Novel production of beta-lactoglobulin preparation, related method, use and food product

Abstract

To provide a novel method of producing an isolated beta-lactoglobulin composition and / or a composition containing crystallized beta-lactoglobulin and a novel beta-lactoglobulin composition, the uses of these compositions, and food products containing these compositions.SOLUTION: There is provided an edible beta-lactoglobulin (BLG) composition. The edible BLG composition contains: at least 50% (weight / weight) total protein relative to the total solids of the edible BLG composition; and at least 90% (weight / weight) BLG relative to the total protein.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to novel methods for producing isolated beta-lactoglobulin compositions and / or compositions comprising crystallized beta-lactoglobulin. The present invention further relates to novel beta-lactoglobulin compositions, uses of these compositions and food products comprising these compositions. [Background technology]

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

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

[0004] For example, de Jongh et al. (Mild Isolation Procedure Discloses New Protein Structural Properties of β-Lactoglobulin, J Dairy Sci., vol. 84(3), 2001, pages 562-571) described the purification of BLG from freshly milk by low temperature acid coagulation of casein and subjecting the resulting acid whey to a combination of affinity chromatography (DEAE Sepharose) and gel permeation chromatography. The resulting BLG composition was stated to contain 0.985 g beta-lactoglobulin per g protein.

[0005] Slack et al. (Journal of Food Processing and Preservation, vol. 10, 1986, pages 19-30) investigated various approaches and prepared a BLG-enriched precipitate by adjusting the pH of dehydrochlorinated and sweet whey to pH 4.65 and separating the precipitate that formed by centrifugation and decantation. The resulting precipitate pellet was described as relatively insoluble and contained significant amounts of protein impurities in additional BLG. No crystal formation was observed. It should be noted that the BLG precipitate that may form at pH 4.65 is not BLG crystals.

[0006] Palmer (Crystalline Globulin from Cow's Milk, J. Biol. Chem., Vol. 104, 1934, pages 359-372) reported a laborious and time-consuming process for producing protein crystals based on acid whey using several sequences of salting-out precipitation of undesired proteins, pH adjustment and dialysis to remove other undesired proteins. Finally, when a high purity BLG solution was obtained, BLG was crystallized. This process lasted for more than 12 days and required the addition of further toluene. Thus, the procedure disclosed by Palmer is incompatible with safe food production and results in a product that is obviously inedible.

[0007] Aschaffenburg et al. (Improved Method for the Preparation of Crystalline beta-Lactoglobulin and alpha-Lactalbumin from Cow's Milk, Bioch., vol. 65, 1957, pages 273-277) disclose an improved process compared to Palmer's process, which allows the preparation of beta-lactoglobulin crystals in a matter of days instead of weeks. However, this improved method still requires the removal of undesirable proteins before crystallization, and furthermore, it uses toluene for crystallization, which makes this method incompatible with safe food production.

[0008] JP 10-218755 A discloses the preparation of a cosmetic composition containing a melanogenesis inhibitor containing BLG as an active ingredient. The document further suggests that BLG can be isolated, for example, by the following process: adding hydrochloric acid to milk to precipitate casein, followed by filtration to obtain whey. The pH of the whey is adjusted to 6.0, ammonium sulfate is added in a subsaturated amount, the precipitated protein is removed by salting out, and the filtrate is recovered. The filtrate is saturated with ammonium sulfate, and the precipitated protein is recovered. The recovered protein is redissolved in water and dialyzed at pH 5.2 to separate the crystals, and β-lactoglobulin is prepared at a rate of about 1.8 g per 1 L of whey. However, the general process steps of the proposed process described in JP 10-218755 A are insufficient to form BLG crystals. Thus, the document does not include any disclosure of crystallization of BLG or enabling BLG crystallization.

[0009] US 2790790 discloses a process for precipitation of proteins from solution, more specifically for the fractional precipitation of relatively unconjugated proteins from aqueous solutions by the use of sodium chloride as a precipitant. This process is suggested to be useful for the isolation of BLG by NaCl-induced precipitation at pH 3.6-3.8. Example II of this document suggests that the NaCl precipitate may be dialyzed in a conventional manner to form crystalline B-lactoglobulin. However, US 2790790 does not demonstrate that the formation of BLG crystals at pH 3.6-3.8 is actually possible, nor does it contain any reference to the meaning of "conventional manner" of dialyzing the BLG precipitate. Thus, this document does not contain any disclosure of enabling BLG crystallization and BLG crystallization. Summary of the Invention

[0010] By chance, the inventors have made the surprising discovery that high purity BLG crystals can be prepared directly in a crude whey protein solution that contains significant amounts of other whey proteins in addition to BLG, without the use of organic solvents such as toluene. This is contrary to common conventional wisdom in the art which teaches that a protein must be highly purified before one can hope to crystallize it, and that not all proteins can be crystallized.

[0011] This discovery has the potential to change the way the dairy industry handles and fractionates whey proteins, opening up the efficient and gentle production of high-purity BLG for safe use as a food ingredient.

[0012] Therefore, one aspect of the present invention is a method for preparing an edible composition comprising beta-lactoglobulin (BLG) in crystallized and / or isolated form, comprising the steps of: 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 to 6; b) crystallizing BLG in the supersaturated whey protein solution; c) optionally separating the BLG crystals from the remaining whey protein solution; The present invention relates to a method comprising the steps of:

[0013] The inventors have further discovered that edible whey protein compositions in powder form containing BLG crystals have a significantly higher bulk density than comparable compositions of the prior art, which is advantageous as it makes the powder easier to handle and less dusty.

[0014] Therefore, another aspect of the present invention relates to an edible composition comprising beta-lactoglobulin in crystallized and / or isolated form, for example obtainable by one or more of the methods described herein. The edible composition may for example be a powder comprising BLG crystals and having a bulk density of at least 0.40 g / mL. Alternatively, the edible product may be a liquid suspension or slurry comprising BLG crystals.

[0015] In the context of the present invention, a dry product (e.g., powder) comprising "BLG crystals" includes a product obtained from drying a suspension of BLG crystals, the crystal structure of which may be distorted during the drying process and may at least partially lose its X-ray diffraction characteristics. In the same manner, the terms "dried BLG crystals" and "dried BLG crystals" refer to particles obtained from drying of wet BLG crystals, which do not necessarily have a crystal structure themselves. However, the inventors have observed that when dry BLG crystals are resuspended in cold (4°C) demineralized water in a weight ratio of 2 parts water to 1 part dry BLG crystals, the BLG crystals rehydrate and regain substantially the same crystal structure (space group type and unit cell dimensions) as before drying.

[0016] BLG is known to be a large source of essential amino acids, including, for example, leucine, and therefore, the edible BLG compositions provided by the present invention have several interesting nutritional uses.

[0017] A further aspect of the invention is the orthorhombic space group P2 1 2 1 2 1 and for an isolated BLG crystal having unit cell dimensions a=68.68(±5%) Å, b=68.68(±5%) Å and c=156.65(±5%) Å, the crystal having unit cell integer angles α=90°, β=90° and γ=90°.

[0018] Furthermore, an aspect of the present invention relates to the use of an edible composition as defined herein as a food ingredient.

[0019] A further aspect of the present invention relates to a food product comprising an edible composition as defined herein and a fat source and / or a carbohydrate source. [Brief description of the drawings]

[0020] [Figure 1] Figure 1 shows two overlaid chromatograms of a crude whey protein solution based on sweet whey (solid line) and the mother liquor obtained after crystallization (dashed line). The difference between the solid and dashed lines is due to the removed BLG crystals. [Diagram 2] FIG. 2 is a micrograph of recovered BLG crystals from Example 1. [Diagram 3] FIG. 3 is a chromatogram of the BLG crystals recovered from Example 1. [Figure 4] FIG. 4 is a plot of the relationship between the conductivity of a whey protein solution and the resulting yield of recovered BLG crystals. [Diagram 5] FIG. 5 is a plot of the relationship between temperature and conductivity of a whey protein solution and the resulting yield of recovered BLG crystals. [Figure 6] FIG. 6 shows the relationship between the total protein content of a whey protein solution (indirectly indicated by the Brix degree, which is proportional to this protein content) and the obtained yield of recovered BLG crystals. [Figure 7]FIG. 7 shows the chromatograms of feed 1 of Example 3 (solid line) and the mother liquor obtained after crystallization and removal of BLG crystals (dashed line). [Figure 8] FIG. 8 is a photomicrograph of a sample taken during the early stages of crystallization of Raw Material 1 in Example 3. [Figure 9] FIG. 9 is a micrograph of a sample taken after completion of crystallization of Raw Material 1 of Example 3. [Figure 10] FIG. 10 shows the chromatogram of washed BLG crystals obtained from Source 1 in Example 3. [Figure 11] FIG. 11 shows the chromatograms of the starting material 2 of Example 3 (solid line) and the mother liquor obtained after crystallization and removal of the BLG crystals (dashed line). [Figure 12] FIG. 12 shows a photograph of raw material 2 of Example 3 before (left photograph) and after (right photograph) crystallization. [Figure 13] FIG. 13 shows micrographs of BLG crystals (both whole and fragmented) obtained from Raw Material 2 in Example 3. [Figure 14] FIG. 14 shows that increasing the conductivity or changing the pH of the BLG crystal slurry dissolves the BLG crystals. [Figure 15] FIG. 15 shows that increasing the conductivity or changing the pH of the BLG crystal slurry dissolves the BLG crystals. [Figure 17] FIG. 17 shows a photograph of Raw Material 3 of Example 3 before (left photo) and after (right photo) crystallization. [Figure 18] FIG. 18 is a micrograph of BLG crystals recovered from Raw Material 3 in Example 3. [Figure 19] FIG. 19 shows the chromatogram of recovered BLG crystals of Source 3 of Example 3 without any washing step. [Figure 20] FIG. 20 shows the effect of increasing conductivity on the yield of recovered BLG crystals. [Figure 21] FIG. 21 shows a micrograph of BLG crystals formed at a conductivity of 4.20 mS / cm. [Figure 22]FIG. 22 shows a micrograph of BLG crystals from an early stage of crystallization of an SPC-based whey protein solution. [Diagram 23] FIG. 23 shows the difference in bulk density between a standard whey protein isolate (WPI) and a high purity BLG composition of the present invention that includes BLG crystals. [Figure 24] FIG. 24 is a photograph of a spin filter in which BLG crystals of Example 3, Raw Material 1, are separated from the mother liquor. [Diagram 25] 25 is a photograph of aliquots of the six low phosphorus beverage samples of Example 8. From left to right, the aliquots are Samples A, B, C, D, E and F. [Figure 26] FIG. 26 is a schematic diagram showing a variation of the crystallization process of Example 10 using DCF for separation of BLG crystals from the mother liquor. [Figure 27] FIG. 27 shows three photographs of the filter cake obtained from the separation of BLG crystals and mother liquor using a filter centrifuge. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] As mentioned above, one aspect of the present invention is a method for preparing an edible composition comprising beta-lactoglobulin (BLG) in crystallized and / or isolated form, comprising the steps of: 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 to 6; b) crystallizing BLG in the supersaturated whey protein solution; c) optionally separating the BLG crystals from the remaining whey protein solution; The present invention relates to a method comprising the steps of:

[0022] In the context of the present invention, the term "edible composition" refers to a composition that is safe for human consumption and use as a food ingredient and does not contain problematic amounts of toxic components (e.g., toluene or other undesirable organic solvents). BLG is the most predominant protein in bovine whey and milk serum and is present in several genetic variants, the main ones in cow's milk being labeled A and B. BLG is a lipocalin protein and can bind many hydrophobic molecules, suggesting a role in the transport of these molecules. BLG has also been found to be able to bind iron via siderophores, which may have a role in combating pathogens. Homologs of BLG are absent in human breast milk.

[0023] Bovine BLG is a relatively small protein of approximately 162 amino acid residues with a molecular weight of approximately 18.3-18.4 kDa. Under physiological conditions, this bovine BLG is primarily a dimer, but dissociates into monomers below approximately pH 3 and retains this monomeric native state as determined using NMR. Conversely, BLG also occurs in tetramers, octamers and other multimeric aggregate forms under a variety of native conditions.

[0024] If the native structure is sufficiently destabilized to allow aggregation, BLG solutions can form gels under a variety of conditions: upon prolonged heating at low pH and low ionic strength, clear "fine-chain" gels form in which the protein molecules assemble into long, rigid fibers.

[0025] In the context of the present invention, the term "BLG" or "beta-lactoglobulin" relates to BLG from mammalian species, for example in native and / or glycosylated form, including naturally occurring genetic variants.

[0026] In the context of the present invention, the term "crystal" refers to a solid material in which the components (e.g., atoms, molecules, or ions) are arranged in a highly ordered microscopic structure, forming a crystal lattice that extends in all directions. BLG crystals are protein crystals that primarily contain BLGs arranged in a highly ordered microscopic structure, forming a crystal lattice that extends in all directions. BLG crystals can be, for example, monolithic or polycrystalline, and can be, for example, intact crystals, crystal fragments, or combinations thereof. Crystal fragments are formed, for example, when intact crystals are subjected to mechanical shear during processing. Crystal fragments also have the highly ordered microscopic structure of crystals, but may lack the uniform surface and / or uniform edges or corners of intact crystals. See, for example, FIG. 18 for an example of many intact BLG crystals and FIG. 13 for an example of a BLG crystal fragment. In both cases, the BLG crystals or crystal fragments can be visually identified as well-defined, compact, coherent structures using a light microscope. BLG crystals or crystal fragments are often at least partially transparent. Protein crystals are further known to be birefringent, and this optical property can be used to identify unknown particles with crystalline structure. On the other hand, non-crystalline BLG aggregates appear as loose or porous masses that are poorly defined, opaque, and irregularly sized.

[0027] In the context of the present invention, the term "crystallizing" relates to the formation of protein crystals. Crystallization can, for example, occur spontaneously or can be initiated by the addition of crystallizing seeds.

[0028] The edible composition comprises BLG in a crystallized and / or isolated form. An edible composition comprising BLG in isolated form comprises at least 80% (w / w) BLG based on total solids. An edible composition comprising BLG in crystallized form comprises at least some BLG crystals, preferably a substantial amount of BLG crystals.

[0029] BLG crystals can often be observed under a microscope and may even reach a size visible to the naked eye.

[0030] 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 exceeds the saturation point of BLG in this liquid under given physical and chemical conditions. The term "supersaturation" is known in the field of crystallization (see, for example, Gerard Coquerela, "Crystallization of molecular systems from solution: phase diagrams, supersaturation and other basic concepts", Chemical Society Reviews, p. 2286-2300, Issue 7, 2014), and supersaturation can be determined by many different measurement techniques (e.g., by spectroscopy or particle size analysis). In the context of the present invention, supersaturation with respect to BLG is determined by the following procedure.

[0031] Procedure to test if a liquid is supersaturated at specific conditions for BLG: a) Transfer a 50 ml sample of the liquid to be tested into a centrifuge tube of 115 mm height, 25 mm internal diameter and 50 mL capacity (VWR catalogue no. 525-0402). During steps a) to h), care should be taken to preserve the sample and subsequent fractions thereof in the original physical and chemical conditions of the liquid. b) The sample is immediately centrifuged at 3000 g for 3.0 minutes with a maximum acceleration of 30 seconds and a maximum deceleration of 30 seconds. c) Immediately after centrifugation, transfer as much of the supernatant as possible (without disturbing the pellet, if one has formed) to a second centrifuge tube (same type as in step a). d) Take a 0.05 mL aliquot of this supernatant (aliquot A). e) To a second centrifuge tube, add 10 mg of BLG crystals (at least 98% pure BLG based on total solids) having a particle size of up to 200 microns and vortex the mixture. f) Leave this second centrifuge tube at the original temperature for 60 minutes. g) Immediately after step f), centrifuge this second centrifuge tube at 500 g for 10 minutes and then take another 0.05 mL aliquot of the supernatant (aliquot B). h) If a centrifugation pellet is present from step g), recover the pellet, resuspend the pellet in milliQ water and immediately inspect the suspension for the presence of crystals visible by microscope. i) Determine the concentration of BLG in aliquot A and in aliquot B using the method outlined in Example 9.9 and express the results as wt / wt% BLG relative to the total weight of the aliquot. The concentration of BLG in aliquot A is C BLG,A The concentration of BLG in the sample B is called C BLG,B It is called. j)c BLG,B C BLG,A and crystals are observed in step i), the liquid from which the sample was taken in step a) was supersaturated (for these particular conditions).

[0032] In the context of the present invention, the terms "liquid" and "solution" encompass compositions that include a combination of a liquid and solid or semi-solid particles (e.g., protein crystals or other protein particles). Thus, a "liquid" or "solution" may be a suspension or even a slurry. However, a "liquid" or "solution" is preferably pumpable.

[0033] In some preferred embodiments of the invention, the method does not include the separation of step c) and an edible composition is produced that includes both BLG crystals and additional whey protein. When a variation of this method further includes drying of step f), a dry composition is produced that includes BLG crystals and additional whey protein (i.e., a WPC or WPI in which at least a portion of the BLG is present in the form of BLG crystals). Preferably, the method includes steps a), b) and f) in direct order.

[0034] When the whey protein raw material is a whey protein concentrate (WPC), a whey protein isolate (WPI), a serum protein concentrate (SPC) or a serum protein isolate (SPI), a variation of the above method allows for the preparation of the WPC, WPI, SPC or SPI in liquid or dry form in which at least a portion of the BLG is in crystalline form.

[0035] The terms "whey protein concentrate" and "serum protein concentrate" relate to a dry or aqueous composition comprising a total amount of protein of between 20 and 89% (w / w) relative to the total solids.

[0036] The WPC or SPC is preferably 20-89% (w / w) protein on total solids; 15–70% (w / w) BLG relative to total protein; 8–50% (w / w) ALA based on total protein, and 0-40% (w / w) CMP on protein Includes.

[0037] Alternatively, but also preferably, the WPC or SPC is 20-89% (w / w) protein on total solids; 15–90% (w / w) BLG relative to total protein; 4-50% (wt / wt) ALA based on total protein, and 0-40% (w / w) CMP on protein may include.

[0038] Preferably, the WPC or SPC is 20-89% (w / w) protein on total solids; 15–80% (w / w) BLG relative to total protein; 4-50% (wt / wt) ALA based on total protein, and 0-40% (w / w) CMP on protein Includes.

[0039] More preferably, the WPC or SPC is 70-89% (w / w) protein on total solids; 30–90% (w / w) BLG relative to total protein; 4-35% (wt / wt) ALA based on total protein, and 0-25% (w / w) CMP on protein Includes.

[0040] The terms "whey protein isolate" and "serum protein isolate" relate to a dry or aqueous composition comprising a total amount of protein of 90-100% (w / w) relative to the total solids.

[0041] The WPI or SPI is preferably 90-100% (w / w) protein on total solids; 15–70% (w / w) BLG relative to total protein; 8–50% (w / w) ALA based on total protein, and CMP of 0–40% (w / w) of total protein Includes.

[0042] Alternatively, but also preferably, the WPI or SPI comprises: 90-100% (w / w) protein on total solids; 30–95% (w / w) BLG relative to total protein; 4-35% (wt / wt) ALA based on total protein, and CMP of 0–25% (w / w) of total protein may include.

[0043] More preferably, the WPI or SPI is 90-100% (w / w) protein on total solids; 30–90% (w / w) BLG relative to total protein; 4-35% (wt / wt) ALA based on total protein, and CMP of 0–25% (w / w) of total protein may include.

[0044] In some preferred embodiments of the invention, the method further comprises a step d) of washing the BLG crystals (eg, the separated BLG crystals obtained from step c).

[0045] In some preferred embodiments of the present invention, the method further comprises a step e) of recrystallizing the BLG crystals (eg, the BLG crystals obtained from step c) or d).

[0046] The method may, for example, comprise or consist of steps a), b), c), d) and e). Alternatively, the method may comprise or consist of steps a), b), c) and e).

[0047] In some particularly preferred embodiments of the invention, the method further comprises a step f) of drying the BLG-containing composition derived from steps b), c), d) or e).

[0048] The method may, for example, comprise or consist of steps a), b) and f). Alternatively, the method may comprise or consist of steps a), b), c) and f). Alternatively, the method may comprise or consist of steps a), b), c), d) and f). Alternatively, the method may comprise or consist of steps a), b), c), d), e) and f).

[0049] As mentioned above, step a) of the present invention involves providing a whey protein solution comprising BLG and at least additional whey protein.

[0050] In the context of the present invention, the term "whey protein" refers to the protein found in whey or milk serum. The whey protein of the present whey protein solution may be a subset of the protein species found in whey or milk serum, or may be the complete set of protein species found in whey and / or milk serum. However, the whey protein solution always contains BLG.

[0051] In the context of the present invention, the term "additional protein" refers to a protein that is not BLG. The additional protein present in the whey protein solution typically includes one or more of the non-BLG proteins found in whey or milk serum. Non-limiting examples of such proteins are alpha-lactalbumin, bovine serum albumin, immunoglobulins, caseino-macropeptide (CMP), osteopontin, lactoferrin and milk fat globule membrane protein.

[0052] Thus, the whey protein solution may preferably comprise at least one additional whey protein selected from the group consisting of alpha-lactalbumin, bovine serum albumin, immunoglobulins, caseinomacropeptide (CMP), osteopontin, lactoferrin, milk fat globule membrane protein and combinations thereof.

[0053] Alpha-lactalbumin (ALA) is a protein present in the milk of almost all mammalian species. ALA forms the regulatory subunit of the lactose synthase (LS) heterodimer, and beta-1,4-galactosyltransferase (beta4Gal-T1) forms the catalytic component. Together, these proteins enable LS to produce lactose by introducing a galactose moiety into glucose. As a multimer, alpha-lactalbumin binds strongly to calcium and zinc ions and may have bactericidal or antitumor activity. One of the main structural differences with beta-lactoglobulin is that ALA does not have any thiol groups that could act as starting points for covalent aggregation reactions. As a result, pure ALA will not form a gel upon denaturation and acidification.

[0054] In the context of the present invention, the term "ALA" or "alpha-lactalbumin" relates to alpha-lactalbumin from mammalian species, for example in native and / or glycosylated form, including naturally occurring genetic variants.

[0055] In some embodiments of the invention, the whey protein solution comprises up to 10% (w / w) casein based on the total amount of protein, preferably up to 5% (w / w), more preferably up to 1% (w / w), even more preferably up to 0.5% casein based on the total amount of protein. In some preferred embodiments of the invention, the whey protein solution does not contain any detectable amount of casein.

[0056] The term "whey" refers to the liquid remaining when casein and milk fat globules are removed from milk, for example by microfiltration or large pore ultrafiltration. Whey may also be referred to as "ideal whey".

[0057] The term "whey protein" or "serum protein" relates to proteins present in whey.

[0058] The term "whey" refers to the liquid supernatant remaining after the precipitation and removal of casein from milk. Casein precipitation can be accomplished, for example, by acidification of the milk and / or the use of rennet enzymes.

[0059] There are several types of whey, including "sweet whey", which is a whey product made by rennet-based precipitation of casein, and "acid whey" or "sour whey", which is a whey product made by acid-based precipitation of casein. Acid-based precipitation of casein can be accomplished, for example, by the addition of food acids or by bacterial culture.

[0060] In some preferred embodiments of the present invention, the whey protein solution of step a) comprises at least 5% (wt / wt) additional whey protein based on the total amount of protein. Preferably, the whey protein solution of step a) comprises at least 10% (wt / wt) additional whey protein based on the total amount of protein. More preferably, the whey protein solution of step a) comprises at least 15% (wt / wt) additional whey protein based on the total amount of protein. Even more preferably, the whey protein solution of step a) comprises at least 20% (wt / wt) additional whey protein based on the total amount of protein. Most preferably, the whey protein solution of step a) may comprise at least 30% (wt / wt) additional whey protein based on the total amount of protein.

[0061] In another preferred embodiment of the present invention, the whey protein solution of step a) comprises at least 1% (wt / wt) additional whey protein based on the total amount of protein. Preferably, the whey protein solution of step a) comprises at least 2% (wt / wt) additional whey protein based on the total amount of protein. Even more preferably, the whey protein solution of step a) comprises at least 3% (wt / wt) additional whey protein based on the total amount of protein. Most preferably, the whey protein solution of step a) may comprise at least 4% (wt / wt) additional whey protein based on the total amount of protein.

[0062] In yet another preferred embodiment of the present invention, the whey protein solution of step a) comprises at least 35% (wt / wt) additional whey protein relative to the total amount of protein. Preferably, the whey protein solution of step a) may comprise at least 40% (wt / wt) additional whey protein relative to the total amount of protein. More preferably, the whey protein solution of step a) may comprise, for example, at least 45% (wt / wt) additional whey protein relative to the total amount of protein. Even more preferably, the whey protein solution of step a) may comprise at least 50% (wt / wt) additional whey protein relative to the total amount of protein.

[0063] In some preferred embodiments of the present invention, the whey protein solution of step a) comprises additional whey protein in the range of 5-90% (wt / wt) based on the total amount of protein. Preferably, the whey protein solution of step a) may comprise additional whey protein in the range of 10-80% (wt / wt) based on the total amount of protein. The whey protein solution of step a) may, for example, comprise additional whey protein in the range of 20-70% (wt / wt) based on the total amount of protein. Preferably, the whey protein solution of step a) comprises additional whey protein in the range of 30-70% (wt / wt) based on the total amount of protein.

[0064] As mentioned above, the inventors have discovered that BLG can be crystallized without the use of organic solvents. This purification approach can also be used to purify preparations containing whey protein (which preparations have already been subjected to several rounds of BLG purification), providing a simple way to further increase the purity of BLG. Therefore, in some preferred embodiments of the present invention, the whey protein solution of step a) comprises additional whey protein in the range of 1-20% (wt / wt) relative to the total amount of protein. Preferably, the whey protein solution of step a) may comprise additional whey protein in the range of 2-15% (wt / wt) relative to the total amount of protein. Even more preferably, the whey protein solution of step a) may comprise additional whey protein in the range of, for example, 3-10% (wt / wt) relative to the total amount of protein.

[0065] In some embodiments of the present invention, the whey protein solution of step a) comprises at least 5% (wt / wt) ALA based on the total amount of protein. Preferably, the whey protein solution of step a) comprises at least 10% (wt / wt) ALA based on the total amount of protein. Even more preferably, the whey protein solution of step a) comprises at least 15% (wt / wt) ALA based on the total amount of protein. Alternatively, the whey protein solution of step a) may comprise at least 20% (wt / wt) ALA based on the total amount of protein.

[0066] In some preferred embodiments of the present invention, the whey protein solution of step a) comprises at least 25% (wt / wt) ALA based on the total amount of protein. Preferably, the whey protein solution of step a) comprises at least 30% (wt / wt) ALA based on the total amount of protein. Preferably, the whey protein solution of step a) comprises at least 35% (wt / wt) ALA based on the total amount of protein. Even more preferably, the whey protein solution of step a) may comprise at least 40% (wt / wt) ALA based on the total amount of protein.

[0067] In some preferred embodiments of the present invention, the whey protein solution of step a) comprises ALA in the range of 5-95% (wt / wt) based on the total amount of protein. Preferably, the whey protein solution of step a) comprises ALA in the range of 5-70% (wt / wt) based on the total amount of protein. Even more preferably, the whey protein solution of step a) may comprise ALA in the range of 10-60% (wt / wt) based on the total amount of protein. The whey protein solution of step a) preferably comprises ALA in the range of 12-50% (wt / wt) based on the total amount of protein. Even more preferably, the whey protein solution of step a) may comprise ALA in the range of 20-45% (wt / wt) based on the total amount of protein.

[0068] In some preferred embodiments of the present invention, the whey protein solution in step a) has a weight ratio between BLG and ALA of at least 0.01. Preferably, the whey protein solution in step a) has a weight ratio between BLG and ALA of at least 0.5. Even more preferably, the whey protein solution in step a) has a weight ratio between BLG and ALA of at least 1, such as at least 2. For example, the whey protein solution in step a) may have a weight ratio between BLG and ALA of at least 3.

[0069] The amounts and concentrations of BLG and other proteins in whey protein solutions and whey protein raw materials refer to all dissolved protein and do not include precipitated or crystallized protein.

[0070] In the context of the present invention, the term "weight ratio" between component X and component Y is calculated X / m Y (In the formula, m X is the amount (weight) of component X, and m Y is the amount (by weight) of component Y.

[0071] In some preferred embodiments of the present invention, the whey protein solution of step a) has a weight ratio between BLG and ALA in the range of 0.01 to 20. Preferably, the whey protein solution of step a) has a weight ratio between BLG and ALA in the range of 0.2 to 10. Even more preferably, the whey protein solution of step a) has a weight ratio between BLG and ALA in the range of 0.5 to 4. For example, the whey protein solution of step a) has a weight ratio between BLG and ALA in the range of 1 to 3.

[0072] In some preferred embodiments of the present invention, the whey protein solution of step a) comprises at least 1% (wt / wt) BLG based on the total amount of protein. Preferably, the whey protein solution of step a) comprises at least 2% (wt / wt) BLG based on the total amount of protein. Even more preferably, the whey protein solution of step a) comprises at least 5% (wt / wt) BLG based on the total amount of protein. Preferably, the whey protein solution of step a) may comprise at least 10% (wt / wt) BLG based on the total amount of protein.

[0073] In some preferred embodiments of the present invention, the whey protein solution of step a) comprises at least 12% (wt / wt) BLG based on the total amount of protein. For example, the whey protein solution of step a) may comprise at least 15% (wt / wt) BLG based on the total amount of protein. The whey protein solution of step a) may comprise at least 20% (wt / wt) BLG based on the total amount of protein. Alternatively, the whey protein solution of step a) may comprise at least 30% (wt / wt) BLG based on the total amount of protein.

[0074] In some particularly preferred embodiments of the present invention, the whey protein solution of step a) comprises up to 95% (wt / wt) BLG based on the total amount of protein. Preferably, the whey protein solution of step a) may comprise up to 90% (wt / wt) BLG based on the total amount of protein. More preferably, the whey protein solution of step a) may comprise, for example, up to 85% (wt / wt) BLG based on the total amount of protein. Even more preferably, the whey protein solution of step a) may comprise, for example, up to 80% (wt / wt) BLG based on the total amount of protein. Preferably, the whey protein solution of step a) may comprise up to 78% (wt / wt) BLG based on the total amount of protein. Preferably, the whey protein solution of step a) may comprise up to 75% (wt / wt) BLG based on the total amount of protein.

[0075] In some preferred embodiments of the present invention, the whey protein solution of step a) comprises BLG in the range of 1-95% (wt / wt) based on the total amount of protein. Preferably, the whey protein solution of step a) may comprise BLG in the range of 5-90% (wt / wt) based on the total amount of protein. More preferably, the whey protein solution of step a) comprises BLG in the range of 10-85% (wt / wt) based on the total amount of protein. Even more preferably, the whey protein solution of step a) comprises BLG in the range of 10-80% (wt / wt) based on the total amount of protein. Most preferably, the whey protein solution of step a) may comprise BLG in the range of 20-70% (wt / wt) based on the total amount of protein.

[0076] In another preferred embodiment of the present invention, the whey protein solution of step a) comprises BLG in the range of 10-95% (wt / wt) relative to the total amount of protein. Preferably, the whey protein solution of step a) may comprise BLG in the range of 12-90% (wt / wt) relative to the total amount of protein. More preferably, the whey protein solution of step a) comprises BLG in the range of 15-85% (wt / wt) 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% (wt / wt) relative to the total amount of protein. Most preferably, the whey protein solution of step a) may comprise BLG in the range of 30-70% (wt / wt) relative to the total amount of protein.

[0077] In some preferred embodiments of the present invention, the whey protein solution of step a) comprises at least 0.4% (wt / wt) BLG based on the weight of the whey protein solution. Preferably, the whey protein solution comprises at least 1.0% (wt / wt) BLG. More preferably, the whey protein solution comprises at least 2.0% (wt / wt) BLG. Even more preferably, the whey protein solution comprises at least 4% (wt / wt) BLG.

[0078] Even more preferred is a higher concentration of BLG, 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 preferred is that the whey protein solution comprises at least 15% (w / w) BLG.

[0079] In some preferred embodiments of the present invention, the whey protein solution of step a) comprises BLG in the range of 0.4-40% (wt / wt) based on the weight of the whey protein solution. Preferably, the whey protein solution comprises BLG in the range of 1-35% (wt / wt). More preferably, the whey protein solution comprises BLG in the range of 4-30% (wt / wt). Even more preferably, the whey protein solution comprises BLG in the range of 10-25% (wt / wt).

[0080] Any suitable whey protein source may be used to prepare the whey protein solution. In some preferred embodiments of the present invention, the whey protein solution comprises or consists of whey protein concentrate, whey protein concentrate, whey protein isolate, whey protein isolate or combinations thereof.

[0081] Preferably, the whey protein solution is a desalted whey protein solution.

[0082] In this context, the term desalted means that the whey protein solution has a conductivity of at most 15 mS / cm, preferably at most 10 mS / cm, even more preferably at most 8 mS / cm. The conductivity of the UF permeate of the desalted whey protein solution is preferably at most 7 mS / cm, more preferably at most 4 mS / cm, even more preferably at most 1 mS / cm.

[0083] It is particularly preferred that the whey protein solution is a desalted whey protein concentrate, a desalted whey protein isolate, a desalted whey protein concentrate or a desalted whey protein isolate.

[0084] In some particularly preferred embodiments of the present invention, the whey protein solution comprises or consists of desalted, pH adjusted whey protein concentrate, whey protein concentrate, whey protein isolate, whey protein isolate, or combinations thereof.

[0085] The whey protein solution may, for example, comprise or consist of a desalted whey protein concentrate. Alternatively, the whey protein solution may comprise or consist of a desalted whey protein concentrate. Alternatively, the whey protein solution may comprise or consist of a desalted whey protein isolate. Alternatively, the whey protein solution may comprise or consist of a desalted whey protein isolate.

[0086] In the context of the present invention, the terms "whey protein concentrate" and "whey protein concentrate" relate to preparations of whey or whey, which preparations contain in the range of about 20-89% (w / w) protein based on total solids.

[0087] In the context of the present invention, the terms "whey protein isolate" and "whey protein isolate" relate to a preparation of whey or whey, which preparation comprises at least 90% (w / w) protein based on the total solids.

[0088] The terms "consisting essentially of" and "consisting essentially of" mean that the subject claim or feature includes specified materials or steps that do not materially affect the basic and novel characteristics of the claimed invention.

[0089] The proteins of the whey protein solution are preferably derived from mammalian milk, preferably from ruminant milk (e.g., cow, sheep, goat, buffalo, camel, llama, mare and / or deer). Proteins derived from bovine (cow) milk are particularly preferred. Thus, the BLG and additional whey proteins are preferably bovine BLG and bovine whey protein.

[0090] The proteins of the whey protein solution are preferably as close to their native state as possible and preferably have been subjected to only mild, if any, heat treatment.

[0091] In some preferred embodiments of the present invention, the BLG of the whey protein solution has a lactosylation degree of at most 1. Preferably, the BLG of the whey protein solution has a lactosylation degree of at most 0.6. More preferably, the BLG of the whey protein solution has a lactosylation degree of at most 0.4. Even more preferably, the BLG of the whey protein solution has a lactosylation degree of at most 0.2. Most preferably, the BLG of the whey protein solution has a lactosylation degree of at most 0.1, such as preferably at most 0.01.

[0092] The degree of lactosylation of BLG is determined according to Czerwenka et al. (J. Agric. Food Chem., Vol. 54, No. 23, 2006, pages 8874-8882).

[0093] In some preferred embodiments of the present invention, the whey protein solution has a furosine value of up to 80 mg / 100 g protein. Preferably, the whey protein solution has a furosine value of up to 40 mg / 100 g protein. More preferably, the whey protein solution has a furosine value of up to 20 mg / 100 g protein. Even more preferably, the whey protein solution has a furosine value of up to 10 mg / 100 g protein. Most preferably, the whey protein solution has a furosine value of up to 5 mg / 100 g protein, for example, preferably has a furosine value of 0 mg / 100 g protein.

[0094] The whey protein solution typically contains other ingredients in addition to protein. The whey protein solution may contain other ingredients that are normally found in whey or whey, such as minerals, carbohydrates and / or lipids. Alternatively or in addition, the whey protein solution may contain ingredients that are not native to whey or whey. However, such non-natural ingredients should preferably be safe for use in food production, and also preferably be safe for human consumption.

[0095] The method is particularly advantageous for separating BLG from a crude whey protein solution that contains solids other than BLG.

[0096] 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).

[0097] In some preferred embodiments of the invention, the whey protein solution comprises up to 20% (wt / wt) carbohydrates, preferably up to 10% (wt / wt) carbohydrates, more preferably up to 5% (wt / wt) carbohydrates, and even more preferably up to 2% (wt / wt) carbohydrates.

[0098] The whey protein solution may also include lipids, for example in the form of triglycerides and / or other lipid types (eg, phospholipids).

[0099] In some embodiments of the present invention, the whey protein solution of step a) comprises a total amount of lipids of up to 15% (w / w) based on total solids. Preferably, the whey protein solution of step a) comprises a total amount of lipids of up to 10% (w / w) based on total solids. More preferably, the whey protein solution of step a) comprises a total amount of lipids of up to 6% (w / w) based on total solids. Even more preferably, the whey protein solution of step a) comprises a total amount of lipids of up to 1.0% (w / w) based on total solids. Most preferably, the whey protein solution of step a) comprises a total amount of lipids of up to 0.5% (w / w) based on total solids.

[0100] The total amount of protein in the whey protein solution is typically at least 1% (w / w) based on the weight of the whey protein solution. Preferably, the total amount of protein in the whey protein solution is at least 5% (w / w). More preferably, the total amount of protein in the whey protein solution is at least 10% (w / w). Even more preferably, the total amount of protein in the whey protein solution is at least 15% (w / w).

[0101] In some preferred embodiments of the present invention, the total amount of protein in the whey protein solution is in the range of 1-50% (wt / wt). Preferably, the total amount of protein in the whey protein solution is in the range of 5-40% (wt / wt). More preferably, the total amount of protein in the whey protein solution is in the range of 10-30% (wt / wt). Even more preferably, the total amount of protein in the whey protein solution is in the range of 15-25% (wt / wt).

[0102] The total protein content of the whey protein solution is determined according to Example 9.2.

[0103] The whey protein solutions are typically prepared by subjecting a whey protein raw material to one or more conditioning procedures that result in the formation of a whey protein solution that is supersaturated with respect to BLG.

[0104] The raw material is preferably WPC, WPI, SPC, SPI or a combination thereof.

[0105] In the context of the present invention, the term "whey protein raw material" refers to a composition that is converted into a whey protein solution that is supersaturated with respect to BLG. This whey protein raw material is typically an aqueous liquid that contains BLG and at least one whey protein, but is not usually supersaturated with respect to BLG.

[0106] The embodiments relating to the chemical composition of the whey protein solution apply equally to the whey protein raw material, although typically at least one parameter of the whey protein raw material is set to avoid supersaturation or at least spontaneous crystallization.

[0107] In some preferred embodiments of the present invention, the supersaturated whey protein solution is prepared by subjecting the whey protein raw material to the following conditioning: - adjusting the pH, - reducing electrical conductivity; - lowering the temperature; - increasing protein concentration; - adding an agent that reduces water activity; - Changing the ionic composition The composition is prepared by subjecting the composition to one or more of the following:

[0108] In some preferred embodiments of the present invention, preparing the whey protein solution involves adjusting the pH of the whey protein raw material to a pH in the range of 5-6.

[0109] All pH values ​​are measured using a pH glass electrode and normalized to 25°C.

[0110] The whey protein solution may have a pH in the range of, for example, 4.9 to 6.1. The pH of the whey protein solution may be in the range of, for example, 5.0 to 6.1. Alternatively, the pH of the whey protein solution may be in the range of 5.1 to 6.1. Preferably, the pH of the whey protein solution is in the range of 5.1 to 6.0.

[0111] In some preferred embodiments of the present invention, the whey protein solution has a pH in the range of 5.0 to 6.0. Preferably, the whey protein solution has a pH in the range of 5.1 to 6.0. More preferably, the whey protein solution has a pH in the range of 5.1 to 5.9. Even more preferably, the whey protein solution has a pH in the range of 5.2 to 5.9. Most preferably, the whey protein solution has a pH in the range of 5.2 to 5.8.

[0112] The pH is preferably adjusted using a food acceptable acid and / or base. Food acceptable acids, such as carboxylic acids, are particularly preferred. Useful examples of such acids are, for example, hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, maleic acid, tartaric acid, lactic acid, citric acid or gluconic acid and / or mixtures thereof.

[0113] In some preferred embodiments of the invention, the pH is adjusted using a lactone (e.g., D-glucono-delta-lactone), which slowly hydrolyzes and simultaneously lowers the pH of an aqueous liquid containing the lactone. The target pH after hydrolysis of the lactone is complete can be accurately calculated.

[0114] Useful examples of food acceptable bases are, for example, hydroxide sources such as sodium hydroxide, potassium hydroxide, calcium hydroxide, salts of food acids (eg, trisodium citrate), and / or combinations thereof.

[0115] In another preferred embodiment of the present invention, the pH is adjusted to the H + The addition of beaded / large particle cation exchange material is easily removed from the whey protein solution before or after crystallization. + Adjusting the pH by addition of moulds is particularly advantageous in the present invention because it lowers the pH without adding negative counter ions that would significantly affect the conductivity of the whey protein raw material.

[0116] In some preferred embodiments of the present invention, preparing the whey protein solution involves reducing the conductivity of the whey protein raw material.

[0117] Conductivity values ​​referred to herein are normalized to 25° C. unless otherwise stated.

[0118] The inventors have found that reducing the conductivity of the whey protein solution leads to higher yields of BLG crystals. The minimum conductivity of the whey protein solution that can be obtained depends on the composition of protein fraction and lipid fraction (if present). Some protein species, such as caseinomacropeptide (CMP), contribute more to conductivity than other protein species. Therefore, it is preferred to approach the conductivity of the whey protein raw material to a level where the protein and its counterions are the main contributors to conductivity. This reduction in conductivity is often accompanied by the removal of at least a portion of the small free ions that are present in the liquid phase and are not tightly bound to the protein.

[0119] It is often preferred that the whey protein solution has a conductivity of at most 10 mS / cm. In some preferred embodiments of the invention, the whey protein solution has a conductivity of at most 5 mS / cm. Preferably, the whey protein solution has a conductivity of at most 4 mS / cm.

[0120] The lower the conductivity, the higher the yield of BLG crystals. Therefore, the whey protein solution preferably has a conductivity of up to 3 mS / cm. In some preferred embodiments of the present invention, the whey protein solution has a conductivity of up to 1 mS / cm. Preferably, the whey protein solution has a conductivity of up to 0.5 mS / cm.

[0121] Preferably, the conductivity of the whey protein raw material is reduced by dialysis or diafiltration. Diafiltration by ultrafiltration is particularly preferred as it is possible to wash out salts and small charged molecules while retaining the protein. In some preferred embodiments of the present invention, the same UF unit is used for UF / diafiltration and subsequent concentration of the whey protein raw material.

[0122] The inventors have found indications that it is advantageous that the ratio between the electrical conductivity (expressed in mS / cm) and the total amount of protein in the whey protein solution (expressed as % by weight of total protein relative to the total weight of the whey protein solution) can be kept below a certain threshold value in order to promote crystallization of BLG.

[0123] In some preferred embodiments of the present invention, the ratio between the conductivity of the whey protein solution and the total amount of protein is at most 0.3. Preferably, the ratio between the conductivity of the whey protein solution and the total amount of protein is at most 0.25. Preferably, the ratio between the conductivity of the whey protein solution and the total amount of protein is at most 0.20. More preferably, the ratio between the conductivity of the whey protein solution and the total amount of protein is at most 0.18. Even more preferably, the ratio between the conductivity of the whey protein solution and the total amount of protein is at most 0.12. Most preferably, the ratio between the conductivity of the whey protein solution and the total amount of protein is at most 0.10.

[0124] For example, the ratio between the conductivity and the total amount of protein of the whey protein solution is preferably about 0.07 or less.

[0125] The inventors have further discovered that the whey protein raw material may be advantageously conditioned to produce a whey protein solution having a UF permeate conductivity of up to 10 mS / cm. The UF permeate conductivity is a measure of the conductivity of the small molecule fraction of the liquid, and is measured according to Example 9.10. When the term "conductivity" is so 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 the liquid, and is measured according to Example 9.10.

[0126] Preferably, the conductivity of the UF permeate of the whey protein solution is at most 7 mS / cm. More preferably, the conductivity of the UF permeate of the whey protein solution may be at most 5 mS / cm. Even more preferably, the conductivity of the UF permeate of the whey protein solution may be at most 3 mS / cm.

[0127] Even lower UF permeate conductivities can be used, which is particularly preferred when a high yield of BLG is to be obtained. Thus, preferably, the conductivity of the UF permeate of the whey protein solution is at most 1.0 mS / cm. More preferably, the conductivity of the UF permeate of the whey protein solution can be at most 0.4 mS / cm. Even more preferably, the conductivity of the UF permeate of the whey protein solution can be at most 0.1 mS / cm. Most preferably, the conductivity of the UF permeate of the whey protein solution can be at most 0.04 mS / cm.

[0128] For example, even lower UF permeate conductivity can be achieved using MilliQ water (MilliQ has a conductivity of about 0.06 μS / cm) as a diluent during diafiltration. Thus, the conductivity of the UF permeate of the whey protein solution can be at most 0.01 mS / cm. Alternatively, the conductivity of the UF permeate of the whey protein solution can be at most 0.001 mS / cm. Alternatively, the conductivity of the UF permeate of the whey protein solution can be at most 0.0001 mS / cm.

[0129] In some preferred embodiments of the present invention, preparing the whey protein solution involves reducing the temperature of the whey protein raw material.

[0130] For example, preparing the whey protein solution may involve reducing the temperature of the whey protein raw material to at least 5° C., preferably at least 10° C., and even more preferably at least 15° C. For example, preparing the whey protein solution may involve reducing the temperature of the whey protein raw material to at least 20° C.

[0131] The temperature of the whey protein raw material may be reduced, for example, to a maximum of 30° C., preferably to a maximum of 20° C., even more preferably to a maximum of 10° C. The inventors have found that lower temperatures result in a higher degree of supersaturation, and therefore the temperature of the whey protein raw material may be reduced, for example, to a maximum of 5° C., preferably to a maximum of 2° C., even more preferably to a maximum of 0° C. This temperature may even be lower than 0° C., but preferably the whey protein solution should remain pumpable, for example in the form of an ice slurry.

[0132] In some preferred embodiments of the present invention, the whey protein solution is an ice slurry prior to the initiation of BLG crystallization. Alternatively or additionally, the whey protein solution to be crystallized may be converted to or maintained as an ice slurry during the BLG crystallization of step b).

[0133] In some particularly preferred embodiments of the present invention, the preparation of the whey protein solution involves increasing the total protein concentration of the whey protein raw material, which can be, for example, subjected to one or more protein concentration steps (e.g., ultrafiltration, nanofiltration, reverse osmosis and / or evaporation) to thereby concentrate the whey protein solution.

[0134] Ultrafiltration is particularly preferred as it allows for the selective concentration of proteins while leaving the salt and carbohydrate concentrations largely unaffected. As noted above, ultrafiltration is preferably used for both the diafiltration and concentration of the whey protein raw material.

[0135] In some preferred embodiments of the present invention, the concentration of BLG in the whey protein solution is lower than the level at which spontaneous crystallization of BLG occurs.Therefore, it is often preferable to stop modifying the whey protein raw material when the whey protein solution is in the metastable region, i.e., in the supersaturated region where BLG crystals can grow when seeding is used, but crystallization does not begin spontaneously.

[0136] In some preferred embodiments of the present invention, preparing a solution of whey protein involves the addition of one or more water activity reducing agents to the whey protein raw material.

[0137] Useful non-limiting examples of such water activity reducing agents are polysaccharides and / or polyethylene glycol (PEG).

[0138] In some preferred embodiments of the present invention, preparation of the whey protein solution involves modifying the ionic composition of the whey protein raw material, for example by ion exchange, by adding new ionic species, by dialysis or diafiltration.

[0139] Typically, the whey protein solution is prepared by combining two or more of the above process steps to produce supersaturation.

[0140] In some preferred embodiments of the present invention, the whey protein solution is prepared by mixing the whey protein raw material with at least: - Concentrating, for example using ultrafiltration, nanofiltration or reverse osmosis, at a temperature above 10°C; and - followed by cooling to a temperature below 10°C This involves placing a

[0141] In another preferred embodiment of the present invention, the preparation of the whey protein solution comprises the steps of: - Concentrating at a pH above 6.0; and - followed by an acid (e.g., GDL or H + Lowering the pH by adding cation exchange material ( This involves placing a

[0142] In yet another preferred embodiment of the present invention, the whey protein solution is prepared by mixing the whey protein raw material with at least: - Reducing the conductivity, for example by diafiltration using a membrane that retains at least the BLG This involves placing a

[0143] In a further preferred embodiment of the invention, the whey protein solution is prepared by mixing the whey protein raw material with at least: - Adjust the pH to 5-6; - Reducing the conductivity by diafiltration using a membrane that retains at least the BLG; - concentrating the protein using, for example, ultrafiltration, nanofiltration or reverse osmosis at a temperature above 10°C; and - Finally, cool to a temperature below 10°C. This involves subjecting the material to a combination of:

[0144] The inventors have further discovered that the BLG yield of the present process can be improved by controlling the molar ratio of the sum of sodium plus potassium to the sum of calcium and magnesium. Higher relative amounts of calcium and magnesium surprisingly increase the yield of BLG and thus appear to increase the efficiency of BLG recovery of the present process.

[0145] In some preferred embodiments of the present invention, the whey protein solution of step a) has a molar ratio between Na+K and Ca+Mg of at most 4. More preferably, the whey protein solution of step a) has a molar ratio between Na+K and Ca+Mg of at most 2. Even more preferably, the whey protein solution of step a) has a molar ratio between Na+K and Ca+Mg of at most 1.5, even more preferably at most 1.0. Most preferably, the whey protein solution of step a) has a molar ratio between Na+K and Ca+Mg of at most 0.5, such as at most 0.2.

[0146] The molar ratio between Na+K and Ca+Mg is (m Na +m K ) / (m Ca +m Mg ) where m Na is the content of element Na in moles, m K is the content of element K in mol, m Ca is the content of element Ca in mol, m Mg is the content of elemental Mg in mol

[0147] It is particularly preferred that the whey protein solution is supersaturated with respect to BLG by salting out, and therefore BLG can be crystallized from this whey protein solution in the salting out mode.

[0148] In some embodiments of the present invention, especially when the edible BLG product of the present invention should also have some 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 up to 2%, preferably up to 1.5%, more preferably up to 1.0%, most preferably up to 0.8%.

[0149] Step b) of the method involves crystallizing at least a portion of the BLG of the supersaturated whey protein solution.

[0150] It is particularly preferred that the crystallization in step b) occurs in a salting-in mode (i.e. in a liquid of low ionic strength and conductivity), as opposed to a salting-out mode, in which a significant amount of salt is added to the solution to induce crystallization.

[0151] The crystallization of BLG in step b) can be carried out, for example, as follows: - waiting for crystallization to occur, - addition of crystallization seeds, - Further increasing the degree of supersaturation of the BLG, and / or - Mechanical stimulation may involve one or more of:

[0152] In some preferred embodiments of the present invention, step b) involves adding crystallization seeds to the whey protein solution.The inventors have discovered that adding crystallization seeds can control when and where BLG crystallization occurs, so as to avoid sudden clogging of process equipment and unintended stoppage during production.For example, it is often desirable to concentrate the whey protein raw material while avoiding the onset of crystallization.

[0153] In principle, any seed material that initiates the crystallization of BLG may be used, however, it is preferred to use hydrated or dry BLG crystals for seeding in order to avoid adding further impurities to the whey protein solution.

[0154] The crystallization seeds may be in dry form or may form part of a suspension when added to the whey protein solution. Addition of a suspension containing the crystallization seeds (e.g., BLG crystals) is currently preferred as it appears to initiate crystallization sooner. Such suspensions containing crystallization seeds have a pH in the range of 5-6 and a conductivity of up to 10 mS / cm.

[0155] In some embodiments of the invention, at least a portion of the crystallization seeds are located on a solid phase in contact with the whey protein solution.

[0156] The crystallization seeds are preferably small in size compared to the desired size of the BLG crystals. The size of the crystallization seeds may be altered by removing the largest seeds by sieving or other size fractionation processes. Particle size reduction, for example by grinding, prior to size fractionation may also be used.

[0157] In some embodiments of the invention, at least 90% (wt / wt) of the crystallization seeds have a particle size (as measured by sieving analysis) in the range of 0.1 to 600 microns. For example, at least 90% (wt / wt) of the crystallization seeds may have a particle size in the range of 1 to 400 microns. Preferably, at least 90% (wt / wt) of the crystallization seeds may have a particle size in the range of 5 to 200 microns. More preferably, at least 90% (wt / wt) of the crystallization seeds may have a particle size in the range of 5 to 100 microns.

[0158] The particle size and dosage of the crystallization seeds may be adjusted to provide optimal crystallization of BLG.

[0159] In some preferred embodiments of the present invention, the crystallization seeds are added to the whey protein raw material before obtaining supersaturation with respect to BLG, but preferably such that when supersaturation is achieved, at least some crystallization seeds are still present. This can be achieved, for example, by adding crystallization seeds when the whey protein raw material is close to supersaturation (e.g., during cooling, concentration, and / or pH adjustment) and is likely to reach supersaturation before the crystallization seeds are completely dissolved.

[0160] In some preferred embodiments of the present invention, step b) preferably involves further increasing the degree of supersaturation of BLG to such an extent that crystallization of BLG begins immediately (i.e., within up to 20 minutes, preferably within up to 5 minutes), also referred to as the nucleation zone where crystals spontaneously form and initiate the crystallization process.

[0161] The degree of supersaturation can be, for example, as follows: - further increasing the protein concentration of the whey protein solution; - further cooling the whey protein solution; - bringing the whey protein solution closer to the optimal pH for BLG crystallization; - Further reducing the conductivity may be increased by one or more of:

[0162] In some preferred embodiments of the present invention, step b) involves waiting for BLG crystals to form, which may take several hours and is typically for whey protein solutions that are only slightly supersaturated with respect to BLG and that have not been seeded with added crystallization seeds.

[0163] In some preferred embodiments of the present invention, providing the whey protein solution (step a) and crystallizing BLG (step b) occur as two separate steps.

[0164] However, in another preferred embodiment of the invention, step b) involves further adjustment of the crystallization of the whey protein solution to increase or at least maintain the degree of supersaturation of BLG, which further adjustment increases the yield of BLG crystals.

[0165] Such further adjustments may include: - further increasing the protein concentration of the whey protein solution to be crystallized; - cooling the crystallizing whey protein solution to a lower temperature; - bringing the whey protein solution to be crystallized closer to the optimal pH for BLG crystallization; - Further reducing the conductivity of the crystallizing whey protein solution may involve one or more of:

[0166] In some preferred embodiments of the present invention, the crystallizing whey protein solution is maintained within the metastable region during step b) to avoid the spontaneous formation of new crystals.

[0167] The present inventors have determined the crystal lattice structure of isolated BLG crystals by X-ray crystallography and have not found similar crystals in the prior art.

[0168] In some preferred embodiments of the present invention, at least a portion of the BLG crystals obtained in step b) are orthorhombic in space group P2 1 2 1 2 1 has.

[0169] Preferably, at least a portion of the obtained BLG crystals is in the orthorhombic space group P2 1 2 1 2 1 and unit cell dimensions a=68.68(±5%) Å, b=68.68(±5%) Å and c=156.65(±5%) Å, and unit cell integer angles α=90°, β=90° and γ=90°.

[0170] In some preferred embodiments of the present invention, at least a portion of the obtained BLG crystals are orthorhombic in space group P2 1 2 1 2 1 and unit cell dimensions a=68.68(±2%) Å, b=68.68(±2%) Å and c=156.65(±2%) Å, and unit cell integer angles α=90°, β=90° and γ=90°.

[0171] Even more preferably, at least a portion of the obtained BLG crystals is in the orthorhombic space group P2 1 2 1 2 1 and unit cell dimensions a=68.68(±1%) Å, b=68.68(±1%) Å and c=156.65(±1%) Å, and unit cell integer angles α=90°, β=90° and γ=90°.

[0172] Most preferably, at least a portion of the resulting BLG crystals are orthorhombic space group P2. 1 2 1 2 1 and unit cell dimensions a=68.68 Å, b=68.68 Å and c=156.65 Å, and unit cell integer angles α=90°, β=90° and γ=90°.

[0173] In some particularly preferred embodiments of the invention, the method comprises a step c) of separating at least a portion of the BLG crystals from the remaining whey protein solution, which is particularly preferred when purification of BLG is desired.

[0174] Step c) may, for example, comprise separating the BLG crystals to a solids content of at least 30% (wt / wt). Preferably, step c) comprises separating the BLG crystals to a solids content of at least 40% (wt / wt). Even more preferably, step c) comprises separating the BLG crystals to a solids content of at least 50% (wt / wt).

[0175] The inventors have discovered that a high solids content is advantageous for the purification of BLG, since the aqueous portion adhering to the separated BLG crystals generally contains impurities that should be avoided. In addition, this high solids content reduces the energy consumption for converting the separated BLG crystals to a dry product (e.g., a powder) and increases the BLG yield obtained from a given capacity of drying equipment.

[0176] In some preferred embodiments of the invention, step c) comprises isolating the BLG crystals to a solids content of at least 60%. Preferably, step c) comprises isolating the BLG crystals to a solids content of at least 70%. Even more preferably, step c) comprises isolating the BLG crystals to a solids content of at least 80%.

[0177] In some preferred embodiments of the present invention, the separation in step c) is carried out by the following procedure: - Centrifugation, - Decanting, - Filtration, - Sedimentation, - A combination of the above accompanied by one or more of the following:

[0178] These unit operations are known to those of skill in the art and are readily performed. Separation by filtration can involve, for example, the use of vacuum filtration, dynamic cross-flow filtration (DCF), a filtration press, or a filter centrifuge.

[0179] Depending on the desired results, various pore sizes may be utilized for filtration. Preferably, the filter allows native whey proteins and small aggregates to pass, but retains BLG crystals. The filter preferably has a nominal pore size of at least 0.1 microns. The filter may, for example, have a nominal pore size of at least 0.5 microns. Even more preferably, the filter may have a nominal pore size of at least 2 microns.

[0180] Filters with larger pore sizes may also be used, and are indeed preferred when primarily large crystals are to be separated from a liquid containing BLG crystals. In some embodiments of the invention, the filter has a nominal pore size of at least 5 microns. Preferably, the filter has a nominal pore size of at least 20 microns. Even more preferably, the filter may have a pore size of at least 40 microns.

[0181] The filter may have a pore size in the range of, for example, 0.03 to 5000 microns, such as 0.1 to 5000 microns. Preferably, the filter may have pores in the range of 0.5 to 1000 microns. Even more preferably, the filter may have pores in the range of 5 to 800 microns, such as 10 to 500 microns or 50 to 500 microns.

[0182] In some embodiments of the present invention, the filter has pores in the range of 0.03 to 100 microns. Preferably, the filter has pores in the range of 0.1 to 50 microns. More preferably, the filter has pores in the range of 4 to 40 microns. Even more preferably, the filter has pores in the range of 5 to 30 microns, such as in the range of 10 to 20 microns.

[0183] The advantage of using filters with pores larger than 1 micron is that bacteria and other microorganisms are at least partially removed during separation and, optionally, during washing and / or recrystallization. Thus, the method allows for the production of highly pure BLG with very low bacterial load while avoiding heat damage to the protein.

[0184] Another advantage of using filters with pores larger than 1 micron is that removal of water and subsequent drying is easier and requires less energy.

[0185] The remaining whey protein solution separated from the BLG crystals can be recycled to the whey protein raw material during preparation of the whey protein solution.

[0186] In some preferred embodiments of the present invention, step c) utilizes a filter centrifuge. In other preferred embodiments of the present invention, step c) utilizes a decanter centrifuge. Initial results (see Example 13) have shown that using a filter centrifuge and / or a decanter centrifuge to separate the BLG crystals from the mother liquor results in a robust operation of the method compared to, for example, vacuum filtration.

[0187] In many cases, it is preferred to dry the formed filter cake with a drying gas to reduce the moisture content of the filter cake and allow it to be peeled from the filter. The use of a drying gas may form part of the separation step, or alternatively may form part of the final drying step if the filter cake is directly converted into a dry edible BLG composition.

[0188] In some preferred embodiments of the present invention, step c) utilises a DCF device.

[0189] Initial testing (see Example 12) has shown that using a DCF device with membrane pore sizes in the range of 0.03 to 5 microns, preferably 0.3 to 1.0 microns, provides efficient separation of BLG crystals, and the inventors have observed that the DCF device can be operated for a sustained period sufficient to separate the crystals from a larger batch of whey protein solution containing BLG crystals.

[0190] In some preferred embodiments of the invention, step c) is carried out using a DCF device equipped with a membrane capable of retaining BLG crystals, the DCF permeate may be recycled to form part of the whey protein solution or whey protein raw material, and the DCF retentate may be recovered or returned to the crystallization tank. Preferably, the DCF permeate is treated, for example, by ultrafiltration / diafiltration, which renders the DCF permeate supersaturated with respect to BLG, before mixing with the whey protein solution or whey protein raw material.

[0191] Advantageously, these embodiments do not require the temperature of the liquid stream to be increased above 15°C, and therefore are less susceptible to microbial contamination than process variations that require higher temperatures. Another industrial advantage of these embodiments is that the supersaturation level can be easily controlled and maintained at a level that does not cause undesirable spontaneous crystallization. Thus, the temperature of the liquid stream in these embodiments of the process is preferably at most 15°C, more preferably at most 12°C, even more preferably at most 10°C, and most preferably at most 5°C.

[0192] These embodiments are exemplified in Example 10 and shown in Figure 26. These embodiments may be carried out as a batch or continuous process.

[0193] In some preferred embodiments of the present invention, the method comprises a step d) of washing the BLG crystals (e.g., the isolated BLG crystals of c). This washing may consist of a single washing step or may consist of multiple washing steps.

[0194] The washing in step d) preferably involves contacting the BLG crystals with a washing solution without completely dissolving the BLG crystals, followed by separating the remaining BLG crystals from the washing solution.

[0195] The wash liquid is preferably selected to avoid complete dissolution of the BLG crystals and may, for example, comprise or consist essentially of cold demineralized water, cold tap water or cold permeate of reverse osmosis.

[0196] This washing solution may have a pH in the range of 5 to 6, preferably in the range of 5.0 to 6.0, even more preferably in the range of 5.1 to 6.0, for example in the range of 5.1 to 5.9.

[0197] The cleaning liquid may have a conductivity of at most 0.1 mS / cm, preferably at most 0.02 mS / cm, even more preferably at most 0.005 mS / cm.

[0198] A cleaning liquid with even lower conductivity may be used. For example, the cleaning liquid may have a conductivity of at most 1 microS / cm. Alternatively, the cleaning liquid may have a conductivity of at most 0.1 microS / cm, such as about 0.05 microS / cm.

[0199] To limit dissolution of the crystallized BLG, the washing step is preferably carried out at low temperature: the temperature of the washing liquid is preferably at most 30°C, more preferably at most 20°C, even more preferably at most 10°C.

[0200] The washing step may be carried out, for example, at up to 5° C., more preferably up to 2° C., for example at about 0° C. Temperatures below 0° C. may be used, so long as the washing solution does not freeze, for example due to the presence of one or more freezing point depressants.

[0201] In some embodiments of the invention, the washing solution comprises BLG, for example in an amount of at least 1% (w / w), preferably in an amount of at least 3% (w / w), such as in an amount of 4% (w / w).

[0202] The washing of step d) generally dissolves at most 80% (wt / wt) of the initial amount of BLG crystals, preferably at most 50% (wt / wt), even more preferably at most 20% (wt / wt) of the initial amount of BLG crystals. Preferably, the washing of step d) dissolves at most 15% (wt / wt), more preferably at most 10% (wt / wt), even more preferably at most 5% (wt / wt) of the initial amount of BLG crystals.

[0203] The weight ratio between the total amount of washing liquid and the initial amount of separated BLG crystals is often at least 1, preferably at least 2, 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 may be at least 10. Alternatively, the weight ratio between the total amount of washing liquid and the initial amount of separated BLG crystals may be at least 20, such as at least 50 or at least 100.

[0204] The term "total amount of washing liquid" relates to the total amount of washing liquid used during the entire process.

[0205] In some preferred embodiments of the invention, one or more washing sequences are performed in the same or a similar filter arrangement as the BLG crystal separation: the filter cake containing primarily BLG crystals is subjected to one or more sequences of washing liquid that is removed through the filter, and the remaining portion of the BLG crystals remains in the filter cake.

[0206] In a particularly preferred embodiment of the present invention, the separation of step c) is carried out using a filter that retains BLG crystals. The filter cake is then contacted with one or more volumes of washing liquid that move through the filter cake and the filter. It is often preferred that each volume 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 time the volume of the filter cake, even more preferably at most 0.5 times the volume of the filter cake, for example at most 0.2 times the volume of the filter cake. The volume of the filter cake includes both the solids and the fluids (liquid and gas) of the filter cake. The filter cake is preferably washed in this way at least twice, preferably at least 4 times, even more preferably at least 6 times.

[0207] The spent wash solution from step d) may for example be recycled to the whey protein raw material or the whey protein washed free of BLG may be re-isolated.

[0208] The method comprises: - dissolving the separated BLG crystals in a recrystallization solution; - adjusting the recrystallization solution to obtain a supersaturation with respect to BLG; - crystallizing BLG in the supersaturated adjusted recrystallization solution; and - Separating the BLG crystals from the remaining conditioned recrystallization solution. The method may further comprise step e) comprising a recrystallization step comprising:

[0209] Step e) may involve either a single recrystallization sequence or multiple recrystallization sequences.

[0210] In some embodiments of the invention, the BLG crystals of step c) or d) are recrystallized at least twice, for example, the BLG crystals may be recrystallized at least three times, such as at least four times.

[0211] The washing and recrystallization steps may be combined in any order and may be carried out multiple times if necessary. The separated BLG crystals of step c) may be, for example, purified by the following process sequence: - one or more washing steps (step d), followed by - one or more steps of recrystallization (step e) may be subjected to.

[0212] Alternatively, the isolated BLG crystals of step c) may be prepared according to the following process sequence: - one or more steps of recrystallization (step e), followed by - one or more washing steps (step d) may be subjected to.

[0213] It is also possible to combine multiple washing and recrystallization steps, for example in the following order: - one or more washing steps (step d), - one or more steps of recrystallization (step e), - one or more washing steps (step d), and - one or more steps of recrystallization (step e).

[0214] Or, for example, the following sequence: - one or more steps of recrystallization (step e), - one or more washing steps (step d), - one or more steps of recrystallization (step e), - one or more washing steps (step d) It is.

[0215] In some embodiments of the present invention, the method further involves subjecting the separated BLG to a further BLG enrichment step, for example based on chromatography or selective filtration. However, in other preferred embodiments of the present invention, the method does not include a further BLG enrichment step after step b). The term "further BLG enrichment step" refers to a process step that enriches BLG relative to the total amount of protein, and this step does not involve crystallization of BLG or 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 are not considered as "further BLG enrichment steps".

[0216] In some particularly preferred embodiments of the invention, the method involves a drying step f) in which the BLG-containing composition derived from steps b), c), d) or e) is converted into a dry composition.

[0217] In the context of the present invention, the term "dry" means that the composition or product in question contains a maximum of 6% (w / w) water, preferably even less.

[0218] In the context of the present invention, the term "BLG-containing composition" is used to describe the composition that is subjected to drying in step f).

[0219] In the context of the present invention, a "BLG-containing composition derived from step b), c), d) or e)" means a composition comprising at least a portion of the BLG derived from step b), c), d) or e). In some preferred embodiments of the present invention, the "BLG-containing composition derived from step b), c), d) or e)" is obtained directly from step b), c), d) or e). However, in other preferred embodiments of the present invention, the "BLG-containing composition derived from step b), c), d) or e)" is the result of further processing of the composition obtained directly from step b), c), d) or e).

[0220] It is often preferred that this BLG-containing composition comprises a substantial amount of the BLG present in the composition directly obtained from steps b), c), d) or e). In some preferred embodiments of the invention, the BLG-containing composition derived from steps 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 steps b), c), d) or e).

[0221] Preferably, the BLG-containing composition derived from step b), c), d) or e) comprises at least 85% (wt / wt) of the BLG obtained from step b), c), d) or e). More preferably, the BLG-containing composition derived from step b), c), d) or e) comprises at least 90% (wt / wt) of the BLG obtained from step b), c), d) or e). Even more preferably, the BLG-containing composition derived from step b), c), d) or e) comprises at least 95% (wt / wt) of the BLG obtained from step b), c), d) or e). Most preferably, the BLG-containing composition derived from step b), c), d) or e) comprises 100% (wt / wt) of the BLG obtained from step b), c), d) or e).

[0222] In some preferred embodiments of the invention, the drying step involves one or more of spray drying, freeze drying, spin flash drying, rotary drying and / or fluidized bed drying.

[0223] In some particularly preferred embodiments of the invention, the drying step involves a BLG-containing composition in which the BLG crystals are dissolved, and the resulting powder does not contain BLG crystals formed by step b) or recrystallization prior to this drying step. This embodiment is preferred when the edible BLG composition is to resemble that of, for example, a conventional dried whey protein powder.

[0224] The BLG crystal is, for example, - increasing the temperature; - increasing the conductivity, for example by the addition of one or more salts; - changing the pH, for example, outside the range of 5-6; - Reducing the concentration of BLG, for example by dilution, or - A combination of the above It can be dissolved by

[0225] Spray drying is the currently preferred method of drying BLG-containing compositions that are free of BLG crystals.

[0226] In another particularly preferred embodiment of the present invention, the drying step involves a BLG-containing composition that still contains BLG crystals, and the resulting powder contains BLG crystals. This embodiment is preferred when the edible BLG composition is to have a higher density than conventional dried whey protein powders.

[0227] In some particularly preferred embodiments of the present invention, the drying step involves a BLG-containing composition that still contains BLG crystals, and the resulting powder contains BLG crystals. This embodiment is preferred when the edible BLG composition is to be denser than conventional dried whey protein powders.

[0228] As demonstrated in Example 7, the inventors have discovered that a slurry of BLG crystals can be spray-dried and that the dried BLG crystals can retain at least a portion of their crystal structure when resuspended in cold demineralized water. It is particularly advantageous to avoid exposing the BLG-containing composition containing BLG crystals to a heat treatment regime that dissolves a significant amount of the BLG crystals prior to spraying. Therefore, if pre-heating of the BLG-containing composition containing BLG crystals prior to spraying is used, it is preferred that the heat load be carefully controlled.

[0229] In some embodiments of the invention, the BLG-containing composition comprising BLG crystals has a temperature of up to 70° C., preferably up to 60° C., more preferably up to 50° C. when it reaches the outlet of the spraying device (e.g., nozzle or sprayer). In some preferred embodiments of the invention, the BLG-containing composition comprising BLG crystals has a temperature of up to 40° C., preferably up to 30° C., more preferably up to 20° C., even more preferably up to 10° C., and most preferably up to 5° C. when it reaches the outlet of the spraying device.

[0230] The atomizing device for spray drying is the device (eg, nozzle or atomizer) that converts the solution or suspension to be dried into small droplets that enter the drying chamber of the spray dryer.

[0231] It is particularly preferred that the BLG-containing composition containing BLG crystals has a temperature in the range of 0 to 50°C, preferably in the range of 2 to 40°C, more preferably in the range of 4 to 35°C, and most preferably in the range of 5 to 10°C when it reaches the outlet of the spray device.

[0232] In some preferred embodiments of the invention, the BLG-containing composition has a 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%, of BLG when it reaches the outlet of the spray device. The BLG-containing composition may be, for example, a BLG isolate containing BLG in an amount of more than 90% (w / w) of total protein, or may contain substantial amounts of other proteins and thus contain BLG in an amount of up to 90% (w / w) of total protein.

[0233] In some preferred embodiments of the invention, the BLG-containing composition may have the protein composition of a conventional liquid WPC or WPI or a conventional liquid SPC or SPI as described herein, but the crystallinity of the BLG when it reaches the outlet of the spray device may be at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, and most preferably at least 90%, for example preferably 97-100%.

[0234] The inlet temperature of the gas in the spray dryer is preferably in the range of 140 to 220° C., more preferably in the range of 160 to 200° C., even more preferably in the range of 170 to 190° C., for example, preferably about 180° C. The outlet temperature of the gas from this spray dryer is preferably in the range of 50 to 95° C., more preferably in the range of 70 to 90° C., even more preferably in the range of 80 to 88° C., for example, preferably about 85° C. As a rule of thumb, it is said that the solid subjected to spray drying is heated to a temperature 10 to 15° C. lower than the outlet temperature of the gas.

[0235] In some preferred embodiments of the present invention, the spray dryer is preferably at a temperature in the range of 50 to 85°C, more preferably in the range of 60 to 80°C, even more preferably in the range of 65 to 75°C, for example preferably about 70°C.

[0236] The concept of spray drying a suspension of BLG crystals has not been disclosed in the prior art and is itself another aspect of the present invention.

[0237] Accordingly, one aspect of the invention is a method for producing a spray-dried edible powder composition comprising BLG, said composition comprising dried BLG crystals, - providing a liquid BLG-containing composition comprising BLG crystals and preferably having a crystallinity of at least 20% BLG, said liquid BLG-containing composition preferably comprising at least 10% (w / w) total solids and preferably comprising at least 5% (w / w) BLG; - spraying the liquid BLG-containing composition into a drying chamber of a running spray dryer to convert the liquid BLG-containing composition containing BLG crystals into a powder. The present invention relates to a method comprising the steps of:

[0238] In some preferred embodiments of the present invention, the BLG-containing composition to be dried is mixed with dried BLG isolate to raise the solids content to a level where the mixture can be dried by fluidized bed drying. This is also referred to as back-mixing and allows for very cost-effective drying of the BLG product. This embodiment is particularly preferred for BLG-containing compositions that contain BLG crystals.

[0239] An advantage of the method is that the BLG-containing composition to be dried can have a very high solids content before the drying step, and therefore less water has to be removed and less energy is consumed in the drying operation. In some preferred embodiments of the present invention, the BLG-containing composition resulting from step b), c), d) or e) has a solids content of at least 20% (w / w). Preferably, the BLG-containing composition resulting from step b), c), d) or e) has a solids content of at least 30% (w / w). More preferably, the BLG-containing composition resulting from step b), c), d) or e) has a solids content of at least 40% (w / w). Even more preferably, the BLG-containing composition resulting from step b), c), d) or e) has a solids content of at least 50% (w / w), such as at least 60% (w / w).

[0240] In another preferred embodiment of the present invention, the BLG-containing composition derived from step b), c), d) or e) has a solids content in the range of 20-80% (w / w). Preferably, the BLG-containing composition derived 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 derived 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 derived 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).

[0241] The inventors have discovered that the higher the crystallinity of the BLG-containing composition, the less water is bound to the BLG-containing composition and the higher the total solids content of the BLG-containing composition can be achieved prior to the drying step.

[0242] Thus, in some preferred embodiments of the present invention, the BLG-containing composition has a 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).

[0243] Even higher degrees of crystallinity are often preferred. Thus, in some preferred embodiments of the present invention, the BLG of the BLG-containing composition has a degree of crystallinity of at least 50% (w / w). Preferably, the BLG of the BLG-containing composition has a degree of crystallinity of at least 60% (w / w). More preferably, the BLG of the edible BLG composition has a degree of crystallinity of at least 70% (w / w). Even more preferably, the BLG of the BLG-containing composition has a degree of crystallinity of at least 80% (w / w). Most preferably, the BLG of the BLG-containing composition has a degree of crystallinity of at least 90% (w / w), preferably at least 95% (w / w), more preferably at least 97% (w / w), and even more preferably at least 99% (w / w).

[0244] The inventors have discovered that decreasing the water content tends to increase the crystallinity of BLG in a composition, such that compositions with a high water:BLG ratio (e.g., a suspension of 4% BLG crystals in water) tend to have lower crystallinity of BLG than compositions with a lower water:BLG ratio (e.g., filter cake or wet isolated crystals) under the same conditions.

[0245] The methods of the present invention may be carried out using moderate temperatures which do not destroy the nutritional value of either the BLG or the other whey proteins of the whey protein solution.

[0246] In some preferred embodiments of the invention, the BLG is not exposed to temperatures above 90° C. during the process. Preferably, the BLG is not exposed to temperatures above 80° C. during the process. Even more preferably, the BLG is not exposed to temperatures above 75° C. during the process. It should be noted that even though spray drying often employs temperatures above 150° C., the short exposure times and the concomitant evaporation of water mean that the spray dried protein does not experience temperatures above 50-70° C.

[0247] The inventors have found indications that prolonged heating during the drying step reduces the amount of BLG in crystalline form. In some preferred embodiments of the present invention, the heat exposure during the drying step is kept low enough to ensure that the degree of denaturation of BLG is at most 10%, preferably at most 4%, more preferably at most 1%, even more preferably at most 0.4%, even more preferably at most 0.1%. Most preferably, there is no detectable denaturation of BLG due to the drying step.

[0248] The degree of denaturation caused by the drying step was evaluated by comparing the BLG content (relative to the total solids) in the BLG composition to be dried (c in step f). ステップf前 ) and the BLG content (relative to total solids) in the redissolved and dried composition was determined according to the formula: Degree of denaturation = ((c ステップf前 -c ステップf後 ) / c ステップf前 ) * 100% It is calculated by using

[0249] Some preferred embodiments of the present invention provide a method for preparing an edible composition comprising beta-lactoglobulin (BLG) in a crystalline form, comprising the steps of: 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 to 6, said whey protein solution comprising: - 70-100% (w / w) protein on total solids; - 30 to 90% (w / w) BLG, preferably 30 to 70% (w / w) BLG relative to the total protein, - 4-50% (w / w) ALA, preferably 8-35% (w / w) ALA, based on total protein; - CMP from 0 to 25% (w / w) on protein, - At least 10% (w / w) protein based on the total weight of the whey protein solution and b) crystallizing BLG in the supersaturated whey protein solution, preferably by addition of crystallization seeds; f) drying the BLG-containing composition directly obtained from step b), said BLG-containing composition preferably having a crystallinity of at least 30% BLG. and does not include steps c), d) or e).

[0250] The whey protein solution is preferably a desalted whey protein solution, preferably having a ratio between conductivity and total amount of protein of maximum 0.3 and / or a UF permeate conductivity of maximum 7 mS / cm.

[0251] In this embodiment, the BLG crystals that are not separated from the whey protein solution are dried to provide a dense, edible BLG composition in powder form.

[0252] The invention further relates to an edible composition obtainable by this embodiment.

[0253] Another preferred embodiment of the present invention is a method for preparing an edible composition comprising beta-lactoglobulin (BLG) in a crystalline form, comprising the steps of: 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 to 6, said whey protein solution comprising: - 70-100% (w / w) protein on total solids; - 30-90% (w / w) BLG relative to the total protein, preferably 30-70% BLG, - 4-50% (w / w) ALA, preferably 8-35% ALA, based on total protein; - CMP from 0 to 25% (w / w) of total protein, - At least 10% (w / w) protein based on the total weight of the whey protein solution and b) crystallizing BLG in the supersaturated whey protein solution, preferably by addition of crystallization seeds; c) separating the BLG crystals from the remaining whey protein solution; d) optionally washing the separated BLG crystals obtained from step c); e) optionally recrystallizing the BLG crystals obtained from step c) or d); f) drying the BLG-containing composition resulting from step c), d) or e), preferably obtained directly from step c), d) or e), wherein the BLG-containing composition comprises BLG crystals and preferably has a crystallinity of at least 30% BLG. The present invention relates to a method comprising the steps of:

[0254] The whey protein solution is preferably a desalted whey protein solution, preferably having a ratio between conductivity and total protein of at most 0.3 and / or a UF permeate conductivity of at most 7 mS / cm.

[0255] This embodiment is particularly useful for producing edible, low mineral and low phosphorus BLG compositions in the form of a dense powder.

[0256] The invention further relates to an edible composition obtainable by this embodiment.

[0257] Yet another preferred embodiment of the present invention is a method for preparing an edible composition comprising beta-lactoglobulin in isolated form, comprising the steps of: 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 to 6, said whey protein solution comprising: - 70-100% (w / w) protein on total solids; - 30 to 90% (w / w) BLG, preferably 30 to 70% (w / w) BLG relative to the total protein, - 5-50% (w / w) ALA, preferably 8-35% (w / w) ALA, based on total protein; - CMP from 0 to 25% (w / w) of total protein, - At least 10% (w / w) protein based on the total weight of the whey protein solution and b) crystallizing BLG in the supersaturated whey protein solution, preferably by addition of crystallization seeds; c) separating the BLG crystals from the remaining whey protein solution; d) optionally washing the separated BLG crystals obtained from step c); e) optionally recrystallizing the BLG crystals obtained from step c) or d); f) drying the BLG-containing composition resulting from step c), d) or e), wherein the BLG-containing composition is free of BLG crystals. The present invention relates to a method comprising the steps of:

[0258] The whey protein solution is preferably a desalted whey protein solution, preferably having a ratio between conductivity and total amount of protein of at most 0.3 and / or a UF permeate conductivity of at most 7 mS / cm.

[0259] In this embodiment, the BLG crystals are dissolved prior to drying.

[0260] The invention further relates to an edible composition obtainable by this embodiment.

[0261] In some preferred embodiments, the method is carried out as a batch process. Alternatively, and sometimes preferably, the method may be carried out as a semi-batch process. In other preferred embodiments, the method is carried out as a continuous process.

[0262] The advantage of the method is that it is much faster than comparable methods for BLG crystallization in the prior art. The duration from the initial preparation of the whey protein raw material to the completion of the separation in step c can be up to 10 hours, preferably up to 4 hours, more preferably up to 2 hours, even more preferably up to 1 hour.

[0263] A further aspect of the present invention relates to isolated BLG crystals obtainable from the methods described herein.

[0264] In the context of the present invention, the term "isolated BLG crystals" refers to BLG crystals that have been separated from the solution in which they were formed, but which may still contain internal water (i.e., hydrated BLG molecules of the crystals).

[0265] The isolated BLG crystals preferably have an orthorhombic space group P2 1 2 1 2 1 has.

[0266] Preferably, the isolated BLG crystals are in the orthorhombic space group P2 1 2 1 2 1 and unit cell dimensions a=68.68(±5%) Å, b=68.68(±5%) Å and c=156.65(±5%) Å, and unit cell integer angles α=90°(±2%), β=90°(±2%) and γ=90°(±2%).

[0267] In some preferred embodiments of the present invention, the isolated BLG crystals are orthorhombic in space group P2 1 2 1 2 1and unit cell dimensions a=68.68(±2%) Å, b=68.68(±2%) Å and c=156.65(±2%) Å, and unit cell integer angles α=90°(±1%), β=90°(±1%) and γ=90°(±1%).

[0268] Even more preferably, the isolated BLG crystals are in the orthorhombic space group P2 1 2 1 2 1 and unit cell dimensions a=68.68(±1%) Å, b=68.68(±1%) Å, and c=156.65(±1%) Å, and unit cell integer angles α=90°(±0.5%), β=90°(±0.5%), and γ=90°(±0.5%).

[0269] Most preferably, the isolated BLG crystals are in the orthorhombic space group P2 1 2 1 2 1 and unit cell dimensions a=68.68 Å, b=68.68 Å, and c=156.65 Å, and unit cell integer angles α=90°, β=90°, and γ=90°.

[0270] The isolated BLG crystals may contain, for example, at least 20% (weight / weight) BLG and up to 80% (weight / weight) water. Preferably, the isolated BLG crystals may contain at least 40% (weight / weight) BLG and 0-60% (weight / weight) water. Even more preferably, the isolated BLG crystals contain 40-60% (weight / weight) BLG and about 40-60% (weight / weight) water.

[0271] The inventors have discovered that the BLG crystals of the present invention are surprisingly capable of recovering their original crystal structure after drying and rehydration, which is particularly advantageous in applications that benefit from the crystal structure of BLG.

[0272] A further aspect of the present invention relates to an edible composition comprising beta-lactoglobulin (eg an edible composition obtainable by a process as defined herein).

[0273] Another aspect of the present invention relates to an edible BLG composition comprising at least 90% (w / w) BLG based on total solids. Such an edible BLG composition may be obtainable by the method defined herein.

[0274] A further aspect of the present invention relates to an edible BLG composition comprising dried BLG crystals (at least 20% (w / w) BLG on total solids) and preferably having a crystallinity of at least 20% BLG. Such an edible BLG composition comprising dried BLG crystals may be obtainable by the method defined herein.

[0275] In some preferred embodiments of the present invention, the BLG of the edible BLG composition has a lactosylation degree of at most 1. Preferably, the BLG of the edible BLG composition has a lactosylation degree of at most 0.6. More preferably, the BLG of the edible BLG composition has a lactosylation degree of at most 0.4. Even more preferably, the BLG of the edible BLG composition has a lactosylation degree of at most 0.2. Most preferably, the BLG of the edible BLG composition has a lactosylation degree of at most 0.1, such as preferably at most 0.01.

[0276] In some preferred embodiments of the present invention, the BLG of the edible BLG composition comprises at least 90% (wt / w) non-lactosylated BLG, preferably at least 95% (wt / w) non-lactosylated BLG, and even more preferably at least 98% (wt / w) non-lactosylated BLG.

[0277] The percentage of non-lactosylated BLG is determined according to Example 9.1.

[0278] In some preferred embodiments of the present invention, the BLG of the edible BLG composition has a crystallinity of at least 10% (wt / wt). Preferably, the BLG of the edible BLG composition has a crystallinity of at least 20% (wt / wt). More preferably, the BLG of the edible BLG composition has a crystallinity of at least 30% (wt / wt). Even more preferably, the BLG of the edible BLG composition has a crystallinity of at least 40% (wt / wt).

[0279] Even higher degrees of crystallinity are often preferred. Thus, in some preferred embodiments of the present invention, the BLG of the edible BLG-containing composition has a degree of crystallinity of at least 50% (wt / wt). Preferably, the BLG of the edible BLG-containing composition has a degree of crystallinity of at least 60% (wt / wt). More preferably, the BLG of the edible BLG-containing composition has a degree of crystallinity of at least 70% (wt / wt). Even more preferably, the BLG of the edible BLG-containing composition has a degree of crystallinity of at least 80% (wt / wt). Most preferably, the BLG of the edible BLG-containing composition has a degree of crystallinity of at least 90% (wt / wt), preferably at least 95% (wt / wt).

[0280] The crystallinity of BLG in liquids with a pH in the range of 5-6 is measured according to Example 9.7. The crystallinity of BLG in powdered materials is measured according to Example 9.8. If the edible composition is a dry product but not in the form of a powder, it must be converted to a powder, for example by grinding or milling, before being subjected to the method of Example 9.8.

[0281] In some preferred embodiments of the present invention, the edible BLG composition is WPC, WPI, SPC or SPI, where at least a portion of the BLG is in crystalline form. The edible BLG composition may, for example, contain up to 90% (wt / wt) BLG based on the total amount of protein, and have a crystallinity of at least 10% BLG. For example, the edible BLG composition may contain up to 80% (wt / wt) BLG based on the total amount of protein, and have a crystallinity of at least 10% BLG. The edible BLG composition may, for example, contain 30-70% (wt / wt) BLG based on the total amount of protein, and have a crystallinity of at least 10% BLG.

[0282] In another preferred embodiment of the present invention, the edible BLG composition comprises up to 90% (wt / wt) BLG based on the total amount of protein and has a crystallinity of at least 30% BLG. Preferably, the edible BLG composition may comprise up to 80% (wt / wt) BLG based on the total amount of protein and has a crystallinity of at least 30% BLG. Even more preferably, the edible BLG composition may comprise 30-70% (wt / wt) BLG based on the total amount of protein and has a crystallinity of at least 30% BLG.

[0283] The inventors have discovered that the present invention makes it possible to prepare an edible whey protein product having a very low content of phosphorus and other minerals, which is advantageous for patients suffering from renal disease or who otherwise have reduced renal function.

[0284] The edible BLG composition is preferably a low phosphorus composition.

[0285] In the context of the present invention, the term "low phosphorus" relates to a composition (e.g. liquid, powder or another food product) with a total phosphorus content of at most 100 mg phosphorus per 100 g protein. Preferably, the low phosphorus composition has a total content of at most 80 mg phosphorus per 100 g protein. More preferably, the low phosphorus composition may have a total content of at most 50 mg phosphorus per 100 g protein. Even more preferably, the low phosphorus composition may have a total phosphorus content of at most 20 mg phosphorus per 100 g protein. Even more preferably, the low phosphorus composition may have a total phosphorus content of at most 5 mg phosphorus per 100 g protein. The low phosphorus composition according to the present invention may be used as a food ingredient for the manufacture of a food product for a patient group with reduced renal function.

[0286] Thus, in some particularly preferred embodiments of the present invention, the edible BLG composition comprises up to 80 mg phosphorus per 100 g protein. Preferably, the edible BLG composition comprises up to 30 mg phosphorus per 100 g protein. More preferably, the edible BLG composition comprises up to 20 mg phosphorus per 100 g protein. Even more preferably, the edible BLG composition comprises up to 10 mg phosphorus per 100 g protein. Most preferably, the edible BLG composition comprises up to 5 mg phosphorus per 100 g protein.

[0287] The phosphorus content relates to the total amount of elemental phosphorus in the composition of interest and is determined according to Example 9.5.

[0288] In another preferred embodiment of the present invention, the edible BLG composition is a low-mineral composition.

[0289] In the context of the present invention, "low in minerals" refers to: - Ash content of maximum 1.2% (w / w) based on total solids, - Total calcium and magnesium content of maximum 0.3% (w / w) based on total solids; - Total sodium and potassium content of maximum 0.10% (w / w) based on total solids; - Total phosphorus content up to 100mg phosphorus per 100g protein The present invention relates to a composition (e.g., a liquid, powder or another food product) having at least one, preferably two, and even more preferably all of the following:

[0290] Preferably, the low mineral composition comprises: - Ash content of maximum 0.7% (w / w) based on total solids, - Total calcium and magnesium content of maximum 0.2% (w / w) based on total solids; - Total sodium and potassium content of maximum 0.08% (w / w) based on total solids; - Total phosphorus content up to 80mg phosphorus per 100g protein It has at least one, preferably two or more, and even more preferably all of the above.

[0291] Even more preferably, the low mineral composition comprises: - Ash content of maximum 0.5% (w / w) based on total solids, - Total calcium and magnesium content of maximum 0.15% (w / w) based on total solids; - Total sodium and potassium content of maximum 0.06% (w / w) based on total solids; - Total phosphorus content up to 50mg phosphorus per 100g protein It has at least one, preferably two or more, and even more preferably all of the above.

[0292] Low mineral composition: - Ash content of maximum 0.5% (w / w) based on total solids, - Total calcium and magnesium content of maximum 0.15% (w / w) based on total solids; - Total sodium and potassium content of maximum 0.06% (w / w) based on total solids; - Total phosphorus content up to 50mg phosphorus per 100g protein It is particularly preferred that

[0293] In some preferred embodiments of the present invention, the edible BLG composition comprises a total amount of protein of at least 25% (wt / wt) based on the total solids of the edible BLG composition. Preferably, the edible BLG composition comprises a total amount of protein of at least 50% (wt / wt) based on the total solids of the edible BLG composition. More preferably, the edible BLG composition comprises a total amount of protein of at least 75% (wt / wt) based on the total solids of the edible BLG composition. Even more preferably, the edible BLG composition comprises a total amount of protein of at least 90% (wt / wt) based on the total solids of the edible BLG composition.

[0294] In some preferred embodiments of the present invention, the total amount of protein in the edible BLG composition is in the range of 25-100% (wt / wt) based on total solids. Preferably, the total amount of protein in the edible BLG composition is in the range of 50-100% (wt / wt). More preferably, the total amount of protein in the edible BLG composition is in the range of 75-100% (wt / wt) based on total solids. Even more preferably, the total amount of protein in the edible BLG composition is in the range of 90-100% (wt / wt) based on total solids.

[0295] In some preferred embodiments of the present invention, the edible BLG composition comprises at least 75% (wt / wt) BLG based on the total amount of protein. Preferably, the edible BLG composition may comprise at least 90% (wt / wt) BLG based on the total amount of protein. More preferably, the edible BLG composition may comprise at least 95% (wt / wt) BLG based on the total amount of protein. Even more preferably, the edible BLG composition may comprise at least 97% (wt / wt) BLG based on the total amount of protein. Most preferably, the edible BLG composition comprises about 100% (wt / wt) BLG based on the total amount of protein.

[0296] In some preferred embodiments of the present invention, the edible BLG composition comprises up to 10% (wt / wt) carbohydrates, preferably up to 5% (wt / wt) carbohydrates, more preferably up to 1% (wt / wt) carbohydrates, and even more preferably up to 0.1% (wt / wt) carbohydrates.

[0297] The edible BLG composition may also include lipids (eg, in the form of triglycerides and / or other lipid types (eg, phospholipids)).

[0298] In some embodiments of the present invention, the edible BLG composition comprises a total amount of lipids of up to 1% (w / w) based on total solids. Preferably, the edible BLG composition comprises a total amount of lipids of up to 0.5% (w / w) based on total solids. More preferably, the edible BLG composition comprises a total amount of lipids of up to 0.1% (w / w) based on total solids. Even more preferably, the edible BLG composition comprises a total amount of lipids of up to 0.05% (w / w) based on total solids. Most preferably, the edible BLG composition comprises a total amount of lipids of up to 0.01% (w / w) based on total solids.

[0299] In some preferred embodiments of the invention, the edible BLG composition is a dry composition, such as a powder. It is particularly preferred that the edible BLG composition is a spray-dried powder.

[0300] The inventors have observed that edible BLG compositions in powder form, in which at least a portion of the BLG is in crystalline form when dried, have a higher density than comparable BLG compositions that do not contain BLG crystals (see Example 7). Quite surprisingly, this high density effect is also observed in the case of edible BLG compositions in powder form obtained from a spray-dried BLG crystal slurry.

[0301] Thus, in some preferred embodiments of the present invention, the edible BLG composition in powder form has a bulk density of at least 0.40 g / mL. Preferably, the edible BLG composition in powder form has a bulk density of at least 0.45 g / mL. More preferably, the edible BLG composition in powder form has a bulk density of at least 0.50 g / mL. Even more preferably, the edible BLG composition in powder form has a bulk density of at least 0.6 g / mL. The edible BLG composition in powder form may, for example, have a bulk density of at least 0.7 g / mL.

[0302] The bulk density advantage applies to both powders of edible BLG compositions in which BLG is nearly the only protein present, and powders of edible BLG compositions in which the concentration of BLG is not enriched relative to other proteins present in the whey protein solution.Thus, the present invention provides high density powders of both isolated BLG and crude whey protein that contain significant amounts of ALA and other whey proteins in addition to BLG.

[0303] In some preferred embodiments of the present invention, the edible BLG composition in powder form has a bulk density of at least 0.45 g / mL and comprises at least 70% (w / w) protein relative to the total weight of the composition. More preferably, the edible BLG composition in powder form has a bulk density of at least 0.50 g / mL and comprises at least 70% (w / w) protein relative to the total weight of the composition. Even more preferably, the edible BLG composition in powder form has a bulk density of at least 0.6 g / mL and comprises at least 70% (w / w) protein relative to the total weight of the composition. The edible BLG composition in powder form may, for example, have a bulk density of at least 0.7 g / mL and comprises at least 70% (w / w) protein relative to the total weight of the composition.

[0304] In another preferred embodiment of the present invention, the edible BLG composition in powder form has a bulk density of at least 0.45 g / mL and comprises at least 80% (w / w) protein relative to the total weight of the composition. More preferably, the edible BLG composition in powder form has a bulk density of at least 0.50 g / mL and comprises at least 80% (w / w) protein relative to the total weight of the composition. Even more preferably, the edible BLG composition in powder form has a bulk density of at least 0.6 g / mL and comprises at least 80% (w / w) protein relative to the total weight of the composition. The edible BLG composition in powder form may, for example, have a bulk density of at least 0.7 g / mL and comprises at least 80% (w / w) protein relative to the total weight of the composition.

[0305] 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 contain at least 80% (wt / wt) of protein based on the total weight of the composition. Preferably, the edible BLG composition in powder form may have a bulk density in the range of 0.45-1.0 g / mL and contain at least 80% (wt / wt) of protein based on the total weight of the composition. More preferably, the edible BLG composition in powder form may have a bulk density in the range of 0.50-0.9 g / mL and contain at least 80% (wt / wt) of protein based on the total weight of the composition. It is even more preferred that the edible BLG composition in powder form has a bulk density in the range of 0.6-0.9 g / mL and contain at least 80% (wt / wt) of protein based on the total weight of the composition. The powdered edible BLG composition may, for example, have a bulk density in the range of 0.6-0.8 g / mL and contain at least 80% (w / w) protein based on the total weight of the composition.

[0306] The inventors have discovered that the high density powder of the present invention advantageously allows for more cost-efficient packaging and logistics of the powder since less packaging material is required per kg of powder and more powder (mass) can be transported in a given container or truck.

[0307] The present edible BLG composition in powder form may have a bulk density in the range of, for example, 0.40 to 1.5 g / mL. Preferably, the present edible BLG composition in powder form has a bulk density in the range of 0.45 to 1.0 g / mL. More preferably, the present edible BLG composition in powder form may have a bulk density in the range of 0.50 to 0.9 g / mL. It is even more preferable that the present edible BLG composition in powder form has a bulk density in the range of 0.6 to 0.9 g / mL. The present edible BLG composition in powder form may have a bulk density in the range of, for example, 0.6 to 0.8 g / mL.

[0308] In another preferred embodiment of the present invention, the edible BLG composition in powder form has a bulk density in the range of 0.50 to 1.5 g / mL. Preferably, the edible BLG composition in powder form has a bulk density in the range of 0.55 to 1.0 g / mL. More preferably, the edible BLG composition in powder form may have a bulk density in the range of 0.60 to 1.0 g / mL. It is even more preferable that the edible BLG composition in powder form has a bulk density in the range of 0.65 to 1.0 g / mL. Preferably, the edible BLG composition in powder form may have a bulk density in the range of 0.70 to 1.0 g / mL.

[0309] 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 contain at least 70% (wt / wt) protein based on the total weight of the composition. Preferably, the edible BLG composition in powder form may have a bulk density in the range of 0.45-1.0 g / mL and contain at least 70% (wt / wt) protein based on the total weight of the composition. More preferably, the edible BLG composition in powder form may have a bulk density in the range of 0.50-0.9 g / mL and contain at least 70% (wt / wt) protein based on the total weight of the composition. It is even more preferred that the edible BLG composition in powder form has a bulk density in the range of 0.6-0.9 g / mL and contain at least 70% (wt / wt) protein based on the total weight of the composition. The powdered edible BLG composition may, for example, have a bulk density in the range of 0.6-0.8 g / mL and contain at least 70% (w / w) protein based on the total weight of the composition.

[0310] 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 contain at least 80% (wt / wt) of protein based on the total weight of the composition. Preferably, the edible BLG composition in powder form may have a bulk density in the range of 0.45-1.0 g / mL and contain at least 80% (wt / wt) of protein based on the total weight of the composition. More preferably, the edible BLG composition in powder form may have a bulk density in the range of 0.50-0.9 g / mL and contain at least 80% (wt / wt) of protein based on the total weight of the composition. It is even more preferred that the edible BLG composition in powder form has a bulk density in the range of 0.6-0.9 g / mL and contain at least 80% (wt / wt) of protein based on the total weight of the composition. The powdered edible BLG composition may, for example, have a bulk density in the range of 0.6-0.8 g / mL and contain at least 80% (w / w) protein based on the total weight of the composition.

[0311] In another preferred embodiment of the present invention, the edible BLG composition in powder form has a bulk density in the range of 0.50-1.5 g / mL and contains at least 70% (wt / wt) protein based on the total weight of the composition. Preferably, the edible BLG composition in powder form has a bulk density in the range of 0.55-1.0 g / mL and contains at least 70% (wt / wt) protein based on the total weight of the composition. More preferably, the edible BLG composition in powder form may have a bulk density in the range of 0.60-1.0 g / mL and contains at least 70% (wt / wt) protein based on the total weight of the composition. Even more preferably, the edible BLG composition in powder form has a bulk density in the range of 0.65-1.0 g / mL and contains at least 70% (wt / wt) protein based on the total weight of the composition. The powdered edible BLG composition may preferably have a bulk density in the range of 0.70-1.0 g / mL and contain at least 70% (w / w) protein based on the total weight of the composition.

[0312] In another preferred embodiment of the present invention, the edible BLG composition in powder form has a bulk density in the range of 0.50-1.5 g / mL and contains at least 80% (wt / wt) protein based on the total weight of the composition. Preferably, the edible BLG composition in powder form has a bulk density in the range of 0.55-1.0 g / mL and contains at least 80% (wt / wt) protein based on the total weight of the composition. More preferably, the edible BLG composition in powder form may have a bulk density in the range of 0.60-1.0 g / mL and contains at least 80% (wt / wt) protein based on the total weight of the composition. Even more preferably, the edible BLG composition in powder form has a bulk density in the range of 0.65-1.0 g / mL and contains at least 80% (wt / wt) protein based on the total weight of the composition. The powdered edible BLG composition may preferably have a bulk density in the range of 0.70-1.0 g / mL and contain at least 80% (w / w) protein based on the total weight of the composition.

[0313] The bulk density of the powder is measured according to Example 9.3.

[0314] The inventors have found indications that the BLG compositions of the present invention have better long-term stability than similar BLG compositions, particularly when at least a portion of the BLG is present in the form of BLG crystals, which appears to provide better storage stability of the BLG molecule.

[0315] In some preferred embodiments of the invention, the dried BLG composition has a furosine value of at most 80 mg / 100 g protein after 60 days at 30° C., preferably at most 60 mg / 100 g protein, more preferably at most 40 mg / 100 g protein, even more preferably at most 20 mg / 100 g protein. Most preferably, the dried BLG composition has a furosine value of at most 10 mg / 100 g protein after 60 days at 30° C.

[0316] In some preferred embodiments of the present invention, the dry BLG composition has a furosine value of at most 80 mg / 100 g protein, preferably at most 60 mg / 100 g protein, more preferably at most 40 mg / 100 g protein, even more preferably at most 20 mg / 100 g protein. Most preferably, the dry BLG composition has a furosine value of at most 10 mg / 100 g protein. Preferably, the BLG composition has a furosine value of 0 mg / 100 g protein.

[0317] In some preferred embodiments of the present invention, the BLG of the dry BLG composition has a lactosylation degree of at most 1, preferably at most 0.6, more preferably at most 0.2, even more preferably at most 0.1, and most preferably at most 0.01 after 60 days at 30°C.

[0318] In some preferred embodiments of the present invention, the edible BLG composition is a liquid composition. A liquid edible BLG composition preferably comprises at least 20% (w / w) water, more preferably at least 30% (w / w) water, and even more preferably at least 40% (w / w).

[0319] The liquid edible BLG composition may, for example, contain water in the range of 20-90% (wt / wt), more preferably in the range of 30-80% (wt / wt), and even more preferably at least 40% (wt / wt).

[0320] The inventors have discovered that the edible BLG composition of the present invention surprisingly exhibits a low degree of protein denaturation, even when spray drying is used to prepare the edible BLG powder composition (see Example 11).

[0321] Thus, in some preferred embodiments of the present invention, the edible BLG composition has a degree of protein denaturation of up to 2%. Preferably, the edible BLG composition has a degree of protein denaturation of up to 1.5%. More preferably, the edible BLG composition has a degree of protein denaturation of up to 1.0%. Even more preferably, the edible BLG composition has a degree of protein denaturation of up to 0.8%. Even more preferably, the edible BLG composition has a degree of protein denaturation of up to 0.5%.

[0322] In some preferred embodiments of the present invention, the edible BLG composition is a dry powder, preferably a spray-dried powder, and has a degree of protein denaturation of up to 2%, preferably up to 1.5%. More preferably, for example, the dry edible BLG composition in the form of a spray-dried powder has a degree of protein denaturation of up to 1.0%. Even more preferably, for example, the dry edible BLG composition in the form of a spray-dried powder has a degree of protein denaturation of up to 0.8%. Even more preferably, for example, the dry edible BLG composition in the form of a spray-dried powder has a degree of protein denaturation of up to 0.5%.

[0323] In some preferred embodiments of the present invention, the edible BLG composition comprises: - Maximum 6% (w / w) water, - At least 80% total protein based on total solids; - At least 95% BLG based on total protein The edible BLG composition comprises: - It is a dry powder, and - a bulk density of at least 0.50 g / mL, preferably at least 0.60 g / m

[0324] In another preferred embodiment of the present invention, the edible BLG composition comprises: - Maximum 6% (w / w) water, - At least 80% total protein based on total solids; - At least 95% BLG based on total protein The edible BLG composition comprises: - It is a dry powder, - a bulk density of at least 0.50 g / mL, preferably at least 0.60 g / mL; and - The crystallinity of the BLG is at least 20%, preferably at least 40%.

[0325] In a further preferred embodiment of the invention, the edible BLG composition comprises: - Maximum 6% (w / w) water, - At least 80% total protein based on total solids; - at least 95% BLG based on total protein, The edible BLG composition comprises: - It is a dry powder, - a bulk density of at least 0.50 g / mL, preferably 0.60 g / mL; and - the degree of protein denaturation is at most 2%, preferably at most 1.0%.

[0326] In a further preferred embodiment of the invention, the edible BLG composition comprises: - Maximum 6% (w / w) water, - At least 80% total protein based on total solids; - at least 95% BLG based on total protein, - Up to 80mg phosphorus per 100g protein The edible BLG composition comprises: - It is a dry powder.

[0327] In a further preferred embodiment of the invention, the edible BLG composition comprises: - Maximum 6% (w / w) water, - at least 90% total protein based on total solids; - at least 97% BLG based on total protein; - Up to 50mg phosphorus per 100g protein The edible BLG composition comprises: - It is a dry powder.

[0328] In another preferred embodiment of the present invention, the edible BLG composition comprises: - Maximum 6% (w / w) water, - at least 80% total protein based on total solids, preferably at least 90% total protein based on total solids; - 30-70% BLG based on total protein, - 8-25% (w / w) ALA based on total protein The edible BLG composition comprises: - It is a dry powder, and - The crystallinity of the BLG is at least 20%, preferably at least 40%.

[0329] In some preferred embodiments of the present invention, the edible BLG composition comprises: - 20 to 80% (w / w) water, preferably 20 to 60% (w / w) water, - at least 80% total protein, preferably at least 90% total protein, based on total solids; - at least 95% BLG based on total protein, - Up to 80mg phosphorus per 100g protein The edible BLG composition comprises: - the crystallinity of the BLG is at least 20%, preferably at least 40; and Optionally, the degree of protein denaturation is at most 2%, preferably at most 1.0%.

[0330] The edible compositions of these embodiments are particularly useful for preparing edible BLG compositions in dry form, and are particularly suitable for spray drying and preparation of high density whey protein powders having the usual concentration profile of whey protein species in high density whey protein powders, but containing at least a portion of the BLG in the form of dried BLG crystals.

[0331] In another preferred embodiment of the present invention, the edible BLG composition comprises: - 20 to 80% (w / w) water, preferably 20 to 60% (w / w) water, - at least 80% total protein based on total solids, preferably at least 90% total protein based on total solids; - 30-70% BLG based on total protein, - 8-25% (w / w) ALA based on total protein The edible BLG composition comprises: - The crystallinity of the BLG is at least 20%, preferably at least 40%.

[0332] The edible composition according to this embodiment is particularly useful for preparing an edible BLG composition in a dry form, and is particularly suitable for spray drying and preparation of high density whey protein having the normal concentration profile of whey protein species in the high density whey protein, but containing at least a portion of the BLG in the form of dried BLG crystals.

[0333] Furthermore, an embodiment of the present invention relates to the use of an edible BLG composition as defined herein as a food ingredient.

[0334] For example, it may be desirable to use the low phosphorus edible BLG compositions defined herein as a food ingredient in the production of low phosphorus food products.

[0335] A further aspect of the present invention relates to a food product comprising an edible BLG composition as defined herein and at least an additional ingredient, such as a source of fat and / or carbohydrate.

[0336] In some preferred embodiments of the invention, the food product is a dry food (e.g., a bar) comprising carbohydrate and protein, said dry food comprising at least 1% (w / w) BLG, preferably at least 5%, i) the crystallinity of the BLG is at least 20%, preferably at least 40%, and / or ii) At least 90% (wt / wt) of the total protein is composed of BLG.

[0337] In some particularly preferred embodiments of the invention, the food product is a low phosphorus food comprising in the low phosphorus food a maximum of 100 mg phosphorus per 100 g protein, preferably a maximum of 80 mg phosphorus per 100 g protein, more preferably a maximum of 40 mg phosphorus per 100 g protein, and even more preferably a maximum of 20 mg phosphorus per 100 g protein.

[0338] BLG has an advantageous amino acid profile and preferably accounts for a significant portion of the protein of the food. This is particularly interesting when the food is a low-mineral or low-phosphorus food. In some preferred embodiments of the present invention, the edible BLG composition accounts for at least 25% (w / w), or at least 50% (w / w), more preferably at least 80% (w / w), even more preferably at least 90% (w / w) of the total protein of the food. Moreover, it may be most preferred that the edible BLG composition accounts for the total protein of the food.

[0339] In some preferred embodiments of the present invention, the low phosphorus edible BLG composition accounts for at least 25% (w / w), or at least 50% (w / w), more preferably at least 80% (w / w), and even more preferably at least 90% (w / w) of the total protein of the low phosphorus food product. Moreover, it may be most preferred that the low phosphorus edible BLG composition accounts for the total protein of the low phosphorus food product.

[0340] Non-limiting examples of such food products are, for example, dairy products, candy, beverages, protein bars, enteral nutritional compositions, bakery products.

[0341] In some preferred embodiments of the present invention, the food product is a beverage. The beverage preferably comprises: - an edible BLG composition as defined herein for a total amount of BLG of at least 1% (w / w), preferably at least 5% (w / w), more preferably at least 8% (w / w), even more preferably 12% (w / w), - sweeteners, e.g. sugar sweeteners and / or sugar-free sweeteners, at least one edible acid, such as citric acid or another suitable edible acid, - optional flavourings, and - Up to 80mg phosphorus per 100g protein and has a pH in the range of 2.5 to 4.0.

[0342] The present inventors have recognized that it is not easy to prepare an acidic, high protein, low mineral beverage or liquid from a dry edible BLG composition containing BLG crystals. Dry edible BLG compositions containing BLG crystals generally have a pH of 5-6 when suspended in water, and the addition of an acid or salt to change the pH or increase the conductivity also increases the mineral load of the resulting liquid / beverage.

[0343] However, the inventors have discovered that when carboxylic acids, lactones, carboxylic acid anhydrides or combinations thereof are used to lower the pH, unnecessary minerals are not added and better control of the mineral composition of the beverage / liquid is obtained.

[0344] Thus, one aspect of the invention is a method for producing an acidified, low-mineral liquid using an edible BLG composition comprising BLG crystals as an ingredient, the method comprising: - providing one or more acidifying agents selected from the group consisting of carboxylic acids, lactones, carboxylic anhydrides or combinations thereof; - contacting an edible BLG composition comprising BLG crystals with one or more acidifying agents and optional additional ingredients (e.g., water, a fat source and / or a carbohydrate source), the one or more acidifying agents being used in an amount sufficient to adjust the pH to between 2 and 4.5 (preferably between 2.5 and 4.0) and allowing the BLG crystals to dissolve; and forming a liquid.

[0345] This liquid may be used, for example, as a beverage or may be used as an ingredient to prepare another food product.

[0346] If the edible BLG composition used in this method is provided in a dry form, for example as a powder, it is often preferable to rehydrate the edible BLG composition with water before adding the acidifying agent.

[0347] The edible BLG composition used in this method is preferably present in the liquid in an amount sufficient to provide 1-30% (wt / wt) protein, preferably 2-25% (wt / wt) protein, more preferably 4-20% (wt / wt) protein, and even more preferably 5-16% (wt / wt) protein.

[0348] The edible BLG composition used in this method preferably has a crystallinity of at least 30%, preferably at least 50%, even more preferably at least 70% BLG.

[0349] Examples of suitable acidifying agents are: carboxylic acids, such as acetic acid, maleic acid, tartaric acid, lactic acid, citric acid, gluconic acid or mixtures thereof; lactones, such as D-glucono-delta-lactone, - Carboxylic acid anhydrides It is.

[0350] In some preferred embodiments, the edible BLG composition containing BLG crystals used in this method is preferably a low phosphorus composition, and any other ingredients used in the method are preferably selected such that the final liquid is also a low phosphorus composition.

[0351] In another preferred embodiment, the edible BLG composition containing BLG crystals used in this method is preferably a low-mineral composition, and any other ingredients used in the method are preferably selected such that the final liquid is also a low-mineral composition.

[0352] The process is preferably carried out at a temperature in the range of 1 to 65°C, preferably in the range of 2 to 50°C, more preferably in the range of 3 to 20°C, even more preferably in the range of 4 to 15°C.

[0353] The present invention has been described above with reference to specific embodiments. However, other embodiments than those described above are equally possible within the scope of the invention. Unless otherwise specified, the various features and steps of the various embodiments and aspects of the invention may be combined in ways other than those described herein. EXAMPLES

[0354] Example 1: Crystallization of beta-lactoglobulin from crude whey protein concentrate protocol: Lactose-depleted UF retentate from sweet whey from a standard cheese making process and filtered through a 1.2 micron filter was used as the feedstock for the BLG crystallization process. The sweet whey feedstock required an ultrafiltration setup using a Koch HFK-328 type membrane with a 46 mil spacer feed pressure of 1.5-3.0 bar, using a feedstock concentration of 21% TS (total solids) ±5 and refined water (water filtered by reverse osmosis to obtain a conductivity of up to 0.05 mS / cm) as the diafiltration medium. The temperature of the feedstock and retentate during ultrafiltration was about 12°C. The pH was then adjusted by adding HCl to obtain a pH of about 5.40. Diafiltration was continued until the conductivity of the retentate dropped below 0.03 mS / cm over a period of 20 minutes. The retentate was then concentrated to about 30% TS (about 23.1% total protein by total weight of concentrated retentate). A sample of this concentrated retentate was centrifuged at 3000 g for 5 min, but no visible pellet was formed. The supernatant was subjected to HPLC analysis. The composition of this raw material is shown in Table 1.

[0355] The concentrated retentate was seeded with 0.5 g / L of pure BLG crystal material obtained from spontaneous BLG crystallization (described in Example 3 in connection with Feedstock 2). The seed material was prepared by washing the BLG crystal slurry five times with milliQ water and collecting the BLG crystals after each wash. After washing, the BLG crystals were freeze-dried and ground using a pestle and mortar, then passed through a 200 micron sieve. Thus, the crystallization seeds were less than 200 microns in particle size.

[0356] The concentrated retentate was transferred to a 300 L crystallization tank, cooled to approximately 4°C, and held at this temperature overnight with gentle stirring. The following morning, a sample of the cooled concentrated retentate was transferred to a test tube and examined both visually and microscopically. Rapidly settling crystals were evident overnight. A laboratory sample of the mixture containing both 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 3000g for 5 min, and samples of the supernatant and pellet were taken for HPLC analysis. The crystals were washed once with cold refined water and then centrifuged again before the pellet was freeze-dried. TIFF2025003954000002.tif98170

[0357] Quantification of BLG relative yield by HPLC: All samples were diluted to the same extent by adding refined water. The samples were filtered through a 0.22 micron filter. For each sample, the same volume was loaded onto an HPLC system equipped with a Phenomenex Jupiter® 5 μm C4 300 Å, LC column 250×4.6 mm (Part Number: 00G-4167-E0) and detected at 214 nm.

[0358] The samples were run using the following conditions: Buffer A: MilliQ water, 0.1% w / w TFA Buffer B: HPLC grade acetonitrile, 0.085% w / w TFA Flow rate: 1ml / min 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.

[0359] Data Processing: Since all samples were processed in the same way, we can directly compare the areas of the BLG peaks to obtain relative yields. Since the crystals contain only BLG and the samples were all processed in the same way, the concentration of alpha-lactalbumin (ALA) (hence the area of ​​ALA) should be the same in all samples, and therefore the areas of ALA before and after crystallization are used as correction factors (cf) when calculating the relative masses. TIFF2025003954000003.tif21170

[0360] The relative yield was calculated according to the following formula: Calculated using TIFF2025003954000004.tif19170.

[0361] result: Figure 1 shows overlaid chromatograms before and after crystallization of BLG from sweet whey. The "before crystallization" sample is represented by the solid black line and the "after crystallization" sample is represented by the dotted line. It is clear that a significant reduction in BLG concentration occurred and, using the yield calculations already described, the yield of BLG recovered was determined to be 64.5% (wt / wt).

[0362] The crystal slurry was examined under a microscope and, as can be seen in Figure 2, the sample contained hexagonal crystals, many significantly larger than 200 microns in size, indicating that the crystals observed were not just seeded crystals. The crystals were easily crushed when pressed against a needle, confirming that they were protein crystals.

[0363] 3 shows a chromatogram of the washed crystalline product, where BLG accounts for 98.9% of the total area of ​​the chromatogram. The purity of this BLG product can be further increased by additional washing.

[0364] Conclusion: This example demonstrates that it is surprisingly possible to selectively crystallize BLG from a crude whey protein concentrate containing more than 48% non-BLG protein relative to the total protein, and that the resulting BLG crystal isolate has extremely high purity. This discovery opens up a new approach to industrial milk protein isolation, where BLG is preferably separated from other protein components in a gentle manner that avoids prolonged exposure to high temperatures and problematic chemicals.

[0365] Example 2: Effect of Conductivity and Temperature on the Yield of BLG protocol: Using the same raw materials, experimental and analytical setup as in Example 1, samples of the retentate (approximately 13.9% (w / w) total protein) were taken during UF diafiltration at various conductivity levels to examine the effect of conductivity on BLG crystal yield. The samples were cooled to 4° C. and held at this temperature overnight (however, we observed that 30 minutes or less may be sufficient to reach equilibrium), and then three samples were cooled to 0° C. in ice water and held at this temperature for at least 1 hour to demonstrate the effect of temperature on yield. The results for the samples at 4° C. can be seen in FIG. 4.

[0366] After diafiltration was completed, samples were taken during concentration at Brix 21, 24 and 32.5. 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. The yield of BLG crystals was then measured again.

[0367] result: When the relative yield of BLG versus conductivity for these samples is plotted, as shown in FIG. 4, there is a clear correlation between lower conductivity and higher relative yield of BLG.

[0368] In Figure 5, the yields of three samples of various conductivities are shown at two temperatures (4°C and 0°C) and it can be seen that the lower the temperature, the higher the yield of BLG. Reducing the temperature further is expected to increase the yield.

[0369] Figure 6 shows the effect of protein concentration on the relative yield of BLG at both 4° C. and 0° C. The figure shows a clear correlation between protein concentration (shown herein by Brix measurement) and the relative yield of BLG, showing that the relative yield continues to increase as the concentration of protein increases.

[0370] Conclusion: The inventors have observed that several parameters affect the efficiency of the crystallization process: at a given pH value, decreasing the conductivity, increasing the concentration of BLG, and decreasing the temperature can increase the yield of BLG.

[0371] Example 3: Crystallization of BLG in three whey protein solutions protocol: Using the same experimental and analytical setup as in Example 1, three different whey protein containing raw materials were tested as raw materials for crystallization. However, no seeding was used in the experiment carried out with raw material 2. Raw materials 1 and 2 were based on sweet whey and were fat reduced by Synder FR membranes prior to the processing described in Example 1. Raw material 3 was derived from acid whey.

[0372] The compositions of these three feedstocks can be seen below in Tables 2, 3 and 4. Feedstock 3 was crystallized at 21% TS (13.3% w / w total protein based on the total weight of this feedstock), a significantly lower concentration compared to the other two (26.3% w / w total protein in Feedstock 1 and 25.0% w / w in Feedstock 2).

[0373] The crystallized feedstock 1 slurry was centrifuged on a Maxi-Spin filter with a 0.45 micron CA membrane at 1500g for 5 minutes, then centrifuged again after adding 2 volumes of MilliQ water to the filter cake. The resulting filter cake was analyzed by HPLC. A photograph of the Maxi-Spin filter holding the crystallized feedstock 1 pellet (filter cake) is shown in Figure 24. The pellet from feedstock 2 was washed with 2 volumes of MilliQ water and centrifuged again under standard conditions before the pellet was analyzed by HPLC. The pellet from feedstock 3 was analyzed without washing.

[0374] Crystals made from Feedstock 2 were diluted to 10% TS and the pH was adjusted to pH 7 using 1 M NaOH to reverse crystallization. NaCl was added to the crystal slurry from Feedstock 2 (36% TS) to reverse crystallization. TIFF2025003954000005.tif101170TIFF2025003954000006.tif103170TIFF2025003954000007.tif100170

[0375] result: Ingredient 1: In Figure 7, the chromatograms of the protein composition of the feed (solid line) and mother liquor (dashed line) can be seen. It is clear that the process resulted in the majority of the BLG being crystalline. The yield of isolated BLG (calculated as described in Example 1) is about 65% based on the total amount of BLG in this feed.

[0376] Figure 8 is a photomicrograph of a sample taken during the early stages of the crystallization period. Figure 9 is a photomicrograph of a sample taken when crystallization was complete. It is clear from these two photos that the BLG crystals are relatively fragile. Some of the crystals appear to break during stirring and transform from a hexagonal or rhomboid shape into crystal fragments that are still very compact and clearly visible, but have a more irregular shape.

[0377] Figure 10 shows a chromatogram of the BLG crystals separated and washed on the spin filter. As can be seen, the purity is very high and the removal of other whey proteins is extremely efficient.

[0378] Ingredient 2: The protein composition of Feed 2 (actual) and the resulting mother liquor (dashed line) can be seen in Figure 11. It is clear that the majority of the BLG was removed, with a calculated yield of 82% based on the total amount of BLG in Feed 2.

[0379] Figure 12 shows Feedstock 2 before (left photo) and after (right photo) crystallization. During crystallization, the feedstock changed from a clear liquid (stirring magnets were visible in the liquid) to a milky, opaque liquid.

[0380] Figure 13 shows a micrograph of BLG crystals. Most of the crystals are fractured, but the hexagonal shape can be seen.

[0381] Figure 16 is a chromatogram of an isolated pellet of BLG crystals after washing with two volumes of MilliQ water, which clearly shows that the crystals contain BLG in very high purity.

[0382] 14 and 15 show the results of increasing the conductivity (by adding NaCl) or changing the pH (by adjusting the pH to 7 by adding NaOH) so that the environment is no longer favorable for crystalline structures. In both cases, the milky suspension reverts to a clear liquid as the BLG crystals dissolve.

[0383] The mineral composition of the crystal preparation obtained from source 2 is shown in Table 5. We note that the phosphorus to protein ratio is very low, making this crystal preparation suitable as a protein source for patients suffering from renal disease. TIFF2025003954000008.tif61170

[0384] Ingredient 3: In Figure 17, the chromatograms of the protein composition of feed 3 (solid line) and the resulting mother liquor (dashed line) are shown. It is clear that the majority of BLG was isolated (calculated yield of 70.3% based on the total amount of BLG in this feed). If the protein content was higher before crystallization, the yield obtained would have been even higher.

[0385] Figure 18 is a micrograph of BLG crystals isolated from Source 3 (substantially free of CMP). The crystals had a rectangular shape rather than a hexagonal shape. The rectangular crystals appeared more robust than the hexagonal crystals. Figure 19 shows a chromatogram of the unwashed isolated crystal pellet, which clearly shows that the crystals were BLG crystals, albeit with a rectangular rather than hexagonal shape (e.g., compare the rectangular crystal shape in Figure 18 with the hexagonal crystal shape in Figure 2). TIFF2025003954000009.tif62170

[0386] The crystal preparation from source 3 contained 45 mg P / 100 g protein. We note that the phosphorus to protein ratio is very low, making this crystal preparation suitable as a protein source for patients suffering from renal disease.

[0387] Conclusion: All three raw materials were suitable for the BLG crystallization process. The BLG crystals were easily dissolved by adding salts or increasing the pH or temperature. This novel method allows the preparation of a BLG preparation with a very low phosphorus content, which makes it suitable as a protein source for patients suffering from renal disease.

[0388] Example 4 Effect of pH on the yield of BLG crystals protocol: The same protocol and experimental set-up as in Example 1 was used (using reduced fat sweet whey protein concentrate), except that for each experiment the pH was adjusted to the levels described in Table 8. The protein concentration at the start of the crystallization step was approximately 24% (w / w).

[0389] To investigate the effect of pH on the crystallization process and the yield obtained, the pH was adjusted with either dilute NaOH (>4%) or dilute HCl (>3.6%) solutions. After crystallization, the BLG crystals were isolated by centrifugation as described in Example 1. TIFF2025003954000010.tif94170TIFF2025003954000011.tif58170

[0390] result: Yields were 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 this 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 would appear negative. TIFF2025003954000012.tif57170

[0391] Conclusion: This experiment demonstrates that crystallization of BLG in salt-solution mode was possible in the pH range of 5–6.

[0392] Example 5: Investigating the effect of increasing levels of conductivity protocol: The same protocol and experimental setup as in Example 1 was used, except samples were taken at various conductivities. The raw materials listed in Table 10 were required and used as the feedstock for the crystallization process. Samples of the raw materials were taken before UF to add NaCl to increase the conductivity to determine the conductivity level at which BLG crystals could grow. The protein content during crystallization was about 16.7% (w / w). TIFF2025003954000013.tif100170

[0393] result: The samples were processed as described in Example 1. Figure 20 shows the calculated yield at various retentate conductivities. The point at 3.53 mS / cm was the feed after pH adjustment. All points above 3.53 were the result of adding NaCl to increase the conductivity. Points below 3.53 were the result of diafiltration with the UF system. The yield at 4.93 mS / cm was close to zero and was not considered significant.

[0394] The retentate sample with a conductivity of 4.93 mS / cm had a UF permeate conductivity of about 5.7 mS / cm. The retentate sample with a conductivity of 3.53 mS / cm had a UF permeate conductivity of about 4.35 mS / cm.

[0395] From Figure 20, it can be seen that BLG crystals formed in the feedstock at conductivities below 4.93 mS / cm (at 4°C and a total protein content of about 16.7% (w / w)). A BLG yield of about 75% was obtained with a retentate conductivity of about 2 mS / cm and a UF permeate conductivity of about 1.6 mS / cm.

[0396] FIG. 21 is a micrograph of crystals formed at 4.20 mS / cm in the retentate, showing the expected BLG crystal characteristics.

[0397] Conclusion: The specific raw material of Example 5 allows the formation of BLG crystals at less than 4.93 mS / cm (corresponding to a UF permeate conductivity of 5.75 mS / cm and a ratio between conductivity and total protein of 0.057). This upper conductivity limit is expected to depend on the protein concentration and composition. For example, higher protein concentration and / or increased content of highly charged proteins or other macromolecules (e.g., CMPs) are expected to increase the upper conductivity limit at which BLG crystallization is possible.

[0398] Example 6: Crystallization of BLG in serum protein concentrate A serum protein concentrate (SPC) was prepared by microfiltration of skim milk using a Synder FR membrane and a process temperature of about 50°C. The resulting retentate contained substantially all the casein and residual fat, and further contained some serum proteins, lactose and minerals. The permeate contained molecules that could permeate the membrane (e.g., serum proteins, lactose and minerals), but was substantially free of casein or fat. The permeate was then prepared for crystallization as described in Example 1 (see Table 11 for the composition of the feedstock), 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. TIFF2025003954000014.tif106170

[0399] Similar to the crystallizations of Examples 1-5, the BLG of the SPC feedstock formed crystals that could be isolated with very high purity (as confirmed by chromatography as in the previous examples), resulting in a BLG yield of 70% relative to the total amount of BLG in the SPC feedstock. In Figure 22, BLG crystals from an early stage of crystallization are shown. As can be seen, the crystals have a rectangular or square shape, as opposed to the hexagonal shape observed in Example 2, for example.

[0400] Example 7: Preparation of spray-dried BLG crystals and determination of bulk density A portion of the BLG crystals produced in Example 3 (using Raw Material 2) was separated in a decanter centrifuge at 1200g, 5180 RPM, 110 RPM Diff. with a 64 mil spacer (mil means 1 / 1000 inch) and a flow rate of 25-30 L / hr. The BLG crystal phase was then mixed 1:1 with refined water and then separated again in a decanter centrifuge using the same settings. The BLG crystal phase was then mixed with refined water to a slurry containing about 25% dry matter and with a BLG crystallinity of about 80, followed by drying in a pilot plant spray dryer (inlet temperature 180°C and outlet temperature 85°C without preheat). The temperature of the fluid stream until spray drying was 10-12°C. The powder obtained by sampling at the outlet had a moisture content of 4.37% (w / w).

[0401] The crystallinity of the BLG in this slurry was about 90%.

[0402] We also successfully separated the BLG crystal slurry and mother liquor in a decanter centrifuge at 350g, 2750 RPM, 150 RPM Diff. with a 64 mil spacer and a flow rate of 75 L / hr. The BLG crystal phase was then mixed 1:2 with refined water. The BLG crystal phase was then mixed with refined water to make a thin slurry, which was then dried in a pilot plant spray dryer using the same parameters as above.

[0403] The bulk density of the spray-dried powder was then measured according to Example 9.3 and compared to that of standard WPI dried in the same equipment. Standard WPI was found to have a bulk density (based on 625 stampings) of 0.39 g / mL, which is at the upper end of the normal range for WPI powders. However, the spray-dried BLG crystal preparation had a bulk density of 0.68 g / mL, which is 75% higher than that of standard WPI (see, e.g., FIG. 23). This is actually surprising and provides many both logistical and application-related advantages. TIFF2025003954000015.tif102170

[0404] A sample of the spray-dried BLG crystal preparation was subsequently resuspended in cold demineralized water and the BLG crystals were still clearly visible under a microscope. Addition of citric acid or NaCl dissolved the BLG crystals, turning the opaque crystal suspension into a clear liquid.

[0405] The inventors have found indications that prolonged heating during the drying step reduces the amount of BLG in crystalline form, therefore it is preferred that the heat exposure of the BLG crystal preparation is as low as possible.

[0406] Conclusion: This example demonstrates that a slurry containing BLG crystals can be spray dried and that if heating during the drying step is controlled, the BLG crystals will still be present in the resuspended spray-dried powder.

[0407] The inventors have further discovered that the bulk density of whey protein powders containing BLG crystals is significantly higher than that obtained by conventional spray drying of dissolved protein streams. High density powders allow for more cost-effective packaging and logistics of the powders, since less packaging material is required per kg of powder and more powder (mass) can be shipped in a given container or truck.

[0408] This high density powder is also believed to be easier to handle and less fluffy and dusty during manufacture and use.

[0409] Example 8: Low Phosphorus Protein Beverage Six low phosphorus beverage samples were prepared using the purified BLG product (crystal preparation from Raw Material 3) from Example 3. All dry ingredients were mixed with demineralized water to obtain 10 kg of each sample and hydrated at 10° C. for 1 hour. TIFF2025003954000016.tif66170

[0410] Aliquots of these six samples were taken to measure turbidity with a Turbiquant® 3000 IR Turbidimeter and viscosity with a Gilson vicoman, the results of which are shown in the table below. TIFF2025003954000017.tif58170

[0411] Photographs of the test tubes containing aliquots of the six low phosphorus beverage samples are shown in Figure 25. From left to right, the aliquots were samples A, B, C, D, E and F. Visual inspection of the test tubes verified the turbidity measurements and demonstrated that all beverage samples were clear, especially samples C and D (pH 3.0), which were very clear. The low viscosity demonstrates that the beverage samples are easy to drink.

[0412] All ingredients used to prepare the beverages were low in phosphorus and free of unnecessary minerals. Thus, the resulting beverages had phosphorus contents of about 45 mg P / 100 g protein and generally very low mineral contents. Thus, the six beverages were suitable for use as protein beverages for patients with renal disease.

[0413] Example 9 – Method of Analysis Example 9.1 Determination of Lactosylated vs. Non-Lactosylated BLG: Quantification of the amounts of lactosylated and native BLG was performed using LC-MS. The analysis was performed on an Agilent Technologies 6410 Triple Quad MS coupled with an Agilent Technologies HP1200 Series HPLC. A Symmetry300™ C18 column (WAT106172: 5 μm solid phase particles, column dimensions 2.1×150 mm) was applied for separation prior to ionization, and proteins were detected at 214 nm. Samples were filtered through a 0.22 micron filter before analysis. All samples were run in duplicate.

[0414] The analysis was carried out using the following conditions: HPLC Buffer A: 99.9% MilliQ-band and 0.1% TFA Buffer B: 9.9% MilliQ-band, 90% acetonitrile, 0.1% TFA Flow rate: 0.3mL / min gradient: 0-20 min: 85-60% A and 15-40% B 20-45 min: 60-50% A and 40-50% B 45-55 min: 0%A and 100%B 55-70 min 85%A and 15%B Load: 40 μL The column temperature was set at 60°C.

[0415] Mass spectrometry: Ions with m / z between 100 and 2000 were detected and the data obtained were evaluated with MassHunter Workstation Software, Ver. B.04.00. Deconvolution was used to group all forms of the same species (same mass). Masses between 18 kDa and 20 kDa were further investigated. The intact mass of BLG-A is 18.361 kDa and that of BLG-B is 18.276, lactosylation adds 324 Da to the mass of the protein, and by examining this mass region, up to five lactosylations per protein can be detected. Relative quantification is performed by comparing the signal intensity for each mass, ignoring the ionization discrimination of different species.

[0416] Example 9.2: Determination of total protein Determine the total protein content (true protein) of the samples by: 1) Determination of the total nitrogen in the sample according to ISO 8968-1 / 2 | IDF 020-1 / 2-Milk-Determination of nitrogen content-Part 1 / 2: Determination of nitrogen content using the Kjeldahl method. 2) Determination of the non-protein nitrogen of the sample according to ISO 8968-4|IDF 020-4-Milk-Determination of nitrogen content-Part 4: Determination of non-protein-nitrogen content. 3)(m 総窒素 -m 非タンパク質窒素 ) * Calculate the total amount of protein as 6.38.

[0417] Example 9.3: Determination of Loose and Bulk Density The density of a dry powder is defined as the relationship between the weight and volume of the powder analyzed using a special Stampf volumeter (i.e. a graduated cylinder) under specific conditions. This density is generally expressed in g / ml or kg / L.

[0418] In this method, a sample of dry powder is packed into a graduated cylinder and after a specified number of taps, the volume of the product is read and its density calculated.

[0419] By this method, three densities can be defined: Packing density, which is the mass divided by the volume of the powder after it has been transferred to a specified graduated cylinder. Loose density, which is the mass divided by the volume of a powder after 100 taps according to the conditions specified in this standard. Bulk density, which is the mass divided by the volume of a powder after 625 taps according to the conditions specified in this standard.

[0420] This method uses a special measuring cylinder (250 ml, graduations 0-250 ml, weight 190±15 g) (J. Engelsmann AG67059 Ludwigshafen / Rh) and a Stampf volumeter (eg J. Engelsmann AG).

[0421] The loose and bulk densities of the dry products are determined by the following procedure.

[0422] Pretreatment: The sample to be measured is stored at room temperature.

[0423] The sample is then thoroughly mixed by repeatedly rotating and inverting the container (to avoid crushing particles). The container is not filled more than 2 / 3 full.

[0424] procedure: Weigh out 100.0±0.1 grams of powder and transfer to a graduated cylinder. Volume V 0 Read in ml.

[0425] If 100 g of powder will not fit into the graduated cylinder, this amount should be reduced to 50 or 25 grams.

[0426] Attach this graduated cylinder to a Stampf volumeter and tap 100 times. Smooth the surface with a spatula and measure the volume V 100 Read in ml.

[0427] Change the number of tabs to 625 (including 100 taps). After tapping flat on the surface, V 625 Read in ml.

[0428] Density calculation: The formula: Bulk density=M / V The loose and bulk densities, expressed in g / ml, are calculated according to the formula: where M is the weight of the sample in grams and V is the volume after 625 taps in ml.

[0429] Example 9.4: Determination of Moisture Content of Powders Determine the moisture content of foods according to ISO 5537:2004 (Dried milk - Determination of moisture content (Reference method)). NMKL is the abbreviation for "Nordisk Metodikkomite for Naeringsmidler".

[0430] Example 9.5: Determination of total calcium, magnesium, sodium, potassium, and phosphorus A procedure is used to first break down the samples using microwave digestion and then use an ICP instrument to determine the total amounts of minerals to determine the total amounts of calcium, magnesium, sodium, potassium and phosphorus.

[0431] Device: The microwave oven is manufactured by Anton Paar and the ICP is an Optima 2000DV manufactured by PerkinElmer Inc.

[0432] material: 1M HNO 3 2% HNO 3 Yttrium in 5% HNO 3 Suitable standards for calcium, magnesium, sodium, potassium and phosphorus in

[0433] Pretreatment: Weigh out a specific amount of powder and transfer this powder into a microwave digestion tube. 3 Add 5 mL. Microwave digest the samples according to the microwave instructions. Place the digested tubes in a fume cupboard and remove the lids to allow the volatile fumes to evaporate.

[0434] Measurement steps: Transfer the pretreated sample to a digestion tube using a known amount of Milli-Q water. Add 2% HNO 3 Add solution (approximately 0.25 mL per 50 mL of diluted sample) and dilute to a known volume using Milli-Q water. Analyze samples by ICP using procedures described by the manufacturer.

[0435] 1M HNO using Milli-Q water 3 10 mL of 2% HNO 3 Prepare blind samples by diluting the mixture of 0.5 mL of solution to a final volume of 100 mL.

[0436] Prepare at least three standard samples with concentrations bracketing the expected sample concentrations.

[0437] Example 9.6: Determination of Furosine Value: Furosine values ​​are 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 total protein is determined according to Example 9.2. Furosine values ​​are reported in mg furosine per 100 g protein.

[0438] Example 9.7: Determination of Crystallinity of BLG in Liquid The following method is used to determine the crystallinity of BLG in liquids with pH ranging from 5 to 6. a) A 10 mL sample of the liquid of interest is transferred to a Maxi-Spin filter with a 0.45 micron pore size CA membrane. b) Immediately spin the filter at 1500 g for 5 minutes. Keep the centrifuge at 2° C. c) Add 2mL of cold milliQ water (2°C) to the retentate side of the spin filter and immediately spin the filter at 1500g for 5 minutes while keeping the centrifuge at 2°C, collect the permeate (Permeate A), measure the volume and determine the BLG concentration by HPLC using the method outlined in Example 9.9. d) Add 4 mL of 2M NaCl to the retentate side of the filter, mix rapidly, and allow the mixture to stand at 25° C. for 15 minutes. e) Immediately spin the filter at 1500 g for 5 minutes and collect the permeate (Permeate B). f) Determine the total weight of BLG in Permeate A and Permeate B using the method outlined in Example 9.9 and convert this result to total weight of BLG instead of weight percent. 透過液AThe weight of BLG in the permeate B is called m 透過液B It is called. g) The liquid crystallinity for BLG was determined by: Crystallinity=m 透過液B / (m 透過液A +m 透過液B ) * 100% It is determined as follows.

[0439] Example 9.8: Determination of Crystallinity of BLG in Dry Powder This method is used to determine the crystallinity of BLG in dry powders. a) Mix 5.0 grams of powder sample with 20.0 grams of cold milliQ water (2°C) and leave at 2°C for 5 minutes. b) A sample of the liquid of interest is transferred to a Maxi-Spin filter with a 0.45 micron CA membrane. c) Immediately spin the filter at 1500 g for 5 minutes. Keep the centrifuge at 2° C. 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, converting the result to total weight of BLG instead of weight percent. The weight of BLG in Permeate A is m 透過液A It is called. f) The crystallinity of BLG in the powder was then calculated using the following formula: TIFF2025003954000018.tif15170 (in the formula, M 総BLG is the total amount of BLG in the powder sample in step a).

[0440] If the total amount of BLG in a powder sample is unknown, it can be determined by suspending 5 g of another powder sample (from the same powder source) in 20.0 grams of milliQ water, adjusting the pH to 7.0 by adding aqueous NaOH, leaving the mixture at 25° C. for 1 hour with stirring, and finally determining the total amount of BLG in the powder sample using Example 9.9.

[0441] Example 9.9: Determination of the total amount of BLG, ALA and CMP in an aqueous liquid The contents of alpha-lactalbumin, beta-lactoglobulin and CMP were analyzed by HPLC analysis at 0.4 mL / min. 25 microL of the filtered sample was injected into two TSKgel3000PWxl (7.8 mm 30 cm, Tosohass, Japan) columns connected in series with an attached precolumn PWxl (6 mm × 4 cm, Tosohass, Japan) equilibrated with elution solution (consisting of 465 g MilliQ water, 417.3 g acetonitrile and 1 mL trifluoroacetic acid) and a UV detector at 210 nm.

[0442] By comparing the peak areas of this sample with those obtained for the corresponding standard proteins, native alpha-lactalbumin (C アルファ ), beta-lactoglobulin (C ベータ ) and caseinomacropeptide (C CMP ) content was quantitatively determined.

[0443] The total amount of additional proteins (non-BLG proteins) was determined by subtracting the amount of BLG from the amount of total protein (determined according to Example 9.2).

[0444] Example 9.10: Determination of UF permeate conductivity Transfer 15 mL of sample to an Amicon Ultra-15 Centrifugal Filter Unit with a 3 kDa cutoff (3000 NMWL) and centrifuge at 4000 g for 20-30 min or until a sufficient volume of UF permeate accumulates at the bottom of the filter unit to measure conductivity. Conductivity is measured immediately after centrifugation. Sample handling and centrifugation are performed at the temperature of the sample source.

[0445] Example 9.11: Determination of the degree of protein denaturation of whey protein compositions Denatured whey proteins are known to be less soluble at pH 4.6 compared to 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.

[0446] More specifically, the whey protein composition to be analyzed (e.g., a powder or an aqueous solution) is - a first aqueous solution containing 5.0% (w / w) total protein and having a pH of 7.0, and - a second aqueous solution containing 5.0% (w / w) total protein and having a pH of 4.6; Convert to.

[0447] Perform pH adjustment using 3% (wt / wt) NaOH (aqueous) or 5% (wt / wt) HCl (aqueous).

[0448] The total protein content (P pH7.0 ) is determined according to Example 9.2.

[0449] The second aqueous solution is stored at room temperature for 2 hours, followed by centrifugation at 3000 g for 5 minutes. A sample of the supernatant is collected and analyzed according to Example 9.2 to determine total protein (S pH4.6 ) to determine

[0450] The degree of protein denaturation D of the whey protein composition is D=((P pH7.0 -S pH4.6 ) / P pH7.0 ) * 100% It is calculated as follows.

[0451] Example 9.12: Detection of dry BLG crystals in powder The presence of dry BLG crystals in the powder can be confirmed by the following method: The powder sample to be analyzed is resuspended and gently mixed with demineralized water having a temperature of 4°C in a weight ratio of 2 parts water to 1 part powder and allowed to rehydrate at 4°C for 1 hour.

[0452] The rehydrated sample is examined under a microscope to confirm the presence of crystals, preferably using plane polarized light to detect birefringence.

[0453] The crystalline material is isolated and subjected to X-ray crystallography to verify the presence of crystalline structure, and preferably also to verify that the crystal lattice (space group and unit cell dimensions) corresponds to that of BLG crystals.

[0454] The chemical composition of the isolated crystal-like material is analyzed to verify that the crystal-like solids consist primarily of BLG.

[0455] Example 10: Crystallization by UF-based dynamic cross-flow filtration The feedstock for the crystallization tank was prepared as described in Example 1, except that the diafiltration was performed at pH 5.92 and the final TS was 20%.

[0456] After the raw material was prepared (the composition of the raw material can be seen in Table 15), it was transferred to a 300 L crystallization tank, the pH was initially adjusted to pH 5.80, and the temperature was kept at 10-12 °C. After the pH adjustment, seeding material, produced in the same way as described in Example 1, but originating from an involuntary crystallization production, was added. The raw material was seeded with seeding material to a concentration of 0.5 g seeding material per liter of raw material. After seeding, the temperature of 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 1 hour, after which a DCF (Dynamic Cross-Flow Filtration) unit was connected to the crystallization tank, as shown in Figure 26. The DCF unit was fitted with a Kerafol ceramic membrane with a pore size of 500 nm, the TMP (Transmembrane Pressure) was set to 0.4 bar, and the rotation speed of the membrane was 32 Hz.

[0457] The retentate from the DCF was returned to the crystallization tank and the permeate was used as the 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 increases but does not exceed 12°C. The amount of diafiltrate added was adjusted so that the retentate leaving the UF and returning to the crystallization tank was about 21% TS, removing minerals from the mother liquor (ML).

[0458] Diafiltration of the ML was continued until the difference in conductivity between the permeate and diafiltrate was less than 50 μS / cm. At this point, the volume of diafiltrate was adjusted so that the retentate was approximately 30% TS. This continuous removal of excess water and minerals allows for a high overall yield, since the amount of TS in the ML is reduced when BLG is removed as crystals, and the concentrations of other proteins during BLG crystallization appear to have limited, if any, effect on BLG solubility in the range investigated.

[0459] The composition of the ML permeate from the DCF can be seen in Table 16. The first 300 L of feed was reduced to approximately 100 L of ML. Based on mass conservation the relative yield of BLG was calculated to be 92%. TIFF2025003954000019.tif106170TIFF2025003954000020.tif57170

[0460] Conclusion: By continuously removing excess minerals and water from the matrix in which BLG crystallization occurs, the yield of BLG can be significantly improved and the process can be carried out at low temperatures.

[0461] Example 11: Degree of Protein Denaturation in Various Whey Protein Products The degree of protein denaturation of four edible BLG compositions of the present invention and commercial products was compared. The samples are described below. TIFF2025003954000021.tif52170

[0462] Samples B through E were prepared in the following manner: A crystal slurry was prepared as described in Example 12 and separated as described in Example 7. A portion of the separated BLG slurry was removed and divided into four portions.

[0463] Sample B: A 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, and then the sample was diluted to Bx6 to make an approximately 5% protein solution.

[0464] Sample C: A second portion of the isolated BLG crystal slurry was freeze-dried. The powder was then resuspended in refined water, the pH was adjusted to 7.09 using 3% NaOH, and the sample was then diluted to Brix 6 to make a protein solution of approximately 5%.

[0465] Sample D: A 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 refined water and the pH was measured to be 7.07. The sample was then diluted to Brix 6 to make an approximately 5% protein solution.

[0466] Sample E: A fourth portion of the isolated BLG crystal slurry was processed and spray dried as described in Example 7. The powder was then resuspended in refined water and the pH was adjusted to 7.04 using 3% NaOH. The sample was then diluted to Brix 6 to make an approximately 5% protein solution.

[0467] The degree of protein denaturation for each sample was determined according to Example 9.11 and the results are shown in Table 17. TIFF2025003954000022.tif82170

[0468] Conclusion: Regardless of the drying method, the edible BLG compositions of the present invention exhibit surprisingly low levels of protein denaturation, only 10-fold lower than that seen in the commercial WPI used for comparison. It is particularly surprising that the spray-dried BLG crystal slurry product still exhibits the lowest level of all protein denaturation.

[0469] Example 12: Separation of crystals by dynamic cross-flow filtration Lactose-depleted UF retentate from sweet whey from a standard cheese-making process and filtered through a 1.2 micron filter was used as the feedstock for the crystallization process. The sweet whey feedstock required 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 feedstock concentration of 10% TS (total solids) ±5 and refined water (water filtered by reverse osmosis to obtain a conductivity of up to 0.05 mS / cm) as the diafiltration medium. The temperature of the feedstock and retentate during ultrafiltration was about 12°C. The pH was then adjusted by adding HCl to obtain a pH of about 5.60. Diafiltration was continued until the conductivity of the retentate was less than 1.30 mS / cm. The feedstock was then heated to 25°C, after which the retentate was concentrated to about 27% TS (about 21% total protein by total weight of concentrated retentate). The conductivity of the permeate was 0.33 mS / cm at the end of concentration. A sample of this concentrated retentate was centrifuged at 3000 g for 5 min, but no visible pellet was formed.

[0470] The concentrated retentate was transferred to a 300 L crystallization tank and the concentrated retentate was cooled to about 6° C. and kept at this temperature overnight with gentle stirring. The next morning the retentate had crystallized. The mother liquor and crystals were separated by centrifugation at 3000 g for 5 min and samples of the supernatant and pellet were taken for HPLC analysis. The yield of BLG from this process was calculated to be 67%.

[0471] The crystal slurry from the 300L tank was used as the feedstock for an Andritz DCF 152S system using one disk membrane with a pore size of 500 nm. The filtration was carried out at 8° C., the rotation speed was 32 Hz, and the transmembrane pressure was 0.4 bar. This system functions as a dead-end filtration where the retentate accumulates in the filtration chamber, unlike larger units where the retentate will be removed continuously. The filtration was carried out in a stable manner for just 40 minutes, when the solids accumulated in the filtration chamber started to affect the filtration.

[0472] During the operation of the DFC, the amount of crystal mass increased significantly.

[0473] Conclusion. The DCF provides a stable and efficient means for separating the crystals from the ML, to which a wash solution could be added if necessary.

[0474] Example 13: Separation of crystals using a filtration centrifuge Using the same raw materials and the same crystallization process as in Example 12, the separation was tested in a Filtration Centrifuge HZ 25 / 0.1 equipped with filter paper with a pore size of about 20 micrometers.

[0475] Test 1: 4 L of raw material was fed to the filter centrifuge running at 60 g. After all the raw material was added, the centrifuge was accelerated to 250 g to dry the filter cake. The cake contained 47.6% TS and the composition of the cake is shown in Table 18.

[0476] After washing, the centrifuge was fed with 7 L of the same feedstock at 60 g. The centrifuge was then accelerated to 250 g to spin for approximately 5 minutes, after which it was slowed down again to 60 g and 0.25 L of refined water was added to wash. After the wash water was added, the centrifuge was accelerated again to 250 g to spin. The TS of the cake was measured to be 47%. The cake is shown in Figure 27A. The composition of the cake, ML fraction and post-wash wash are shown in Table 18. After spinning the cake, an attempt was made to peel it off the side of the centrifuge, but while the top layer smoldered and fell through the intended tube, as shown in Figure 27C, the bottom layer was too wet and sticky to peel properly, as shown in Figure 27B. TIFF2025003954000023.tif105170

[0477] Conclusion: The filter centrifuge offers an interesting option to obtain a BLG cake that is pure enough that ALA and CMP are below the levels required for quantification even without washing. Even a small amount of washing medium applied to the filter cake can further reduce the mineral content in this cake, as can be seen by the protein composition of the washing water in Table 18. The non-BLG protein content of the cake is also reduced by washing, as can be seen from the washing water used. The washing water used contains a larger ratio of ALA:BLG compared to the ratio in the filter cake. This indicates that the washing step has a greater tendency to remove ALA (and possibly other non-BLG proteins) compared to BLG.

[0478] The filter cake produced was not exfoliating, but was still permeable. This allows the option of adding dry gas at a given temperature to reduce the moisture content of the filter cake to a level that allows it to be isolated as an upper layer. Alternatively, the filter cake could be redissolved in this centrifuge by adding an appropriate amount of acid, base or salt in aqueous solution in a siphon centrifuge setup.

[0479] Example 14: Effect of mineral composition of whey protein solutions In this example, the effect of the molar ratio between monovalent and divalent metal cations on the yield of BLG was investigated.

[0480] The following two samples were compared: Sample A: Overweight Na + (Source: Na 2 SO 4 ) Sample B: Overweight Ca 2+ (Source: CaSO 4 ).

[0481] The same raw materials used in Example 1 were adjusted with 2.5% sulfuric acid. The pH of the samples was adjusted to about pH 5.4, and the exact pH is reported in Table 20. The original volume of each sample was 250 mL. These two samples were dialyzed in another 24 L vessel against about 24 L of cold refined water. For all dialysis processes, dialysis tubing OrDial D-Clean MWCO 3500 (part number 63034405) was used. The vessel was continuously stirred during the dialysis process, and the dialysis was performed in a refrigerator at 4° C. The first dialysis was performed overnight.

[0482] To remove excess ions after the first dialysis, the dialysis bag was transferred to a container containing 2 L of salt solution, the concentrations of which were as follows: Sample A: Na 2 SO 4 (sodium sulfate) 0.059M, Sample B: CaSO 4 (Calcium sulfate) 0.059M.

[0483] The first salt dialysis was performed overnight. The conductivity, pH and Brix after the first salt dialysis are reported in Table 20. The salt solution was replaced with fresh one and the dialysis was continued over the weekend.

[0484] After the second salt dialysis, the tube was transferred to a 24 L container filled with approximately 24 L of cold refined water and dialyzed overnight to remove excess ions before crystallization.

[0485] After the final dialysis step, the protein concentration was a little lower than desired. Therefore, the samples were concentrated in a Pellicon XL UF laboratory setup using a 10 kDa cutoff membrane and a peristaltic pump running at 75 mL / hr. The mineral content of the samples is shown in Table 19 along with the raw materials.

[0486] The samples were then seeded with 0.5 g / L of the seed material previously described and allowed to crystallize overnight at 4° C. Then the next day, crystalline precipitates were found in all samples. HPLC samples of each sample were prepared by centrifuging each sample at 3000 g for 5 minutes, followed by analysis of the supernatant samples. The results are shown in Table 21. TIFF2025003954000024.tif69170TIFF2025003954000025.tif96170TIFF2025003954000026.tif62170

[0487] Conclusion: Table 21 demonstrates that if high molar ratios of monovalent to divalent cations are avoided, less residual BLG remains in the mother liquor (and higher yields of isolated BLG crystals are obtained). The molar ratio between monovalent and divalent cations (actually Na+K vs. Ca+Mg) can be controlled to improve the yield of BLG in the present process.

Claims

1. A method for preparing an edible composition comprising beta-lactoglobulin (BLG) in crystallized and / or isolated form, comprising: a) providing a whey protein solution comprising BLG and at least one additional whey protein, said whey protein solution comprising: - supersaturated with respect to BLG and having a pH in the range of 5 to 6; - containing BLG in an amount of up to 90% (w / w), b) crystallizing BLG in said supersaturated whey protein solution, preferably in salt-in mode; c) optionally separating the BLG crystals from the remaining whey protein solution; The method according to claim 1, further comprising:

2. 2. The method of claim 1, further comprising a step d) of washing the BLG crystals, e.g. the separated crystals obtained from step c).

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

4. 4. The method of claim 1, further comprising the step f) of drying the BLG-containing composition resulting from steps b), c), d) or e).

5. 5. The method according to claim 1, wherein the whey protein solution of step a) contains at least 5% (w / w) ALA based on the total amount of protein.

6. 6. The method according to claim 1, wherein the whey protein solution of step a) comprises at least 15% (w / w) additional whey protein based on the total amount of protein.

7. 7. The method according to claim 1, wherein the whey protein solution of step a) comprises at least 1% (w / w) BLG relative to the total amount of protein.

8. 8. The method according to claim 1, wherein the whey protein solution of step a) comprises at least 0.4% (w / w) BLG based on the weight of the whey protein solution.

9. 9. The method according to claim 1, wherein the whey protein solution comprises whey protein concentrate, whey protein concentrate, whey protein isolate and / or whey protein isolate.

10. 10. The method according to claim 1 , wherein the ratio between the conductivity of the whey protein solution and the total amount of protein is at most 0.

3.

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

12. 12. The method of claim 1, wherein the supersaturated whey protein solution is prepared by subjecting a whey protein raw material to the following adjustments: - adjusting the pH, - reducing the electrical conductivity, - lowering the temperature; - increasing the protein concentration, and - Adding agents that reduce water activity The method according to claim 1, wherein the composition is prepared by subjecting the composition to one or more of the following:

13. 13. The method of any one of claims 1 to 12, wherein said preparing the whey protein solution involves adjusting the pH of the whey protein raw material.

14. 14. The method of claim 1, wherein the preparing the whey protein solution involves reducing the conductivity of the whey protein raw material.

15. 15. The method of any one of claims 1 to 14, wherein the preparation of the whey protein involves reducing the temperature of the whey protein raw material.

16. 16. The method of any one of claims 1 to 15, wherein said preparing the whey protein solution involves increasing the total protein concentration of the whey protein raw material.

17. 17. The method according to claim 1, wherein the crystallization of the BLG in step b) comprises the steps of: - waiting for crystallization to occur, - addition of crystallization seeds, Further increasing the degree of supersaturation of the BLG, and / or - Mechanical stimulation The method of claim 1, further comprising:

18. 18. The method according to any one of claims 1 to 17, characterized in that step c) comprises separating the BLG crystals to a solids content of at least 30% (wt / wt), preferably at least 40% (wt / wt), even more preferably at least 50% (wt / wt).

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

20. 20. The method according to claim 19, characterized in that the washing in step d) dissolves up to 80% (wt / wt) of the initial amount of BLG crystals, preferably up to 50% (wt / wt), even more preferably up to 20% (wt / wt) of the initial amount of BLG crystals.

21. 21. The method according to any one of claims 3 to 20, wherein the recrystallization step comprises: - dissolving the separated BLG crystals in a recrystallization liquid; - adjusting the recrystallization solution to obtain a supersaturation with respect to BLG; - crystallizing BLG in said supersaturated adjusted recrystallization solution; and - Separating the BLG crystals from the remaining conditioned recrystallization liquid. The method according to claim 1, further comprising:

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

23. 23. The method of any one of claims 4 to 22, wherein the drying step involves one or more of spray drying, freeze drying, spin flash drying, rotary drying and / or fluidized bed drying.

24. 24. An edible BLG composition, characterized in that it is obtainable by one or more processes according to any one of claims 1 to 23.

25. An edible BLG composition comprising at least 90% (w / w) BLG based on total solids.

26. 26. The edible BLG composition of claim 25, having a crystallinity of at least 10% of the BLG.

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

28. 28. The edible BLG composition according to any one of claims 24 to 27, which is a dry composition.

29. 29. The dry BLG composition according to claim 28, characterized in that it is in the form of a powder, preferably a spray-dried powder, having a bulk density of at least 0.4 g / mL.

30. 30. The dry BLG composition of claim 28 or 29, - at least 20% (w / w) of BLG relative to the total amount of protein, and - Crystallinity of at least 10% of the BLG A dry BLG composition comprising:

31. 28. The edible BLG composition according to any one of claims 24 to 27, which is a liquid composition.

32. 32. The edible BLG composition according to any one of claims 24 to 31, which is a low-mineral composition.

33. 33. The edible BLG composition according to any one of claims 24 to 32, which is a low phosphorus composition.

34. 34. Use of an edible BLG composition according to any one of claims 24 to 33 as a food ingredient.

35. 34. Use of the low phosphorus edible BLG composition according to any one of claims 24 to 33 as a food ingredient in the manufacture of low phosphorus food products.

36. 34. A food product comprising an edible BLG composition according to any one of claims 24 to 33 and at least additional ingredients, such as a source of fat and / or carbohydrates.

37. 37. The food product of claim 36, which is a dry food comprising carbohydrates and protein, said dry food comprising at least 1% (w / w) BLG; i) the BLG has a crystallinity of at least 10%; and / or ii) A food product, characterized in that at least 90% (w / w) of the total protein is constituted by BLG.

38. 38. The food product of claim 36 or 37, which is a low phosphorus food containing up to 80 mg phosphorus per 100 g protein.

39. 39. The food product of any one of claims 36 to 38, which is a dairy product, a candy, a beverage, a protein bar or an enteral nutritional composition.

40. 40. The food product according to any one of claims 36 to 39, - an edible product according to any one of claims 24 to 33, so as to provide a total amount of BLG of at least 1% (w / w); - sweeteners, at least one food acid, - having a pH in the range of 2.5 to 4.0, and - Up to 80 mg phosphorus per 100 g protein A food product in the form of a beverage comprising the above-mentioned.

41. In isolated BLG crystals, the orthorhombic space group is P2 １ 2 １ 2 １ and an isolated BLG crystal characterized by unit cell dimensions a=68.68(±5%) Å, b=68.68(±5%) Å and c=156.65(±5%) Å, and unit cell integer angles α=90°, β=90° and γ=90°.

42. 42. The isolated BLG crystal of claim 41, comprising at least 20% (w / w) BLG and up to about 80% (w / w) water.

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