make them crystallise, and that not all proteins can be crystallised.

[0012] This finding has the potential to change the way whey proteins are handled and fractionated in the dairy industry, and opens up for an 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 of preparing an edible composition comprising beta-lactoglobulin (BLG) in crystalline and / or isolated form, said method comprising the steps of:

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

[0015] b) crystallising BLG in said 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 as it simplifies handling of the powder and makes it less dusty.

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

[0019] In the context of the present application, a dry product, e.g. a powder, comprising "BLG crystals" contains a product obtained by drying a suspension of BLG crystals, whereas the crystal structure of the wet BLG crystals can have been deformed during the drying process and can have lost at least partially its X-ray diffraction characteristics. Likewise, the terms "dried BLG crystals" and "dried BLG crystal" refer to particles obtained by drying wet BLG crystals, and such dried particles do not need to have a crystal structure as such. However, the inventors observed that when dried BLG crystals are resuspended in cold (4°C) demineralized water in a weight ratio of 2 parts water to 1 part dried BLG crystals, the BLG crystals rehydrate and regain essentially the same crystal structure (space group type and cell size) as before drying.

[0020] It is well known that BLG is an important source of essential amino acids, including e.g. leucine, and thus the edible BLG composition provided by the present application has several interesting nutritional uses.

[0021] Another aspect of the present application relates to an isolated BLG crystal having an orthorhombic space group P 21 21 21, and cell dimensions a = 6.9 A, b = 7.9 A, and c = 2.9 A. and and wherein the crystal has cell integral angles a = 90°, b = 90°, and g = 90°.

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

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

[0024] The present application provides the following:

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

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

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

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

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

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

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

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

[0033] 4) The method according to any one of the preceding items, further comprising 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 items, wherein the whey protein solution of step a) comprises at least 5% (w / w) ALA relative to the total amount of protein.

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

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

[0037] 8) The method according to any one of the preceding items, 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 items, wherein the whey protein solution comprises milk serum protein concentrate, whey protein concentrate, milk serum protein isolate and / or whey protein isolate.

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

[0040] 11) The method according to any one of the preceding items, 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, wherein the supersaturated whey protein solution is prepared by one or more of the following adjustments to the whey protein feed:

[0042] - adjusting the pH,

[0043] - reducing the electrical conductivity

[0044] - reducing the temperature

[0045] - increasing the protein concentration, and

[0046] - adding an agent that reduces the water activity.

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

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

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

[0050] 16) The method according to any of the preceding, 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, wherein the BLG crystallization of step b) involves one or more of the following:

[0052] - waiting for crystallization to occur,

[0053] - adding seed crystals,

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

[0055] - mechanical stimulation.

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

[0057] 19) The method according to any of 2) - 19), wherein the 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.

[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) of the initial amount of BLG crystals, 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) - 20), wherein the recrystallization step involves:

[0060] - dissolving the isolated BLG crystals in a recrystallization liquid,

[0061] - adjusting the recrystallization liquid to obtain an over-saturation with respect to BLG,

[0062] - crystallizing BLG in the over-saturated adjusted recrystallization liquid, and

[0063] - separating the BLG crystals from the remaining adjusted recrystallization liquid.

[0064] 22) The method according to any one of 3) - 21), wherein the BLG crystals of step d) are recrystallized at least 2 times.

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

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

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

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

[0069] 27) The edible BLG composition according to any one of 24) - 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) - 27), wherein the composition is a dry composition.

[0071] 29) The dry BLG composition according to 28) 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] - at least 10% BLG crystallinity.

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

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

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

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

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

[0080] 36) A food product comprising the edible BLG composition according to any of 24) - 33) and at least one additional ingredient, such as e.g. 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, the dry food product comprising at least 1% (w / w) BLG, wherein:

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

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

[0084] 38) The food product according to any of 36) - 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 of 36) - 38), which is a dairy product, a confectionary, a beverage, a protein bar or an enteral nutritional composition.

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

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

[0088] - a sweetener,

[0089] - at least one edible acid,

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

[0091] - at most 80 mg of phosphorus per 100 g of protein.

[0092] 41 ) An isolated BLG crystal having an orthorhombic space group P 21 21 21 and a unit cell size of a = 2.5 nm, b = 2.5 nm, and c = 2.5 nm. and and having unit cell integral angles a = 90°, b = 90°, and g = 90°.

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

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

[0095] Figure 2 is a microscope picture of recovered BLG crystals from Example 1.

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

[0097] Figure 4 is a plot of the conductivity of the whey protein solution versus the yield of recovered BLG crystals obtained.

[0098] Figure 5 is a plot of the temperature and conductivity of the whey protein solution versus the yield of recovered BLG crystals obtained.

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

[0100] Figure 7 shows the chromatogram 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 sample microscope picture taken during the early stage of crystallization of feed 1 of Example 3.

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

[0103] Figure 10 shows a chromatogram of the washed BLG crystals obtained from feed 1 of Example 3.

[0104] Figure 11 shows a chromatogram 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 shows pictures of feed 2 of Example 3 before (left picture) and after (right picture) crystallization.

[0106] Figure 13 shows microscope pictures of the whole and the pieces of BLG crystals obtained from feed 2 of Example 3.

[0107] Figure 14 and Figure 15 shows that increasing the conductivity or changing the pH of the BLG crystal slurry results in dissolution of the BLG crystals.

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

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

[0110] Figure 18 is a microscope picture of BLG crystals recovered from feed 3 of Example 3.

[0111] Figure 19 shows a chromatogram of the recovered BLG crystals of feed 3 of Example 3 (without any washing step).

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

[0113] Figure 21 is a microscope picture of BLG crystals formed at a conductivity of 4.20 mS / cm.

[0114] Figure 22 shows a microscope picture of BLG crystals from the early stage of crystallization of SPC based whey protein solutions.

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

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

[0117] Figure 25 is a photo of the sub-samples of the six low phosphorus beverage samples of Example 8. From left to right, the sub-samples are samples A, B, C, D, E and F.

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

[0119] Figure 27 Three photos showing the filter cake obtained using a filter centrifuge to separate BLG crystals from the mother liquor. DETAILED DESCRIPTION

[0120] As mentioned above, one aspect of the present invention relates to a method of preparing an edible composition comprising beta-lactoglobulin (BLG) in crystallized and / or isolated form, said 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 said 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 as well as for 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 exists in several genetic variants, the predominant proteins in bovine milk being labeled A and B. BLG is a lipocalin and can bind many hydrophobic molecules, indicating that BLG plays a role in the transport of said hydrophobic molecules. BLG has also been shown to be able to bind iron via a transferrin-like mechanism and can have a role in combating pathogens. Homologues of BLG are absent in human breast milk.

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

[0126] BLG solutions can form gels under various conditions when the native structure is unstable enough to allow aggregation. Upon prolonged heating at low pH and low ionic strength, a transparent 'fine-stranded' gel is formed in which the protein molecules assemble into long, hard fibres.

[0127] In the context of the present invention, the term "BLG" or "beta-lactoglobulin" relates to BLG from a mammalian species, e.g. 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 microscopic structure, forming a crystal lattice that extends in all directions. A BLG crystal is a protein crystal that mainly contains BLG arranged in a highly ordered microscopic structure, forming a crystal lattice that extends in all directions. A BLG crystal can be e.g. single- or polycrystalline, and can e.g. be a complete crystal, a crystal fragment or a combination thereof. A crystal fragment is e.g. formed when a complete crystal is subjected to mechanical shearing during processing. A crystal fragment also has a highly ordered crystal microstructure, but can lack the uniform surface and / or uniform edges or corners of a complete crystal. See e.g. https: / / en.wikipedia.org / wiki / Single-crystal. Figure 18 is an example of a complete BLG crystal, and Figure 13 is an example of a BLG crystal fragment. In both cases, the BLG crystal or crystal fragment can be visually identified as a distinct, compact and coherent structure using an optical microscope. The BLG crystal or crystal fragment is typically at least partially transparent. Furthermore, protein crystals are known to be birefringent, and this optical property can be used to identify unknown particles as having a crystal structure. On the other hand, non-crystalline BLG aggregates appear as non-distinct, opaque, and as irregularly sized open or porous lumps.

[0129] In the context of the present invention, the term "crystallization" relates to the formation of a protein crystal. Crystallization can e.g. occur spontaneously or be initiated by the addition of seeds.

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

[0131] The BLG crystals can typically be observed by microscopy, and can even reach a size that makes the eye see them.

[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 higher than the saturation point of BLG in that liquid under the given physical and chemical conditions. The term "supersaturation" is well known in the field of crystallization (see for example Gérard Coquerela, "Crystallization of molecular systems from solution: phase diagrams, supersaturation and other basic concepts", Chemical Society Reviews, pages 2286-2300, Issue 7, 2014), and supersaturation can be determined by many different measurement techniques, for example 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 to test if 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 with a height of 115 mm, an inner diameter of 25 mm, and a capacity of 50 mL (VWR catalog number 525-0402). During steps a) - h), care should be taken to keep the sample and its subsequent fractions under 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 of 30 seconds acceleration and a maximum of 30 seconds deceleration.

[0136] c) Immediately after centrifugation, transfer as much of the supernatant as possible (if a precipitate has formed, do not disturb the precipitate) 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 (BLG at least 98% pure with respect to total solids) having a particle size of at most 200 microns are added to a second centrifuge tube and the mixture is stirred.

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

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

[0141] h) If present, the centrifugation pellet of step g) is recovered, resuspended in milliQ water and the suspension is immediately checked for the presence of crystals visible by microscope.

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

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

[0144] In the context of the present application, the terms "liquid" and "solution" encompass compositions containing a combination of a liquid and solid or semi-solid particles, such as protein crystals or other protein particles. Thus, the "liquid" or "solution" can be a suspension or even a slurry. However, the "liquid" and "solution" are preferably pumpable.

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

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

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

[0148] The 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, it can also be preferred that the WPC or SPC contains:

[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 a dry or aqueous composition containing a total amount of protein of 90-100% (w / w) relative to total solids.

[0169] The 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, it can also be preferred that the WPI or SPI can 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 can 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 application, the method further comprises a step d) of washing the BLG crystals, e.g. the isolated BLG crystals obtained from step c).

[0185] In some preferred embodiments of the application, the method further comprises a step e) of recrystallizing the BLG crystals, e.g. the BLG crystals obtained from step c) or d).

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

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

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

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

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

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

[0192] As mentioned, step a) of the application involves providing a whey protein solution comprising BLG and at least one further whey protein.

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

[0194] In the context of the present application, the term "further protein" means a protein that is not BLG. The further proteins present in the whey protein solution typically comprise one or more of the non-BLG proteins found in milk serum or whey. Non-limiting examples of such proteins are a-lactalbumin, bovine serum albumin, immunoglobulin, casein macropeptide (CMP), osteopontin, lactoferrin and milk fat globule membrane proteins.

[0195] Thus, the whey protein solution can preferably contain at least one further whey protein selected from the group consisting of a-lactalbumin, bovine serum albumin, immunoglobulin, caseinomacropeptide (CMP), osteopontin, lactoferrin, milk fat globule membrane proteins, and combinations thereof.

[0196] a-lactalbumin (ALA) is a protein found in the milk of almost all mammalian species. ALA forms the regulatory subunit of the lactose synthase (LS) heterodimer, and the b-1,4-galactosyltransferase (b4Gal-T1 ) forms the catalytic component. Together, these proteins enable LS to produce lactose by transferring a galactose moiety to glucose. As a multimer, a-lactalbumin binds strongly to calcium and zinc ions and can have bactericidal or antitumor activity. One of the major structural differences from b-lactoglobulin is that ALA does not have any free thiol groups that can serve as a starting point for covalent aggregation reactions. As a result, pure ALA does not form a gel upon denaturation and acidification.

[0197] In the context of the present invention, the term "ALA" or "a-lactalbumin" relates to a-lactalbumin from a mammalian species, e.g. 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 "milk serum" refers to the liquid that remains when casein and milk fat globules are removed from milk, e.g. by microfiltration or by ultrafiltration through large pores. Milk serum can also be referred to as "ideal whey".

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

[0201] The term "whey" relates to the liquid supernatant that remains after the casein of milk has been precipitated and removed. Casein precipitation can be accomplished, e.g. by acidifying the milk and / or using rennet.

[0202] There are several types of whey: e.g. "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, e.g. by adding a food acid or by bacterial culturing.

[0203] In some preferred embodiments of the application, 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) can comprise at least 30% (w / w) additional whey protein relative to the total amount of protein.

[0204] In other preferred embodiments of the application, 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) can comprise at least 4% (w / w) additional whey protein relative to the total amount of protein.

[0205] In yet other preferred embodiments of the application, 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) can 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) can 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) can comprise at least 50% (w / w) additional whey protein relative to the total amount of protein.

[0206] In some preferred embodiments of the application, 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) can 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) can 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 mentioned, the inventors found that BLG can be crystallized without the use of organic solvents. This purification approach can also be used to refine preparations containing whey proteins that have already undergone some BLG purification; and provides a simple way to further increase the purity of BLG. Thus, in some preferred embodiments of the present application, the whey protein solution of step a) comprises in the range of 1-20% (w / w) additional whey protein relative to the total amount of protein. Preferably, the whey protein solution of step a) can comprise in the range of 2-15% (w / w) additional whey protein relative to the total amount of protein. Even more preferably, the whey protein solution of step a) can for example comprise in the range of 3-10% (w / w) additional whey protein relative to the total amount of protein.

[0208] In some embodiments of the present application, 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) can comprise at least 20% (w / w) ALA relative to the total amount of protein.

[0209] In some preferred embodiments of the present application, 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) can comprise at least 40% (w / w) ALA relative to the total amount of protein.

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

[0211] In some preferred embodiments of the application, 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, e.g. at least 2. For example, the weight ratio between BLG and ALA in the whey protein solution of step a) can be at least 3.

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

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

[0214] In some preferred embodiments of the application, 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 application, the whey protein solution of step a) comprises at least 1 % (w / w) BLG relative to the total amount of proteins. Preferably, the whey protein solution of step a) comprises at least 2 % (w / w) BLG relative to the total amount of proteins. Even more preferably, the whey protein solution of step a) comprises at least 5 % (w / w) BLG relative to the total amount of proteins. Preferably, the whey protein solution of step a) can comprise at least 10 % (w / w) BLG relative to the total amount of proteins.

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

[0217] In some particularly preferred embodiments of the application, the whey protein solution of step a) comprises at most 95% (w / w) BLG relative to the total amount of protein. Preferably, the whey protein solution of step a) can comprise at most 90% (w / w) BLG relative to the total amount of protein. More preferably, the whey protein solution of step a) can for example comprise at most 85% (w / w) BLG relative to the total amount of protein. Even more preferably, the whey protein solution of step a) can for example comprise at most 80% (w / w) BLG relative to the total amount of protein. Preferably, the whey protein solution of step a) can comprise at most 78% (w / w) BLG relative to the total amount of protein. Preferably, the whey protein solution of step a) can comprise at most 75% (w / w) BLG relative to the total amount of protein.

[0218] In some preferred embodiments of the application, the whey protein solution of step a) comprises BLG in the range of 1-95% (w / w) relative to the total amount of protein. Preferably, the whey protein solution of step a) can comprise 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) comprises 10-85% (w / w) BLG relative to the total amount of protein. Even more preferably, the whey protein solution of step a) comprises 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) can comprise BLG in the range of 20-70% (w / w) relative to the total amount of protein.

[0219] In other preferred embodiments of the application, the whey protein solution of step a) comprises BLG in the range of 10-95% (w / w) relative to the total amount of protein. Preferably, the whey protein solution of step a) can comprise 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) comprises 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) comprises 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) can comprise BLG in the range of 30-70% (w / w) relative to the total amount of protein.

[0220] In some preferred embodiments of the application, 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] Even 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 application, 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 source of whey protein can be used to prepare the whey protein solution. In some preferred embodiments of the application, the whey protein solution comprises or even consists of milk serum protein concentrate, whey protein concentrate, milk serum protein isolate, whey protein isolate, or a combination thereof.

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

[0225] In the context of the present document, the term demineralized means that the 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 milk serum protein concentrate, a demineralized milk serum protein isolate, a demineralized whey protein concentrate, or a demineralized whey protein isolate.

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

[0228] The whey protein solution may, for example, comprise or even consist of a demineralized milk serum 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 milk serum 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 application, the terms "whey protein concentrate" and "milk serum protein concentrate" relate to a preparation of whey or milk serum containing in the range of about 20-89% (w / w) protein relative to total solids.

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

[0231] The terms "consists essentially of and "consisting essentially of mean including the materials or steps specified and those that do not materially affect the basic and novel characteristic(s) of the claimed application.

[0232] The proteins of the whey protein solution are preferably from mammalian milk, and preferably from ruminant milk, e.g. cow, sheep, goat, buffalo, camel, llama, mare and / or deer milk. Proteins from bovine (cow) milk are particularly preferred. Thus, the BLG and the further whey proteins are preferably bovine BLG and bovine whey proteins.

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

[0234] In some preferred embodiments of the application, 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, e.g. preferably at most 0.01.

[0235] The degree of lactosylation of BLG is determined according to Czerwenka et al. (J. Agric. Food Chem. [Journal of Agricultural and Food Chemistry], Vol. 54, Issue 23, 2006, pp. 8874-8882).

[0236] In some preferred embodiments of the application, the whey protein solution has a furfural amine value of at most 80 mg / 100 g protein. Preferably, the whey protein solution has a furfural amine value of at most 40 mg / 100 g protein. More preferably, the whey protein solution has a furfural amine value of at most 20 mg / 100 g protein. Even more preferably, the whey protein solution has a furfural amine value of at most 10 mg / 100 g protein. Most preferably, the whey protein solution has a furfural amine value of at most 5 mg / 100 g protein, e.g. preferably a furfural amine value of 0 mg / 100 g protein.

[0237] In addition to proteins, the whey protein solution typically also contains other components. The whey protein solution can contain other components normally found in whey or milk serum, e.g. minerals, carbohydrates and / or lipids. Alternatively or additionally, the whey protein solution can contain non-native components of whey or milk serum. However, such non-native components should preferably be safe for food production use, and preferably also safe for human consumption.

[0238] The present method is particularly advantageous for isolating 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 hydrolysis products 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 application, 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 can also comprise lipids, such as lipids in the form of triglycerides and / or other lipid types, such as phospholipids.

[0242] In some embodiments of the application, 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 of 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 of the whey protein solution is at least 5% (w / w). More preferably, the total amount of protein of the whey protein solution is at least 10% (w / w). Even more preferably, the total amount of protein of the whey protein solution is at least 15% (w / w).

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

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

[0246] The whey protein solution is typically prepared by one or more adjustments to a whey protein feed, which adjustments form 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 application, the term "whey protein feed" relates to a composition that is converted into a whey protein solution that is supersaturated with respect to BLG. The whey protein feed is typically an aqueous liquid comprising BLG and at least one additional whey protein, but is generally not supersaturated with respect to BLG.

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

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

[0251] - adjusting the pH,

[0252] - reducing the electrical conductivity

[0253] - reducing the temperature

[0254] - increasing the protein concentration

[0255] - adding an agent that reduces the water activity

[0256] - modifying the ionic composition

[0257] In some preferred embodiments of the present application, 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 are 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 can 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 application, 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 can 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] It is preferred to adjust the pH using a food acceptable acid and / or base. A food acceptable acid is particularly preferred, such as a carboxylic acid. 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 application, the pH is adjusted using a lactone, such as D-gluconic acid-delta-lactone, which hydrolyzes slowly and simultaneously decreases the pH of the aqueous liquid containing it. The target pH after the end of the lactone hydrolysis can be calculated precisely.

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

[0264] In other preferred embodiments of the application, the pH is adjusted by adding a cation exchange material in the form of H + prior to crystallization or even after crystallization. The bead / large particle type cation exchange material is easily removed from the whey protein solution. The pH is adjusted by adding a cation exchange material in the form of H + The adjustment of the pH by adding a cation exchange material in the form of H

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

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

[0267] The inventors have found that reducing the conductivity of the whey protein solution results in a higher yield of BLG crystals. The minimum obtainable 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 caseinomacropeptide (CMP), contribute more to the conductivity than other protein species. It is therefore preferred to bring the conductivity of the whey protein feed close to the level where the proteins and the counterions of the proteins are the main contributors to the conductivity. The reduction of the conductivity typically involves the removal of at least some small free ions that are present in the liquid phase and are not tightly bound to the proteins.

[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 application, 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] Even lower conductivity is even more preferred and results in even higher BLG crystal yield. Thus, it is preferred that the whey protein solution has a conductivity of at most 3 mS / cm. In some preferred embodiments of the application, 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 conductivity of the whey protein feed is preferably reduced by dialysis or by ultrafiltration. Ultrafiltration by ultrafiltration is particularly preferred as it allows for washing away of salts and small charged molecules while retaining the proteins. In some preferred embodiments of the application, the same UF unit is used for the UF / ultrafiltration as well as for the subsequent concentration of the whey protein feed.

[0271] The inventors have found indications that the ratio between the conductivity (in mS / cm) and the total amount of protein (in %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 to facilitate crystallization of BLG.

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

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

[0274] The inventors have also found 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. The UF permeate conductivity is a measure of the conductivity of the small molecule fraction of a liquid and is measured according to Example 9.10. 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 can be at most 5 mS / cm. Even more preferably, the UF permeate conductivity of the whey protein solution can be at most 3 mS / cm.

[0276] If a high yield of BLG should be obtained, even lower UF permeate conductivities can be used and are particularly preferred. Thus, 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 can be at most 0.4 mS / cm. Even more preferably, the UF permeate conductivity of the whey protein solution can be at most 0.1 mS / cm. Most preferably, the UF permeate conductivity of the whey protein solution can be at most 0.04 mS / cm.

[0277] Even lower UF permeate conductivities can be achieved, e.g. using MilliQ water as diluent during diafiltration (MilliQ water has a conductivity of about 0.06 μ8 / cm). Thus, the UF permeate conductivity of the whey protein solution can be at most 0.01 mS / cm. Alternatively, the UF permeate conductivity of the whey protein solution can be at most 0.001 mS / cm. Alternatively, the UF permeate conductivity of the whey protein solution can be at most 0.0001 mS / cm.

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

[0279] For example, the preparation of the whey protein solution can 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, the preparation of the whey protein solution can 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 reduced 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, thus, the temperature of the whey protein feed can for example be reduced 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 be kept pumpable, e.g. in the form of an ice slurry.

[0281] In some preferred embodiments of the application, the whey protein solution is an ice slurry prior to the initiation of BLG crystallization. Alternatively or additionally, the crystallization of the whey protein solution can be converted to an ice slurry or kept as an ice slurry during the BLG crystallization of step b).

[0282] In some particularly preferred embodiments of the application, 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 be subjected to 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 the proteins, while the concentration of salts and carbohydrates is hardly affected. As mentioned above, ultrafiltration is preferably used for both diafiltration and concentration of the whey protein feed.

[0284] In some preferred embodiments of the application, the BLG concentration of the whey protein solution is below the level at which spontaneous crystallization of BLG occurs. It is thus generally preferred that the modification of the whey protein feed is terminated when the whey protein solution is in the metastable zone, i.e. in the supersaturation zone, in which BLG crystals can grow but crystallization does not spontaneously start using inoculation.

[0285] In some preferred embodiments of the application, 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 application, the preparation of the whey protein solution involves modifying the ionic composition of the whey protein feed, e.g. by ion exchange, by adding 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 create supersaturation.

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

[0290] - concentrating at a temperature above 10°C, e.g. using ultrafiltration, nanofiltration or reverse osmosis, and

[0291] - subsequently cooling to a temperature below 10°C.

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

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

[0294] - subsequently lowering the pH by adding an acid, e.g. GDL or H + In yet other preferred embodiments of the application, the preparation of the whey protein solution involves subjecting the whey protein feed to at least:

[0295] - lowering the conductivity by diafiltration, e.g. using a membrane that retains at least BLG.

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

[0297] - adjusting the pH to 5-6,

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

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

[0300] - finally, cooling to a temperature below 10°C.

[0301] The inventors have also found 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, a relatively higher amount of calcium and magnesium appears to increase the yield of BLG, and thus the BLG recovery efficiency of the present process.

[0302] In some preferred embodiments of the application, 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, e.g. 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 ), wherein m Na is the content of the element Na in mol, m K is the content of the element K in mol, m Ca is the content of the element Ca in mol, and m Mg is the content of the element Mg in mol.

[0304] It is particularly preferred that the whey protein solution is supersaturated with respect to BLG by salt-in, so that BLG can crystallize from the whey protein solution in a salt-in mode.

[0305] In some embodiments of the application, in particular if the edible BLG product of the application 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 method involves crystallizing at least some of the BLG of the supersaturated whey protein solution.

[0307] It is particularly preferred that the crystallization of step b) is performed in a salt-in mode, i.e. in a liquid having a low ionic strength and a low electrical conductivity. This is in contrast to a 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] - waiting for crystallization to occur,

[0310] - adding seeds,

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

[0312] - mechanical stimulation.

[0313] In some preferred embodiments of the application, step b) involves adding seeds to the whey protein solution. The inventors have found that adding seeds can control the time and place of BLG crystallization to avoid sudden clogging of process equipment and unexpected stops in the production process. For example, it is generally desirable to avoid crystallization onset when concentrating the whey protein feed.

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

[0315] The seed crystals can be in dry form or can form part of a suspension when added to the whey protein solution. It is currently preferred to add a suspension containing seed crystals, such as BLG crystals, as this appears to provide a faster start 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 application, at least some of the seed crystals are located on a solid phase which is contacted with the whey protein solution.

[0317] The seed crystals preferably have a particle size which is smaller than the desired size of the BLG crystals. The size of the seed crystals can be altered by sieving or other size fractionation processes to remove the largest seeds. The particle size can also be reduced prior to size fractionation (e.g. by grinding).

[0318] In some embodiments of the application, 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 can have a particle size in the range of 1-400 microns. Preferably, at least 90% (w / w) of the seed crystals can have a particle size in the range of 5-200 microns. More preferably, at least 90% (w / w) of the seed crystals can 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 application, the seed crystals are added to the whey protein feed prior to obtaining supersaturation with respect to BLG, but preferably in such a way that at least some of the seed crystals are still present when supersaturation is reached. This can for example be done by adding the seed crystals when the whey protein feed is close to supersaturation (e.g. during cooling, concentration and / or pH adjustment), supersaturation being reached before the seed crystals are completely dissolved.

[0321] In some preferred embodiments of the application, step b) involves further increasing the supersaturation of BLG, preferably to such an extent that crystallization of BLG starts immediately (i.e. within at most 20 minutes, and preferably within at most 5 minutes). This is also referred to as the nucleation zone, in which microcrystals form spontaneously and the crystallization process starts.

[0322] The supersaturation can for example be increased by one or more of:

[0323] - further increasing the protein concentration of the whey protein solution

[0324] - further cooling the whey protein solution

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

[0326] - further decreasing the conductivity.

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

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

[0329] However, in other preferred embodiments of the application, step b) involves a further adjustment of the whey protein solution to be crystallized to increase the supersaturation of BLG or at least to maintain the supersaturation. The further adjustment results in an increased yield of BLG crystals.

[0330] Such further adjustment can involve one or more of the following:

[0331] - further increasing the protein concentration of the crystallization whey protein solution

[0332] - cooling the crystallization whey protein solution to an even lower temperature

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

[0334] - further decreasing the conductivity of the crystallization whey protein solution.

[0335] In some preferred embodiments of the application, during step b) the crystallization whey protein solution is kept in the metastable zone to avoid spontaneous formation of new microcrystals.

[0336] The inventors have determined the lattice structure of the isolated BLG crystals by X-ray crystallography, and no similar crystals have been found in the prior art.

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

[0338] Preferably, at least some of the BLG crystals obtained have an orthorhombic space group P 21 21 21, and cell dimensions a = 6.9 A, b = 7.9 A, and c = 9.9 A. and and cell integral angles a = 90°, b = 90°, and g = 90°.

[0339] In some preferred embodiments of the application, at least some of the BLG crystals obtained have an orthorhombic space group P 21 21 21, and cell dimensions a = 6.9 A, b = 7.9 A, and c = 9.9 A. and and the unit cell integral angles a = 90°, b = 90°, and g = 90°.

[0340] Even more preferably, at least some of the obtained BLG crystals can have an orthorhombic space group P 21 21 21, and the unit cell size and and the unit cell integral angles a = 90°, b = 90°, and g = 90°.

[0341] Most preferably, at least some of the obtained BLG crystals have an orthorhombic space group P 21 21 21, the unit cell size and and the unit cell integral angles a = 90°, b = 90°, and g = 90°.

[0342] In some particularly preferred embodiments of the application, the method comprises a step c) of separating at least some of the BLG crystals from the remaining whey protein solution. This is particularly preferred when BLG is to be purified.

[0343] Step c) can 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 solid content is advantageous for the purification of BLG, since the aqueous fraction adhering to the separated BLG crystals typically contains impurities that should be avoided. In addition, a high solid content reduces the energy consumption for converting the separated BLG crystals into a dry product, such as a powder, and increases the BLG yield obtained from a drying unit of a given capacity.

[0345] In some preferred embodiments of the application, 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 application, the separation of step c) involves one or more of the following operations:

[0347] - centrifugation,

[0348] - decantation,

[0349] - filtration,

[0350] - sedimentation,

[0351] - a combination of the above.

[0352] These unit operations are well known to the skilled person and are readily implemented. The separation by filtration can for example involve the use of vacuum filtration, dynamic cross-flow filtration (DCF), filter press or filter centrifuge.

[0353] Depending on the desired result, different pore sizes for the filtration can be employed. Preferably, the filter allows native whey proteins and small aggregates to pass but retains the BLG crystals. The filter preferably has a nominal pore size of at least 0.1 micrometer. The filter may, for example, have a nominal pore size of at least 0.5 micrometer. Even more preferably, the filter can have a nominal pore size of at least 2 micrometers.

[0354] Filters with larger pore sizes can also be used and are in fact preferred if the main separation is to be between large crystals and liquid containing BLG crystals. In some embodiments of the present application, the filter has a nominal pore size of at least 5 micrometers. Preferably, the filter has a nominal pore size of at least 20 micrometers. Even more preferably, the filter can have a pore size of at least 40 micrometers.

[0355] The filter may, for example, have a pore size in the range of 0.03-5000 micrometers, such as 0.1-5000 micrometers. Preferably, the filter can have a pore size in the range of 0.5-1000 micrometers. Even more preferably, the filter can have a pore size in the range of 5-800 micrometers, such as a pore size in the range of 10-500 micrometers or in the range of 50-500 micrometers.

[0356] In some preferred embodiments of the present application, the filter has a pore size in the range of 0.03-100 micrometers. Preferably, the filter can have a pore size in the range of 0.1-50 micrometers. More preferably, the filter can have a pore size in the range of 4-40 micrometers. Even more preferably, the filter can have a pore size in the range of 5-30 micrometers, such as a pore size in the range of 10-20 micrometers.

[0357] An advantage of using a filter with a pore size larger than 1 micrometer is that bacteria and other microorganisms are also at least partly removed during the separation process and optionally during the washing and / or recrystallization process. Thus, the present process can produce high purity BLG at very low bacterial load but avoiding thermal damage of the protein.

[0358] Another advantage of using a filter with a pore size larger than 1 micrometer is that removal of water and subsequent drying becomes easier and less energy consuming.

[0359] The remaining whey protein solution separated from the BLG crystals can be recycled into the whey protein feed during the preparation of the whey protein solution.

[0360] In some preferred embodiments of the present application, step c) uses a filter centrifuge. In other preferred embodiments of the present application, step c) uses a decanter centrifuge. Preliminary results (see Example 13) have shown that the use of a filter centrifuge and / or a decanter centrifuge to separate the BLG crystals from the mother liquor provides a more robust process operation 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 enable the filter cake to be stripped from the filter. The use of a drying gas can form part of the separation step or, alternatively, if the filter cake is to be directly converted into a dry edible BLG composition, can form the final drying step.

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

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

[0364] In some preferred embodiments of the present application, step c) is performed using a DCF unit equipped with a membrane capable of retaining the 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 crystallisation tank. Preferably, the DCF permeate is treated, for example by ultrafiltration / diafiltration, to oversaturate it with respect to BLG before it is mixed 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 at which unwanted spontaneous crystallisation does not occur. Thus, 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 exemplified in Example 10 and demonstrated in Figure 26 These embodiments can be implemented as a batch process or as a continuous process.

[0367] In some preferred embodiments of the application, the method comprises a step d) of washing the BLG crystals, such as the separated BLG crystals of c). The washing can comprise 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 complete dissolution of the BLG crystals, and subsequently separating the remaining BLG crystals from the washing liquid.

[0369] The washing liquid is preferably selected to avoid complete dissolution of the BLG crystals, and can for example comprise or even consist essentially of cold demineralized water, cold tap water or cold reverse osmosis permeate.

[0370] The pH of the washing liquid can 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 washing liquid can 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] Washing liquids with even lower conductivity can be used. For example, the washing liquid can have a conductivity of at most 1 μ8 / cm. Alternatively, the conductivity of the washing liquid can be at most 0.1 μ8 / cm, such as about 0.05 μ8 / 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 can be performed, for example, at at most 5 °C, more preferably at at most 2 °C, such as at about 0 °C. So far, temperatures below 0 °C can be used, such 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 application, the washing liquid contains BLG in an amount of at least 1 % (w / w), and preferably in an amount of at least 3 % (w / w), such as in an amount of 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 washing 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 washing liquid and the initial amount of separated BLG crystals can be at least 10. Alternatively, the weight ratio between the total amount of washing liquid and the initial amount of separated BLG crystals can be at least 20, such as at least 50 or at least 100.

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

[0379] In some preferred embodiments of the present application, one or more washing sequences are performed with the same filter arrangement or a similar filter arrangement as the separation of BLG crystals. The filter cake containing mainly BLG crystals is added to one or more washing liquid sequences, the washing liquid is removed through the filter, while the remaining part of the BLG crystals remains in the filter cake.

[0380] In particularly preferred embodiments of the present application, the separation of step c) is performed using a filter that retains the BLG crystals. Subsequently, the filter cake is contacted with one or more amounts of washing liquid, which is moved 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, such as at most 0.2 times the volume of the filter cake. The volume of the filter cake includes the solids and the fluids (liquids and gases) of the filter cake. The filter cake is preferably washed in this way at least 2 times, preferably at least 4 times, and even more preferably at least 6 times.

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

[0382] The method can further comprise a step e) involving a recrystallization step, said recrystallization step comprising:

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

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

[0385] - crystallizing BLG in the oversaturated adjusted recrystallization liquid, and

[0386] - separating the BLG crystals from the remaining adjusted recrystallization liquid.

[0387] Step e) can comprise a single recrystallization sequence or a plurality of recrystallization sequences.

[0388] In some embodiments of the application, 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 can be performed multiple times if desired.

[0390] The isolated BLG crystals of step c) can for example be subjected to a processing sequence of:

[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) can be subjected to a processing sequence of:

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

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

[0396] Multiple steps of washing and recrystallization can also be combined, for example in the 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 the order:

[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 application, the method further involves subjecting the isolated BLG to a further BLG enrichment step, e.g. based on chromatography or selective filtration. However, in other preferred embodiments of the present application, the method does not comprise a further BLG enrichment step after step b). The term "further BLG enrichment step" means a process step that enriches BLG relative to the total amount of proteins, which step is independent of the crystallization of BLG or the handling of BLG crystals. An example of such a further BLG enrichment step is ion exchange chromatography. Washing of BLG crystals and / or recrystallization of BLG is not considered a "further BLG enrichment step".

[0407] In some particularly preferred embodiments of the present application, 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 application, the term "dry" means that the composition or product in question comprises at most 6% (w / w) and preferably even less water.

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

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

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

[0414] In some particularly preferred embodiments of the present application, the drying step involves a BLG containing composition wherein the BLG crystals have been dissolved, and wherein 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 should resemble edible BLG compositions with e.g. conventional dried whey protein powder.

[0415] The BLG crystals can be dissolved, e.g. by:

[0416] - increasing the temperature,

[0417] - increasing the electrical conductivity, e.g. by adding one or more salts,

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

[0419] - decreasing the concentration of BLG, e.g. by dilution,

[0420] - or a combination of the above.

[0421] Spray drying is presently the preferred method of drying the BLG containing composition free of BLG crystals.

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

[0423] In some particularly preferred embodiments of the application, the drying step involves a BLG containing composition which still contains BLG crystals, and wherein the resulting powder contains BLG crystals. These embodiments are preferred if the edible BLG composition should have a higher density than conventional dried whey protein powder.

[0424] As documented in Example 7, the inventors found that when dried BLG crystals are resuspended in cold demineralized water, the slurry of BLG crystals can be spray dried and retain at least some of the crystal structure. It is particularly advantageous to avoid exposing the BLG containing composition containing BLG crystals to a heat treatment regime which dissolves a significant amount of the BLG crystals prior to spraying. Thus, if a pre-heat of the BLG containing composition containing BLG crystals is employed prior to spraying, it is preferred to carefully control the heat load.

[0425] In some embodiments of the application, 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 when it reaches the outlet of the spraying device, e.g. a nozzle or an atomizer. In some preferred embodiments of the application, 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 when it reaches the outlet of the spraying device.

[0426] The spraying device of the spray dryer is e.g. a device such as a nozzle or an atomizer which transforms 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 spraying device, preferably 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 spraying device.

[0428] In some preferred embodiments of the application, the BLG containing composition 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 it reaches the outlet of the spraying device. The BLG containing composition can be a BLG isolate, e.g. containing BLG in an amount of more than 90% (w / w) relative to total protein, or it can contain a significant amount of other proteins and thus BLG in an amount of at most 90% (w / w) relative to total protein.

[0429] In some preferred embodiments of the present application, the BLG containing composition can have a protein composition as the traditional liquid WPC or WPI or traditional liquid SPC or SPI as described herein, but with 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 of the spray dryer is preferably in the range of 140-220°C, more preferably in the range of 160-200°C, and even more preferably in the range of 170-190°C, e.g. preferably about 180°C. The outlet temperature of the gas from the spray dryer is preferably in the range of 50-95°C, more preferably in the range of 70-90°C, and even more preferably in the range of 80-88°C, e.g. preferably about 85°C. It is empirically stated that the solids subjected to spray drying are heated to a temperature of 10-15°C lower than the gas outlet temperature.

[0431] In some preferred embodiments of the present application, 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, e.g. preferably about 70°C.

[0432] The concept of spray drying a BLG crystal suspension has not been disclosed in the prior art, and is as such a separate aspect of the present application.

[0433] An aspect of the present application thus 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 application, the BLG containing composition to be dried is mixed with dried BLG isolate to increase the solids content to a level at which the mixture can be dried by fluid bed drying. This is also referred to as back mixing, and allows for a 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 method is that the BLG containing composition to be dried can have a very high solids content prior to the drying step, thus less water has to be removed and less energy consumed in the drying operation.

[0438] In some preferred embodiments of the present application, the BLG containing composition from step b), c), d), or e) has a solids content of at least 20% (w / w). Preferably, the BLG containing composition from step b), c), d), or e) has a solids content of at least 30% (w / w). More preferably, the BLG containing composition from step b), c), d), or e) has a solids content of at least 40% (w / w). Even more preferably, the BLG containing composition from step b), c), d), or e) has a solids content of at least 50% (w / w), such as at least 60% (w / w).

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

[0440] The inventors have found that the higher the crystallinity of the BLG containing composition, the less water is bound to the BLG containing composition, and a higher total solids content of the BLG containing composition can be achieved prior to the drying step.

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

[0442] Even higher degrees of crystallinity are generally preferred. Thus, in some preferred embodiments of the application, 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 found that a reduced water content tends to increase the BLG crystallinity of the 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 moist separated crystals).

[0444] The method of the application can be operated at 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 application, the BLG is not subjected to a temperature higher than 90°C during the method. Preferably, the BLG is not subjected to a temperature higher than 80°C during the method. Even more preferably, the BLG is not subjected to a temperature higher than 75°C during the method. It is noted that even though spray drying typically employs temperatures in excess of 150°C, the short exposure time and the simultaneous evaporation of water means that the protein subjected to spray drying does not experience temperatures in excess of 50-70°C.

[0446] The inventors have seen indications that prolonged heating during the drying step reduces the amount of BLG in crystalline form. In some preferred embodiments of the application, 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 detectable denaturation of the BLG at all.

[0447] The degree of denaturation caused by the drying step is calculated by determining the BLG content (relative to total solids) in the BLG-composition to be dried in step f) and the BLG content (relative to total solids) in the resolubilized dried composition and using the following formula: 步骤f前

[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 application relate to a method of preparing an edible composition comprising beta-lactoglobulin (BLG) in crystalline form, said method comprising the steps of:

[0450] a) providing a whey protein solution comprising BLG and at least one additional 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] - 4-50% (w / w) ALA relative to total protein, and preferably 8-35% (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 the supersaturated whey protein solution, preferably by adding seed crystals, 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 demineralized whey protein solution, and preferably has a ratio between conductivity and total amount of protein of at most 0.3 and / or a UF permeate conductivity of at most 7 mS / cm.

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

[0461] The present application also relates to the edible composition obtainable by these embodiments.

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

[0463] a) providing a whey protein solution comprising BLG and at least one additional 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 the 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 a BLG-containing composition, said BLG-containing composition being 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 demineralized whey protein solution and preferably has a ratio between conductivity and total amount of protein of at most 0.3 and / or a UF permeate conductivity of at most 7 mS / cm.

[0475] These embodiments are in particular suitable for preparing low-mineral and low-phosphorous edible BLG compositions in the form of a high-density powder

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

[0477] Yet other preferred embodiments of the present invention relate to a method of preparing an edible composition comprising beta-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] - 5-50% (w / w) ALA relative to total protein, and preferably 8-35% (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 the 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) which does not comprise BLG crystals.

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

[0490] In these embodiments, the BLG crystals are dissolved prior to drying.

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

[0492] In some preferred embodiments, the present 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] One advantage of the present method is that it is much faster than comparable methods of BLG crystallization of the prior art. The duration from the initial adjustment of the whey protein feed to the completion of the separation of step c can be at most 10 hours, preferably at most 4 hours, more preferably at most 2 hours, and even more preferably at most 1 hour.

[0494] A further aspect of the present invention relates to isolated BLG crystals obtainable from the method 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 in which it was formed but can still contain internal water, i.e. 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 cell dimensions a = 6.9 A (± 0.1 A), b = 6.9 A (± 0.1 A), and c = 6.9 A (± 0.1 A). and and have cell integral angles a = 90° (± 2%), b = 90° (± 2%), and g = 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 cell dimensions a = 6.9 A (± 0.1 A), b = 6.9 A (± 0.1 A), and c = 6.9 A (± 0.1 A). and and have cell integral angles a = 90° (± 1%), b = 90° (± 1%), and g = 90° (± 1%).

[0499] Even more preferably, the isolated BLG crystals can have an orthorhombic space group P 21 21 21, and cell dimensions a = 6.9 A (± 0.1 A), b = 6.9 A (± 0.1 A), and c = 6.9 A (± 0.1 A). and and have cell integral angles a = 90° (± 0.5%), b = 90° (± 0.5%), and g = 90° (± 0.5%).

[0500] Most preferably, the isolated BLG crystals have an orthorhombic space group P 21 21 21, and cell dimensions a = 6.9 A (± 0.1 A), b = 6.9 A (± 0.1 A), and c = 6.9 A (± 0.1 A). and and have cell integral angles a = 90°, b = 90°, and g = 90°.

[0501] The isolated BLG crystals can for example comprise at least 20% (w / w) BLG and at most 80% (w / w) water. Preferably, the isolated BLG crystals can 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 water in the range of about 40% to about 60% (w / w).

[0502] The inventors have found that the BLG crystals of the present application surprisingly have the ability to recover their original crystal structure upon drying and rehydration. This is particularly advantageous in applications that benefit from the BLG crystal structure.

[0503] A further aspect of the present application relates to an edible composition comprising beta-lactoglobulin, for example an edible composition obtainable by a method as defined herein.

[0504] A further aspect of the present application 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 method as defined herein.

[0505] A further aspect of the present application 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 method as defined herein.

[0506] In some preferred embodiments of the present application, 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, for example preferably at most 0.01.

[0507] In some preferred embodiments of the present application, 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 is determined according to Example 9.1.

[0509] In some preferred embodiments of the application, 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 application, 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 a liquid having a pH in the range of 5-6 is measured according to Example 9.7. The crystallinity of BLG in a powder material is measured according to Example 9.8. If the edible composition is a dry product and not in the form of a powder, it must be converted into a powder, e.g. by grinding or milling, before the method of Example 9.8 is performed.

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

[0513] In other preferred embodiments of the application, the edible BLG composition comprises at most 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 can comprise at most 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 can 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 application makes it possible to prepare edible whey protein products with very low phosphorus and other mineral content, which is advantageous 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 application, the term "low phosphorus" relates to a composition, such as a liquid, powder or other food product, having a total phosphorus content of at most 100 mg phosphorus per 100 g protein. Preferably, the low phosphorus composition has a total phosphorus content of at most 80 mg per 100 g protein. More preferably, the low phosphorus composition can have a total phosphorus content of at most 50 mg per 100 g protein. Even more preferably, the low phosphorus composition can have a total phosphorus content of at most 20 mg phosphorus per 100 g protein. Even more preferably, the low phosphorus composition can have a total phosphorus content of at most 5 mg phosphorus per 100 g protein. The low phosphorus composition according to the present application can be used as a food product ingredient for the production of food products for a reduced kidney function patient group.

[0517] Thus, in some particularly preferred embodiments of the present application, the edible BLG composition comprises at most 80 mg phosphorus per 100 g protein. Preferably, the edible BLG composition comprises at most 30 mg phosphorus per 100 g protein. More preferably, the edible BLG composition comprises at most 20 mg phosphorus per 100 g protein. Even more preferably, the edible BLG composition comprises at most 10 mg phosphorus per 100 g protein. Most preferably, the edible BLG composition comprises at most 5 mg phosphorus per 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 application, the edible BLG composition is a low mineral composition.

[0520] In the context of the present application, the term "low mineral" 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] - a total ash content of at most 1.2% (w / w) relative to total solids,

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

[0523] - a total sodium and potassium content of at most 0.10% (w / w) relative to 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] - a total ash content of at most 0.7% (w / w) relative to total solids,

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

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

[0529] - a total phosphorus content of at most 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] - a total ash content of at most 0.5% (w / w) relative to total solids,

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

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

[0534] - a total phosphorus content of at most 50 mg phosphorus per 100 g protein.

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

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

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

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

[0539] - a total phosphorus content of at most 50 mg phosphorus per 100 g protein.

[0540] In some preferred embodiments of the application, the edible BLG composition comprises at least 25% (w / w) of the total amount of protein relative to the total solids of the edible BLG composition. Preferably, the edible BLG composition comprises at least 50% (w / w) of the total amount of protein relative to the total solids of the edible BLG composition. More preferably, the edible BLG composition comprises at least 75% (w / w) of the total amount of protein relative to the total solids of the edible BLG composition. Even more preferably, the edible BLG composition comprises at least 90% (w / w) of the total amount of protein relative to the total solids of the edible BLG composition.

[0541] In some preferred embodiments of the application, the total amount of protein of the edible BLG composition is in the range of 25-100% (w / w) relative to the total solids. Preferably, the total amount of protein of the edible BLG composition is in the range of 50-100% (w / w). More preferably, the total amount of protein of 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 of 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 application, the edible BLG composition comprises at least 75% (w / w) BLG relative to the total amount of protein. Preferably, the edible BLG composition can comprise at least 90% (w / w) BLG relative to the total amount of protein. More preferably, the edible BLG composition can comprise at least 95% (w / w) BLG relative to the total amount of protein. Even more preferably, the edible BLG composition can 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 application, 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] The edible BLG composition can also comprise lipids, e.g. in the form of triglycerides and / or other lipid types such as phospholipids.

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

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

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

[0548] Thus, in some preferred embodiments of the application, 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 the almost sole protein present, and to edible BLG composition powders in which the BLG concentration is not enriched relative to other proteins present in the whey protein solution. Thus, the present application provides high density powders of both isolated BLG and of crude whey protein comprising significant amounts of ALA and other whey proteins in addition to BLG.

[0550] In some preferred embodiments of the present application, 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 application, 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 edible BLG composition in powder form 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 edible BLG composition in powder form has a bulk density in the range of 0.50-0.9 g / mL and comprises at least 80% (w / w) protein relative to the total weight of the composition. Even more preferably, the edible BLG composition in powder form 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 edible BLG composition in powder form 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 application advantageously allows for more cost efficient packaging and logistics of the powder, as less packaging material is needed per kg of powder and more powder (mass) can be transported by a given container or truck.

[0554] The edible BLG composition in powder form can 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 can 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 can for example have a bulk density in the range of 0.6-0.8 g / mL.

[0555] In other preferred embodiments of the present application, 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 can 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 can preferably have a bulk density in the range of 0.70-1.0 g / mL.

[0556] The edible BLG composition in powder form can 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 comprise at least 70% (w / w) protein relative to the total weight of the composition. More preferably, the powdered edible BLG composition can 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 comprise at least 70% (w / w) protein relative to the total weight of the composition. The powdered edible BLG composition can 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 can 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 can have a bulk density in the range of 0.50-0.9 g / mL and comprises at least 80% (w / w) protein relative to the total weight of the composition. Even more preferably, the powdered edible BLG composition 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 can for example have a bulk density in the range of 0.6-0.8 g / mL and comprises at least 80% (w / w) protein relative to the total weight of the composition.

[0558] In other preferred embodiments of the present application, 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 can have a bulk density in the range of 0.60-1.0 g / mL and comprises at least 70% (w / w) protein relative to the total weight of the composition. Even more preferably, the powdered edible BLG composition has a bulk density in the range of 0.65-1.0 g / mL and comprises at least 70% (w / w) protein relative to the total weight of the composition. The powdered edible BLG composition can preferably have a bulk density in the range of 0.70-1.0 g / mL and comprises at least 70% (w / w) protein relative to the total weight of the composition.

[0559] In other preferred embodiments of the present application, 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 can have a bulk density in the range of 0.60-1.0 g / mL and comprises at least 80% (w / w) protein relative to the total weight of the composition. Even more preferably, the powdered edible BLG composition has a bulk density in the range of 0.65-1.0 g / mL and comprises at least 80% (w / w) protein relative to the total weight of the composition. The powdered edible BLG composition can preferably have a bulk density in the range of 0.70-1.0 g / mL and comprises at least 80% (w / w) protein relative to the total weight of the composition.

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

[0561] The inventors have seen indications that the BLG composition according to the present application has a better long-term stability than similar BLG compositions. This is especially the case when at least some of the BLG is present in the form of BLG crystals, which seems to provide a better storage stability of the BLG molecules.

[0562] In some preferred embodiments of the present application, the dried BLG composition has a furanone 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 dried BLG composition has a furanone value of at most 10 mg / 100 g protein, after 60 days at 30°C.

[0563] In some preferred embodiments of the present application, the dried BLG composition has a furanone 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 dried BLG composition has a furanone value of at most 10 mg / 100 g protein. Preferably, the dried BLG composition has a furanone value of 0 mg / 100 g protein.

[0564] In some preferred embodiments of the present application, 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 application, 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, even more preferably at least 40% (w / w) water.

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

[0567] The inventors have found that the edible BLG composition according to the present application has a surprisingly low degree of protein denaturation, even if spray drying has been used for the preparation of the edible BLG powder composition (see Example 11).

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

[0569] In some preferred embodiments of the present application, the edible BLG composition is a dried powder, and preferably a spray-dried powder, and has a degree of protein denaturation of at most 2%, and preferably at most 1.5%. More preferably, for example, the dried edible BLG composition in the form of a spray-dried powder has a degree of protein denaturation of at most 1.0%. Even more preferably, for example, the dried edible BLG composition in the form of a spray-dried powder has a degree of protein denaturation of at most 0.8%. Even more preferably, for example, the dried edible BLG composition in the form of a spray-dried powder has a degree of protein denaturation of at most 0.5%.

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

[0571] - at most 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] - has 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 application, the edible BLG composition comprises:

[0578] - at most 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] - has a BLG crystallinity of at least 20%, and preferably at least 40%.

[0585] In further preferred embodiments of the application, the edible BLG composition comprises:

[0586] - at most 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 further preferred embodiments of the application, the edible BLG composition comprises:

[0593] - at most 6% (w / w) water

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

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

[0596] - at most 80 mg phosphorous per 100 g protein.

[0597] The edible BLG composition:

[0598] - is a dry powder.

[0599] In yet preferred embodiments of the application, the edible BLG composition comprises:

[0600] - at most 6% (w / w) water

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

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

[0603] - at most 50 mg phosphorous per 100 g protein.

[0604] The edible BLG composition:

[0605] - is a dry powder.

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

[0607] - at most 6% (w / w) water

[0608] - at least 80% total protein relative to total solids, and preferably at least 90% total protein relative to 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] - has a BLG crystallinity of at least 20%, and preferably at least 40%.

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

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

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

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

[0618] - at most 80 mg phosphorous per 100 g protein.

[0619] The edible BLG composition:

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

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

[0622] The edible composition according to these embodiments is particularly useful for preparing an edible BLG composition in dry form, and is particularly suitable for spray drying and for preparing a high density whey protein powder having the normal concentration profile of the whey protein species whey protein but containing at least some BLG in the form of dry BLG crystals.

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

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

[0625] - at least 80% total protein, and preferably at least 90% total protein relative to 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] - has a BLG crystallinity of at least 20%, and preferably at least 40%.

[0630] The edible composition according to these embodiments is particularly useful for preparing an edible BLG composition in dry form, and is particularly suitable for spray drying and for preparing a high density whey protein powder having the normal concentration profile of the whey protein species whey protein but containing at least some BLG in the form of dry BLG crystals.

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

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

[0633] Another aspect of the present application 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 application, the food product is a dry food product comprising carbohydrates and proteins, such as a bar, 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) the BLG comprises at least 90% (w / w) of the total amount of proteins.

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

[0638] BLG has a favourable amino acid profile and preferably contributes a significant portion of the proteins of the food product. This is particularly interesting if the food product is a low-mineral or low-phosphorus food product. In some preferred embodiments of the present application, 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 amount of proteins of the food product. It can even be most preferred that the edible BLG composition contributes all of the proteins of the food product.

[0639] In some preferred embodiments of the present application, 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 amount of proteins of the low-phosphorus food product. It can even be most preferred that the low-phosphorus edible BLG composition contributes all of the proteins of the low-phosphorus food product.

[0640] Non-limiting examples of food products are e.g. dairy products, confectionary, beverages, protein bars, enteral nutritional compositions, bakery products.

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

[0642] - an edible BLG composition as defined herein, to provide 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) of the total amount of BLG,

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

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

[0645] - optionally, a flavouring agent, and

[0646] - at most 80 mg phosphor per 100 g protein

[0647] The pH value ranges from 2.5-4.0.

[0648] The inventors have realized that it is not unimportant 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 result in a pH in the range of 5-6 when resuspended in water, and adding acid or salt to change the pH or increase the conductivity also increases the mineral load of the resulting liquid / beverage.

[0649] However, the inventors have found that if a carboxylic acid, lactone, carboxylic anhydride or combination thereof is used to lower the pH, no unnecessary minerals are 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, said process comprising the steps of:

[0651] - providing one or more acidifying agents selected from the group consisting of a carboxylic acid, a lactone, a carboxylic anhydride or a combination thereof,

[0652] - contacting the edible BLG composition comprising BLG crystals with one or more acidifying agents and optionally other ingredients (such as 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] thereby forming a liquid.

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

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

[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 one or more 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-gluconic acid-delta-lactone,

[0661] - carboxylic anhydrides.

[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 such 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 application has been described above with reference to a number of specific embodiments. However, other embodiments than the above described are equally possible within the scope of the application. The different features and steps of the various embodiments and aspects of the application can be combined with each other as long as no contradiction arises.

[0666] Examples

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

[0668] Protocol:

[0669] Lactose depleted UF retentate derived from sweet whey from a standard cheese production process and filtered through a 1.2 micron filter was used as feed for the BLG crystallization process. The sweet whey feed was conditioned using an ultrafiltration setup using a Koch HFK-328 type membrane with a 46 mil spacer with a feed pressure of 1.5-3.0 bar, using a feed concentration of 21% TS (total solids) ± 5 and polishing water (water filtered through reverse osmosis to obtain a conductivity of at most 0.05 mS / cm) as diafiltration medium. The temperature of the feed and retentate during ultrafiltration was about 12°C. The pH was then adjusted by addition of HC1 to obtain a pH of about 5.40. Diafiltration was continued until the conductivity of the retentate decreased below 0.03 mS / cm within a period of 20 min. The retentate was then concentrated to about 30% TS (about 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] To the concentrated retentate 0.5 g / L of pure BLG crystal material obtained from spontaneous BLG crystallization (as described in Example 3 under feed 2) was inoculated. The inoculum material was prepared by washing the BLG crystal slurry 5 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 was less than 200 microns.

[0671] The concentrated retentate was transferred to a 300 L crystallization tank, where it was cooled to about 4°C and kept at this temperature overnight with gentle agitation. The next morning, a sample of the cooled concentrated retentate was transferred to a test tube and inspected visually and under a microscope. Rapidly settling crystals were clearly formed during the night. A laboratory sample comprising the mixture of 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 the precipitate were taken for HPLC analysis. The crystals were washed once in cold polishing water and then centrifuged again and the precipitate was freeze-dried.

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

[0673]

[0674] BLG relative yield quantification by HPLC:

[0675] All samples were diluted to the same extent by adding finishing water. The samples were filtered through a 0.22 micron filter. For each sample, the same volume was loaded onto a HPLC system with a Phenomenex 5 μιη C4 LC column 250 x 4.6 mm (part number: 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: 1 ml / 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 treated in the same way, we can directly compare the area of the BLG peak to obtain the relative yield. Since the crystals only contain BLG and the samples were all treated in the same way, the concentration of a-lactalbumin (ALA) and thus the area of ALA should be the same in all samples, and thus the area of ALA before and after crystallization was used as a correction factor (cf) when calculating the relative yield.

[0683]

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

[0685]

[0686] Results:

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

[0688] The crystal slurry was investigated by microscopy; from Figure 2As can be seen, the sample contained hexagonal crystals, many of which were much larger than 200 microns in size, indicating that the observed crystals were not only seeding crystals. The crystals were easily broken when pressed with a needle, confirming that they were protein crystals.

[0689] Figure 3 A chromatogram of the washed crystal product is shown, and in this case, BLG accounted for 98.9% of the total area of the chromatogram. Further purification of the BLG product can be achieved by further washing.

[0690] Conclusions:

[0691] This example surprisingly demonstrates that BLG can be selectively crystallized from a crude whey protein concentrate that contains more than 48% non-BLG proteins relative to total protein, and that the obtained BLG crystal isolate has an extremely high purity. This finding opens up new ways of industrial milk protein separation, where BLG is separated from other protein components in a gentle manner that preferably avoids long exposure to high temperatures and problematic chemicals.

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

[0693] Protocol:

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

[0695] After the diafiltration was completed, samples were taken at Brix 21, 24 and 32.5 during the concentration. These samples were first cooled to 4°C and kept 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 kept at this temperature for at least 1 hour. Subsequently, the yield of BLG crystals was measured again.

[0696] Results:

[0697] When the relative yield of BLG is plotted against the conductivity in the sample (as shown in Figure 4 , there is a clear correlation between lower conductivity and higher relative yield of BLG.

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

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

[0700] Conclusions:

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

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

[0703] Protocol:

[0704] Using the same experimental and analytical set-up as in Example 1, three different types of whey protein containing feedstocks were tested as crystallisation feed. However, no seeding was used in the experiment with feed 2. Feed 1 and feed 2 are based on sweet whey and were fat reduced by a Synder FR membrane prior to the treatment as described in Example 1. Feed 3 is from acid whey.

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

[0706] The slurry of crystallised feed 1 was centrifuged at 1500g for 5 minutes on a Maxi- spin filter with a 0.45 micron CA membrane, then 2 volumes of MilliQ water was added to the cake, after which it was centrifuged again. The resulting cake was analysed by HPLC. A photo of the Maxi-spin filter holding the precipitate (cake) of crystallised 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, then the precipitate was analysed by HPLC. The precipitate from feed 3 was analysed without washing.

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

[0708] Table 2 shows the concentrations of selected components in Feed 1 (whey protein concentrate based on sweet whey). BDL in wet samples was below the detection limit.

[0709]

[0710] Table 3 shows the concentrations of selected components in Feed 2 (ALA-reduced whey protein concentrate based on sweet whey). In wet, non-standard samples, BDL was below the detection limit.

[0711]

[0712]

[0713] Table 4 shows the concentrations of selected components in Feed 3 (whey protein concentrate based on acid whey).

[0714]

[0715] result:

[0716] Feed 1:

[0717] exist Figure 7 The chromatograms of protein composition in the feed (solid line) and mother liquor (dashed line) are shown in the diagram. Clearly, the process recovers most of the BLG as crystals. The yield of the separated BLG (calculated as described in Example 1) is approximately 65% ​​relative to the total amount of BLG in the feed.

[0718] Figure 8 These are microscope photographs of the sample taken during the early stages of crystallization. Figure 9 These are microscope images of the samples taken at the end of crystallization. From these two images, it is clear that the BLG crystals are relatively fragile. Some crystals showed signs of cracking during stirring and transformed from hexagonal or rhomboid shapes into crystal fragments, which still appear very compact and well-defined but with more irregular shapes.

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

[0720] Feed 2:

[0721] exist Figure 11The diagram shows the protein composition of feed 2 (solid line) and the obtained mother liquor (dashed line). Clearly, most of the BLG has been removed, and the calculated yield is 82% relative to the total amount of BLG in feed 2.

[0722] Figure 12 The image shows feed 2 before (left) and after (right) crystallization. During crystallization, the feed changes from a clear liquid (in which the stirring magnet is visible) to a milky, opaque liquid.

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

[0724] Figure 16 This is a chromatogram of the BLG crystal precipitate separated after washing with two volumes of MilliQ water. The chromatogram clearly shows that the crystals contain very high purity BLG.

[0725] Figure 14 and Figure 15 The results show the effects of increasing conductivity (by adding NaCl) or changing pH (by adjusting pH to 7 by adding NaOH) to make the environment no longer favorable for crystal structure. In both cases, the milky white suspension turned into a transparent liquid when the BLG crystals dissolved.

[0726] The mineral composition of the crystal formulation obtained from feed 2 is provided in Table 5. 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.

[0727]

[0728] Feed 3:

[0729] exist Figure 17 The image shows chromatograms of the protein composition of feed 3 (solid line) and the resulting mother liquor (dashed line). Clearly, most of the BLG was separated (the calculated yield is 70.3% relative to the total BLG in the feed). A higher yield would have been obtained if the protein content had been higher prior to crystallization.

[0730] Figure 18 These are microscopic images of BLG crystals separated from feed 3 (which is essentially CMP-free). Unlike hexagonal crystals, the crystals have a rectangular shape. The rectangular crystals appear to be more stable than the hexagonal crystals. Figure 19 The chromatogram of the crystalline precipitate separated without washing is shown; the chromatogram clearly shows that the crystals are BLG crystals, but with a rectangular shape rather than a hexagonal shape (compare to example). Figure 18 The rectangular crystal shape and Figure 2 (The hexagonal crystal shape).

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

[0732]

[0733]

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

[0735] Conclusion:

[0736] All three feeds were suitable for the BLG crystallization process. By adding salt or increasing the pH or temperature, the BLG crystals were easily dissolved. The new method makes it possible to prepare a BLG preparation with a very low phosphorous content, which makes the preparation suitable as a protein source for patients with kidney disease.

[0737] Example 4 Influence of pH on BLG crystal yield

[0738] Protocol:

[0739] The same protocol and experimental setup as in Example 1 was used (using a fat-reduced sweet whey protein concentrate), with the difference that for each experiment the pH was adjusted to the level described in Table 8. The protein concentration at the start of the crystallization step was about 24% (w / w).

[0740] The pH was adjusted with a dilute NaOH solution (>4%) or a dilute HC1 solution (>3.6%) to investigate the influence of the pH on the crystallization process and the yield obtained. After crystallization, the BLG crystals were separated by centrifugation as described in Example 1.

[0741] Table 7 Concentration range of selected components of the feed used for Example 4.

[0742]

[0743]

[0744] Table 8 Target pH of samples

[0745] Sample Target pH 1 4.80 2 5.20 3 5.50 4 5.80 5 6.00 6 6.20

[0746] Results:

[0747] The yield was calculated as described in Example 1. It should be noted that the starting sample was taken before the addition of the seeding material. Therefore, if the sample was not supersaturated with respect to BLG, the seeding material would dissolve and contribute to the total BLG concentration, in which case the BLG yield appears to be negative.

[0748] Table 9 Sample yields calculated based on HPLC measurements.

[0749]

[0750]

[0751] Conclusions:

[0752] This experiment confirmed that in the pH range of 5-6, crystallization of BLG can be achieved in a salt-solubility mode.

[0753] Example 5: Study of the effect of increasing conductivity levels

[0754] Protocol:

[0755] The same protocol and experimental setup as in Example 1 was used, with the difference 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, samples of the feedstock were taken and NaCI was added to increase the conductivity, and it was studied at what conductivity level BLG crystals could grow. The protein content during crystallization was about 16.7% (w / w).

[0756] Table 10 Composition ranges of the feed used in Example 5.

[0757]

[0758]

[0759] Results:

[0760] The samples were treated as described in Example 1. Figure 20 The calculated yields in the retentate at different conductivities are shown. The point at 3.53 mS / cm is the feedstock after pH adjustment. All points larger than 3.53 are the result of adding NaCI to increase the conductivity. Points smaller 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.

[0761] 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.

[0762] From Figure 20As can be seen, BLG crystals formed in the feed at a conductivity below 4.93 mS / cm (at 4°C, total protein content of about 16.7% (w / w)). A BLG yield of about 75% was obtained at a conductivity in the retentate of about 2 mS / cm and a UF permeate conductivity of about 1.6 mS / cm.

[0763] Figure 21 Figure 4 is a microscope picture of crystals formed in the retentate at 4.20 mS / cm, showing the expected BLG crystal characteristics.

[0764] Conclusions:

[0765] The specific feed of Example 5 allowed for BLG crystals to form at a conductivity below 4.93 mS / cm (corresponding to a UF permeate conductivity of 5.75 mS / cm, and a ratio between conductivity and total amount of protein of 0.057). The upper limit of the conductivity is expected to depend on the protein concentration and the protein composition. For example, higher protein concentrations and / or an increased content of highly charged proteins or other macromolecules (e.g. CMP) are expected to increase the upper limit of the conductivity at which BLG can crystallize.

[0766] Example 6: Crystallization of BLG in a serum protein concentrate

[0767] A serum protein concentrate (SPC) was prepared by microfiltration of skim milk using a Synder FR membrane and subjected to a process temperature of about 50°C. The retentate obtained contained essentially all of the caseins and residual fat, and also some serum proteins, lactose and minerals. The permeate contained molecules capable of passing through the membrane, including serum proteins, lactose and minerals, but essentially no caseins 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.

[0768] Table 11 Concentrations of selected components of the feed (serum protein concentrate). BDL = below detection limit in wet non-standard sample.

[0769]

[0770]

[0771] Similar to the crystallization of Examples 1-5, BLG formation of the SPC feed can be isolated as crystals at very high purity (confirmed by chromatography as in the previous examples) and provides a BLG yield of 70% relative to the total amount of BLG of the SPC feed. In Figure 22In this case, BLG crystals from the early stage of crystallization are shown. As shown before, the crystals have a rectangular or square shape, as opposed to the hexagonal shape observed for example in Example 2.

[0772] Example 7: Preparation and determination of bulk density of spray-dried BLG crystals

[0773] 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 crystal phase was then mixed 1 : 1 with polishing water and then separated again on a decanter centrifuge using the same settings. The BLG crystal phase was then mixed with polishing water to make it into a slurry containing about 25% dry matter and having a BLG crystallinity of about 80, which was subsequently 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 until the spray drying was 10-12 °C. The moisture content of the resulting powder sampled at the outlet was 4.37% (w / w).

[0774] The BLG crystallinity in the slurry was about 90%.

[0775] 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 1 :2 with polishing water. The BLG crystal phase was then mixed with polishing water to make it into a thinner slurry, which was subsequently dried on a pilot plant spray dryer using the same parameters as above.

[0776] 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 preparation 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 is indeed surprising and provides many advantages related to logistics and applications.

[0777] Table 12 Concentrations of selected components of the spray-dried BLG crystal preparation of Example 7. BDL = below detection limit

[0778]

[0779] Subsequently samples of the spray-dried BLG crystal preparation were re-suspended in cold demineralised water and the BLG crystals were still clearly visible by microscopy. Addition of citric acid or NaCI dissolved the BLG crystals and transformed the opaque crystal suspension into a transparent liquid.

[0780] The inventors have seen indications that prolonged heating during the drying step reduces the amount of BLG in crystal form. It is therefore preferred that the heat exposure of the BLG crystal preparation is as low as possible.

[0781] Conclusion:

[0782] This example demonstrates that a slurry comprising BLG crystals can be spray-dried and that if the heating during the drying step is controlled, the BLG crystals are still present in the re-suspended spray-dried powder.

[0783] The inventors have also found that whey protein powders containing BLG crystals have a much higher bulk density than normally obtained by spray-drying of a dissolved protein stream. The high density powder allows for more cost-efficient packaging and logistics of the powder, as less packaging material is needed per kg of powder and more powder (mass) can be transported through a given container or truck.

[0784] The high density powder also appears to be easier to handle and less fluffy and dusty during manufacturing and use.

[0785] Example 8: Low phosphorus protein drinks

[0786] Six low phosphorus drink samples were prepared using the purified BLG product from example 3 (crystal preparation obtained from feed 3). All dry ingredients were mixed with demineralised water to obtain 10 kg of each sample and hydrated for 1 hour at 10 °C.

[0787] Table 13 Composition of the six drink samples.

[0788]

[0789] Sub-samples of the six samples were taken to measure Turbidity was measured on a 3000 IR Turbidimeter and viscosity was measured on a vicoman from Gilson. The results are shown in the table below.

[0790] Table 14 Measured viscosity and turbidity of the six drink samples.

[0791] 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

[0792] The tube photos of the sub-samples comprising the six low phosphorus drink samples show in Figure 25The sub-samples were visually inspected from left to right, samples A, B, C, D, E and F. Visual inspection of the test tubes verified the turbidity measurements and it was noted that all beverage samples were clear and in particular samples C and D (pH 3.0) were very clear. The low viscosity confirmed that the beverage samples were easy to drink.

[0793] All ingredients used for the preparation of the beverages were low in phosphorus and did not contain unnecessary minerals. Thus, the resulting beverages had a phosphorus content of about 45 mg P / 100 g protein and in general a very low mineral content. Therefore, the six beverages were suitable for use as protein beverages for patients with kidney disease.

[0794] Example 9 - Analytical methods

[0795] Example 9.1 Determination of lactosylated BLG vs. non-lactosylated BLG

[0796] The amount of lactosylated BLG and native BLG was quantified using LC-MS.

[0797] The analysis was performed on a HP1200 series HPLC coupled to a 6410 Triple Quad MS also from Agilent Technologies. For separation prior to ionization a Symmetry300 TM C18 column (WAT106172: 5 μm solid phase particles, column size 2.1 x 150 mm) was applied and proteins were detected at 214 nm. Prior to analysis of the samples they were filtered through a 0.22 micron filter. All samples were run in duplicate.

[0798] The analysis was performed using the following conditions:

[0799] HPLC

[0800] Buffer A: 99.9% MilliQ-vand with 0.1% TFA

[0801] Buffer B: 9.9% MilliQ-vand, 90% acetonitrile, 0.1% TFA

[0802] Flow: 0.3 mL / min

[0803] Gradient:

[0804] 0-20 min: 85%-60% A and 15%-40% B

[0805] 20-45 min: 60%-50% A and 40%-50% B

[0806] 45-55 min: 0% A and 100% B

[0807] 55-70 min 85% A and 15% B

[0808] Load: 40 pL

[0809] The column temperature was set to 60 °C.

[0810] Mass spectrum:

[0811] Ions with m / z 100-2000 were detected and the resulting data were evaluated in MassHunter Workstation software (version B.04.00). All forms of the same species (mass) were grouped using deconvolution. Further query was made for masses between 18 kDa and 20 kDa. The intact mass of BLG-A is 18.361 kDa and for BLG-B 18.276 kDa, and the lactosylation adds 324 Da to the protein mass, by inspecting this mass region, up to 5 lactosylated proteins can be detected. By comparing the signal strength of each mass, the ionization differences of different species were ignored for relative quantification.

[0812] Example 9.2: Determination of total protein

[0813] The total protein content (true protein) of the sample was determined by:

[0814] 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: determination of nitrogen content by Kjeldahl determination.

[0815] 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.

[0816] 3) The total amount of protein was calculated as (m 总氮 - m 非蛋白氮 )* 6.38.

[0817] Example 9.3: Determination of loose and bulk density

[0818] The density of a dry powder is defined as the relationship between the weight and the volume of the powder, which is analyzed under specific conditions using a special Stampf volumeter (i.e. a measuring cylinder). Density is typically expressed in g / ml or kg / L.

[0819] In this method, a sample of dry powder is tamped in a measuring cylinder. After a specified number of tamps, the volume of the product is read and the density is calculated.

[0820] This method can define three types of density:

[0821] ■Tap density, i.e. mass divided by the volume of the powder transferred into a specified graduated cylinder after 100 taps.

[0822] ■Tap density, i.e. mass divided by the volume of the powder transferred into a specified graduated cylinder after 100 taps.

[0823] ■Tap density, i.e. mass divided by the volume of the powder transferred into a specified graduated cylinder after 100 taps.

[0824] 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 A.G.

[0825] The tap density and the bulk density of the dry product are determined by the following procedure.

[0826] Pre-treatment:

[0827] The sample to be measured is stored at room temperature.

[0828] The sample is then thoroughly mixed by repeated rotation and turning of the container (avoiding breaking of the particles). The filling of the container is not more than 2 / 3.

[0829] Procedure:

[0830] 100.0 ± 0.1 g of powder are weighed and transferred into the graduated cylinder. The volume Vo is read in ml.

[0831] If 100 g of powder cannot be placed into the graduated cylinder, it should be reduced to 50 or 25 g.

[0832] The graduated cylinder is fixed to the Stampf volumeter and then tapped 100 times. The surface is leveled with a spatula and the volume V 100 (ml) is read.

[0833] The number of taps is changed to 625 (including 100 taps). After tapping the surface, the volume V 625 (ml) is read.

[0834] Density calculation:

[0835] The tap density and the bulk density in g / ml are calculated according to the following formula:

[0836] Bulk density = M / V

[0837] where M represents the weighed sample in g and V represents the volume after 625 taps in ml.

[0838] Example 9.4: Determination of moisture content of the powder

[0839] The moisture content of the food product was determined according to ISO 5537:2004 (Milk powder - Determination of moisture content (reference method)). NMKL is an abbreviation for "Nordisk Metodikkomité for ” (Nordic Committee for Food Analysis).

[0840] Example 9.5: Determination of calcium, magnesium, sodium, potassium, phosphorous total

[0841] The following procedure was used to determine the total amount of calcium, magnesium, sodium, potassium and phosphorus: The sample was first decomposed using microwave digestion, and then the total amount of one or more minerals was determined using an ICP device.

[0842] Equipment:

[0843] The microwave oven was from Anton Paar, and the ICP was an Optima 2000DV from PerkinElmer Inc.

[0844] Materials:

[0845] 1M HNO3

[0846] Yttrium in 2% HNO3

[0847] Suitable standards for calcium, magnesium, sodium, potassium and phosphorus in 5% HNO3

[0848] Pre-treatment:

[0849] An amount of powder was weighed out and transferred to a microwave digestion tube. 5 mL of 1M HNO3 was added. The sample in the microwave oven was digested according to the microwave instructions. The digested tube was placed in a fume hood, the cap was removed and the volatile fumes were allowed to evaporate.

[0850] Measurement procedure:

[0851] The pre-treated sample was transferred to a digestion tube using a known amount of Milli-Q water. A solution of yttrium in 2% HNO3 was added to the digestion tube (about 0.25 mL per 50 mL of diluted sample), and diluted to a known volume using Milli-Q water. The sample was analysed on the ICP using the manufacturer’s described procedure.

[0852] A blind sample was prepared by diluting a mixture of 10 mL of 1M HNO3 and 0.5 mL of a solution of yttrium in 2% HNO3 to a final volume of 100 mL using Milli-Q water.

[0853] Prepare at least three standard samples with concentrations including the expected sample concentrations.

[0854] Example 9.6: Determination of Furosine value

[0855] The furosine value was 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 protein content was determined according to Example 9.2. The furosine value per mg of furosine per 100 g of protein was reported.

[0856] Example 9.7: Determination of BLG crystallinity in liquid

[0857] The following method is used to determine the crystallinity of BLG in liquids with a pH range of 5-6.

[0858] a) Transfer a 10 mL sample of the liquid in question to a Maxi-rotary filter with a CA membrane having a pore size of 0.45 μm.

[0859] b) Immediately rotate the filter at 1500g for 5 minutes. Keep the centrifuge at 2°C.

[0860] c) Add 2 mL of cold milliQ water (2°C) to the permeate side of the rotary filter and immediately rotate the filter at 1500 g for 5 min while maintaining centrifugation at 2°C to cool. Collect the permeate (permeate A), measure the volume, and determine the BLG concentration by HPLC using the method outlined in Example 9.9.

[0861] d) Add 4 mL of 2M NaCl to the effluent side of the filter, stir rapidly, and let the mixture stand at 25°C for 15 minutes.

[0862] e) Immediately rotate the filter at 1500g for 5 minutes and collect the permeate (permeate B).

[0863] f) Determine the total weight of BLG in permeate A and permeate B using the method outlined in Example 9.9, and convert the result to the total weight of BLG rather than a 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 .

[0864] g) The crystallinity of the liquid with respect to BLG is determined as follows:

[0865] Crystallinity = m 渗透物B / (m 渗透物A +m 渗透物B )*100%

[0866] Example 9.8: Determination of BLG crystallinity in dry powder

[0867] This method is used to determine the BLG crystallinity in a dry powder.

[0868] a) Mix 5.0 g of the powder sample with 20.0 g of cold milliQ water (2°C) and let stand for 5 minutes at 2°C.

[0869] b) Transfer a sample of the liquid in question to a Maxi-Spin filter with a 0.45 micron CA membrane.

[0870] c) Immediately spin the filter at 1500 g for 5 min. Keep centrifuge at 2°C

[0871] 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. Convert the result to total weight of BLG instead of weight percentage. The weight of BLG in permeate A is called m 渗透物A

[0872] f) Then calculate the BLG crystallinity in the powder using the following formula:

[0873]

[0874] Where m 总BLG is the total amount of BLG in the powder sample of step a).

[0875] If the total amount of BLG in the powder sample is not known, this can be determined by suspending an additional 5 g of the powder sample (from the same powder source) in 20.0 g of milliQ water, adjusting the pH to 7.0 by the addition of aqueous NaOH, letting the mixture stand for 1 hour at 25°C under stirring, and finally determining the total amount of BLG in the powder sample using Example 9.9.

[0876] Example 9.9: Determination of total amount of BLG, ALA and CMP in aqueous liquid

[0877] The content of a-lactalbumin, b-lactoglobulin and CMP was analyzed by HPLC at 0.4 mL / min. 25 microliter of filtered sample was injected on 2 TSKgel3000 PWxl (7.8 mm 30 cm, Tosohass, Japan) columns connected in series with a pre-column PWxl (6 mm x 4 cm, Tosohass, Japan) equilibrated in eluent (consisting of 465 g MilliQ water, 417,3 g acetonitrile and 1 mL trifluoroacetic acid) and using a UV detector at 210 nm.

[0878] Quantitative determination of the content of native a-lactalbumin (C α ), b-lactoglobulin (C β ) and caseinomacropeptide (C CMP ) was performed by comparing the peak areas obtained for the respective standard proteins to those of the sample.

[0879] The total amount of additional proteins (non-BLG proteins) was determined by subtracting the amount of BLG from the total amount of proteins (determined according to Example 9.2)

[0880] Example 9.10: Determination of UF permeate conductivity

[0881] A 15 mL sample was transferred to an Amicon Ultra-15 centrifugal filter unit with a 3 kDa cut-off (3000 NMWL) and centrifuged at 4000 g for 20-30 minutes, or until a sufficient volume of UF permeate for measuring conductivity had accumulated in the bottom of the filter unit. The conductivity was measured immediately after centrifugation. Sample handling and centrifugation were performed at the temperature of the sample source.

[0882] Example 9.11: Determination of the degree of protein denaturation of whey protein composition

[0883] It is known that denatured whey proteins have a 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.

[0884] More specifically, the whey protein composition to be analyzed (e.g. a powder or an aqueous solution) is converted to:

[0885] - a first aqueous solution containing 5.0% (w / w) total protein and having a pH of 7.0, and

[0886] - a second aqueous solution containing 5.0% (w / w) total protein and having a pH of 4.6.

[0887] The pH adjustment is performed using 3% (w / w) NaOH (aq) or 5% (w / w) HC1 (aq).

[0888] The total protein content (P pH 7.0 ) of the first aqueous solution was determined according to Example 9.2.

[0889] The second aqueous solution was stored at room temperature for 2 h, followed by centrifugation at 3000 g for 5 min. The sample of the supernatant was recovered and analysed according to Example 9.2 to determine the total protein (S pH 4.6 ).

[0890] The degree of denaturation of the proteins of the whey protein composition, D, was calculated as follows:

[0891] D = ((P pH 7.0 - S pH 4.6 ) / P pH 7.0 )* 100%

[0892] Example 9.12: Detection of dry BLG crystals in powder

[0893] The presence of dry BLG crystals in the powder can be identified by:

[0894] The powder sample to be analysed was resuspended and gently mixed in demineralised water at a temperature of 4°C in a weight ratio of 2 parts water to 1 part powder and allowed to rehydrate for 1 hour at 4°C.

[0895] The rehydrated sample was examined by microscopy to identify the presence of crystals, preferably using plane polarised light to detect birefringence.

[0896] The crystalline material was isolated and subjected to X-ray crystallography to verify the presence of the crystal structure and, preferably, also to verify that the lattice (space group and cell size) corresponds to that of BLG crystals.

[0897] The chemical composition of the isolated crystalline material was analysed to verify that it consists mainly of BLG.

[0898] Example 10: Crystallisation by UF-based dynamic cross-flow filtration

[0899] The feed to the crystallisation tank was prepared as described in Example 1, except that the diafiltration was performed at pH 5.92 and the final TS was 20%.

[0900] After the feed was adjusted (the feed composition can be seen in Table 15), it was transferred to a 300 L crystallization tank and initially the pH was adjusted to pH 5.80 and the temperature was kept at 10-12 °C. After the pH was adjusted, inoculum material was added, which was produced in the same way as described in Example 1, but originating from a non-spontaneous crystallization production. The feed was inoculated with the inoculum material to a concentration of 0.5 g inoculum material per liter feed. After inoculation, the temperature on the cooling mantle 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 Figure 26 Figure 1. The DCF unit was equipped with a Kerafol ceramic membrane with a pore size of 500 nm, the TMP (trans-membrane pressure) was set to 0.4 bar and the rotational speed of the membrane was 32 Hz.

[0901] 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 type membrane with a 46 mil spacer. In the UF unit, the temperature was allowed to rise to but not above 12 °C. The amount of diafiltration water added was adjusted so that the retentate from the UF, which was returned to the crystallization tank, was about 21% TS when removing minerals from the mother liquor (ML).

[0902] Diafiltration on the ML was continued until the conductivity difference between the permeate and the diafiltration water was below 50 μ8 / cm. At this point, the amount of diafiltration water was adjusted so that the retentate was about 30% TS. When BLG is removed as crystals, the amount of TS in the ML decreases; this continuous removal of excess water and minerals makes it possible to increase the overall yield, since the concentration of other proteins that manifest themselves during BLG crystallization have limited, if any, influence on the solubility of BLG in the range that has been explored.

[0903] 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 about 100 L of ML. Based on mass conservation, the relative yield of BLG was calculated to be 92%.

[0904] Table 15 Selected components of the feed used in Example 10.

[0905]

[0906] Table 16 Protein composition of the final mother liquor obtained in Example 10.

[0907]

[0908]

[0909] Conclusion:

[0910] By continuously removing excess minerals and water from the substrate where BLG crystallization occurs, BLG yield can be significantly improved and the process can be performed at low temperatures.

[0911] Example 11: Degree of protein denaturation of different whey protein products

[0912] The degree of protein denaturation of four edible BLG compositions of the application was compared to a commercial product. The samples are described below.

[0913] Sample A: BiPro (commercially available WPI; Davisco, USA) B: BLG crystal slurry as-is - no drying (Invention) C: BLG crystal slurry freeze-dried (Invention) D: BLG crystals re-dissolved (pH 7) and freeze-dried E: BLG crystal slurry spray-dried (Invention)

[0914] Samples B-E were prepared as follows:

[0915] A crystal slurry was prepared as described in Example 12 and separated as described in Example 7. Some of the separated BLG slurry was removed and divided into four portions.

[0916] Sample B: The first portion of the separated BLG crystal slurry was redissolved without any drying by adjusting the pH of the BLG crystal slurry to 7.01 using 3% NaOH; the sample was then diluted to Brix 6 to make a ~5% protein solution.

[0917] Sample C: The second portion of the separated BLG crystal slurry was freeze-dried. The powder was then resuspended in finisher water, the pH adjusted to 7.09 using 3% NaOH, and the sample was then diluted to Brix 6 to make a ~5% protein solution.

[0918] Sample D: The third portion of the separated BLG crystal slurry was redissolved by adjusting the pH to 7.0 using 3% NaOH and then freeze-dried. The freeze-dried powder was then resuspended in finisher water and the pH was measured to be 7.07. The sample was then diluted to Brix 6 to make a ~5% protein solution.

[0919] Sample E: The fourth portion of the separated BLG crystal slurry was treated and spray-dried as described in Example 7. The powder was then resuspended in finisher water and the pH was adjusted to 7.04 using 3% NaOH. The sample was then diluted to Brix 6 to make a ~5% protein solution.

[0920] The degree of protein denaturation of each sample was determined according to Example 9.11 and the results are presented in Table 17.

[0921] Table 17 compares the degree of protein denaturation of a commercially available WPI product (Bipro) to four BLG products of the application.

[0922]

[0923] Conclusions:

[0924] Regardless of the drying method, the edible BLG composition of the present application has a surprisingly low degree of denatured protein; only one tenth of the above degree can be found in commercially available WPI used for comparison. It is particularly surprising that the spray-dried BLG crystal slurry product still has the lowest degree of denaturation of all products.

[0925] Example 12: Isolation of crystals by dynamic cross-flow filtration

[0926] A lactose depleted UF retentate derived from sweet whey from a standard cheese production process and filtered through a 1.2 micron filter was used as feed for the crystallization process. The sweet whey feed was conditioned using ultrafiltration setup using a Koch HFK-328 type membrane with a 46 mil spacer with a feed pressure of 1.5-3.0 bar, using a feed concentration of 10% TS (total solids) ± 5 and polishing water (water filtered through reverse osmosis to obtain a conductivity of at most 0.05 mS / cm) as diafiltration medium. The temperature of the feed and retentate during ultrafiltration was about 12°C. The pH was then adjusted by addition of HC1 to obtain a pH of about 5.60. Diafiltration was continued until the conductivity of the retentate was below 1.30 mS / cm. The feed was then heated to 25°C after which the retentate was concentrated to about 27% TS (about 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 was formed.

[0927] The concentrated retentate was transferred to a 300 L crystallization tank where it was cooled to about 6°C and kept at this temperature overnight with gentle agitation. 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 the precipitate were taken for HPLC analysis. The BLG yield of this process was calculated to be 67%.

[0928] The crystal slurry from the 300 L tank was used to feed an Andritz DCF 152S system using one disc membrane with a 500 nm pore size. The filtration was run at 8°C with a rotation speed of 32 Hz and a transmembrane pressure of 0.4 bar. The system was run as a dead-end filtration where the retentate accumulated in the filtration chamber, unlike in larger units where the retentate would be continuously removed. The filtration was run in a steady manner for just over 40 minutes at which point the solids accumulated in the filtration chamber started to affect the filtration.

[0929] During the DFC operation the amount of crystal mass increased significantly.

[0930] Conclusions.

[0931] The DCF provides a stable and efficient means of separating the crystals from the ML. If desired, a wash liquid can be added to the DCF.

[0932] Example 13: Crystal separation using filter centrifuge

[0933] The separation was tested on a filter centrifuge HZ 25 / 0.1, fitted with filter cloth having a pore size of about 20 microns, using the same feed and the same crystallization process as in Example 12.

[0934] Test 1 : 4 L of feed was fed to the filter centrifuge operating at 60 g. After all the feed was added, the centrifuge was accelerated to 250 g for drying the filter cake. The filter cake contained 47.6% TS; the composition of the filter cake is shown in Table 18.

[0935] After cleaning, 7 L of the same feed as above was fed to the centrifuge (60 g). The centrifuge was then accelerated to 250 g for about 5 minutes of dewatering, after which it was again decelerated to 60 g and 0.25 L of finishing water was added for washing. After the wash water was added, the centrifuge was again accelerated to 250 g for dewatering. The TS of the filter cake was measured at 47%. The filter cake is shown in Figure 27 .A. The composition of the filter cake, ML fraction, and wash liquid after washing is shown in Table 18. After the filter cake was dewatered, it was attempted to peel it off the side of the centrifuge; as shown in Figure 27 .C, the top layer did accumulate and drop through the predetermined tube, but the lower layer was too wet and sticky to properly peel off, as shown in Figure 27 .B.

[0936] Table 18 Concentrations of selected components of the compositions provided in Example 13.

[0937]

[0938]

[0939] 1) Protein composition % (w / w) relative to the weight of the solution

[0940] 2) Concentrations of other selected components (normalized to % w / w of 95% total solids relative to the total weight of the composition)

[0941] Conclusion:

[0942] A filter centrifuge offers an interesting option to obtain a BLG cake that is so pure that ALA and CMP are below the levels required for quantification even without washing. By applying even a small amount of washing medium to the cake, the mineral content in the cake can be further reduced as shown by the protein composition of the wash water in Table 18. By washing, the content of non-BLG proteins of the cake is also reduced as shown from the wash water used. The wash water used contained a ratio between ALA:BLG that was larger than the ratio in the cake. This indicates that the washing step has a greater tendency to remove ALA (and possibly other non-BLG proteins) compared to BLG.

[0943] The cake produced this way is not peelable, but still permeable. This enables the option to add drying gas at a given temperature to reduce the moisture content of the cake to a degree that it is peelable like the top layer. Alternatively, the cake can be redissolved in the centrifuge by adding an appropriate amount of acid, base or salt to the water solution in a siphon centrifuge type set-up.

[0944] Example 14: Influence of mineral composition of whey protein solution

[0945] In this example the influence of the molar ratio between monovalent and divalent metal cations on the BLG yield was investigated.

[0946] Two samples were compared:

[0947] Sample A: with superweight Na + (origin: Na2SO4)

[0948] Sample B: with superweight Ca 2+ (origin: CaSO4)

[0949] 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 about 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 vessel. For all dialysis processes, dialysis tubing OrDial D-Clean MWCO 3500 (item number 63034405) was used. The vessels were continuously stirred during dialysis and dialysis was performed in a cooler at 4°C. The first dialysis was performed overnight.

[0950] To remove the excess ions after the first dialysis, the dialysis bag was transferred to a vessel containing 2 L of a salt solution. The concentrations were as follows:

[0951] Sample A: Na2SO4 (sodium sulfate) 0.059 M,

[0952] Sample B: CaSO4 (calcium sulfate) 0.059 M.

[0953] 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 dialysis was continued over the weekend.

[0954] After the second salt dialysis, the tubes were transferred to a 24 L vessel with approximately 24 L of cold polishing water and dialyzed overnight to remove excess ions before crystallization.

[0955] After the last dialysis step, the protein concentration was slightly lower than the preferred concentration. Therefore, the sample was concentrated on a Pellicon XL UF lab set using a 10 kDa cut-off membrane and a peristaltic pump running at 75 mL / h. The mineral content of the sample is shown in Table 19 together with the feedstock.

[0956] The samples were then inoculated with 0.5 g / L of the aforementioned inoculum material and crystallized overnight at 4 °C. The next day, crystal precipitation could be seen in all samples. An HPLC sample of each sample was prepared by centrifuging each sample at 3000 g for 5 minutes, and the sample of the supernatant was analyzed. The results are shown in Table 21.

[0957] Table 19 Concentration of selected mineral components in the feedstock of Example 14 and in samples A and B.

[0958]

[0959]

[0960] 1) The change in relation to the concentration of a given component in the feedstock

[0961] Table 20 pH, conductivity and Brix at various stages during the preparation of samples A and B.

[0962]

[0963] Table 21 Concentration of BLG in samples with different ratios between monovalent and divalent cations.

[0964]

[0965]

[0966] Conclusion:

[0967] Table 21 documents that less residual amount of BLG is left in the mother liquor (and higher yield of isolated BLG crystals is obtained) if high molar ratios between monovalent and divalent cations are avoided. The molar ratio between monovalent and divalent cations, and in practice Na+K and Ca+Mg, can be controlled to improve the BLG yield of the process of the present application.

Claims

1. A method of preparing an edible composition comprising beta-lactoglobulin (BLG) in a crystalline form, said method comprising the steps of: a) providing a whey protein solution comprising native BLG and at least one further whey protein, said whey protein solution being supersaturated with respect to BLG, having a pH in the range of 5-6, comprising at least 0.4% w / w native BLG relative to the weight of the whey protein solution, and having: - a conductivity of at most 5 mS / cm, and / or - a ratio between conductivity expressed in mS / cm and 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 salt-solution mode, and c) optionally, separating BLG crystals from the remaining whey protein solution.

2. The method according to claim 1, further comprising step d) washing the separated BLG crystals obtained from step c).

3. The method according to claim 1, further comprising step e) recrystallizing the BLG crystals obtained from step c).

4. The method according to claim 1, further comprising step f) drying the BLG containing composition from step b) or c). The whey protein solution of step a) comprises at least 5% w / w alpha-lactalbumin relative to the total amount of protein. The whey protein solution of step a) comprises at least 15% w / w further whey protein relative to the total amount of protein. The whey protein solution of step a) comprises at least 1% w / w native BLG relative to the total amount of protein. The whey protein solution of step a) comprises at least 4% w / w native BLG relative to the weight of the whey protein solution. The whey protein solution comprises milk serum protein concentrate, whey protein concentrate, milk serum protein isolate and / or whey protein isolate.

5. The method according to any one of the preceding claims, wherein, The ratio of conductivity to total amount of protein of the whey protein solution is at most 0.

25.

6. The method of claim 1, wherein, The UF permeate conductivity of the whey protein solution is at most 7 mS / cm.

7. The method of claim 1, wherein, The supersaturated whey protein solution is prepared by one or more of the following adjustments to the whey protein feed:

8. The method of claim 1, wherein, - adjusting the pH, 9. The method of claim 1, wherein, - decreasing the conductivity 10. The method of claim 1, wherein, - decreasing the temperature 11. The method of claim 1, wherein, - increasing the protein concentration, and 12. The method of claim 1, wherein, - adding an agent that decreases the water activity. The preparation of the supersaturated whey protein solution involves adjusting the pH of the whey protein feed. The preparation of the supersaturated whey protein solution involves decreasing the conductivity of the whey protein feed. The preparation of the supersaturated whey protein solution involves decreasing the temperature of the whey protein feed. The preparation of the supersaturated whey protein solution involves increasing the total protein concentration of the whey protein feed. The BLG crystallization of step b) involves one or more of the following:

13. The method of claim 12, wherein, - waiting for crystallization to occur, 14. The method of claim 12, wherein, - adding seeds, 15. The method of claim 12, wherein, - further increasing the supersaturation of BLG, and / or 16. The method of claim 12, wherein, - mechanical stimulation.

17. The method of claim 1, wherein, Step c) comprises separating the BLG crystals to a solid content of at least 30% w / w. ​ ​ ​ ​ 18. The method of claim 1, wherein, ​ 19. The method of claim 18, wherein, Step c) comprises isolating the BLG crystals to a solid content of at least 40% w / w.

20. The method of claim 18, wherein, Step c) comprises isolating the BLG crystals to a solid content of at least 50% w / w.

21. The method of claim 2, wherein, The washing in step d) involves contacting the isolated BLG crystals with a washing liquid without completely dissolving the BLG crystals, and subsequently separating the remaining BLG crystals from the washing liquid.

22. The method of claim 21, wherein, The washing of step d) dissolves at most 80% w / w of the initial amount of BLG crystals.

23. The method of claim 3, wherein, The recrystallization step involves: - dissolving the isolated BLG crystals in a recrystallization liquid, - adjusting the recrystallization liquid to obtain an oversaturation with respect to BLG, - crystallizing BLG in the oversaturated adjusted recrystallization liquid, and - separating the BLG crystals from the remaining adjusted recrystallization liquid.

24. The method of claim 3, wherein, The BLG crystals of step d) are recrystallized at least 2 times.

25. The method of claim 4, wherein, The drying step involves one or more of spray drying, freeze drying, spin flash dryer, spin drying and / or fluid bed drying.

26. The method of claim 4, wherein, The drying step is performed by spray drying.

27. The method of claim 4, wherein, The drying step is performed by fluid bed drying.

28. The method of claim 4, wherein, The BLG of the BLG containing composition has a crystallinity of at least 20% w / w.

29. The method of claim 28, wherein, The BLG of the BLG containing composition has a crystallinity of at least 40% w / w.

30. The method of claim 28, wherein, The BLG of the BLG containing composition has a crystallinity of at least 60% w / w.

31. The method of any one of claims 28-30, wherein, The drying step is performed by spray drying.

32. The method of claim 4, wherein, The BLG containing composition from step b), c), d) or e) has a solid content of at least 20% w / w.

33. The method of claim 32, wherein, The BLG containing composition from step b), c), d) or e) has a solid content of at least 30% w / w.

34. The method of claim 32, wherein, The BLG containing composition from step b), c), d) or e) has a solid content of at least 40% w / w.

35. The method of claim 4, wherein, The heat exposure during the drying step is kept low enough to keep the degree of denaturation of the BLG at most 10%.

36. The method of claim 35, wherein, The heat exposure during the drying step is kept low enough to keep the degree of denaturation of the BLG at most 4%.

37. The method of claim 35, wherein, The heat exposure during the drying step is kept low enough to keep the degree of denaturation of the BLG at most 1%.

38. A method of producing a spray dried edible powder composition comprising native BLG, the composition comprising dried BLG crystals, the method comprising the steps of: - providing a liquid BLG containing composition comprising BLG crystals, and the liquid BLG containing composition has a BLG crystallinity of at least 20%, the liquid BLG containing composition comprising at least 10% w / w total solids, and comprising at least 5% w / w native BLG, and - 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.

39. An edible BLG composition in the form of a spray dried powder, the edible BLG composition having a bulk density of at least 0.45 g / mL, and the edible BLG composition comprising: - a total amount of protein of at least 50% w / w relative to the total solids of the edible BLG composition, and - at least 50% w / w native BLG relative to the total amount of protein; and wherein the BLG of the edible BLG composition has a crystallinity of at least 20% w / w.

40. The edible BLG composition according to claim 39, wherein 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.

41. The edible BLG composition according to claim 39, wherein 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.

42. The edible BLG composition according to any one of claims 39-41, comprising at least 95% w / w native BLG relative to the total amount of protein.

43. The edible BLG composition according to claim 42, comprising at least 97% w / w native BLG relative to the total amount of protein.

44. The edible BLG composition according to claim 42, comprising about 100% w / w native BLG relative to the total amount of protein.

45. The edible BLG composition according to any one of claims 39-41, comprising at most 10% w / w carbohydrates.

46. The edible BLG composition according to claim 45, comprising at most 1% w / w carbohydrates.

47. The edible BLG composition according to claim 45, comprising at most 0.1% w / w carbohydrates.

48. The edible BLG composition according to any one of claims 39-41, comprising at most 0.5% w / w total amount of lipids relative to total solids.

49. The edible BLG composition according to claim 48, comprising at most 0.1% w / w total amount of lipids relative to total solids.

50. The edible BLG composition according to claim 48, comprising at most 0.01% w / w total amount of lipids relative to total solids.

51. The edible BLG composition according to any one of claims 39-41, having a bulk density of at least 0.50 g / mL.

52. The edible BLG composition according to claim 51, having a bulk density of at least 0.6 g / mL.

53. The edible BLG composition according to any of the claims 39-41, wherein, the BLG of the edible BLG composition has a crystallinity of at least 40% w / w.

54. The edible BLG composition according to claim 53, wherein, the BLG of the edible BLG composition has a crystallinity of at least 60% w / w.

55. The edible BLG composition according to claim 53, wherein, the BLG of the edible BLG composition has a crystallinity of at least 80% w / w.

56. The edible BLG composition according to any of the claims 39-41, wherein, the BLG of the edible BLG composition has a degree of lactosylation of at most 1.

57. The edible BLG composition according to claim 56, wherein, the BLG of the edible BLG composition has a degree of lactosylation of at most 0.

6.

58. The edible BLG composition according to claim 56, wherein, the BLG of the edible BLG composition has a degree of lactosylation of at most 0.

4.

59. The edible BLG composition according to claim 56, wherein, The BLG of the edible BLG composition has a degree of lactosylation of at most 0.

2.

60. The edible BLG composition according to any of the claims 39-41, wherein, The edible BLG composition comprises at most 80 mg phosphorous per 100 g protein.

61. The edible BLG composition according to claim 60, wherein, The edible BLG composition comprises at most 20 mg phosphorous per 100 g protein.

62. The edible BLG composition according to claim 60, wherein, The edible BLG composition comprises at most 5 mg phosphorous per 100 g protein.

63. The edible BLG composition according to any one of claims 39-41 having a furanone value of at most 80 mg / 100 g protein after 60 days at 30°C.

64. The edible BLG composition according to claim 63 having a furanone value of at most 40 mg / 100 g protein after 60 days at 30°C.

65. The edible BLG composition according to claim 63 having a furanone value of at most 10 mg / 100 g protein after 60 days at 30°C.

66. The edible BLG composition according to any one of claims 39-41 having a degree of protein denaturation of at most 2%.

67. The edible BLG composition according to claim 66 having a degree of protein denaturation of at most 1%.

68. The edible BLG composition according to any one of claims 39-41 comprising: - at most 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 0.50 g / mL.

69. The edible BLG composition according to any one of claims 39-41 comprising: - at most 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%.

70. The edible BLG composition according to any one of claims 39-41 comprising: - at most 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, - at most 80 mg phosphorous per 100 g protein.

71. The edible BLG composition according to any one of claims 39-41 comprising: - at most 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 furanone value of at most 80 mg / 100 g protein after 60 days at 30°C.

72. The edible BLG composition according to any one of claims 39-41 comprising: - at most 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 1.

73. The edible BLG composition according to any one of claims 39-41, comprising: - at most 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%.

74. The edible BLG composition according to any one of claims 39-41, comprising: - at most 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 - at most 80 mg phosphorus per 100 g protein.

75. The edible BLG composition according to any one of claims 39-41, comprising: - at most 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 furanone value of at most 80 mg per 100 g protein after 60 days at 30°C.

76. The edible BLG composition according to any one of claims 39-41, comprising: - at most 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 1.

77. The edible BLG composition according to any one of claims 39-76, obtainable by the method according to one or more of claims 1-38.

78. Use of the edible BLG composition according to any one of claims 39-77 as a food ingredient.

79. The use of claim 78, wherein, The edible BLG composition is low in phosphorus, and the edible BLG composition is used as a food ingredient in the production of a low-phosphorus food product containing at most 100 mg phosphorus per 100 g protein.

80. The use of claim 79, wherein, The edible BLG composition is used as a food ingredient in the production of a low-phosphorus food product containing at most 20 mg phosphorus per 100 g protein.

81. A food product comprising the edible BLG composition according to any one of claims 39-76 and at least one further ingredient, the food product being a dry food product comprising a carbohydrate and a protein, the dry food product comprising at least 1% w / w native BLG, and wherein the BLG has a degree of crystallinity of at least 10%.

82. The food product according to claim 81, wherein native BLG constitutes at least 90% w / w of the total amount of protein.

83. The food product according to claim 81, which is a low phosphorus food product comprising at most 80 mg phosphorus per 100 g protein.

84. The food product of claim 81, wherein, The BLG of the food product has a crystallinity of at least 40% w / w.

85. The food product of claim 84, wherein, The BLG of the food product has a crystallinity of at least 60% w / w.

86. The food product according to claim 81, which is a dairy product, a confectionary, a protein bar, an enteral nutritional composition or a bakery product.

87. The food product of claim 81, wherein, The edible BLG composition contributes at least 25% w / w of the total amount of protein of the food product.

88. The food product of claim 87, wherein, The edible BLG composition contributes at least 50% w / w of the total amount of protein of the food product.

89. The food product of claim 87, wherein, The edible BLG composition contributes at least 90% w / w of the total amount of protein of the food product.

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