Process for producing beta-lactoglobulin isolates, as well as related methods and uses.

Abstract

To provide a novel β-lactoglobulin (BLG) isolated matter, a method for producing the isolated matter, and use of powders for a beverage, for example.SOLUTION: BLG isolated powders are prepared by atomization dehydration, have pH in a range of 6.1 to 8.5, and include at least 30 wt.% of total protein, at least 85 wt.% of β-lactoglobulin (BLG) with respect to the total protein, and at most 10 wt.% of water. The BLG isolated powders include one or more features of the following: at least 0.2 g / cm3 of bulk density; at least 1.11 of proper tryptophan fluorescence emission ratio (I330 / I350); at most 10% of protein denaturation degree; at most 200 NTU of heat stability at pH 3.9; and at most 15000 colony forming unit / g.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a novel beta-lactoglobulin (BLG) isolate, a method for producing such isolate, and the use of the powder in beverage applications, for example. [Background technology]

[0002] BLG is known as a gel-forming component of whey protein and tends to undergo undesirable aggregation and gel formation even at pasteurization temperatures. BLG isolates are recognized as being more heat-sensitive than conventional whey protein isolates that contain alpha-lactalbumin (ALA) and caseinomacropeptide (CMP), which are more heat-resistant proteins in addition to BLG. Due to the high cost of BLG isolates and the challenges of handling freeze-dried BLG isolates, the industrial food use of BLG isolates has previously been limited.

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

[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) describe the purification of BLG from fresh milk by temperature acid coagulation of casein and by subjecting the resulting acidic whey to a combination of affinity chromatography (DEAE Sepharose) and gel permeation chromatography. The obtained BLG composition was stated to contain 0.985 g of beta-lactoglobulin per 1 g of protein. The BLG composition was dried by freeze-drying.

[0005] Vyas et al (Scale-Up of Native β-Lactoglobulin Affinity Separation Process, J. Dairy Sci. 85:1639-1645, 2002) disclosed a scale-up method for producing native BLG based on affinity chromatography. The BLG composition was dried by lyophilization.

[0006] U.S. Patent No. 2,790,790, A1 describes a process for isolating BLG from whey by adding NaCl at pH 3.6–4.0, but it does not mention that the isolated BLG was dried.

[0007] Palmer (Crystalline Globulin from Cow's Milk, J. Biol. Chem., Vol. 104, 1934, pages 359-372) reported a cumbersome and time-consuming process for producing protein crystals based on acidic whey, using several successive salting-outs of unwanted proteins, protein, pH adjustment, and dialysis to remove other unwanted proteins. Ultimately, BLG crystallized when a highly purified BLG solution was obtained. This process lasted for more than 12 days and required the addition of toluene. Therefore, the procedure disclosed by Palmer is incompatible with safe food production and clearly results in an inedible product. Palmer reported that BLG crystals could be dried with alcohol and ether.

[0008] European Patent No. 0 604 684A1 discloses a process for recovering a whey protein concentrate rich in alpha-lactalbumin and / or beta-lactoglobulin from a whey protein product. The process included: a) incubating a solution containing the whey protein product in its acidic form with a calcium-bound ion exchange resin to initiate the destabilization of alpha-lactalbumin; b) adjusting the pH of the treated protein product solution to 4.3-4.8 after separation of the resin; c) incubating the protein product solution at a temperature of 10-50°C to promote the coagulation of alpha-lactalbumin; d) fractionating the protein in the protein product solution at pH 4.3-4.8 to provide an alpha-lactalbumin-rich fraction and a beta-lactoglobulin-rich fraction; e) sufficiently raising the pH of the alpha-lactalbumin-rich fraction to solubilize the alpha-lactalbumin fraction; and f) optionally raising the pH of the beta-lactoglobulin-rich fraction to sufficiently neutralize the beta-lactoglobulin fraction.

[0009] International Publication No. 2010 / 037736 A1 discloses the isolation of whey protein, and the preparation of whey products and whey isolates, particularly the isolation of beta-lactoglobulin products from whey obtained from animals and the isolation of alpha-lactalbumin-rich whey protein isolates. The alpha-lactalbumin-rich whey protein isolates provided by the present invention are low in beta-lactoglobulin, as well as high in alpha-lactalbumin and immunoglobulin G.

[0010] International Publication No. 2011 / 112695 (A1) discloses a nutritional composition comprising whey protein micelles and leucine. The nutritional composition provides a sufficient amount of leucine to improve protein synthesis in humans while maintaining a low-viscosity fluid matrix and acceptable sensory perception properties. [Overview of the project]

[0011] The inventors have discovered that high-performance BLG isolation powder can be obtained by drying, preferably by spray drying, a high-BLG or liquid BLG isolate having a pH in the range of i) 2 to 4.9, ii) 6.1 to 8.5, or iii) 5.0 to 6.0.

[0012] Therefore, an embodiment of the present invention is a BLG isolation powder, preferably prepared by spray drying, having a pH in the range of i) 2.5 to 4.9, ii) 6.1 to 8.5, or iii) 5.0 to 6.0. - At least 30% of total protein by weight / weight, - At least 85% by weight of beta-lactoglobulin (BLG) relative to total protein and -Includes up to 10% by weight / weight of water, The BLG isolated powder is -At least 0.2 g / cm³ 3 bulk density, - At least an intrinsic tryptophan fluorescence emission ratio of 1.11 (I330 / I350) -Maximum protein denaturation degree of 10%, -Thermal stability of up to 200 NTU at pH 3.9, and - Relating to BLG isolation powder having one or more colony-forming units / g up to 15,000.

[0013] Another aspect of the present invention is a liquid BLG isolate having a pH in the range of i) 2 to 4.9, ii) 6.1 to 8.5, or iii) 5.0 to 6.0. - at least 10% by weight / weight of total protein, - Containing at least 85% by weight / weight of beta-lactoglobulin (BLG) relative to total protein, The BLG isolated powder is, - At least an intrinsic tryptophan fluorescence emission ratio of 1.11 (I330 / I350) -Maximum protein denaturation degree of 10%, -Thermal stability of up to 200 NTU at pH 3.9, and - Relating to liquid BLG isolates having one or more colony-forming units / g up to 1000.

[0014] A further aspect of the present invention relates to a method for producing a dried BLG isolation powder containing BLG in an amount of at least 85% by weight relative to total protein, a) Liquid BLG isolate having the following, i) pH in the range of 2 to 4.9 ii) pH in the range of 6.1 to 8.5, iii) A step of providing a liquid BLG isolate having a pH in the range of 5.0 to 6.0, The liquid BLG isolate contains BLG in an amount of at least 85% by weight relative to the total protein. b) Optionally, a step of subjecting the liquid BLG isolate to physical microbial reduction. c) The method includes a step of drying the liquid BLG isolate by spray drying.

[0015] A further aspect of the present invention is a food product, for example, a beverage or instant beverage powder having a pH in the range of 2 to 4.7, and further, - A decrease in the level of dry mouthfeel. -Improved transparency, and / or The present invention relates to the use of BLG isolated powder or liquid BLG isolated product as defined herein as a raw material for the production of heat-treated beverages having one or more of the following: an increase in protein content, preferably at least 3-45% by weight, more preferably 11-40% by weight, even more preferably 15-38% by weight, and most preferably 20-36% by weight. [Brief explanation of the drawing]

[0016] [Figure 1] Figure 1 provides a schematic diagram of the present invention's method for producing BLG isolation powder. [Figure 2]Figure 2 provides a schematic diagram of the present invention's method for producing BLG isolation powder starting from a whey protein feed and illustrates the terminology used in the patent description. [Figure 3] Figure 3 is a micrograph of a BLG crystal. [Figure 4] Figure 4 shows micrographs of both the entire BLG crystal and fragmented versions. [Figure 5] Figure 5 shows that spray-dried BLG isolates have a higher bulk density than equivalent WPI dried under the same conditions. [Figure 6] Figure 6 shows that the viscosity of the high-protein liquid BLG isolate is lower than that of the equivalent WPI solution. [Modes for carrying out the invention]

[0017] definition In the context of this invention, the terms “beta-lactoglobulin” or “BLG” refer to beta-lactoglobulin derived from mammalian species, for example, in native, unfolded and / or glycosylated forms, including naturally occurring genetic variants. The terms further include agglutinating BLG, precipitated BLG, and crystalline BLG. When referring to the amount of BLG, the total amount of BLG, including agglutinating BLG, is referred to. The total amount of BLG is determined according to Example 1.31. The term “agglutinating BLG” refers to BLG that is at least partially unfolded and, moreover, is typically agglutinating with other denatured BLG molecules and / or other denatured whey proteins by hydrophobic interactions and / or covalent bonds.

[0018] BLG is the most dominant protein in bovine whey and milk, and is present in several genetic variants, with the main ones in milk being labeled A and B. BLG is a lipocalin protein that can bind to many hydrophobic molecules, suggesting a role in their transport. BLG has also been shown to bind to iron via siderofoam, potentially playing a role in combating pathogens. Homologs of BLG are absent in human breast milk.

[0019] Bovine BLG is a relatively small protein consisting of approximately 162 amino acid residues, with a molecular weight of approximately 18.3–18.4 kDa. Under physiological conditions, bovine BLG is primarily dimerized, but dissociates into monomers with a pH of less than approximately 3 while retaining its native state, as determined by nuclear magnetic resonance spectroscopy. Conversely, BLG also occurs in aggregated forms of tetramers, octamers, and other multimers under various natural conditions.

[0020] In the context of this invention, the terms “non-aggregating beta-lactoglobulin” or “non-aggregating BLG” refer to beta-lactoglobulin derived from mammalian species, for example, in native, unfolded and / or glycosylated forms, including naturally occurring genetic variants. However, the terms do not include agglomerating BLG, precipitated BLG, or crystallized BLG. The amount or concentration of non-aggregating BLG is determined according to Example 1.6.

[0021] The percentage of non-aggregating BLGs relative to total BLGs is calculated (m 総BLG -m 非凝集性BLG ) / m 総BLG *Determined by 100%. 総BLG m is the concentration or amount of BLG determined according to Example 1.31. 非凝集性BLG This is the concentration or amount of non-aggregating BLG determined according to Example 1.6.

[0022] In the context of this invention, the term "crystal" refers to a solid material in which its constituent elements (atoms, molecules, or ions, etc.) are arranged in a highly ordered microscopic structure, forming a crystal lattice that extends in all directions.

[0023] In the context of this invention, the term "BLG crystal" refers to a protein crystal containing primarily non-aggregated, preferably native BLG, which is arranged in a highly ordered microscopic structure and forms a crystal lattice extending in all directions. BLG crystals are, for example, monolithic or polycrystalline, and are, for example, intact crystals, crystal fragments, or combinations thereof. Crystal fragments are formed, for example, when intact crystals are subjected to mechanical shearing during processing. Crystal fragments also have the highly ordered microscopic structure of the crystal, but may lack the uniform surface and / or uniform edges or corners of the intact crystal. For example, see Figure 3 for many examples of intact BLG crystals and Figure 4 for examples of BLG crystal fragments. In either case, BLG crystals or crystal fragments can be visually identified as clearly defined, compact, and coherent structures using an optical microscope. BLG crystals or crystal fragments are often at least partially transparent. Protein crystals are also known to be birefringent, and this optical property can be used to identify unknown particles having a crystalline structure. On the other hand, amorphous BLG aggregates are poorly defined and opaque, often appearing as open or porous lumps of irregular size.

[0024] In the context of this invention, the term "crystallization" refers to the formation of protein crystals. Crystallization may occur spontaneously or be initiated by the addition of a crystallization seed, for example.

[0025] "Mother liquor" In the context of this invention, the term "mother liquor" refers to the whey protein solution remaining after BLG has crystallized and the BLG crystals have been at least partially removed. The mother liquor may still contain some BLG crystals, but usually only small BLG crystals that escaped separation.

[0026] 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 such as toluene or other undesirable organic solvents.

[0027] In the context of the present invention, "ALA" or "alpha-lactalbumin" relates to alpha-lactalbumin derived from mammalian species, for example, in native and / or glycosylated forms, including naturally occurring genetic variants. The term further includes aggregated ALA and precipitated BLG. When referring to the amount of ALA, for example, the total amount of ALA including aggregated ALA is referred to. The total amount of ALA is determined according to Example 1.31. The term "aggregated ALA" typically relates to ALA that is not at least partially folded and that is further aggregated with other denatured ALA molecules and / or other denatured whey proteins, typically by hydrophobic interactions and / or covalent bonds.

[0028] Alpha-lactalbumin (ALA) is a protein contained 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 the production of lactose by LS transferring the galactose moiety to glucose. One of the main structural differences from beta-lactoglobulin is that ALA does not have a free thiol group that can function as a starting point for covalent aggregation reactions.

[0029] In the context of the present invention, "non-aggregated ALA" also relates to ALA derived from mammalian species, for example, in native, non-folded and / or glycosylated forms, including naturally occurring genetic variants. However, the term does not include aggregated ALA or precipitated ALA. The amount or concentration of non-aggregated BLG is determined according to Example 1.6.

[0030] The percentage of non-aggregated ALA relative to total ALA is determined by calculation (m 総ALA -m 非凝集性ALA ) / m 総ALA *100%. m 総ALAm is the concentration or amount of ALA determined according to Example 1.31, 非凝集性ALA This is the concentration or amount of non-aggregating ALA determined according to Example 1.6.

[0031] In the context of the present invention, the terms “caseinomacropeptide” or “CMP” refer to hydrophilic peptides, residues 106-169, derived from the hydrolysis of “κ-CN” or “coppercasein” from mammalian species, including native and / or glycosylated forms, naturally occurring genetic variants by aspartate proteinase, e.g., kymoshi.

[0032] In the context of the present invention, the term "BLG isolate" means a composition comprising BLG in an amount of at least 85% by weight relative to total protein. The BLG isolate preferably has a total protein content of at least 30% by weight, preferably at least 80% by weight, relative to total solids.

[0033] In the context of the present invention, the term "BLG isolated powder" refers to a BLG isolate in powder form, preferably a free-flowing powder.

[0034] In the context of this invention, the term "BLG separation solution" refers to a BLG isolate in liquid form, preferably an aqueous liquid.

[0035] The term "whey" refers to the liquid phase remaining after the casein in milk has precipitated and been removed. Casein precipitation can be achieved, for example, by acidifying the milk and / or using rennet enzymes. There are several types of whey, such as "sweet whey," which is a whey product produced by rennet-based precipitation of casein, and "acid whey" or "sour whey," which is a whey product produced by acid-based precipitation of casein. Acid-based precipitation of casein can be achieved, for example, by adding food acid or by bacterial culture.

[0036] The term "whey" refers to the liquid remaining after casein and milk fat globules have been removed from milk, for example, by microfiltration or large-pore ultrafiltration. Whey is sometimes referred to as "ideal whey."

[0037] The terms "whey protein" or "serum protein" refer to proteins present in whey.

[0038] In the context of this invention, the term "whey protein" refers to proteins found in whey or whey serum. Whey protein may be a subset of protein species found in whey or whey serum, or it may be a single whey protein species, or it may be the complete set of protein species found in whey and / or whey serum.

[0039] In the context of this invention, the major non-BLG proteins in a standard whey protein concentrate derived from sweet whey are ALA, CMP, bovine serum albumin, immunoglobulin, osteopontin, lactoferrin, and lactoperoxidase. In the context of this invention, the weight percentages of the major non-BLG whey proteins in a standard whey protein concentrate derived from sweet whey are as follows: ALA in an amount of 18% by weight relative to total protein. CMP in an amount of 18% by weight relative to total protein. BSA in an amount of 4% by weight relative to total protein, Casein species in an amount of 5% by weight / weight relative to total protein, Immunoglobulin in an amount equivalent to 6% by weight of total protein, 0.5% by weight of osteopontin relative to total protein, Lactoferrin in an amount of 0.1% by weight relative to total protein, and Lactoperoxidase in an amount of 0.1% by weight relative to total protein.

[0040] The term casein refers to the casein protein found in milk and includes both native micellar casein and casein, which are individual casein species found in raw milk.

[0041] In the context of this invention, a liquid that is “supersaturated” or “supersaturated with respect to BLG” contains a concentration of dissolved non-agglomerating BLG that exceeds the saturation point of non-agglomerating BLG in the liquid under given physical and chemical conditions. The term “supersaturated” is well known in the field of crystallization (see, for example, Gerard Coquerela, “Crystallization of molecular systems from solution: phase diagrams, supersaturation and other basic concepts”, Chemical Society Reviews, pp. 2286-2300, Issue 7, 2014), and supersaturation can be determined by various measurement techniques (e.g., spectroscopy or particle size analysis). In the context of this invention, supersaturation with respect to BLG is determined by the following procedure.

[0042] Procedure for testing whether a liquid under specific conditions is supersaturated with respect to BLG: a) Transfer 50 ml of the liquid sample to be tested into a centrifuge tube (VWR catalog number 525-0402) with a height of 115 mm, an inner diameter of 25 mm, and a capacity of 50 ml. During steps a) to h), care must be taken to maintain the sample and subsequent fractions in the original physical and chemical conditions of the liquid: b) Immediately centrifuge the sample at 3000g 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 (the same type as in step a). d) Take a 0.05 mL subsample of the supernatant (Subsample A). e) Add 10 mg of BLG crystals with a maximum particle size of 200 microns (at least 98% pure, non-aggregating BLG relative to the total solids) to the second centrifuge tube and stir the mixture. f) Leave the second centrifuge tube at its original temperature for 60 minutes. g) Immediately after step f), the second centrifuge tube is centrifuged at 500g for 10 minutes, and another 0.05 ml subsample (subsample B) is collected from the supernatant. h) Collect the centrifugation pellet from step g), and if present, resuspend it in milliQ water and immediately examine the suspension for the presence of crystals visible under a microscope. i) Determine the concentrations of non-aggregating BLG in subsamples A and B using the method outlined in Example 1.6. Express the results as %BLG weight / weight relative to the total weight of the subsamples. The concentration of non-aggregating BLG in subsample A is C BLG,A It is referred to as such, and the concentration of non-aggregating BLG in subsample B is C BLG,B It is called that. j) The liquid sample taken in step a) is C BLG,B C BLG,A If the value was lower than and crystals were observed in step i), then it was supersaturated (under specific conditions).

[0043] In the context of the present invention, the terms “liquid” and “solution” encompass both compositions that do not contain particulate matter, such as protein crystals or other protein particles, and compositions that include a combination of liquid and solid and / or semi-solid particles. Therefore, the “liquid” or “solution” may be a suspension or even a slurry. However, the “liquid” and “solution” are preferably pumpable.

[0044] In the context of the present invention, the terms "whey protein concentrate" (WPC) and "serum protein concentrate" (SPC) refer to dry or aqueous compositions in which the total amount of protein is 20-89% by weight relative to the total solids.

[0045] The WPC or SPC should preferably include the following: 20-89% by weight of protein relative to total solids. BLG at 15-70% by weight relative to total protein. ALA at 8-50% by weight relative to total protein, and CMP at 0-40% by weight relative to the protein.

[0046] Alternatively, however, preferably, the WPC or SPC may include the following: 20-89% by weight of protein relative to total solids. BLG at 15-90% by weight relative to total protein. ALA at 4-50% by weight relative to total protein, and CMP at 0-40% by weight relative to the protein.

[0047] Preferably, the WPC or SPC includes: 20-89% by weight of protein relative to total solids. BLG at 15-80% by weight relative to total protein. ALA at 4-50% by weight relative to total protein, and CMP at 0-40% by weight relative to the protein.

[0048] More preferably, the WPC or SPC includes: 70-89% by weight of protein relative to total solids. BLG at 30-90% by weight relative to total protein. ALA at 4-35% by weight relative to total protein, and CMP is 0-25% by weight / weight compared to protein.

[0049] SPC typically contains no CMP or only trace amounts of CMP.

[0050] The terms "whey protein isolate" (WPI) and "serum protein isolate" (SPI) refer to dry or aqueous compositions containing a total amount of protein at 90–100% by weight relative to the total solids.

[0051] The WPI or SPI should preferably include the following: 90-100% of total solids by weight / weight of protein, BLG at 15-70% by weight relative to total protein. ALA at 8-50% by weight relative to total protein, and CMP at 0-40% by weight relative to total protein.

[0052] Alternatively, however, preferably, WPI or SPI may include: 90-100% of total solids by weight / weight of protein, BLG at 30-95% by weight relative to total protein, ALA at 4-35% by weight relative to total protein, and CMP at 0-25% by weight relative to total protein.

[0053] More preferably, WPI or SPI may include: 90-100% of total solids by weight / weight of protein, BLG at 30-90% by weight relative to total protein. ALA at 4-35% by weight relative to total protein, and CMP at 0-25% by weight relative to total protein.

[0054] SPI typically contains no CMP or only trace amounts of CMP.

[0055] In the context of this invention, the term “additional proteins” means proteins that are not BLGs. Additional proteins present in a whey protein solution typically include one or more non-BLG proteins found in whey or milk serum. Non-limiting examples of such proteins include alpha-lactalbumin, bovine serum albumin, immunoglobulins, caseinomacropeptides (CMPs), osteopontin, lactoferrin, and milk fat globule membrane proteins.

[0056] The terms “essentially consisting of” and “essentially consisting of” mean that the claim or feature in question does not substantially affect the basic and novel characteristics (if any) of the particular material or process and the invention described in the claims.

[0057] In the context of this invention, the phrase "Y and / or X" means "Y" or "X" or "Y and X". Following the same logic, "n1, n2, ..., n i-1 , and / or n i The phrase "" is either "n1" or "n2" or ... or "n i-1 " or "n i ", or any combination of components: n1, n2, ... n i-1 , and n i It means...

[0058] In the context of the present invention, the terms “dry” or “dried” mean that the composition or product in question contains up to 10% by weight of water, preferably up to 6% by weight of water, and more preferably even less.

[0059] In the context of the present invention, the term "physical microbial reduction" refers to a physical interaction with the composition that results in a reduction in the total amount of viable microorganisms in the composition. The term does not include the addition of chemicals that result in the killing of microorganisms. The term further includes possible preheating before spray drying, but does not include the thermal exposure to which the sprayed droplets are subjected during spray drying.

[0060] In the context of this invention, the pH of the powder refers to the pH of 10 g of powder mixed with 90 g of desalted water, and is measured according to Example 1.16.

[0061] In the context of the present invention, the weight percentage (% weight / weight) of a component, product, or material of a particular composition means the weight percentage of that component relative to the weight of the particular composition, product, or material, unless another criterion is specifically mentioned (e.g., total solids or total protein).

[0062] In the context of this invention, the process step "concentration" and the verb "to concentrate" relate to the concentration of protein, encompassing both the concentration of protein on a total solids basis and the concentration of protein on a gross weight basis. This means, for example, that the concentration does not necessarily require an increase in the absolute concentration weight / weight of protein in the composition, as long as the protein content increases relative to the total solids.

[0063] In the context of this invention, the term "weight ratio" between component X and component Y is used in calculations m X / m Y This refers to the value obtained by, in the formula, m X m is the amount (weight) of component X, Y This represents the amount (weight) of component Y.

[0064] In the context of this invention, the term "at least pasteurization" refers to a heat treatment that has a microbial killing effect of 10 seconds or more at 70°C. The criterion for determining the sterilization effect is Escherichia coli (E. coli) O157:H7.

[0065] In the context of this invention, the term "whey protein feed" refers to a whey protein source derived from liquid BLG isolate. Whey protein feed has a lower BLG content relative to total protein than liquid BLG isolate and is typically WPC, WPI, SPC, or SPI.

[0066] In the context of this invention, the term "BLG-concentrated composition" refers to a BLG-concentrated composition obtained by isolating BLG from a whey protein feed. A BLG-concentrated composition typically contains the same whey protein as the whey protein feed, but BLG is present at a significantly higher concentration relative to the total protein than in the whey protein feed. BLG-concentrated compositions are prepared from whey protein feed, for example, by chromatography, protein crystallization, and / or membrane-based protein fractionation. A BLG-concentrated composition contains BLG in an amount of at least 85% by weight, preferably at least 90% by weight, relative to the total protein. In some cases, a BLG-concentrated composition can be used directly as a liquid BLG isolate. However, additional processing is often required to convert a BLG-rich composition into a liquid BLG isolate.

[0067] In the context of the present invention, the term "whey protein solution" is used to describe a special aqueous whey protein composition that is supersaturated with respect to BLG in a salt-soluble manner and is useful for preparing BLG crystals.

[0068] In the context of this invention, the term "sterilized" means that the sterilized composition or product in question does not contain viable microorganisms and therefore does not allow for microbial growth during storage at room temperature. A sterilized composition is sterile.

[0069] When liquids such as beverage preparations are sterilized and aseptically packaged in sterile containers, they typically have a shelf life of at least 6 months at room temperature. Sterilization kills spores and microorganisms that could cause the liquid to spoil.

[0070] In the context of the present invention, the term "protein fraction" refers to the protein of the composition in question, for example, the protein of a protein powder or a beverage formulation.

[0071] In the context of this invention, the term "dry mouthfeel" refers to a sensation in the mouth where the mouth and teeth feel dry, and saliva production is minimized.

[0072] Therefore, a dry mouthfeel is not a taste in itself, but rather a physical mouthfeel and a time-dependent sensation in the mouth.

[0073] In the context of this invention, the term “mineral” as used herein refers, unless otherwise specified, to any one of the following: major minerals, trace or minority minerals, other minerals, or combinations thereof. Major minerals include calcium, phosphorus, potassium, sulfur, sodium, chlorine, and magnesium. Trace or minority minerals include iron, cobalt, copper, zinc, molybdenum, iodine, selenium, and manganese, while other minerals include chromium, fluorine, boron, lithium, and strontium.

[0074] In the context of the present invention, the terms “lipid,” “fat,” and “oil” as used herein are used interchangeably to refer to lipid materials derived from or processed from plants or animals, unless otherwise specified. These terms also include synthetic lipid materials, insofar as they are suitable for human consumption.

[0075] In the context of this invention, the term "transparent" encompasses beverage preparations that have a visually transparent appearance, allowing light to pass through and revealing a clear image. The turbidity of a transparent beverage is up to 200 NTU.

[0076] In the context of this invention, the term "opaque" includes beverage preparations having a visibly indistinct appearance and having a turbidity greater than 200 NTU.

[0077] One aspect of the present invention is prepared preferably by spray drying and has a pH in the range of i) 2 to 4.9, ii) 6.1 to 8.5, or iii) 5.0 to 6.0. - At least 30% by weight / total protein, - BLG in an amount of at least 85% by weight / weight relative to total protein, - Relating to beta-lactoglobulin (BLG) isolated powder containing up to 10% by weight / weight of water.

[0078] The BLG isolation powder preferably has one or more of the following: -At least 0.2 g / cm³ 3 bulk density, - At least an intrinsic tryptophan fluorescence emission ratio of 1.11 (I330 / I350) -Maximum protein denaturation degree of 10%, -Thermal stability of up to 200 NTU at pH 3.9, and - Maximum 1000 colony-forming units / g.

[0079] The isolated BLG powder is preferably an edible composition.

[0080] In some preferred embodiments of the present invention, the isolated BLG powder has a pH in the range of 2 to 4.9. Such powder is particularly useful in acidic food products, especially acidic beverages.

[0081] In another preferred embodiment of the present invention, the BLG isolation powder has a pH in the range of 6.1 to 8.5.

[0082] In some preferred embodiments of the present invention, the BLG isolation powder contains a total protein amount of at least 40% by weight, preferably at least 50% by weight, at least 60% by weight, more preferably at least 70% by weight, and even more preferably at least 80% by weight.

[0083] In some cases, an even higher protein content may be required, and in some preferred embodiments of the present invention, the BLG isolation powder contains at least 85% by weight, preferably at least 90% by weight, at least 92% by weight, more preferably at least 94% by weight, and even more preferably at least 95% by weight of total protein.

[0084] Total protein is measured according to Example 1.5.

[0085] In some preferred embodiments of the present invention, the BLG isolation powder contains at least 96% by weight, preferably at least 96.5% by weight, more preferably at least 97% by weight, and even more preferably at least 98% of the total protein amount of BLG, most preferably at least 99.5% by weight of the total protein amount of BLG.

[0086] In some preferred embodiments of the present invention, the BLG isolation powder contains an amount of BLG that is at least 97.5% by weight, preferably at least 98.0% by weight, more preferably at least 98.5% by weight, and even more preferably at least 99.0% of the total protein, most preferably at least 99.7% by weight, for example, about 100.0% of the total protein.

[0087] In some preferred embodiments of the present invention, the total of alpha-lactalbumin (ALA) and caseinomacropeptide (CMP) constitutes at least 40% by weight of the powdered non-BLG protein, preferably at least 60% by weight, more preferably at least 70% by weight, and most preferably at least 90% by weight of the powdered non-BLG protein.

[0088] In other preferred embodiments of the present invention, each major non-BLG whey protein is present as a weight percentage of the total protein, which is up to 25%, preferably up to 20%, more preferably up to 15%, even more preferably up to 10%, and most preferably up to 6% of the total protein in a standard whey protein concentrate derived from sweet whey.

[0089] Even lower concentrations of major non-BLG whey proteins may be desirable. Therefore, in an additional preferred embodiment of the present invention, each major non-BLG whey protein is present as a weight percentage of the total protein, which is up to 4%, preferably up to 3%, more preferably up to 2%, and even more preferably up to 1%, in terms of its weight percentage of the total protein in a standard whey protein concentrate derived from sweet whey.

[0090] In some preferred embodiments of the present invention, ALA constitutes up to 80% by weight of non-BLG proteins in the BLG isolated powder, preferably up to 60% by weight of non-BLG proteins in the BLG isolated powder, more preferably up to 40% by weight, and most preferably up to 30% by weight.

[0091] Even lower ALA content may be preferred, and in some preferred embodiments of the present invention, ALA constitutes up to 20% by weight of non-BLG proteins in the BLG isolation powder, preferably up to 15% by weight of non-BLG proteins in the BLG isolation powder, more preferably up to 10% by weight, and most preferably up to 5% by weight.

[0092] The inventors have observed indications that the reduction of lactoferrin and / or lactoperoxidase is particularly advantageous for obtaining a color-neutral whey protein product.

[0093] Therefore, in some preferred embodiments of the present invention, lactoferrin is present as a weight percentage of total protein, which is up to 25%, preferably up to 20%, more preferably up to 15%, even more preferably up to 10%, and most preferably up to 6% of its weight percentage of total protein in a standard whey protein concentrate derived from sweet whey. Even lower concentrations of lactoferrin may be desirable. Therefore, in an additional preferred embodiment of the present invention, lactoferrin is present as a weight percentage of total protein, which is up to 4%, preferably up to 3%, more preferably up to 2%, and even more preferably up to 1% of its weight percentage of total protein in a standard whey protein concentrate derived from sweet whey.

[0094] Similarly, in some preferred embodiments of the present invention, lactoperoxidase is present as a weight percentage of total protein, which is up to 25%, preferably up to 20%, more preferably up to 15%, even more preferably up to 10%, and most preferably up to 6% of its weight percentage of total protein in a standard whey protein concentrate derived from sweet whey. Even lower concentrations of lactoperoxidase may be desirable. Therefore, in an additional preferred embodiment of the present invention, lactoperoxidase is present as a weight percentage of total protein, which is up to 4%, preferably up to 3%, more preferably up to 2%, and even more preferably up to 1% of its weight percentage of total protein in a standard whey protein concentrate derived from sweet whey.

[0095] Lactoferrin and lactoperoxidase are quantified according to Example 1.29.

[0096] In some preferred embodiments of the present invention, the BLG isolated powder has a water content of up to 10% by weight, preferably up to 7% by weight, more preferably up to 6% by weight, and even more preferably up to 4% by weight, with the most preferred being up to 2% by weight.

[0097] In some preferred embodiments of the present invention, the BLG isolation powder contains carbohydrates in an amount of up to 60% by weight, preferably up to 50% by weight, more preferably up to 20% by weight, even more preferably up to 10% by weight, even more preferably up to 1% by weight, and most preferably up to 0.1% by weight. For example, the BLG isolation powder may contain carbohydrates such as lactose, oligosaccharides and / or hydrolysis products of lactose (i.e., glucose and galactose), sucrose and / or maltodextrin.

[0098] In some preferred embodiments of the present invention, the BLG isolated powder contains lipids in an amount of up to 10% by weight, preferably up to 5% by weight, more preferably up to 2% by weight, and even more preferably up to 0.1% by weight.

[0099] The amount of lipids is determined according to ISO 1211:2010 (Determination of fat content - Rose-Gottlieb gravimetric method).

[0100] The inventors have found that it may be advantageous to control the mineral content of a portion of the isolated BLG powder to achieve desired properties.

[0101] In some preferred embodiments of the present invention, the total amount of Na, K, Mg, and Ca in the BLG isolated powder is up to 10 mmol / g protein. Preferably, the total amount of Na, K, Mg, and Ca in the BLG isolated powder is up to 6 mmol / g protein, more preferably up to 4 mmol / g protein, and even more preferably up to 2 mmol / g protein.

[0102] In another preferred embodiment of the present invention, the total amount of Na, K, Mg, and Ca in the BLG isolated powder is a maximum of 1 mmol / g protein. Preferably, the total amount of Na, K, Mg, and Ca in the BLG isolated powder is a maximum of 0.6 mmol / g protein, more preferably a maximum of 0.4 mmol / g protein, even more preferably a maximum of 0.2 mmol / g protein, and most preferably a maximum of 0.1 mmol / g protein.

[0103] In another preferred embodiment of the present invention, the total amount of Mg and Ca in the BLG isolated powder is a maximum of 5 mmol / g protein. Preferably, the total amount of Mg and Ca in the BLG isolated powder is a maximum of 3 mmol / g protein, more preferably a maximum of 1.0 mmol / g protein, and even more preferably a maximum of 0.5 mmol / g protein.

[0104] In another preferred embodiment of the present invention, the total amount of Mg and Ca in the BLG isolated powder is a maximum of 0.3 mmol / g protein. Preferably, the total amount of Mg and Ca in the BLG isolated powder is a maximum of 0.2 mmol / g protein, more preferably a maximum of 0.1 mmol / g protein, even more preferably a maximum of 0.03 mmol / g protein, and most preferably a maximum of 0.01 mmol / g protein.

[0105] The inventors have found that it is possible to produce a low-phosphorus / low-potassium variant BLG isolate powder that is particularly useful for patients with kidney disease. To produce such a product, the phosphorus and potassium content of the BLG isolate powder must be equally low.

[0106] Therefore, in some preferred embodiments of the present invention, the BLG isolated powder has a total phosphorus content of up to 100 mg per 100 g of protein. Preferably, the BLG isolated powder has a total phosphorus content of up to 80 mg per 100 g of protein. More preferably, the BLG isolated powder has a total phosphorus content of up to 50 mg per 100 g of protein. Even more preferably, the BLG isolated powder has a total phosphorus content of up to 20 mg per 100 g of protein. The total phosphorus content of the BLG isolated powder is up to 5 mg per 100 g of protein.

[0107] In some preferred embodiments of the present invention, the BLG isolated powder contains up to 600 mg of potassium per 100 g of protein. More preferably, the BLG isolated powder contains up to 500 mg of potassium per 100 g of protein. More preferably, the BLG isolated powder contains up to 400 mg of potassium per 100 g of protein. More preferably, the BLG isolated powder contains up to 300 mg of potassium per 100 g of protein. Even more preferably, the BLG isolated powder is isolated with up to 200 mg of potassium per 100 g of protein. Even more preferably, the BLG isolated powder contains up to 100 mg of potassium per 100 g of protein. Even more preferably, the BLG isolated powder contains up to 50 mg of potassium per 100 g of protein, and even more preferably, the BLG isolated powder contains up to 10 mg of potassium per 100 g of protein.

[0108] The phosphorus content is determined according to Example 1.19 with respect to the total amount of elemental phosphorus in the composition in question. Similarly, the potassium content is determined according to Example 1.19 with respect to the total amount of elemental potassium in the composition in question.

[0109] In some preferred embodiments of the present invention, the BLG isolation powder contains a maximum of 100 mg of phosphorus per 100 g of protein and a maximum of 700 mg of potassium per 100 g of protein, preferably a maximum of 80 mg of phosphorus per 100 g of protein and a maximum of 600 mg of potassium per 100 g of protein, more preferably a maximum of 60 mg of phosphorus per 100 g of protein and a maximum of 500 mg of potassium per 100 g of protein, more preferably a maximum of 50 mg of phosphorus per 100 g of protein and a maximum of 400 mg of potassium per 100 g of protein, or more preferably a maximum of 20 mg of phosphorus per 100 g of protein and a maximum of 200 mg of potassium per 100 g of protein, or even more preferably a maximum of 10 mg of phosphorus per 100 g of protein and a maximum of 50 mg of potassium per 100 g of protein. In some preferred embodiments of the present invention, the BLG isolation powder contains a maximum of 100 mg of phosphorus per 100 g of protein and a maximum of 340 mg of potassium per 100 g of protein.

[0110] The low-phosphorus and / or low-potassium composition according to the present invention can be used as a food ingredient for producing food products for a group of patients with impaired renal function.

[0111] The inventors have found that for several applications, for example, in the case of acidic food products, particularly acidic beverages, having an acidic BLG isolated powder with a pH of up to 4.9, and more preferably up to 4.3, is particularly advantageous. This is especially true for high-protein, clear acidic beverages.

[0112] In the context of the present invention, a clear liquid has a turbidity of up to 200 NTU as measured according to Example 1.7.

[0113] Therefore, in some preferred embodiments of the present invention, the BLG isolated powder has a pH in the range of 2 to 4.9. Preferably, the BLG isolated powder has a pH in the range of 2.5 to 4.7, more preferably 2.8 to 4.3, even more preferably 3.2 to 4.0, and most preferably 3.4 to 3.9. Alternatively, however also preferably, the BLG isolated powder may have a pH in the range of 3.6 to 4.3.

[0114] The inventors have found that for several applications, for example, in pH-neutral food products, particularly pH-neutral beverages, it is especially advantageous to use pH-neutral BLG isolated powder. This is particularly true for high-protein, clear or opaque pH-neutral beverages.

[0115] Therefore, in some preferred embodiments of the present invention, the BLG isolation powder has a pH in the range of 6.1 to 8.5. Preferably, the powder has a pH in the range of 6.1 to 8.5, more preferably 6.2 to 8.0, even more preferably 6.3 to 7.7, and most preferably 6.5 to 7.5.

[0116] In another preferred embodiment of the present invention, the BLG isolation powder has a pH in the range of 5.0 to 6.0. Preferably, the powder has a pH in the range of 5.1 to 5.9, more preferably 5.2 to 5.8, even more preferably 5.3 to 5.7, and most preferably 5.4 to 5.6.

[0117] Advantageously, the BLG isolation powder of the present invention contains at least 0.20 g / cm³ 3 Preferably at least 0.30 g / cm³ 3 , more preferably at least 0.40 g / cm³ 3 More preferably, at least 0.45 g / cm³ 3 , more preferably at least 0.50 g / cm³ 3 Most preferably at least 0.6 g / cm³ 3 It may have a bulk density.

[0118] Low-density powders such as freeze-dried BLG isolates are fluffy and easily drawn into the air in the manufacturing environment during use. This is problematic because freeze-dried powders increase the risk of cross-contamination with other food products, and dusty environments are known to cause hygiene problems. In extreme cases, dusty environments also increase the risk of dust explosions.

[0119] The high-density deformable material of the present invention is easier to handle and less likely to flow into the surrounding air.

[0120] An additional advantage of the high-density variants of the present invention is that they occupy less space during transport, thereby increasing the weight of BLG isolation powder that can be transported in one volume unit.

[0121] However, the advantage of the high-density variants of the present invention is that they can be used as raw materials for other powdered food products, such as powdered sugar (bulk density approximately 0.56 g / cm³). 3 ), granulated sugar (bulk density approximately 0.71 g / cm³) 3 ), citric acid powder (bulk density approximately 0.77 g / cm³) 3 When used in powder mixtures with other substances, etc., the substances are less likely to separate.

[0122] The BLG isolation powder of the present invention has, for example, a bulk density of 0.2 to 1.0 g / cm³. 3 The range is preferably 0.30 to 0.9 g / cm³. 3 The range is, more preferably 0.40 to 0.8 g / cm³. 3 The range, more preferably 0.45 to 0.75 g / cm³ 3 More preferably 0.50 to 0.75 g / cm³ 3 The range is most preferably 0.6 to 0.75 g / cm³. 3 It may have a range of possibilities.

[0123] In some preferred embodiments of the present invention, the BLG isolation powder is 0.45 to 1.2 g / cm³. 3 The range is preferably 0.46 to 1.0 g / cm³. 3 The range is, more preferably 0.47 to 0.8 g / cm³. 3 The range, more preferably 0.48 to 0.75 g / cm³ 3 The range is, more preferably, 0.48 to 0.6 g / cm³. 3 The range is most preferably 0.50 to 0.6 g / cm³. 3 It has a bulk density within the range of [value].

[0124] The bulk density of the powder is measured according to Example 1.17.

[0125] The inventors have found it advantageous to maintain the native stereostructure of BLG and have observed that when BLG is used in acidic beverages, increased unfolding of BLG results in an increased level of dry mouthfeel.

[0126] The intrinsic tryptophan fluorescence emission ratio (I330 / I350) is a measure of the degree of BLG unfolding, and the inventors found that a high intrinsic tryptophan fluorescence emission ratio, which correlates with low or no BLG unfolding, was observed to be associated with less dry mouthfeel. The intrinsic tryptophan fluorescence emission ratio (I330 / I350) is measured according to Example 1.1.

[0127] In some preferred embodiments of the present invention, the BLG isolation powder has an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.11.

[0128] In some preferred embodiments of the present invention, the BLG isolation powder has an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.12, preferably at least 1.13, more preferably at least 1.15, even more preferably at least 1.17, and most preferably at least 1.19.

[0129] If the BLG isolated powder contains a significant amount of non-protein material, it is preferable to isolate the protein fraction before measuring the intrinsic tryptophan fluorescence emission ratio. Therefore, in some preferred embodiments of the present invention, the protein fraction of the BLG isolated powder has an intrinsic tryptophan fluorescence emission ratio of at least 1.11.

[0130] In some preferred embodiments of the present invention, the protein fraction of the BLG isolation powder has an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.12, preferably at least 1.13, more preferably at least 1.15, even more preferably at least 1.17, and most preferably at least 1.19.

[0131] For example, the protein fraction can be separated from the BLG isolation powder by dissolving the BLG isolation powder in desalinated water and subjecting the solution to dialysis or ultrafiltration-based dialysis using a protein-retaining filter. If the BLG isolation powder contains interfering levels of lipids, such lipids are removed, for example, by microfiltration. By combining the microfiltration and ultrafiltration / diafiltration steps, both lipids and small molecules can be removed from the protein fraction.

[0132] It is often preferable that a significant amount of BLG in the isolated BLG powder is non-aggregating BLG. Preferably, at least 50% of the BLG is non-aggregating BLG. More preferably, at least 80% of the BLG is non-aggregating BLG. It is even more preferable that at least 90% of the BLG is non-aggregating BLG. It is most preferable that at least 95% of the BLG is non-aggregating BLG. It is even more preferable that approximately 100% of the BLG in the isolated BLG powder is non-aggregating BLG.

[0133] In some preferred embodiments of the present invention, the BLG isolated powder has a protein denaturation degree of up to 10%, preferably up to 8%, more preferably up to 6%, even more preferably up to 3%, even more preferably up to 1%, and most preferably up to 0.2%.

[0134] However, it may also be preferable for the BLG isolate powder to have a significant level of protein denaturation, for example, when an opaque beverage is required. Therefore, in another preferred embodiment of the present invention, the BLG isolate powder has a degree of protein denaturation of at least 11%, preferably at least 20%, more preferably at least 40%, even more preferably at least 50%, even more preferably at least 75%, and most preferably at least 90%.

[0135] If the BLG isolated powder has a considerable level of protein denaturation, it is often preferable to maintain a low level of insoluble protein material, i.e., precipitated protein material that settles in the beverage during storage. The level of insoluble material is measured according to Example 1.10.

[0136] In some preferred embodiments of the present invention, the BLG isolation powder contains up to 20% by weight of insoluble protein material, preferably up to 10% by weight of insoluble protein material, more preferably up to 5% by weight of insoluble protein material, even more preferably up to 3% by weight of insoluble protein material, and most preferably up to 1% by weight of insoluble protein material. It may be even more preferable that the BLG isolation powder contains no insoluble protein material at all.

[0137] The inventors found that the thermal stability of BLG isolated powder at pH 3.9 is a good indicator of its usefulness in clear, high-protein beverages. The thermal stability at pH 3.9 is measured according to Example 1.2.

[0138] The BLG isolated powder has a thermal stability of up to 200 NTU at pH 3.9, preferably up to 100 NTU, more preferably up to 60 NTU, and even more preferably up to 40 NTU, with the most preferred being up to 20 NTU. Even better thermal stability is possible, and the BLG isolated powder preferably has a thermal stability of up to 10 NTU at pH 3.9, preferably up to 8 NTU, more preferably up to 4 NTU, and even more preferably up to 2 NTU.

[0139] The microbial content of BLG isolation powder is preferably kept to a minimum. However, since the microbial reduction process tends to lead to protein unfolding and denaturation, it is difficult to obtain both high protein nativeity and low microbial content. The present invention makes it possible to obtain a very low microbial content while maintaining a high level of BLG nativeity.

[0140] In some embodiments of the present invention, the liquid BLG isolate contains up to 500,000 CFU / g, preferably up to 100,000 CFU / g, more preferably up to 50,000 CFU / g, and even more preferably up to 25,000 CFU / g.

[0141] Even lower microbial content may be preferred; therefore, in some preferred embodiments of the present invention, the BLG isolation powder contains up to 15,000 colony-forming units (CFU) / g. Preferably, the BLG isolation powder contains up to 10,000 CFU / g. More preferably, the BLG isolation powder contains up to 5,000 CFU / g. Even more preferably, the BLG isolation powder contains up to 1,000 CFU / g. Even more preferably, the BLG isolation powder contains up to 300 CFU / g. Most preferably, the BLG isolation powder contains up to 100 CFU / g, for example, up to 10 CFU / g. In particularly preferred embodiments, the powder is sterile. Sterile BLG isolation powder can be prepared by combining several physical microbial reduction processes during the production of the BLG isolation powder, such as microfiltration at an acidic pH and heat treatment. Drying is preferably carried out using a sterile drying system, such as a sterile spray dryer.

[0142] In some preferred embodiments of the present invention, the BLG isolation powder has a pH in the range of i) 2 to 4.9, ii) 6.1 to 8.5, or iii) 5.0 to 6.0. -Total protein in an amount of at least 30% by weight / weight, preferably at least 80% by weight / weight, and more preferably at least 90% by weight / weight, -Beta-lactoglobulin (BLG) in an amount of at least 85% by weight / weight, preferably at least 90% by weight / weight, relative to total protein - Up to 6% by weight / weight of water, - Containing lipids in an amount of up to 2% by weight / weight, preferably up to 0.5% by weight / weight, The BLG isolated powder is, - At least an intrinsic tryptophan fluorescence emission ratio of 1.11 (I330 / I350) -Maximum protein denaturation degree of 10%, and - It has a thermal stability of up to 200 NTU at pH 3.9.

[0143] In some preferred embodiments of the present invention, the BLG isolation powder has a pH in the range of i) 2 to 4.9, or ii) 6.1 to 8.5. -Total protein in an amount of at least 30% by weight / weight, preferably at least 80% by weight / weight, and more preferably at least 90% by weight / weight, -Beta-lactoglobulin (BLG) in an amount of at least 85% by weight, preferably at least 90% by weight, and more preferably at least 94% by weight relative to total protein. - Up to 6% by weight / weight of water, - Containing lipids in an amount of up to 2% by weight / weight, preferably up to 0.5% by weight / weight, The BLG isolated powder is, - At least an intrinsic tryptophan fluorescence emission ratio of 1.11 (I330 / I350) - A maximum of 10%, preferably a maximum of 5%, degree of protein denaturation, -It has a thermal stability of up to 70 NTU at pH 3.9, preferably up to 50 NTU, and more preferably up to 40 NTU.

[0144] In some preferred embodiments of the present invention, the BLG isolation powder has a pH in the range of i) 2 to 4.9, or ii) 6.1 to 8.5. - At least 30% of total protein by weight / weight, -Beta-lactoglobulin (BLG) in an amount of at least 85% by weight / weight, preferably at least 90% by weight / weight, relative to total protein - Contains up to 6% by weight / weight of water, The BLG isolated powder is, -At least 0.2 g / cm³ 3 bulk density, - At least an intrinsic tryptophan fluorescence emission ratio of 1.11 (I330 / I350) -Maximum protein denaturation degree of 10%, and - It has a thermal stability of up to 200 NTU at pH 3.9.

[0145] In another preferred embodiment of the present invention, the BLG isolated powder has a pH in the range of 2 to 4.9. -Total protein in an amount of at least 80% by weight / weight, preferably at least 90% by weight / weight, and more preferably at least 94% by weight / weight, -Beta-lactoglobulin (BLG) in an amount of at least 85% by weight, preferably at least 90% by weight, and more preferably at least 94% by weight of total protein relative to total protein. - Up to 6% by weight / weight of water, - Containing lipids in an amount of up to 2% by weight / weight, preferably up to 0.5% by weight / weight, The BLG isolated powder is, -At least 0.2 g / cm³ 3 Preferably at least 0.3 g / cm³ 3 , more preferably at least 0.4 g / cm³ 3 bulk density, - At least an intrinsic tryptophan fluorescence emission ratio of 1.11 (I330 / I350) - A maximum protein denaturation degree of 10%, preferably 5%, more preferably 2%, and -It has a thermal stability of up to 50 NTU at pH 3.9, preferably up to 30 NTU, and more preferably up to 10 NTU.

[0146] In a further preferred embodiment of the present invention, the BLG isolation powder has a pH in the range of 3.0 to 4.3, preferably in the range of 3.6 to 4.1. -Total protein in an amount of at least 80% by weight / weight, preferably at least 90% by weight / weight, and more preferably at least 94% by weight / weight, -Beta-lactoglobulin (BLG) in an amount of at least 90% by weight / weight, preferably at least 92% by weight / weight, and more preferably at least 94% by weight / weight of total protein and - Up to 6% by weight / weight of water, - Containing lipids in an amount of up to 2% by weight / weight, preferably up to 0.5% by weight / weight, The BLG isolated powder is, -At least 0.2 g / cm³ 3 Preferably at least 0.3 g / cm³ 3 , and more preferably at least 0.4 g / cm³ 3 bulk density, - An intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.11, preferably at least 1.13. - A maximum protein denaturation degree of 10%, preferably 5%, more preferably 2%, and -It has a thermal stability of up to 50 NTU at pH 3.9, preferably up to 30 NTU, and more preferably up to 10 NTU.

[0147] In a more preferred embodiment of the present invention, the BLG isolation powder has a pH in the range of 6.1 to 8.5. -Total protein in an amount of at least 80% by weight / weight, preferably at least 90% by weight / weight, and more preferably at least 94% by weight / weight, -Beta-lactoglobulin (BLG) in an amount of at least 85% by weight, preferably at least 90% by weight, and more preferably at least 94% by weight of total protein relative to total protein. - Up to 6% by weight / weight of water, - Containing lipids in an amount of up to 2% by weight / weight, preferably up to 0.5% by weight / weight, The BLG isolated powder is, -At least 0.2 g / cm³ 3 Preferably at least 0.3 g / cm³ 3 , and more preferably at least 0.4 g / cm³ 3 bulk density, - A maximum protein denaturation degree of 10%, preferably 5%, more preferably 2%, and -It has a thermal stability of up to 50 NTU at pH 3.9, preferably up to 30 NTU, and more preferably up to 10 NTU.

[0148] In a more preferred embodiment of the present invention, the BLG isolation powder has a pH in the range of 6.1 to 8.5. -Total protein in an amount of at least 80% by weight / weight, preferably at least 90% by weight / weight, and more preferably at least 94% by weight / weight, -Beta-lactoglobulin (BLG) in an amount of at least 85% by weight, preferably at least 90% by weight, and more preferably at least 94% by weight of total protein relative to total protein. - Up to 6% by weight / weight of water, - Containing lipids in an amount of up to 2% by weight / weight, preferably up to 0.5% by weight / weight, The BLG isolated powder is, -At least 0.2 g / cm³ 3 Preferably at least 0.3 g / cm³ 3 , more preferably at least 0.4 g / cm³ 3 bulk density, - A maximum protein denaturation degree of 10%, preferably 5%, more preferably 2%, and -It has a thermal stability of up to 50 NTU at pH 3.9, preferably up to 30 NTU, and more preferably up to 10 NTU.

[0149] In a more preferred embodiment of the present invention, the BLG isolation powder has a pH in the range of 5.0 to 6.0. -Total protein in an amount of at least 80% by weight / weight, preferably at least 90% by weight / weight, and more preferably at least 94% by weight / weight, -Beta-lactoglobulin (BLG) in an amount of at least 85% by weight, preferably at least 90% by weight, and more preferably at least 94% by weight of total protein relative to total protein. - Up to 6% by weight / weight of water, - Containing lipids in an amount of up to 2% by weight / weight, preferably up to 0.5% by weight / weight, The BLG isolated powder is, -At least 0.2 g / cm³ 3 Preferably at least 0.3 g / cm³ 3 , more preferably at least 0.4 g / cm³ 3 bulk density, - A maximum protein denaturation degree of 10%, preferably 5%, more preferably 2%. -Thermal stability of up to 50 NTU at pH 3.9, preferably up to 30 NTU, and more preferably up to 10 NTU, and - Preferably, it has a BLG crystallinity of less than 10%.

[0150] In some preferred embodiments of the present invention, the BLG isolation powder has a pH in the range of i) 3.0 to 4.3, ii) 6.5 to 7.5, or iii) 5.0 to 6.0. -Total protein in an amount of at least 90% by weight / weight, preferably at least 92% by weight / weight, and more preferably at least 94% by weight / weight, -Beta-lactoglobulin (BLG) in an amount of at least 92% by weight / weight, preferably at least 94% by weight / weight, relative to total protein - Up to 6% by weight / weight of water, - Containing lipids in an amount of up to 0.5% by weight / weight, preferably up to 0.1% by weight / weight, The BLG isolated powder is, - At least an intrinsic tryptophan fluorescence emission ratio of 1.15 (I330 / I350) -Maximum protein denaturation degree of 5%, -Thermal stability of up to 40 NTU at pH 3.9, and -Having a maximum of 15,000 colony-forming units / g, preferably a maximum of 1,000 colony-forming units / g, more preferably a maximum of 100 colony-forming units / g, and most preferably the BLG isolation powder is sterile.

[0151] In some preferred embodiments of the present invention, the BLG isolation powder has a pH in the range of i) 3.0 to 4.3, ii) 6.5 to 7.5, or iii) 5.0 to 6.0. -Total protein in an amount of at least 90% by weight / weight, preferably at least 92% by weight / weight, and more preferably at least 94% by weight / weight, -Beta-lactoglobulin (BLG) in an amount of at least 92% by weight / weight, preferably at least 94% by weight / weight, relative to total protein - Up to 6% by weight / weight of water, - Containing lipids in an amount of up to 0.5% by weight / weight, preferably up to 0.1% by weight / weight, The BLG isolated powder is, -Maximum protein denaturation degree of 5%, -Thermal stability of up to 40 NTU at pH 3.9, and -Having a maximum of 15,000 colony-forming units / g, preferably a maximum of 1,000 colony-forming units / g, more preferably a maximum of 100 colony-forming units / g, and most preferably the BLG isolation powder is sterile.

[0152] In some preferred embodiments of the present invention, the BLG isolation powder has a pH in the range of i) 3.0 to 4.3, ii) 6.5 to 7.5, or iii) 5.0 to 6.0. -Total protein in an amount of at least 90% by weight / weight, preferably at least 92% by weight / weight, and more preferably at least 94% by weight / weight, -Beta-lactoglobulin (BLG) in an amount of at least 92% by weight / weight, preferably at least 94% by weight / weight, relative to total protein - Up to 6% by weight / weight of water, - Containing lipids in an amount of up to 0.5% by weight / weight, preferably up to 0.1% by weight / weight, The BLG isolated powder is, - At least an intrinsic tryptophan fluorescence emission ratio of 1.15 (I330 / I350) -Thermal stability of up to 40 NTU at pH 3.9, and -Having a maximum of 15,000 colony-forming units / g, preferably a maximum of 1,000 colony-forming units / g, more preferably a maximum of 100 colony-forming units / g, and most preferably the BLG isolation powder is sterile.

[0153] In some preferred embodiments of the present invention, the BLG isolation powder has a pH in the range of i) 3.0 to 4.3, ii) 6.5 to 7.5, or iii) 5.0 to 6.0. -Total protein in an amount of at least 90% by weight / weight, preferably at least 92% by weight / weight, and more preferably at least 94% by weight / weight, -Beta-lactoglobulin (BLG) in an amount of at least 92% by weight / weight, preferably at least 94% by weight / weight, relative to total protein - Up to 6% by weight / weight of water, - Containing lipids in an amount of up to 0.5% by weight / weight, preferably up to 0.1% by weight / weight, The BLG isolated powder is, - At least an intrinsic tryptophan fluorescence emission ratio of 1.15 (I330 / I350), -Maximum protein denaturation degree of 5%, and -Having a maximum of 15,000 colony-forming units / g, preferably a maximum of 1,000 colony-forming units / g, more preferably a maximum of 100 colony-forming units / g, and most preferably the BLG isolation powder is sterile.

[0154] In some preferred embodiments of the present invention, the BLG isolation powder has a pH in the range of i) 3.0 to 4.3, ii) 6.5 to 7.5, or iii) 5.0 to 6.0. -Total protein in an amount of at least 90% by weight / weight, preferably at least 92% by weight / weight, and more preferably at least 94% by weight / weight, -Beta-lactoglobulin (BLG) in an amount of at least 92% by weight, preferably at least 94% by weight, relative to total protein. - Up to 6% by weight / weight of water, - Containing lipids in an amount of up to 0.5% by weight / weight, preferably up to 0.1% by weight / weight, The BLG isolated powder is, - At least an intrinsic tryptophan fluorescence emission ratio of 1.15 (I330 / I350), -Maximum protein denaturation degree of 5%, and - It has a thermal stability of up to 40 NTU at pH 3.9.

[0155] In some preferred embodiments of the present invention, the BLG isolation powder has a pH in the range of i) 3.0 to 4.3, ii) 6.5 to 7.5, or iii) 5.0 to 6.0. -Total protein in an amount of at least 90% by weight / weight, preferably at least 92% by weight / weight, and more preferably at least 94% by weight / weight, -Beta-lactoglobulin (BLG) in an amount of at least 92% by weight / weight, preferably at least 94% by weight / weight, relative to total protein - Up to 6% by weight / weight of water, - Containing lipids in an amount of up to 0.5% by weight / weight, preferably up to 0.1% by weight / weight, The BLG isolated powder is, - At least an intrinsic tryptophan fluorescence emission ratio of 1.15 (I330 / I350) -Thermal stability of up to 40 NTU at pH 3.9, and -Having a maximum of 15,000 colony-forming units / g, preferably a maximum of 1,000 colony-forming units / g, more preferably a maximum of 100 colony-forming units / g, and most preferably the BLG isolation powder is sterile.

[0156] In some preferred embodiments of the present invention, the BLG isolation powder has a pH in the range of i) 3.0 to 4.3 or ii) 6.3 to 7.5, and the following: -Total protein in an amount of at least 30% by weight / weight, preferably at least 50% by weight / weight, and more preferably at least 80% by weight / weight, -Beta-lactoglobulin (BLG) in an amount of at least 90% by weight / weight, more preferably at least 94% by weight / weight relative to total protein, - Up to 6% by weight / weight of water, - Containing lipids in an amount of up to 0.5% by weight / weight, preferably up to 0.1% by weight / weight, The BLG isolated powder is, - At least an intrinsic tryptophan fluorescence emission ratio of 1.15 (I330 / I350), - A maximum protein denaturation degree of 5%, preferably a maximum of 2%, and -It has a thermal stability of up to 40 NTU at pH 3.9, preferably up to 20 NTU, and more preferably up to 10 NTU.

[0157] In some preferred embodiments of the present invention, the BLG isolation powder has a pH in the range of i) 3.0 to 4.3, or ii) 6.3 to 7.5. - At least 30% of total protein by weight / weight, -Beta-lactoglobulin (BLG) in an amount of at least 85% by weight / weight, preferably at least 90% by weight / weight, relative to total protein - Contains up to 6% by weight / weight of water, The BLG isolated powder is, - At least an intrinsic tryptophan fluorescence emission ratio of 1.15 (I330 / I350), -Maximum protein denaturation degree of 5%, and - It has a thermal stability of up to 40 NTU at pH 3.9.

[0158] In another preferred embodiment of the present invention, the BLG isolated powder has a pH in the range of 3.0 to 4.3. -Total protein in an amount of at least 90% by weight / weight, preferably at least 94% by weight / weight, -Beta-lactoglobulin (BLG) in an amount of at least 94% by weight / weight, preferably at least 96% by weight / weight, relative to total protein, - Up to 6% by weight / weight of water, - Contains lipids in an amount of up to 0.5% by weight / weight, preferably up to 0.1% by weight / weight. The BLG isolated powder is, -0.45~0.8 g / cm³ 3 Preferably 0.50 to 0.6 g / cm³ 3 bulk density, - At least an intrinsic tryptophan fluorescence emission ratio of 1.15 (I330 / I350), - A maximum protein denaturation degree of 5%, preferably a maximum of 2%, and - Preferably has a thermal stability of up to 30 NTU at pH 3.9, and more preferably up to 10 NTU.

[0159] In yet another preferred embodiment of the present invention, the BLG isolated powder has a pH in the range of 6.3 to 7.5. -Total protein in an amount of at least 90% by weight / weight, more preferably at least 94% by weight / weight, -Beta-lactoglobulin (BLG) in an amount of at least 94% by weight / weight, more preferably at least 96% by weight / weight, relative to total protein, - Up to 6% by weight / weight of water, - Containing lipids in an amount of up to 0.5% by weight / weight, preferably up to 0.1% by weight / weight, The BLG isolated powder is, - Bulk density 0.45~0.8 g / cm³ 3 Preferably 0.50 to 0.6 g / cm³ 3 , - A maximum protein denaturation degree of 10%, preferably 5%, more preferably 2%, and -It has a thermal stability of up to 50 NTU at pH 3.9, preferably up to 30 NTU, and more preferably up to 10 NTU.

[0160] In a more preferred embodiment of the present invention, the BLG isolation powder has a pH in the range of 5.0 to 6.0. -Total protein in an amount of at least 90% by weight / weight, more preferably at least 94% by weight / weight, -Beta-lactoglobulin (BLG) in an amount of at least 90% by weight / weight, more preferably at least 94% by weight / weight, relative to total protein, - Up to 6% by weight / weight of water, - Containing lipids in an amount of up to 0.5% by weight / weight, preferably up to 0.1% by weight / weight, The BLG isolated powder is, - Bulk density 0.45~0.8 g / cm³ 3, preferably 0.50 - 0.6 g / cm 3 , - A degree of protein denaturation of up to 10%, preferably up to 5%, more preferably up to 2%, - A heat stability of up to 50 NTU at pH 3.9, preferably up to 30 NTU, even more preferably up to 10 NTU, and - A degree of BLG crystallization of preferably less than 10%, more preferably at most 1%.

[0161] A further aspect of the present invention relates to a method for producing a dry BLG isolate powder comprising BLG in an amount of at least 85% weight / weight based on the total protein, a) Providing a liquid BLG isolate having: i) A pH in the range of 2 - 4.9, ii) A pH in the range of 6.1 - 8.5, or iii) A pH in the range of 5.0 - 6.0, wherein the liquid BLG isolate comprises BLG in an amount of at least 85 weight / weight based on the total protein, wherein the liquid BLG isolate comprises BLG in an amount of at least 85 weight / weight based on the total protein, b) Optionally, subjecting the liquid BLG isolate to physical microbial reduction, c) Drying the liquid BLG isolate by spray drying.

[0162] The liquid BLG isolate is preferably an edible composition.

[0163] The liquid BLG isolate is preferably prepared from mammalian milk, preferably from ruminant milk such as bovine, sheep, goat, buffalo, camel, llama, horse and / or deer milk. Proteins derived from bovine milk are particularly preferred. Thus, BLG is preferably bovine BLG.

[0164] In some preferred embodiments of the present invention, the liquid BLG isolate comprises BLG in an amount of at least 92% w / w, preferably at least 95% w / w, more preferably at least 97% w / w, even more preferably at least 98%, most preferably at least 99.5% w / w, based on the total protein.

[0165] In some preferred embodiments of the present invention, the liquid BLG isolate comprises BLG in an amount of at least 97.5% w / w, preferably at least 98.0% w / w, more preferably at least 98.5% w / w, even more preferably at least 99.0%, most preferably at least 99.7% w / w, for example, about 100.0% w / w, based on the total protein.

[0166] In some preferred embodiments of the present invention, the liquid BLG isolate comprises total protein in an amount of at least 5% w / w, preferably at least 10% w / w, more preferably at least 15% w / w, even more preferably at least 20%, most preferably at least 30% w / w.

[0167] In some preferred embodiments of the present invention, the liquid BLG isolate comprises total protein in the range of 5 - 40% w / w, preferably 10 - 35% w / w, more preferably 15 - 30% w / w, even more preferably 20 - 25% w / w.

[0168] The inventors have observed that an increase in the BLG concentration in the liquid BLG isolate results in a spray-dried powder having a higher bulk density, and thus it is preferred to have a relatively high concentration of BLG in the liquid BLG isolate.

[0169] Therefore, in another preferred embodiment of the present invention, the liquid BLG isolate contains an amount of total protein in the range of 10-40% by weight / weight, preferably 20-38% by weight / weight, more preferably 24-36% by weight / weight, and even more preferably 28-34% by weight / weight.

[0170] In some preferred embodiments of the present invention, the total of alpha-lactalbumin (ALA) and caseinomacropeptide (CMP) constitutes at least 40% by weight of non-BLG proteins in the liquid BLG isolate, preferably at least 60% by weight of non-BLG proteins in the liquid BLG isolate, more preferably at least 70% by weight, and most preferably at least 90% by weight.

[0171] In some preferred embodiments of the present invention, ALA constitutes up to 80% by weight of non-BLG proteins of the liquid BLG isolate, preferably up to 60% by weight of non-BLG proteins of the liquid BLG isolate, more preferably up to 40% by weight, and most preferably up to 30% by weight.

[0172] Even lower ALA content may be preferred, and in some preferred embodiments of the present invention, ALA constitutes up to 20% by weight of non-BLG proteins in the liquid BLG isolate, preferably up to 15% by weight of non-BLG proteins in the liquid BLG isolate powder, more preferably up to 10% by weight, and most preferably up to 5% by weight.

[0173] In another preferred embodiment of the present invention, each major non-BLG whey protein in the liquid BLG isolate is present as a weight percentage of the total protein, which is up to 25%, preferably up to 20%, more preferably up to 15%, even more preferably up to 10%, and most preferably up to 6% of its weight percentage of the total protein in a standard whey protein concentrate derived from sweet whey.

[0174] Even lower concentrations of major non-BLG whey proteins may be desirable. Therefore, in an additional preferred embodiment of the present invention, each major non-BLG whey protein in the liquid BLG isolate is present as a weight percentage of the total protein, which is up to 4%, preferably up to 3%, more preferably up to 2%, and even more preferably up to 1%, of its weight percentage of the total protein in a standard whey protein concentrate derived from sweet whey.

[0175] The inventors have observed that low levels of lactoferrin and / or lactoperoxidase are particularly advantageous in obtaining achromatic whey protein products.

[0176] Therefore, in some preferred embodiments of the present invention, lactoferrin is present in the liquid BLG isolate as a weight percentage of total protein, which is up to 25%, preferably up to 20%, more preferably up to 15%, even more preferably up to 10%, and most preferably up to 6% of its weight percentage of total protein in a standard whey protein concentrate derived from sweet whey. Even lower concentrations of lactoferrin may be desirable. Therefore, in an additional preferred embodiment of the present invention, lactoferrin is present as a weight percentage of total protein, which is up to 4%, preferably up to 3%, more preferably up to 2%, and even more preferably up to 1% of its weight percentage of total protein in a standard whey protein concentrate derived from sweet whey.

[0177] Similarly, in some preferred embodiments of the present invention, lactoperoxidase is present in the liquid BLG isolate as a weight percentage of total protein, which is up to 25%, preferably up to 20%, more preferably up to 15%, even more preferably up to 10%, and most preferably up to 6% of its weight percentage of total protein in a standard whey protein concentrate derived from sweet whey. Even lower concentrations of lactoperoxidase may be desirable. Therefore, in an additional preferred embodiment of the present invention, lactoperoxidase is present as a weight percentage of total protein, which is up to 4%, preferably up to 3%, more preferably up to 2%, and even more preferably up to 1% of its weight percentage of total protein in a standard whey protein concentrate derived from sweet whey.

[0178] In some preferred embodiments of the present invention, the liquid BLG isolate comprises a total solid content in the range of 5-50% by weight, preferably 10-40% by weight, more preferably 15-35% by weight, and even more preferably 20-30% by weight.

[0179] The fraction of liquid BLG isolate that does not contribute to the total solids is preferably essentially water, or even more specifically, water.

[0180] In some preferred embodiments of the present invention, the liquid BLG isolate contains a water content in the range of 50-95% by weight, preferably 60-90% by weight, more preferably 65-85% by weight, and even more preferably 70-80% by weight.

[0181] In some preferred embodiments of the present invention, the liquid BLG isolate contains up to 60% by weight, preferably up to 50% by weight, more preferably up to 20% by weight, even more preferably up to 10% by weight, even more preferably up to 1% by weight, and most preferably up to 0.1% of carbohydrates. For example, the liquid BLG isolate may contain carbohydrates such as lactose, oligosaccharides and / or hydrolysis products of lactose (i.e., glucose and galactose), sucrose and / or maltodextrin.

[0182] In some preferred embodiments of the present invention, the liquid BLG isolate contains lipids in an amount of up to 10% by weight, preferably up to 5% by weight, more preferably up to 2% by weight, and even more preferably up to 0.1% by weight.

[0183] The inventors have found that it may be advantageous to control the mineral content of some of the liquid BLG isolates to achieve desired properties.

[0184] In some preferred embodiments of the present invention, the total amount of Na, K, Mg, and Ca in the liquid BLG isolate is up to 10 mmol / g protein. Preferably, the total amount of Na, K, Mg, and Ca in the liquid BLG isolate is up to 6 mmol / g protein, more preferably up to 4 mmol / g protein, and even more preferably up to 2 mmol / g protein.

[0185] In another preferred embodiment of the present invention, the total amount of Na, K, Mg, and Ca in the liquid BLG isolate is a maximum of 1.0 mmol / g protein. Preferably, the total amount of Na, K, Mg, and Ca in the liquid BLG isolate is a maximum of 0.6 mmol / g protein, more preferably a maximum of 0.4 mmol / g protein, even more preferably a maximum of 0.2 mmol / g protein, and most preferably a maximum of 0.1 mmol / g protein.

[0186] In other preferred embodiments of the present invention, the total amount of Mg and Ca in the liquid BLG isolate is at most 5 mmol / g protein. Preferably, the total amount of Mg and Ca in the liquid BLG isolate is at most 3 mmol / g protein, more preferably at most 1.0 mmol / g protein, even more preferably at most 0.5 mmol / g protein.

[0187] In other preferred embodiments of the present invention, the total amount of Mg and Ca in the liquid BLG isolate is at most 0.3 mmol / g protein. Preferably, the total amount of Mg and Ca in the liquid BLG isolate is at most 0.2 mmol / g protein, more preferably at most 0.1 mmol / g protein, even more preferably at most 0.03 mmol / g protein, and most preferably at most 0.01 mmol / g protein.

[0188] The inventors have found that it is possible to produce a low-phosphorus / low-potassium variant of BLG isolate powder that is particularly useful for patients with kidney disease. To produce such a product, the phosphorus and potassium content of the liquid BLG isolate must be equally low.

[0189] Therefore, in some preferred embodiments of the present invention, the liquid BLG isolate has a total phosphorus content of at most 100 mg per 100 g of protein. Preferably, the liquid BLG isolate has a total phosphorus content of at most 80 mg per 100 g of protein. Preferably, the liquid BLG isolate has a total phosphorus content of at most 50 mg per 100 g of protein. Even more preferably, the liquid BLG isolate has a total phosphorus content of at most 20 mg per 100 g of protein. The liquid BLG isolate has a total phosphorus content of at most 5 mg per 100 g of protein.

[0190] In some preferred embodiments of the present invention, the liquid BLG isolate contains up to 600 mg of potassium per 100 g of protein. More preferably, the liquid BLG isolate contains up to 500 mg of potassium per 100 g of protein. More preferably, the liquid BLG isolate contains up to 400 mg of potassium per 100 g of protein. More preferably, the liquid BLG isolate contains up to 300 mg of potassium per 100 g of protein. Even more preferably, the liquid BLG isolate contains up to 200 mg of potassium per 100 g of protein. Even more preferably, the liquid BLG isolate contains up to 100 mg of potassium per 100 g of protein. Even more preferably, the liquid BLG isolate contains up to 50 mg of potassium per 100 g of protein, and even more preferably, the liquid BLG isolate contains up to 10 mg of potassium per 100 g of protein.

[0191] The phosphorus content is determined according to Example 1.19 with respect to the total amount of elemental phosphorus in the composition in question. Similarly, the potassium content is determined according to Example 1.19 with respect to the total amount of elemental potassium in the composition in question.

[0192] In some preferred embodiments of the present invention, the liquid BLG isolate contains a maximum of 100 mg of phosphorus / 100 g of protein and a maximum of 700 mg of potassium / 100 g of protein, preferably a maximum of 80 mg of phosphorus / 100 g of protein and a maximum of 600 mg of potassium / 100 g of protein, more preferably a maximum of 60 mg of phosphorus / 100 g of protein and a maximum of 500 mg of potassium / 100 g of protein, more preferably a maximum of 50 mg of phosphorus / 100 g of protein and a maximum of 400 mg of potassium / 100 g of protein, or more preferably a maximum of 20 mg of phosphorus / 100 g of protein and a maximum of 200 mg of potassium / 100 g of protein, or even more preferably a maximum of 10 mg of phosphorus / 100 g of protein and a maximum of 50 mg of potassium / 100 g of protein. In some preferred embodiments of the present invention, the liquid BLG isolate contains a maximum of 100 mg of phosphorus / 100 g of protein and a maximum of 340 mg of potassium / 100 g of protein.

[0193] The low-phosphorus and / or low-potassium composition according to the present invention can be used as a food ingredient for producing food products for a group of patients with impaired renal function.

[0194] In some preferred embodiments of the present invention, the liquid BLG isolate has a pH in the range of 2 to 4.9, preferably 2.5 to 4.7, more preferably 2.8 to 4.4, even more preferably 3.0 to 4.0, and most preferably 3.4 to 3.9.

[0195] In another preferred embodiment of the present invention, the liquid BLG isolate has a pH in the range of 6.1 to 8.5, preferably 6.2 to 8.0, more preferably 6.3 to 7.7, and even more preferably 6.5 to 7.5.

[0196] In yet another preferred embodiment of the present invention, the liquid BLG isolate has a pH in the range of 5.0 to 6.0, preferably 5.1 to 5.9, more preferably 5.2 to 5.8, and even more preferably 5.3 to 5.7. When the liquid BLG isolate is in the pH range of 5.0 to 6.0, it is often preferable that the liquid BLG isolate does not contain BLG crystals. This can be achieved, for example, by increasing the temperature and / or adding salt, to ensure that the liquid BLG isolate is below the saturation point of BLG. Alternatively, it is possible to keep the liquid BLG isolate crystal-free even if it is supersaturated with respect to BLG, as long as it is kept in a metastable zone and no crystallization accelerator comes into contact with the liquid BLG isolate.

[0197] Liquid BLG isolates preferably have a low microbial content, which is especially possible when the BLG concentrate composition already contains few microorganisms.

[0198] In some embodiments of the present invention, the liquid BLG isolate contains up to 500,000 CFU / g, preferably up to 100,000 CFU / g, more preferably up to 50,000 CFU / g, and even more preferably up to 10,000 CFU / g.

[0199] Therefore, in some preferred embodiments of the present invention, the liquid BLG isolate contains up to 1000 colony-forming units (CFU) / g. Preferably, the liquid BLG isolate contains up to 600 CFU / g. More preferably, the liquid BLG isolate contains up to 300 CFU / g. Even more preferably, the liquid BLG isolate contains up to 100 CFU / g. Even more preferably, the liquid BLG isolate contains up to 50 CFU / g. Most preferably, the liquid BLG isolate contains up to 20 CFU / gm, for example, up to 10 CFU / g. In particularly preferred embodiments, the powder is sterile. Sterile liquid BLG isolate can be prepared, for example, by combining several physical microbial reduction processes during the production of the BLG isolate powder, such as microfiltration at a low pH (e.g., up to pH 4.0) and heat treatment.

[0200] Since BLG unfolding appears to be an irreversible process, preparing a BLG isolation powder with a low degree of protein unfolding requires that the liquid BLG isolate already has a low degree of protein unfolding.

[0201] When a BLG isolation powder or liquid BLG isolate with a low degree of BLG unfolding is required, the liquid BLG isolate preferably has an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.11.

[0202] In some preferred embodiments of the present invention, the liquid BLG isolate has an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.12, preferably at least 1.13, more preferably at least 1.15, even more preferably at least 1.17, and most preferably at least 1.19.

[0203] If the liquid BLG isolate contains a significant amount of non-protein material, it is preferable to isolate the protein fraction before measuring the intrinsic tryptophan fluorescence emission ratio. Therefore, in some preferred embodiments of the present invention, the protein fraction of the liquid BLG isolate has an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.11.

[0204] Preferably, the protein fraction of the liquid BLG isolate may have an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.12, more preferably at least 1.13, even more preferably at least 1.15, even more preferably at least 1.17, and most preferably at least 1.19.

[0205] For example, the protein fraction is separated from the BLG isolation powder by dialysis or ultrafiltration-based dialysis of the solution using a filter that retains proteins.

[0206] Since BLG protein denaturation appears to be an irreversible process, the preparation of BLG isolation powder with a low degree of protein unfolding requires that the liquid BLG isolate already has a low degree of protein denaturation. Therefore, in some preferred embodiments of the present invention, the liquid BLG isolate has a degree of protein denaturation of up to 10% by weight, preferably up to 6% by weight, more preferably up to 4% by weight, even more preferably up to 2%, and most preferably up to 1% by weight.

[0207] It is often preferable that a substantial amount of BLG in the liquid BLG isolate is non-aggregating BLG. Preferably, at least 50% of the BLG is non-aggregating BLG. More preferably, at least 80% of the BLG is non-aggregating BLG. Even more preferably, at least 90% of the BLG is non-aggregating BLG. Most preferably, at least 95% of the BLG is non-aggregating BLG. Even more preferably, approximately 100% of the BLG in the liquid BLG isolate is non-aggregating BLG.

[0208] However, it may also be preferable for the liquid BLG isolate powder to have a significant level of protein denaturation, for example, when an opaque beverage is required. Therefore, in another preferred embodiment of the present invention, the BLG isolate powder has a degree of protein denaturation of at least 11%, preferably at least 20%, more preferably at least 40%, even more preferably at least 50%, even more preferably at least 75%, and most preferably at least 90%.

[0209] If the liquid BLG isolate has a significant level of protein denaturation, it is often preferable to maintain a low level of insoluble protein material, i.e., precipitated protein material that settles in the beverage during storage. The level of insoluble material is measured according to Example 1.10.

[0210] In some preferred embodiments of the present invention, the liquid BLG isolate contains up to 20% by weight of insoluble protein material, preferably up to 10% by weight of insoluble protein material, more preferably up to 5% by weight of insoluble protein material, even more preferably up to 3% by weight of insoluble protein material, and most preferably up to 1% by weight of insoluble protein. It may be even more preferable that the liquid BLG isolate contains no insoluble protein material at all.

[0211] As described above, the inventors found that the thermal stability of the liquid BLG isolate at pH 3.9 is a good indicator of its usefulness in clear, high-protein beverages. The thermal stability at pH 3.9 is measured according to Example 1.2.

[0212] The liquid BLG isolate has a thermal stability of up to 200 NTU at pH 3.9, preferably up to 100 NTU, more preferably up to 60 NTU, even more preferably up to 40 NTU, and most preferably up to 20 NTU. Even better thermal stability is possible, and the liquid BLG isolate preferably has a thermal stability of up to 10 NTU at pH 3.9, preferably up to 8 NTU, more preferably up to 4 NTU, and even more preferably up to 2 NTU.

[0213] In some preferred embodiments of the present invention, the liquid BLG isolate has a turbidity of up to 200 NTU, preferably up to 100 NTU, more preferably up to 50 NTU, even more preferably up to 20 NTU, even more preferably up to 10 NTU, and most preferably up to 2 NTU.

[0214] In another preferred embodiment of the present invention, the liquid BLG isolate has a turbidity of more than 200 NTU, preferably at least 400 NTU, more preferably at least 800 NTU, even more preferably at least 1000 NTU, even more preferably at least 2000 NTU, and most preferably at least 5000 NTU. Such a liquid BLG isolate is particularly preferred for the production of opaque beverages.

[0215] The inventors observed that the liquid BLG isolate of the present invention surprisingly has a lower viscosity than comparable liquid WPI. The inventors found that this makes it possible to obtain a high protein content without experiencing unpleasant high viscosity, and therefore the liquid BLG isolate is particularly suitable as a high-protein beverage.

[0216] In some preferred embodiments of the present invention, the liquid BLG isolate has a viscosity (p) ±50%, more preferably (p) ±40%, even more preferably (p) ±30%, and most preferably (p) ±25% at 15 degrees Celsius and 300 s. -1 It has a shear rate of .

[0217] Viscosity (p) is defined as follows: When viscosity (p) = p ≤ 23%: 0.3556e 0.1262*p; If p > 23%: 0.0254 * e 0.24*p

[0218] Since p is the total protein content of the liquid BLG isolate expressed as % weight / weight, if the protein content is 31% weight / weight, then p is 31.

[0219] This is because the liquid BLG isolate (having protein content p) has a viscosity (p) of ±25% at 15 degrees Celsius and 300 s. -1 If the shear measure is such that the viscosity of the liquid BLG isolate is at least viscosity(p)-25% and the maximum viscosity(p)+25%. If the protein content p of the liquid BLG isolate is, for example, 31% wt / kg, then the minimum and maximum viscosities in this example are as follows: Minimum viscosity (cP): 0.0254*e 0.24*31 -25% = 43 cP - 25% = 32 cP Maximum viscosity (cP): 0.0254*e 0.24*31 +25% = 43cP - 25% = 54cP

[0220] In some other preferred embodiments of the present invention, the liquid BLG isolate has a viscosity (p) ±20%, more preferably (p) ±15%, even more preferably (p) ±10%, and most preferably (p) ±5% at 15 degrees Celsius and 300 s. -1 It has a shear rate of .

[0221] The viscosity of the liquid BLG isolate is measured according to Example 1.8, but at a temperature of 15 degrees Celsius and 300 seconds. -1 Use the following shear rate.

[0222] In some preferred embodiments of the present invention, the liquid BLG isolate has the following viscosity at 15 degrees Celsius and 300 s -1 Having a shear rate of: -at least viscosity (p) -20%, and -Maximum viscosity 最大 (p) -20%.

[0223] In another preferred embodiment of the present invention, the liquid BLG isolate has the following viscosity at 15 degrees Celsius and 300 s -1 Having a shear rate of: -at least viscosity (p)-10%, and -Maximum viscosity 最大 (p) -40%.

[0224] In another preferred embodiment of the present invention, the liquid BLG isolate has the following viscosity at 15 degrees Celsius and shear rate of 300 s -1 : - at least viscosity (p) - 10%, and - maximum viscosity 最大 (p) - 50%.

[0225] Viscosity 最大 (p) is defined as follows: Viscosity 最大 (p) ≦ 0.611 * e (0.1494*p) cP.

[0226] In another preferred embodiment of the present invention, the liquid BLG isolate has a viscosity at 15 degrees Celsius and shear rate of 300 s of maximum viscosity 最大 (p) - 10%, more preferably maximum viscosity 最大 (p) - 20% and has a shear measure at 15 degrees Celsius, and viscosity -1 (p) - 30% is even more preferred, and viscosity 最大 (p) - 50% is most preferred.

[0227] In some preferred embodiments of the present invention, the liquid BLG isolate has a pH in the range of 2.8 to 4.3, preferably 3.0 to 4.0, and the following: - a total protein in an amount of 20 to 34% weight / weight, more preferably 24 to 32% weight / weight, even more preferably 28 to 32% weight / weight, and - at least 90% weight / weight, more preferably at least 94% weight / weight of beta-lactoglobulin (BLG) based on the total protein, and comprises The BLG isolate powder preferably has the following: - an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.15, - a maximum protein denaturation degree of 2%, - a thermal stability of a maximum of 20 NTU at pH 3.9, - is sterile, and - a viscosity at 15 degrees Celsius of viscosity (p) ± 25%, where p is the protein content in % weight / weight, and viscosity (p) = 0.3556e when p ≦ 23% 0.1262*p ​​;If p>23%, then 0.0254*e 0.24*p It has one or more of the following.

[0228] In another preferred embodiment of the present invention, the liquid BLG isolate has a pH in the range of 6.3 to 8.0, preferably 6.5 to 7.5, and the following: -20-34% by weight / weight, more preferably 24-32% by weight / weight, and even more preferably 28-30% by weight / weight of total protein, - comprising at least 90% by weight, more preferably at least 94% by weight, of beta-lactoglobulin (BLG) relative to total protein, The BLG isolation powder is preferably as follows: - At least an intrinsic tryptophan fluorescence emission ratio of 1.15 (I330 / I350), -Maximum protein denaturation degree of 5%, -Thermal stability of up to 40 NTU at pH 3.9, - Sterile, and -Viscosity(p) ±25% at 15 degrees Celsius, where p is the protein content in units of % by weight / weight, and viscosity(p) = 0.3556e when p ≤ 23%. 0.1262*p ;If p>23%, then 0.0254*e 0.24*p It has one or more of the following.

[0229] Such acidic, high-protein liquid BLG isolates are particularly useful for producing high-quality BLG isolate powders, and their remarkably low viscosity compared to equivalent WPIs reduces energy consumption for processes such as microfiltration. Furthermore, acidic, high-protein liquid BLG isolates enable the production of acidic whey protein powders with a much higher bulk density than can be achieved with conventional WPIs having the same protein content (see, for example, Example 7).

[0230] Liquid BLG isolates can be provided in several different ways.

[0231] Typically, providing a liquid BLG isolate involves, or even consists of, separating BLG from a whey protein feed to provide a BLG-enriched composition by one or more of the following methods: - Crystallization or precipitation of BLG by salt dissolution, - Crystallization or precipitation of BLG by salting out, - Ion exchange chromatography, and - Fractionation of whey protein by ultrafiltration.

[0232] A particularly preferred method for providing a BLG concentrated composition is by crystallization of BLG, preferably by salt solubility or by salting out.

[0233] The whey protein feed is preferably WPC, WPI, SPC, SPI, or a combination thereof.

[0234] The term "whey protein feed" refers to compositions derived from BLG concentrates and subsequent liquid BLG isolates. The chemical characteristics and embodiments described in the context of whey protein solutions also apply to whey protein feed, except that whey protein feed is typically not supersaturated with respect to BLG and the pH of the feed is not limited to the range of 5–6.

[0235] In some embodiments of the present invention, the preparation of the BLG concentrated composition includes, or comprises, the crystallization of high-salt BLG in the pH range of 3.6 to 4.0 according to U.S. Patent No. 2,790,790 A1.

[0236] In other embodiments of the present invention, the preparation of the BLG concentrated composition includes, or comprises, the method described by 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) or Vyas et al (Scale-Up of Native β-Lactoglobulin Affinity Separation Process, J. Dairy Sci. 85:1639-1645, 2002).

[0237] However, in a particularly preferred embodiment of the present invention, the BLG concentrate is prepared by crystallization at pH 5-6 under the salt solubility conditions described in PCT application PCT / EP2017 / 084553, which is incorporated herein by reference for all purposes.

[0238] In some preferred embodiments of the present invention, the BLG concentrate is an edible BLG composition according to PCT / EP2017 / 084553, comprising at least 90% BLG relative to total protein, and preferably containing BLG crystals.

[0239] Preferably, the BLG concentrate composition is as follows: 1) A step of providing a whey protein solution comprising non-aggregating BLG and at least one additional whey protein, wherein the whey protein solution is supersaturated with respect to BLG and has a pH in the range of 5 to 6. 2) A step of crystallizing non-aggregating BLG in a supersaturated whey protein solution. 3) A step to separate BLG crystals from the remaining whey protein solution. 4) Optionally, a step of washing BLG crystals, for example, BLG crystals obtained from step 3) or 5), and 5) Optionally, the BLG crystals are prepared by a process that includes, for example, a step of recrystallizing the BLG crystals obtained in step 3) or 4).

[0240] This process for preparing the BLG concentrated composition comprises the essential steps 1), 2), and 3) in that order, and optionally includes steps 4) and / or 5) in any order and number of iterations. However, steps 4) and 5) often follow step 3). Alternatively, or further, washing water may be added to the crystal-containing whey protein solution before separation.

[0241] This process may further include a step of drying the BLG concentrate. However, currently, it is preferable to use the BLG concentrate without drying to avoid the risk of damaging the protein during drying.

[0242] As described above, step 1) of the crystallization process includes providing a whey protein solution containing non-aggregating BLG and at least additional whey protein.

[0243] The whey protein solution preferably comprises at least one additional non-aggregating whey protein selected from the group consisting of alpha-lactalbumin, bovine serum albumin, immunoglobulin, caseinomacropeptide (CMP), osteopontin, lactoferrin, lactoperoxidase, milk fat globule membrane protein, and combinations thereof.

[0244] In some embodiments of the present invention, the whey protein solution contains up to 10% by weight of casein, preferably up to 5% by weight of casein, more preferably up to 1% by weight of casein, and even more preferably up to 0.5% by weight of casein, relative to the total amount of protein. In some preferred embodiments of the present invention, the whey protein solution contains no detectable amount of casein at all.

[0245] In some preferred embodiments of the present invention, the whey protein solution of step 1) contains at least 5% by weight of additional whey protein relative to the total amount of protein. Preferably, the whey protein solution of step 1) contains at least 10% by weight of additional whey protein relative to the total amount of protein. More preferably, the whey protein solution of step 1) contains at least 15% by weight of additional whey protein relative to the total amount of protein.

[0246] More preferably, the whey protein solution of step 1) contains at least 20% by weight of additional whey protein relative to the total amount of protein. Most preferably, the whey protein solution of step 1) may contain at least 30% by weight of additional whey protein relative to the total amount of protein.

[0247] In another preferred embodiment of the present invention, the whey protein solution of step 1) contains at least 1% by weight of additional whey protein relative to the total amount of protein. Preferably, the whey protein solution of step 1) contains at least 2% by weight of additional whey protein relative to the total amount of protein. Even more preferably, the whey protein solution of step 1) contains at least 3% by weight of additional whey protein relative to the total amount of protein. Most preferably, the whey protein solution of step 1) may contain at least 4% by weight of additional whey protein relative to the total amount of protein.

[0248] In yet another preferred embodiment of the present invention, the whey protein solution of step 1) contains at least 35% by weight of additional whey protein relative to the total amount of protein. Preferably, the whey protein solution of step 1) may contain at least 40% by weight of additional whey protein relative to the total amount of protein. More preferably, the whey protein solution of step 1) may contain, for example, at least 45% by weight of additional whey protein relative to the total amount of protein. Even more preferably, the whey protein solution of step 1) may contain at least 50% by weight of additional whey protein relative to the total amount of protein.

[0249] In some preferred embodiments of the present invention, the whey protein solution of step 1) contains additional whey protein in the range of 5 to 90% by weight relative to the total amount of protein. Preferably, the whey protein solution of step 1) may contain additional whey protein in the range of 10 to 80% by weight relative to the total amount of protein. The whey protein solution of step 1) may contain, for example, 20 to 70% by weight relative to the total amount of protein. Preferably, the whey protein solution of step 1) contains additional whey protein in the range of 30 to 70% by weight relative to the total amount of protein.

[0250] As described above, the inventors have found that it is possible to crystallize non-aggregating BLG without using organic solvents. This purification approach can also be used to purify preparations containing whey protein, which have already been subjected to BLG purification and provide a simple process to further increase the purity of non-aggregating BLG. Therefore, in some preferred embodiments of the present invention, the whey protein solution of step 1) contains additional whey protein in the range of 1 to 20% by weight relative to the total amount of protein. Preferably, the whey protein solution of step 1) may contain additional whey protein in the range of 2 to 15% by weight relative to the total amount of protein. Even more preferably, the whey protein solution of step 1) may contain, for example, 3 to 10% by weight relative to the total amount of protein.

[0251] In some embodiments of the present invention, the whey protein solution of step 1) contains at least 5% by weight of non-aggregating ALA relative to the total amount of protein. Preferably, the whey protein solution of step 1) contains at least 10% by weight of non-aggregating ALA relative to the total amount of protein. More preferably, the whey protein solution of step 1) contains at least 15% by weight of non-aggregating ALA relative to the total amount of protein. Alternatively, the whey protein solution of step 1) may contain at least 20% by weight of non-aggregating ALA relative to the total amount of protein.

[0252] In some preferred embodiments of the present invention, the whey protein solution of step 1) contains at least 25% by weight of non-aggregating ALA relative to the total amount of protein. Preferably, the whey protein solution of step 1) contains at least 30% by weight of non-aggregating ALA relative to the total amount of protein. Preferably, the whey protein solution of step 1) contains at least 35% by weight of non-aggregating ALA relative to the total amount of protein. Even more preferably, the whey protein solution of step 1) may contain at least 40% by weight of non-aggregating ALA relative to the total amount of protein.

[0253] In some preferred embodiments of the present invention, the whey protein solution of step 1) contains non-aggregating ALA in an amount ranging from 5 to 95% by weight relative to the total amount of protein. Preferably, the whey protein solution of step 1) contains non-aggregating ALA in an amount ranging from 5 to 70% by weight relative to the total amount of protein. More preferably, the whey protein solution of step 1) may contain non-aggregating ALA in an amount ranging from 10 to 60% by weight relative to the total amount of protein. Preferably, the whey protein solution of step 1) contains non-aggregating ALA in an amount ranging from 12 to 50% by weight relative to the total amount of protein. More preferably, the whey protein solution of step 1) may contain non-aggregating ALA in an amount ranging from 20 to 45% by weight relative to the total amount of protein.

[0254] In some preferred embodiments of the present invention, the whey protein solution of step 1) has a weight ratio of at least 0.01 between non-aggregating BLG and non-aggregating ALA. Preferably, the whey protein solution of step 1) has a weight ratio of at least 0.5 between non-aggregating BLG and non-aggregating ALA. Even more preferably, the whey protein solution of step 1) has a weight ratio of at least 1, for example, at least 2, between non-aggregating BLG and non-aggregating ALA. For example, the whey protein solution of step 1) may have a weight ratio of at least 3 between non-aggregating BLG and non-aggregating ALA.

[0255] In some preferred embodiments of the present invention, the whey protein solution of step 1) has a weight ratio of non-aggregating BLG to non-aggregating ALA in the range of 0.01 to 20. Preferably, the whey protein solution of step 1) has a weight ratio of non-aggregating BLG to non-aggregating ALA in the range of 0.2 to 10. Even more preferably, the whey protein solution of step 1) has a weight ratio of non-aggregating BLG to non-aggregating ALA in the range of 0.5 to 4. For example, the whey protein solution of step 1) may have a weight ratio of non-aggregating BLG to non-aggregating ALA in the range of 1 to 3.

[0256] In some preferred embodiments of the present invention, the whey protein solution of step 1) contains at least 1% by weight of non-aggregating BLG relative to the total amount of protein. Preferably, the whey protein solution of step 1) contains at least 2% by weight of non-aggregating BLG relative to the total amount of protein. More preferably, the whey protein solution of step 1) contains at least 5% by weight of non-aggregating BLG relative to the total amount of protein. Preferably, the whey protein solution of step 1) may contain at least 10% by weight of non-aggregating BLG relative to the total amount of protein.

[0257] In some preferred embodiments of the present invention, the whey protein solution of step 1) contains at least 12% by weight of non-aggregating BLG relative to the total amount of protein. For example, the whey protein solution of step 1) may contain at least 15% by weight of non-aggregating BLG relative to the total amount of protein. The whey protein solution of step 1) may contain, for example, at least 20% by weight of non-aggregating BLG relative to the total amount of protein. Alternatively, the whey protein solution of step 1) may contain at least 30% by weight of non-aggregating BLG relative to the total amount of protein.

[0258] In some particularly preferred embodiments of the present invention, the whey protein solution of step 1) contains non-aggregating BLG at a maximum weight / weight of 95% of the total protein. Preferably, the whey protein solution of step 1) may contain non-aggregating BLG at a maximum weight / weight of 90% of the total protein. More preferably, the whey protein solution of step 1) may contain, for example, non-aggregating BLG at a maximum weight / weight of 85% of the total protein. Even more preferably, the whey protein solution of step 1) may contain, for example, non-aggregating BLG at a maximum weight / weight of 80% of the total protein. Preferably, the whey protein solution of step 1) may contain non-aggregating BLG at a maximum weight / weight of 78% of the total protein. Preferably, the whey protein solution of step 1) may contain non-aggregating BLG at a maximum weight / weight of 75% of the total protein.

[0259] In some preferred embodiments of the present invention, the whey protein solution of step 1) contains non-aggregating BLG in an amount ranging from 1 to 95% by weight relative to the total amount of protein. Preferably, the whey protein solution of step 1) may contain non-aggregating BLG in an amount ranging from 5 to 90% by weight relative to the total amount of protein. More preferably, the whey protein solution of step 1) contains non-aggregating BLG in an amount ranging from 10 to 85% by weight relative to the total amount of protein. Even more preferably, the whey protein solution of step 1) contains non-aggregating BLG in an amount ranging from 10 to 80% by weight relative to the total amount of protein. Most preferably, the whey protein solution of step 1) may contain non-aggregating BLG in an amount ranging from 20 to 70% by weight relative to the total amount of protein.

[0260] In another preferred embodiment of the present invention, the whey protein solution of step 1) contains non-aggregating BLG in an amount ranging from 10 to 95% by weight relative to the total amount of protein. Preferably, the whey protein solution of step 1) may contain non-aggregating BLG in an amount ranging from 12 to 90% by weight relative to the total amount of protein. More preferably, the whey protein solution of step 1) contains non-aggregating BLG in an amount ranging from 15 to 85% by weight relative to the total amount of protein. Even more preferably, the whey protein solution of step 1) contains non-aggregating BLG in an amount ranging from 15 to 80% by weight relative to the total amount of protein. Most preferably, the whey protein solution of step 1) may contain non-aggregating BLG in an amount ranging from 30 to 70% by weight relative to the total amount of protein.

[0261] In some preferred embodiments of the present invention, the whey protein solution of step 1) contains at least 0.4% by weight of non-aggregating BLG relative to the weight of the whey protein solution. Preferably, the whey protein solution contains at least 1.0% by weight of non-aggregating BLG. More preferably, the whey protein solution contains at least 2.0% by weight of non-aggregating BLG. It is even more preferable that the whey protein solution contains at least 4% by weight of non-aggregating BLG.

[0262] A higher concentration of non-aggregating BLG is even more preferable, and preferably the whey protein solution contains at least 6% by weight of non-aggregating BLG. More preferably, the whey protein solution contains at least 10% by weight of non-aggregating BLG. It is even more preferable that the whey protein solution contains at least 15% by weight of non-aggregating BLG.

[0263] In some preferred embodiments of the present invention, the whey protein solution of step 1) contains non-aggregating BLG in an amount ranging from 0.4 to 45% by weight relative to the weight of the whey protein solution. Preferably, the whey protein solution contains non-aggregating BLG in an amount ranging from 1 to 35% by weight. More preferably, the whey protein solution contains non-aggregating BLG in an amount ranging from 4 to 30% by weight. It is even more preferable that the whey protein solution contains non-aggregating BLG in an amount ranging from 10 to 25% by weight.

[0264] A higher BLG content is particularly preferred, and therefore, in some preferred embodiments of the present invention, the whey protein solution of step 1) contains non-aggregating BLG in the range of 10-45% by weight / weight relative to the weight of the whey protein solution. Preferably, the whey protein solution contains non-aggregating BLG in the range of 15-40% by weight / weight. More preferably, the whey protein solution contains non-aggregating BLG in the range of 20-39% by weight / weight. It is even more preferable that the whey protein solution contains non-aggregating BLG in the range of 25-38% by weight / weight.

[0265] It is often preferable that the substantial amount of BLG in the whey protein solution is non-aggregating BLG. Preferably, at least 50% of the BLG is non-aggregating BLG. More preferably, at least 80% of the BLG is non-aggregating BLG. Even more preferably, at least 90% of the BLG is non-aggregating BLG. Most preferably, at least 95% of the BLG is non-aggregating BLG. Even more preferably, approximately 100% of the BLG in the whey protein solution is non-aggregating BLG.

[0266] A whey protein solution can be prepared using any suitable whey protein source. 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 a combination thereof.

[0267] The whey protein solution is preferably a desalted whey protein solution.

[0268] In this context, the term "desalted" means that the conductivity of the whey protein solution is at most 15 mS / cm, preferably at most 10 mS / cm, and more preferably at most 8 mS / cm. The UF permeability conductivity of the desalted whey protein solution is preferably at most 7 mS / cm, more preferably at most 4 mS / cm, and more preferably at most 1 mS / cm.

[0269] The whey protein solution is preferably a desalted whey protein concentrate, a desalted whey protein isolate, a desalted whey protein concentrate, or a desalted whey protein isolate.

[0270] In some particularly preferred embodiments of the present invention, the whey protein solution comprises or consists of a desalted and pH-adjusted whey protein concentrate, a whey protein concentrate, a whey protein isolate, a whey protein isolate, or a combination thereof.

[0271] The whey protein solution may, for example, contain, or consist of, a desalted whey protein concentrate. Alternatively, the whey protein solution may contain, or consist of, a desalted whey protein concentrate. Alternatively, the whey protein solution may contain, or consist of, a desalted whey protein isolate. Alternatively, the whey protein solution may contain, or consist of, a desalted whey protein isolate.

[0272] BLG concentrate compositions are preferably prepared from the milk of mammals, preferably from the milk of ruminants such as cows, sheep, goats, buffalo, camels, llamas, horses, and / or deer. Proteins derived from cow's milk are particularly preferred.

[0273] The proteins in the whey protein solution are preferably as close as possible to their native state, and preferably have been subjected to only mild heat treatment, if any.

[0274] In some preferred embodiments of the present invention, the whey protein solution has a maximum furosine value of 80 mg / 100g of protein. Preferably, the whey protein solution has a maximum furosine value of 40 mg / 100g of protein. More preferably, the whey protein solution has a maximum furosine value of 20 mg / 100g of protein. Even more preferably, the whey protein solution has a maximum furosine value of 10 mg / 100g of protein. Most preferably, the whey protein solution has a maximum furosine value of 5 mg / 100g of protein, for example, preferably 0 mg / 100g of protein.

[0275] Whey protein solutions typically contain other components in addition to protein. Whey protein solutions may contain other components commonly found in whey or milk serum, such as minerals, carbohydrates, and / or lipids. Alternatively or additionally, whey protein solutions may contain components not inherent to whey or milk serum. However, such non-native components must be safe, preferably for use in food product production and preferably for human consumption.

[0276] This process is particularly advantageous for separating BLG from crude whey protein solutions that contain solid matter other than BLG.

[0277] Whey protein solutions may contain carbohydrates such as lactose, oligosaccharides, and / or hydrolysis products of lactose (i.e., glucose and galactose). Whey protein solutions may contain carbohydrates in the range of 0-40% by weight, for example, in the range of 1-30% by weight or 2-20% by weight.

[0278] In some preferred embodiments of the present invention, the whey protein solution contains up to 20% by weight of carbohydrates, preferably up to 10% by weight of carbohydrates, more preferably up to 5% by weight of carbohydrates, and even more preferably up to 2% by weight of carbohydrates.

[0279] The whey protein solution may also contain lipids in the form of other lipid types, such as triglycerides and / or phospholipids.

[0280] In some embodiments of the present invention, the whey protein solution of step 1) contains a total amount of lipids up to 15% by weight relative to the total solids. Preferably, the whey protein solution of step 1) contains a total amount of lipids up to 10% by weight relative to the total solids. More preferably, the whey protein solution of step 1) contains a total amount of lipids up to 6% by weight relative to the total solids. Even more preferably, the whey protein solution of step 1) contains a total amount of lipids up to 1.0% by weight relative to the total solids. Most preferably, the whey protein solution of step 1) contains a total amount of lipids up to 0.5% by weight relative to the total solids.

[0281] The total amount of protein in a whey protein solution is typically at least 1% by weight relative to the weight of the whey protein solution. Preferably, the total amount of protein in the whey protein solution is at least 5% by weight. More preferably, the total amount of protein in the whey protein solution is at least 10% by weight. Even more preferably, the total amount of protein in the whey protein solution is at least 15% by weight.

[0282] In some preferred embodiments of the present invention, the total amount of protein in the whey protein solution is in the range of 1 to 50% by weight. Preferably, the total amount of protein in the whey protein solution is in the range of 5 to 40% by weight. More preferably, the total amount of protein in the whey protein solution is in the range of 10 to 30% by weight. Even more preferably, the total amount of protein in the whey protein solution is in the range of 15 to 25% by weight.

[0283] The total amount of protein in the whey protein solution is determined according to Example 1.5.

[0284] Whey protein solutions are typically prepared by subjecting a whey protein feed to one or more preparations to form a whey protein solution that is supersaturated with respect to BLG.

[0285] The whey protein feed is preferably WPC, WPI, SPC, SPI, or a combination thereof.

[0286] The term "whey protein feed" refers to compositions derived from a BLG concentrate and a subsequent liquid BLG isolate. A whey protein feed is, for example, converted to a whey protein solution supersaturated with respect to BLG. A whey protein feed is typically an aqueous liquid containing BLG and at least one additional whey protein, but typically not supersaturated with respect to BLG.

[0287] Embodiments relating to the chemical composition of whey protein solutions are similarly applicable to whey protein feeds. However, typically, at least one parameter of the whey protein feed is set to avoid supersaturation or at least spontaneous crystallization.

[0288] In some preferred embodiments of the present invention, a supersaturated whey protein solution is prepared by subjecting a whey protein feed to one or more of the following adjustments: - Adjust pH - Reduce conductivity - Lower the temperature - Increase protein concentration - Add a drug that reduces water activity. - Changes in ionic composition.

[0289] In some preferred embodiments of the present invention, the preparation of the whey protein solution includes adjusting the pH of the whey protein feed to a pH in the range of 5 to 6.

[0290] All pH values ​​are measured using a pH glass electrode and normalized to 25°C. Normalization to 25°C is typically performed using a pH meter, or by adjusting the sample temperature to 25°C.

[0291] 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.

[0292] In some preferred embodiments of the present invention, the pH of the whey protein solution is in the range of 5.0 to 6.0. Preferably, the pH of the whey protein solution is in the range of 5.1 to 6.0. More preferably, the pH of the whey protein solution is in the range of 5.1 to 5.9. Even more preferably, the pH of the whey protein solution may be in the range of 5.2 to 5.9. Most preferably, the pH of the whey protein solution is in the range of 5.2 to 5.8.

[0293] The pH is preferably adjusted using an acid and / or base acceptable for food products. For example, an acid acceptable for food products, such as a carboxylic acid, is particularly preferred. Useful examples of such acids include, for example, acetic acid, adipic acid, ascorbic acid, benzoic acid, butyric acid, citric acid, folic acid, fumaric acid, gluconic acid, hydrochloric acid, lactic acid, malic acid, phosphoric acid, propionic acid, sorbic acid, succinic acid, sulfuric acid, tartaric acid, and / or mixtures thereof.

[0294] In some preferred embodiments of the present invention, the pH is adjusted using a lactone such as D-glucono-delta-lactone that hydrolyzes slowly and simultaneously reduces the pH of the aqueous liquid containing it. The target pH after completion of lactone hydrolysis can be accurately calculated.

[0295] Useful examples of bases acceptable in food products are, for example, hydroxide sources such as sodium hydroxide, potassium hydroxide, calcium hydroxide, salts of edible acids such as trisodium citrate, and / or combinations thereof.

[0296] In other preferred embodiments of the present invention, the pH is adjusted by adding a cation exchange material to its H + type. The bead type / large particle type cation exchange material can be easily removed from the whey protein solution either before or after crystallization. Adjusting the pH by adding a cation exchange material to its H + type is particularly advantageous in the present invention because it reduces the pH without adding negative counterions that significantly affect the conductivity of the whey protein feed.

[0297] In some preferred embodiments of the present invention, the preparation of the whey protein solution involves reducing the conductivity of the whey protein feed.

[0298] The conductivity values described herein are normalized to 25°C unless otherwise specified.

[0299] It has been found that lowering the conductivity of a whey protein solution increases the yield of BLG crystals. The minimum conductivity obtainable from a whey protein solution depends on the composition of the protein fraction and the lipid fraction (if any). Some protein species, such as caseinomacropeptides (CMP), contribute more to conductivity than others. Therefore, it is preferable to bring the conductivity of the whey protein feed closer to a level where proteins and their counterions are the main contributors to conductivity. Lowering conductivity often involves removing at least some of the small free ions present in the liquid phase that are not firmly bound to the proteins.

[0300] Whey protein solutions often preferably have a conductivity of up to 10 mS / cm. In some preferred embodiments of the present invention, the whey protein solution has a conductivity of up to 5 mS / cm. Preferably, the whey protein solution has a conductivity of up to 4 mS / cm.

[0301] A lower conductivity is even more preferable, resulting in a higher 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.

[0302] The conductivity of the whey protein feed is preferably reduced by dialysis or diafiltration. Diafiltration by ultrafiltration is particularly preferred because it can wash away 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 feed.

[0303] The ratio between the conductivity (expressed as mS / cm) in the whey protein solution and the total amount of protein (expressed as a weight % of total protein relative to the total weight of the whey protein solution) can be advantageously kept below a certain threshold to promote the crystallization of BLG.

[0304] In some preferred embodiments of the present invention, the ratio of the conductivity of the whey protein solution to the total amount of protein is up to 0.3. Preferably, the ratio of the conductivity of the whey protein solution to the total amount of protein is up to 0.25. Preferably, the ratio of the conductivity of the whey protein solution to the total amount of protein is up to 0.20. More preferably, the ratio of the conductivity of the whey protein solution to the total amount of protein is up to 0.18. Even more preferably, the ratio of the conductivity of the whey protein solution to the total amount of protein is up to 0.12. Most preferably, the ratio of the conductivity of the whey protein solution to the total amount of protein is up to 0.10.

[0305] For example, the ratio between the conductivity of the whey protein solution and the total amount of protein is preferably about 0.07 or even lower.

[0306] The inventors have further found that the whey protein feed can be advantageously adjusted to provide a whey protein solution having a UF permeable conductivity of up to 10 mS / cm. UF permeable conductivity is a measure of the conductivity of the small molecule fraction of a liquid. When the term "conductivity" is used herein in this way, it refers to the conductivity of the liquid in question. When the term "UF permeable conductivity" is used, it refers to the conductivity of the small molecule fraction of the liquid, and is measured according to Example 1.23.

[0307] Preferably, the UF permeability conductivity of the whey protein solution is up to 7 mS / cm. More preferably, the UF permeability conductivity of the whey protein solution may be up to 5 mS / cm. Even more preferably, the UF permeability conductivity of the whey protein solution may be up to 3 mS / cm.

[0308] Even lower UF permeability conductivity can be used, which is particularly preferable when it is necessary to obtain high-yield BLG. Therefore, preferably, the UF permeability conductivity of the whey protein solution is up to 1.0 mS / cm. More preferably, the UF permeability conductivity of the whey protein solution may be up to 0.4 mS / cm. Even more preferably, the UF permeability conductivity of the whey protein solution may be up to 0.1 mS / cm. Most preferably, the UF permeability conductivity of the whey protein solution may be up to 0.04 mS / cm.

[0309] For example, if Milli-Q water is used as a diluent during diafiltration (Milli-Q water has a conductivity of approximately 0.06 μS / cm), even lower UF permeability can be achieved. Therefore, the UF permeability of the whey protein solution may be as low as 0.01 mS / cm. Alternatively, the UF permeability of the whey protein solution may be as low as 0.001 mS / cm. Or, the UF permeability of the whey protein solution may be as low as 0.0001 mS / cm.

[0310] In some preferred embodiments of the present invention, the preparation of the whey protein solution includes lowering the temperature of the whey protein feed.

[0311] For example, the preparation of the whey protein solution may include lowering the temperature of the whey protein feed to at least 5°C, preferably at least 10°C, and more preferably at least 15°C. For example, the preparation of the whey protein solution may include lowering the temperature of the whey protein feed to at least 20°C.

[0312] The temperature of the whey protein feed may be reduced, for example, to a maximum of 30°C, preferably to a maximum of 20°C, and more preferably to a maximum of 10°C. The inventors have found that even lower temperatures provide a greater degree of supersaturation, and therefore the temperature of the whey protein may be reduced, for example, to a maximum of 5°C, preferably to a maximum of 2°C, and more preferably to a maximum of 0°C. The temperature may be lower than 0°C. However, preferably, the whey protein solution must remain pumpable, for example, in the form of an ice slurry.

[0313] In some preferred embodiments of the present invention, the whey protein solution is an ice slurry prior to the initiation of BLG crystallization. Alternatively, the crystallized whey protein solution may be converted to an ice slurry during BLG crystallization in step 2) or maintained as an ice slurry.

[0314] In some particularly preferred embodiments of the present invention, the preparation of the whey protein solution includes increasing the total protein concentration of the whey protein feed. The whey protein feed is subjected to one or more protein concentration steps, such as ultrafiltration, nanofiltration, reverse osmosis, and / or evaporation, thereby being concentrated to obtain the whey protein solution.

[0315] Ultrafiltration is particularly preferred because it allows for the selective concentration of proteins with minimal impact on salt and carbohydrate concentrations. As described above, ultrafiltration is preferably used for both diafiltration and concentration of whey protein feed.

[0316] 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 feed when the whey protein solution is in a metastable region, i.e., in a supersaturated region where BLG crystals can grow when seeding is used, but crystallization does not spontaneously begin.

[0317] In some preferred embodiments of the present invention, the preparation of a whey protein solution involves adding one or more water activity reducing agents (which may be more) to a whey protein feed.

[0318] Useful but non-limiting examples of such water activity-reducing agents include polysaccharides and / or polyethylene glycol (PEG).

[0319] In some preferred embodiments of the present invention, the preparation of the whey protein solution includes, for example, altering the ionic composition of the whey protein feed by ion exchange, addition of new ion species, dialysis, or diafiltration.

[0320] Typically, whey protein solutions are prepared by combining two or more of the above process steps to produce supersaturation.

[0321] In some preferred embodiments of the present invention, the preparation of the whey protein solution involves using at least: - For example, concentration using ultrafiltration, nanofiltration or reverse osmosis at temperatures above 10°C, and -This includes subsequently cooling the temperature to below 10°C.

[0322] In another preferred embodiment of the present invention, the preparation of the whey protein solution involves using a whey protein feed to at least Concentration at a pH above -6.0, and -Then, an acid (GDL or H + This includes lowering the pH by adding a cation exchange substance of a certain type (such as a cation exchange substance).

[0323] In yet another preferred embodiment of the present invention, the preparation of the whey protein solution involves using a whey protein feed to at least - For example, this includes subjecting the material to deconductivity by diafiltration using a film that retains at least non-aggregated BLGs.

[0324] In a more preferred embodiment of the present invention, the preparation of the whey protein solution involves using a whey protein feed to at least - Adjust the pH to 5-6. - Reducing conductivity by diafiltration using a film that retains at least non-aggregated BLG, - For example, protein concentration using ultrafiltration, nanofiltration or reverse osmosis at temperatures above 10°C, -Finally, this includes subjecting the combination to cooling to a temperature below 10°C.

[0325] The inventors further found that the BLG yield of this process can be improved by controlling the molar ratio between the total sodium + potassium and the total calcium and magnesium. Surprisingly, higher relative amounts of calcium and magnesium appear to increase the yield of non-aggregating BLG, and therefore improve the efficiency of BLG recovery in the process of the present invention.

[0326] In some preferred embodiments of the present invention, the whey protein solution of step 1) has a molar ratio of Na+K to Ca+Mg of up to 4. More preferably, the whey protein solution of step 1) has a molar ratio of Na+K to Ca+Mg of up to 2. Even more preferably, the whey protein solution of step 1) has a molar ratio of Na+K to Ca+Mg of up to 1.5, and even more preferably, up to 1.0. Most preferably, the whey protein solution of step 1) has a molar ratio of Na+K to Ca+Mg of up to 0.5, for example, up to 0.2.

[0327] The molar ratio of Na+K to Ca+mg is (m Na +m K ) / (m Ca +m Mg ) is calculated as, in the formula, m Na This is the molar content of element Na, m K This is the molar content of element K, m Ca This refers to the content of element Ca in molar units, and m Mg This represents the molar content of the element Mg.

[0328] It is particularly preferable that the whey protein solution is supersaturated with respect to BLG by salt solubility, and therefore, that BLG can be crystallized from the whey protein solution by salt solubility.

[0329] In some embodiments of the present invention, the whey protein solution has a low denatured protein content, particularly if the edible BLG product of the present invention is also expected to have some degree of protein denaturation. 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%, and most preferably up to 0.8%.

[0330] Step 2 of the process involves crystallizing at least a portion of the BLG in the supersaturated whey protein solution.

[0331] The crystallization in step 2) is particularly preferably carried out by salt dissolution, i.e., in a liquid with low ionic strength and conductivity. This is in contrast to the salting-out method, in which a considerable amount of salt is added to the solution to induce crystallization.

[0332] Step 2) Crystallization of BLG includes, for example, one or more of the following: - Wait for crystallization to occur. - Addition of crystallization seed, - Further increase the supersaturation of BLG, and / or -Mechanical stimulation.

[0333] In some preferred embodiments of the present invention, step 2) includes adding crystallization seeds to the whey protein solution. The inventors have found that by adding crystallization seeds, it is possible to control when and where BLG crystallization occurs, thereby avoiding sudden clogging of process equipment and unintended shutdowns during production. For example, it is often desirable to avoid the initiation of crystallization while the whey protein feed is being concentrated.

[0334] Unless a high-shear system such as a ceramic membrane or DCF is used, it is particularly preferable that the whey protein solution does not come into contact with the UF membrane or MF membrane during step 2).

[0335] In principle, any seed material can be used to initiate BLG crystallization. However, to avoid the addition of impurities to the whey protein solution, it is preferable to use hydrated BLG crystals or dried BLG crystals for seeding.

[0336] The crystallized seeds may be in a dry form or may form part of a suspension when added to a whey protein solution. Adding a suspension containing crystallized seeds, such as BLG crystals, is currently preferred because it appears to provide a faster initiation of crystallization. Such a suspension preferably contains crystallized seeds with a pH in the range of 5–6 and a conductivity of up to 10 mS / cm.

[0337] The inventors of this invention are aware that the unit of conductivity, "mS / cm," means millisiemens per centimeter, and that 1.00 mS corresponds to 1000 micros.

[0338] The crystallization seed is particularly preferably added via a suspension of BLG crystals that have not been dried after BLG crystallization. Such a suspension may be, for example, a portion of the BLG crystals and mother liquor obtained from step 2) of the previous batch, or a portion of the wet BLG crystals obtained from steps 3), 4), or 5) of the previous batch.

[0339] The inventors observed that using wet BLG crystals as crystallization seeds yielded much larger BLG crystals during step 2) than when dry or insufficiently hydrated BLG crystals were used, which also made the separation of BLG from the mother liquor more efficient. In experiments where the whey protein feed, crystallization conditions, mass and particle size of the seed material, cooling profile, and separation method were the same, the inventors found that seeding with undried BLG crystals resulted in a 100% increase in the particle size of the resulting crystals (100-130 microns) compared to BLG crystals obtained by seeding with rehydrated and dried BLG crystals (resulting particle size: 40-60 microns).

[0340] Alternatively, if the crystallization seed is based on dried BLG crystals, it is preferable to resuspend the crystals in an aqueous liquid, such as water, and rehydrate the dried BLG crystals for at least 30 minutes, preferably at least 1.0 hour, and more preferably at least 1.5 hours, before using the resulting BLG crystal suspension to initiate crystallization.

[0341] In some embodiments of the present invention, at least some of the crystallized seeds are located on a solid phase that is in contact with a whey protein solution.

[0342] The crystallization seeds preferably have a particle size smaller than the desired size of the BLG crystals. The size of the crystallization seeds can be modified by removing the largest seeds by sieving or other size separation processes. For example, particle size reduction by grinding can also be used before particle size separation.

[0343] In some embodiments of the present invention, at least 90% by weight of the crystallized seeds have a particle size in the range of 0.1 to 600 microns (measured by sieving analysis). For example, at least 90% by weight of the crystallized seeds may have a particle size in the range of 1 to 400 microns. Preferably, at least 90% by weight of the crystallized seeds may have a particle size in the range of 5 to 200 microns. More preferably, at least 90% by weight of the crystallized seeds may have a particle size in the range of 5 to 100 microns.

[0344] The particle size and dosage of the crystallization seed can be adjusted to provide optimal crystallization of BLG.

[0345] In some preferred embodiments of the present invention, the crystallized seeds are added to the whey protein feed before supersaturation with respect to BLG is achieved, preferably in a manner in which at least some crystallized seeds are still present when supersaturation is reached. This can be achieved by adding the crystallized seeds when the whey protein feed is close to supersaturation, for example, during cooling, concentration, and / or pH adjustment, so that supersaturation is achieved before the crystallized seeds are completely dissolved.

[0346] In some preferred embodiments of the present invention, step 2) further includes increasing the degree of supersaturation of the BLG, preferably to such an extent that crystallization of the BLG begins immediately, i.e., within a maximum of 20 minutes, preferably within a maximum of 5 minutes. This is also referred to as the nucleation zone, in which crystallites spontaneously form and initiate the crystallization process.

[0347] The degree of supersaturation, for example, - Further increase the protein concentration of the whey protein solution. - Further cooling of the whey protein solution, - Bring the whey protein solution to an optimal pH for BLG crystallization. - It can be increased by one or more of the following: further reducing conductivity.

[0348] In some preferred embodiments of the present invention, step 2) includes waiting for BLG crystals to form. This may take several hours and is typically the case for a whey protein solution that is slightly supersaturated with respect to BLG and to which no crystallization seed has been added.

[0349] In some preferred embodiments of the present invention, the provision of a whey protein solution (step 1) and the crystallization of BLG (step 2) are carried out as two separate steps.

[0350] However, in other preferred embodiments of the present invention, step 2) includes additional adjustments to the crystallized whey protein solution to increase the degree of supersaturation of BLG or at least maintain supersaturation. The additional adjustments improve the yield of BLG crystals.

[0351] The following additional adjustments are necessary: - Further increase the protein concentration of the crystallized whey protein solution. - Cooling the crystallized whey protein solution to an even lower temperature, - Further bring the pH of the crystallized whey protein solution closer to the optimal pH for BLG crystallization. - May include one or more of the following: further reducing the conductivity of the crystallized whey protein solution.

[0352] In some preferred embodiments of the present invention, the crystallized whey protein solution is maintained in a metastable zone during step 2) to avoid the spontaneous formation of new crystallites.

[0353] In some preferred embodiments of the present invention, at least some of the BLG crystals obtained during step 2) have orthorhombic space group P 212121.

[0354] Preferably, at least some of the obtained BLG crystals have an orthorhombic space group P 212121, with unit cell dimensions a = 68.68 (±5%) Å, b = 68.68 (±5%) Å, and c = 156.65 (±5%) Å; and unit cell integration angles α = 90°, β = 90°, and γ = 90°.

[0355] In some preferred embodiments of the present invention, at least some of the obtained BLG crystals have an orthorhombic space group P 212121, with unit cell dimensions a = 68.68 (±2%) Å, b = 68.68 (±2%) Å, and c = 156.65 (±2%) Å; and unit cell integration angles α = 90°, β = 90°, and γ = 90°.

[0356] At least some of the obtained BLG crystals have an orthorhombic space group P 212121, with unit cell dimensions a = 68.68 (±1%) Å, b = 68.68 (±1%) Å, and c = 156.65 (±1%) Å; and more preferably, the integral angles of the unit cell are α = 90°, β = 90°, and γ = 90°.

[0357] Most preferably, at least some of the obtained BLG crystals have an orthorhombic space group P 212121, with unit cell dimensions a=68.68 Å, b=68.68 Å, and c=156.65 Å; and unit cell integration angles α=90°, β=90°, and γ=90°.

[0358] In some particularly preferred embodiments of the present invention, the process includes step 3) separating at least some BLG crystals from the remaining whey protein solution. This is particularly preferred when purification of BLG is desired.

[0359] Step 3) may include, for example, separating the BLG crystals into solids with a weight of at least 30%. Preferably, step 3) includes separating the BLG crystals into solids with a weight of at least 40%. More preferably, step 3) includes separating the BLG crystals into solids with a weight of at least 50%.

[0360] The inventors have found that a high solid content is advantageous for BLG purification because the aqueous portion adhering to the separated BLG crystals typically contains impurities that should be avoided. Furthermore, a high solid content reduces the energy consumption required to convert the separated BLG crystals into a dried product such as powder, and increases the BLG yield obtained from a given volume of drying unit.

[0361] In some preferred embodiments of the present invention, step 3) includes separating the BLG crystals into at least 60% solids. Preferably, step 3) includes separating the BLG crystals into at least 70% solids. Even more preferably, step 3) includes separating the BLG crystals into at least 80% solids.

[0362] In some preferred embodiments of the present invention, the separation in step 3) includes one or more of the following operations: -Centrifugal separation, - Decantation, -filtration, - Settlement, - The above combinations.

[0363] These unit operations are well known to those skilled in the art and are easily carried out. Separation by filtration may include, for example, the use of vacuum filtration, dynamic cross-flow filtration (DCF), filtrate press, or filter centrifuge.

[0364] Different pore sizes can be used for filtration based on the desired results. Preferably, the filter allows native whey protein and small aggregates to pass through 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. More preferably, the filter may have a nominal pore size of at least 2 microns.

[0365] Filters with larger pore sizes can also be used, and are actually preferred, mainly when it is necessary to separate larger crystals from a liquid containing BLG crystals. In some embodiments of the present invention, the filter has a nominal pore size of at least 5 microns. Preferably, the filter has a nominal pore size of at least 20 microns. Even more preferably, the filter may have a pore size of at least 40 microns.

[0366] The filter has a pore size in the range of 0.03 to 5000 microns, for example, 0.1 to 5000 microns. Preferably, the filter may have a pore size in the range of 0.5 to 1000 microns. More preferably, the filter may have a pore size in the range of 5 to 800 microns, 10 to 500 microns, or 50 to 500 microns.

[0367] In some preferred embodiments of the present invention, the filter has a pore size in the range of 0.03 to 100 microns. Preferably, the filter may have a pore size in the range of 0.1 to 50 microns. More preferably, the filter may have a pore size in the range of 4 to 40 microns. Even more preferably, the filter may have a pore size in the range of 5 to 30 microns, such as in the range of 10 to 20 microns.

[0368] The advantage of using a filter with a pore size greater than 1 micron is that bacteria and other microorganisms are also removed, at least partially, during separation and optionally during washing and / or recrystallization. Therefore, this process makes it possible to produce high-purity BLG with a very low bacterial load while avoiding thermal damage to proteins.

[0369] Another advantage of using filters with pore sizes larger than 1 micron is that water removal and subsequent drying are easier, resulting in lower energy consumption.

[0370] The remaining whey protein solution separated from the BLG crystals can be recycled into whey protein feed during the preparation of the whey protein solution.

[0371] In some preferred embodiments of the present invention, step 3) utilizes a filter centrifuge. In other preferred embodiments of the present invention, step 3) utilizes a decanter centrifuge. Early results indicate that using a filter centrifuge and / or a decanter centrifuge to separate BLG crystals from the mother liquor provides a more robust process operation than, for example, vacuum filtration.

[0372] In many cases, it is preferable to dry the formed filter cake with a drying gas to reduce its moisture content, and preferably to allow the filter cake to be peeled off the filter. The use of a drying gas may constitute part of the separation process or the final drying process when the filter cake is directly converted into a dried edible BLG composition.

[0373] In some preferred embodiments of the present invention, step 3) utilizes a dynamic cross-flow filtration (DCF) unit.

[0374] Initial tests showed that using DCF units with 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 observed that the DCF units could run for a sufficient amount of time to separate crystals even from large batches of whey protein solution containing BLG crystals.

[0375] In some preferred embodiments of the present invention, step 3) is carried out using a DCF unit equipped with a membrane capable of holding BLG crystals, the DCF permeate is recycled to form part of the whey protein solution or whey protein feed, and the DCF holding liquid can be recovered or returned to the crystallization tank. Preferably, the DCF permeate is treated, for example, by ultrafiltration / diafiltration to supersaturate it with respect to BLG before being mixed with the whey protein solution or whey protein feed.

[0376] Advantageously, these embodiments do not require the liquid flow temperature to rise above 15°C and are therefore less susceptible to microbial contamination than process variations that require higher temperatures. Another industrial advantage of these embodiments is that the level of supersaturation can be easily controlled and maintained at a level where undesirable spontaneous crystallization does not occur. Thus, the liquid flow temperature during these embodiments of the process is preferably up to 15°C, more preferably up to 12°C, even more preferably up to 10°C, and most preferably up to 5°C.

[0377] These embodiments are illustrated in Example 10 and shown in Figure 26 of PCT application PCT / EP2017 / 084553. These embodiments can be carried out as a batch process or a continuous process.

[0378] In some preferred embodiments of the present invention, the process includes a step 4) of washing the separated BLG crystals, for example, 3). The washing may consist of a single washing or a series of washing steps.

[0379] The washing in step 4) preferably involves contacting the BLG crystals with a washing solution without completely dissolving the BLG crystals, and then separating the remaining BLG crystals from the washing solution.

[0380] The washing solution is preferably selected to avoid complete dissolution of the BLG crystals and may include, for example, cold demineralized water, cold tap water, or cold reverse osmosis permeate, or may even consist essentially of these.

[0381] The washing solution may include, for example, cold demineralized water, cold tap water, or cold reverse osmosis permeate, or may essentially consist of these.

[0382] The washing solution may have a pH in the range of 5 to 6, preferably 5.0 to 6.0, more preferably 5.1 to 6.0, for example, 5.1 to 5.9.

[0383] Alternatively, the washing solution may have a pH in the range of 6.1 to 8, preferably 6.4 to 7.6, and more preferably 6.6 to 7.4, for example, 6.8 to 7.2. This is typically the pH of the washing solution in the case of desalinated water, tap water, or reverse osmosis permeate. Generally, it is preferable that the washing solution has low mineral content and low buffering capacity.

[0384] The cleaning solution may have an conductivity of up to 0.1 mS / cm, preferably up to 0.02 mS / cm, and more preferably up to 0.005 mS / cm.

[0385] A cleaning solution with even lower conductivity can be used. For example, the cleaning solution may have a conductivity of up to 1 microS / cm. Alternatively, the cleaning solution may have a conductivity of up to 0.1 microS / cm, for example, approximately 0.05 microS / cm.

[0386] To limit the dissolution of crystallized BLG, it is preferable to carry out the washing process at a low temperature. The temperature of the washing solution is preferably up to 30°C, more preferably up to 20°C, and even more preferably up to 10°C.

[0387] The washing process may be carried out at a maximum temperature of 5°C, more preferably 2°C, for example, around 0°C. For example, temperatures below 0°C can be used, as long as the washing solution does not freeze at that temperature, in the presence of one or more freezing point inhibitors.

[0388] In some embodiments of the present invention, the cleaning solution contains, for example, an amount of BLG of at least 1% by weight, preferably at least 3% by weight, for example 4% by weight.

[0389] The washing in step 4) typically dissolves up to 80% by weight of the initial amount of BLG crystals, preferably up to 50% by weight, and more preferably up to 20% by weight of the initial amount of BLG crystals. Preferably, the washing in step 4) dissolves up to 15% by weight of the initial amount of BLG crystals, more preferably up to 10% by weight, and more preferably up to 5% by weight of the initial amount of BLG crystals.

[0390] The weight ratio between the total amount of washing solution 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 solution and the initial amount of separated BLG crystals may be at least 10. Alternatively, the weight ratio between the total amount of washing solution and the initial amount of separated BLG crystals may be at least 20, for example, at least 50, or at least 100.

[0391] The term "total volume of cleaning solution" refers to the total volume of cleaning solution used throughout the entire process.

[0392] In some preferred embodiments of the present invention, one or more washing sequences are performed in the same or similar filter configuration as the BLG crystal separation. The filter cake, which mainly contains BLG crystals, is further subjected to one or more sequences of washing solutions that are removed through the filter while the remaining portion of the BLG crystals remains in the filter cake.

[0393] In a particularly preferred embodiment of the present invention, the separation in step 3) is carried out using a filter that holds the BLG crystals. Subsequently, the filter cake is brought into contact with one or more amounts of washing solution moving through the filter cake and the filter. Each amount of washing solution is often preferred to be up to 10 times the volume of the filter cake, preferably up to 5 times the volume of the filter cake, more preferably up to 1 time the volume of the filter cake, and even more preferably up to 0.5 times the volume of the filter cake, for example up to 0.2 times the volume of the filter cake. The volume of the filter cake includes both the solid and fluid (liquid and gaseous) parts of the filter cake. The filter cake is preferably washed in this manner at least 2 times, preferably at least 4 times, and more preferably at least 6 times.

[0394] The used washing solution from step 4) can be recycled, for example, into whey protein feed or whey protein solution, and the washed-out BLG can be separated again.

[0395] The process may further include step 5) which involves a recrystallization step, including: - Dissolve the separated BLG crystals in a recrystallization solution. - Prepare the recrystallization solution so that it becomes supersaturated with BLG. - Crystallizing BLG in a supersaturated recrystallization solution, and - Separate the BLG crystals from the remaining prepared recrystallization solution.

[0396] Step 5) may include either a single recrystallization sequence or multiple recrystallization sequences.

[0397] In some embodiments of the present invention, the BLG crystal from step 3) or 4) is recrystallized at least twice. For example, the BLG crystal may be recrystallized at least three times, for example, at least four times. Example.

[0398] The washing and recrystallization steps can be combined in any order and performed multiple times as needed.

[0399] The BLG crystals separated in step 3) follow, for example, the following process sequence: -One or more washing steps (step 4), followed by, - One or more recrystallization steps (step 5).

[0400] Alternatively, the separated BLG crystals from step 3) can be subjected to a process sequence: - One or more recrystallization steps (step 5), followed by, - One or more washing steps (step 4).

[0401] The washing and recrystallization processes can be combined in the following order, for example: - One or more washing steps (step 4), - One or more recrystallization steps (step 5), - One or more washing steps (step 4), and - One or more recrystallization steps (step 5).

[0402] Or, for example, in the following order: - One or more recrystallization steps (step 5), - One or more washing steps (step 4), - One or more recrystallization steps (Step 5) - One or more washing steps (step 4).

[0403] The inventors noticed that the crystallization process, including the preparation of the whey protein solution, is prone to microbial growth, and found it advantageous to modify the process to address this problem.

[0404] From the supply of the whey protein feed until the separation of the BLG molecules in step c), the total time the BLG molecules are at a temperature above 12°C is preferably a maximum of 24 hours, more preferably 20 hours, more preferably 12 hours, even more preferably 6 hours, and most preferably 3 hours.

[0405] The above period can be further reduced, and is often preferred, and therefore, in some preferred embodiments of the present invention, the total time the BLG molecules are at a temperature above 12°C from the provision of the whey protein feed until the BLG molecules are separated in step c) is at most 2 hours, preferably 1 hour, more preferably at most 0.5 hours, even more preferably at most 0.3 hours, and most preferably at most 0.1 hours.

[0406] In some embodiments of the present invention, the process further includes subjecting the separated BLG to an additional BLG concentration step, for example, based on chromatography or selective filtration. However, in other preferred embodiments of the present invention, the process does not include an additional BLG concentration step after step 2). The term “additional BLG concentration step” means a process step that concentrates BLG relative to the total amount of protein, and this step does not involve the crystallization of BLG or the handling of BLG crystals. An example of such an additional BLG concentration step is ion-exchange chromatography. Washing of BLG crystals and / or recrystallization of BLG are not considered “additional BLG concentration steps”.

[0407] In some preferred embodiments of the present invention, the process includes a drying step in which the BLG concentrated composition derived from step 3), 4), or 5) is converted into a dry composition.

[0408] In a particularly preferred embodiment of the present invention, the process for preparing the BLG concentrated composition is as follows: 1) A step of providing a whey protein solution comprising BLG and at least one additional whey protein, wherein the whey protein solution is supersaturated with respect to BLG, has a pH in the range of 5 to 6, and the whey protein solution is as follows: -70-100% by weight / weight protein relative to total solids, - 30-90% by weight, preferably 30-70% by weight, of the total protein, - 4-50% by weight of non-aggregating ALA relative to total protein, preferably 8-35% by weight, - 0-25% by weight / weight of CMP relative to total protein, - A process comprising at least 10% by weight / weight of protein relative to the total weight of the whey protein solution, 2) A step of crystallizing BLG by preferably adding crystallization seeds in a supersaturated whey protein solution. 3) A step to separate BLG crystals from the remaining whey protein solution. 4) Optionally, a step to wash the separated BLG crystals obtained in step 3), 5) Optionally, the process includes a step of recrystallizing the BLG crystal obtained from step 3) or 4).

[0409] The whey protein solution is preferably a desalted whey protein solution, and preferably the ratio of conductivity to total protein amount is up to 0.3 and / or the UF permeable conductivity is up to 7 mS / cm.

[0410] These embodiments are particularly useful for producing low-mineral and low-phosphorus BLG isolates.

[0411] In some preferred embodiments, the process is carried out as a batch process. Alternatively, sometimes preferably, the process may be carried out as a semi-batch process. In other preferred embodiments, the process is carried out as a continuous process.

[0412] The advantage of this process is that it is much faster than the equivalent process for BLG crystallization in prior art. The time from the initial preparation of the whey protein feed to the completion of separation in step 3) can be up to 10 hours, preferably up to 4 hours, more preferably up to 2 hours, and even more preferably up to 1 hour.

[0413] Generally, it is preferable to prepare the BLG concentrate composition using a mild temperature that does not impair the nutritional value of either the non-aggregating BLG or other whey proteins in the whey protein feed.

[0414] In some preferred embodiments of the present invention, the non-aggregating BLG is not exposed to temperatures exceeding 90°C during the process. Preferably, the BLG is not exposed to temperatures exceeding 80°C during the process. Even more preferably, the non-aggregating BLG is not exposed to temperatures exceeding 75°C during the process. It should be noted that while spray drying often uses temperatures exceeding 150°C, the short exposure time and simultaneous evaporation of water mean that the spray-dried protein does not experience temperatures exceeding 50-70°C.

[0415] Regardless of the process used to prepare the BLG concentrate, it may include a step of drying the BLG concentrate. However, currently, it is preferable to use the BLG concentrate without drying it to avoid the risk of damaging the protein during drying.

[0416] If the BLG concentrate composition separated from the whey protein feed does not yet possess the necessary properties for use as a liquid BLG isolate, it can be subjected to one or more steps selected from the following group as part of providing a liquid BLG isolate: - Desalting, - Addition of minerals, -Dilution, -Concentration, -Physical microorganism reduction; -pH adjustment.

[0417] Non-limiting examples of desalting include, for example, dialysis, gel filtration, UF / diafiltration, NF / diafiltration, and ion exchange chromatography.

[0418] Non-limiting examples of mineral supplementation include, for example, the addition of soluble food-grade salts such as salts of Na, K, Ca, and / or Mg. Such salts may be, for example, phosphates, chlorides, or salts of edible acids such as citrates or lactates. Minerals can be added in solid, suspended, or dissolved forms.

[0419] Non-limiting examples of dilution include, for example, the addition of liquid diluents such as water, demineralized water, or aqueous solutions of minerals, acids, or bases.

[0420] Examples of non-limiting concentrations include evaporation, reverse osmosis, nanofiltration, ultrafiltration, and combinations thereof.

[0421] If the concentration needs to be increased relative to the total solids, it is preferable to use a concentration process such as ultrafiltration or, instead, dialysis. If the concentration does not need to be increased relative to the total solids, for example, evaporation, nanofiltration, and / or reverse osmosis may be useful.

[0422] Non-limiting examples of physical microbial reduction include, for example, heat treatment, bacterial filtration, UV irradiation, high-pressure treatment, pulsed electric field treatment, and ultrasound. These methods are well known to those skilled in the art.

[0423] Bacterial filtration typically involves microfiltration or large-pore ultrafiltration, requiring a pore size that can retain microorganisms but allow proteins and other desired components to pass through. Useful pore sizes are typically up to 1.5 microns, preferably up to 1.0 micron, more preferably up to 0.8 microns, even more preferably up to 0.5 microns, and most preferably up to 0.2 microns. The pore size of bacterial filtration is usually at least 0.1 microns.

[0424] Bacterial filtration may include, for example, a membrane having a pore size of 0.02 to 1 micron, preferably 0.03 to 0.8 microns, more preferably 0.04 to 0.6 microns, even more preferably 0.05 to 0.4 microns, and most preferably 0.1 to 0.2 microns.

[0425] In some preferred embodiments of the present invention, the liquid BLG isolate is subjected to bacterial filtration and subsequently to heat treatment using a temperature of up to 80°C, preferably up to 75°C. The combination of temperature and duration of this heat treatment is preferably selected to provide a sterile beverage preparation.

[0426] In another preferred embodiment of the present invention, the liquid BLG isolate is subjected to bacterial filtration and subsequently subjected to heat treatment for a period of up to 0.2 seconds, preferably up to 0.1 seconds, using a temperature of at least 150°C. The combination of the temperature and duration of this heat treatment is preferably selected to provide a sterile beverage preparation.

[0427] Non-limiting examples of pH adjustment include, for example, the addition of a base and / or acid, preferably a base and / or acid acceptable for food products. It is particularly preferable to use an acid and / or base that can chelate divalent metal cations. Examples of such acids and / or bases include citric acid, citrate, EDTA, lactic acid, lactate, phosphoric acid, phosphate, and combinations thereof.

[0428] In the following are some preferred embodiments of providing a liquid BLG isolate from a BLG concentrated composition in step a). The steps of the process referred to in this context are applied to a BLG-containing product stream following the BLG concentrated composition.

[0429] In some preferred embodiments of the present invention, for example, if the BLG concentrate composition contains BLG crystals derived from the above salt dissolution process, providing the liquid BLG isolate in step a) includes subjecting the BLG concentrate composition to the following steps in the following order: -For example, to dissolve the BLG crystals in the BLG concentrated composition, the pH is adjusted to i) 2-4.9 or ii) 6.1-8.5. -Optionally, desalting or addition of minerals, and - One of the following, - Concentration to the desired protein content and subsequent physical microbial reduction, or - Physical reduction of microorganisms followed by concentration of the desired protein content.

[0430] In yet another preferred embodiment of the present invention, for example, if the BLG concentrate composition contains BLG crystals derived from the above salt dissolution process, providing the liquid BLG isolate in step a) includes subjecting the BLG concentrate composition to the following steps in the following order: -For example, to dissolve the BLG crystals in the BLG concentrated composition, the pH is adjusted to i) 2-4.9 or ii) 6.1-8.5. -Optionally, desalting or adding minerals. - Physical reduction of microorganisms followed by concentration of the desired protein content.

[0431] In another preferred embodiment of the present invention, for example, if the BLG concentrate composition contains BLG crystals derived from the above salt dissolution process, providing the liquid BLG isolate in step a) includes subjecting the BLG concentrate composition to the following steps in the following order: - Preferably, the addition of minerals and preferably soluble salts to dissolve the BLG crystals in the BLG concentrated composition, with a pH between 5.0 and 6.0, and - One of the following, - Concentration to the desired protein content and subsequent physical microbial reduction, or - Physical reduction of microorganisms followed by concentration of the desired protein content.

[0432] When handling acidic dissolved BLG concentrated compositions, it is particularly advantageous to use heat treatment as a physical microbial reduction, either alone or in combination with one or more of the other processes for physical microbial reduction described herein. The inventors have found that the use of mild heat treatment under acidic conditions is particularly beneficial because it allows the BLG to remain in its native folded state, while still contributing to a reduction in the microbial load of the treated flow.

[0433] It is particularly preferable to subject the acidic dissolved BLG concentrate composition to a bacterial filtration step while the total protein concentration is increased to a maximum of 27% by weight, preferably a maximum of 22% by weight, and more preferably a maximum of 17% by weight, and then subject the bacterially filtered BLG concentrate composition or liquid BLG isolate to a heat treatment step.

[0434] It is even more preferable to subject the acidic dissolved BLG concentrate composition to a bacterial filtration step while having a total protein concentration of 5-27% by weight, preferably 10-22% by weight, and more preferably 12-17% by weight, and then subject the bacterially filtered BLG concentrate composition or liquid BLG isolate to a heat treatment step.

[0435] The heat treatment step is preferably performed on the liquid BLG isolate, preferably as the final step before spray drying.

[0436] It is often preferable to avoid, or at least limit, the unfolding of BLG during the supply of liquid BLG isolate. When the heat treatment is used in a pH range of 2 to 4.9, it is preferable to maintain the temperature at a maximum of 82°C, preferably a maximum of 80°C, more preferably a maximum of 78°C, in order to limit or even avoid BLG unfolding.

[0437] The heat treatment is preferably at least pasteurization.

[0438] In some preferred embodiments of the present invention, the heat treatment temperature is in the range of 70 to 80°C, preferably 70 to 79°C, more preferably 71 to 78°C, even more preferably 72 to 77°C, most preferably 73 to 76°C, for example, about 75°C.

[0439] Preferably, the duration of the heat treatment is 1 second to 30 minutes when performed in a temperature range of 70 to 80 degrees Celsius. The longest exposure time is optimal at the lowest temperature in the temperature range, and vice versa.

[0440] In a particularly preferred embodiment of the present invention, the heat treatment is provided to be 70-78°C for 1 second to 30 minutes, more preferably 71-77°C for 1 minute to 25 minutes, and even more preferably 72-76°C for 2 minutes to 20 minutes.

[0441] In some embodiments, particularly when unfolding, higher temperatures may also be preferred, and optionally, aggregation of the BLG before drying is also required. For example, the heat treatment temperature may be at least 81°C, preferably at least 91°C, more preferably at least 100°C, even more preferably at least 120°C, and most preferably at least 140°C.

[0442] The heat treatment may be, for example, a UHT type treatment, which typically involves a temperature in the range of 135–144°C and a duration in the range of 2–10 seconds.

[0443] Alternatively, however, the heat treatment may also include a temperature in the range of 145 to 180°C and a duration in the range of 0.01 to 1 second, and more preferably, a temperature in the range of 150 to 180°C and a duration in the range of 0.01 to 0.2 seconds.

[0444] In some preferred embodiments of the present invention, providing the liquid BLG isolate in step a) includes performing microbial reduction by heat treatment while the pH is in the range of 2 to 4.9, preferably 2.5 to 4.7, more preferably 2.8 to 4.3, and even more preferably 3.2 to 4.0.

[0445] In another preferred embodiment of the present invention, the provision of a liquid BLG isolate from a BLG concentrated composition in step a) includes physical microbial reduction by heat treatment while the pH is in the range of 2 to 4.9, preferably 2.5 to 4.7, more preferably 2.8 to 4.3, and even more preferably 3.2 to 4.0.

[0446] In yet another preferred embodiment of the present invention, the provision of a liquid BLG isolate from the BLG concentrated composition in step a) includes desalting while the pH is in the range of 6.1 to 8.5, preferably 6.3 to 8.0, and more preferably 6.5 to 7.5. The inventors have found that such desalting is advantageous in improving the thermal stability of the BLG isolate powder prepared by this method.

[0447] In a particularly preferred embodiment of the present invention, providing a liquid BLG isolate from a BLG concentrated composition in step a) includes: - Physical reduction of microorganisms by heat treatment while the pH is in the range of 2 to 4.9, preferably 2.5 to 4.7, more preferably 2.8 to 4.3, and even more preferably 3.2 to 4.0, and - Desalting while the pH is in the range of 6.1 to 8.5, preferably 6.3 to 8.0, and more preferably 6.5 to 7.5.

[0448] In some preferred embodiments of the present invention, for example, if the BLG concentrate composition contains BLG crystals derived from the above salt dissolution process, providing the liquid BLG isolate in step a) includes subjecting the BLG concentrate composition to the following steps in the following order: - Adjusting the pH to 2-4.9 to dissolve the BLG crystals in the BLG concentrated composition. -Optionally, concentration to a desired protein content while the pH is between 2 and 4.9, preferably 2.5 and 4.0, more preferably 3.0 and 3.9, and - Physical reduction of microorganisms by heat treatment while the pH is 2 to 4.9, preferably 2.5 to 4.7, more preferably 2.8 to 4.3, and even more preferably 3.2 to 4.0.

[0449] If the BLG concentrate already has a pH in the range of 2 to 4.9, providing the liquid BLG isolate in step a) preferably involves subjecting the BLG concentrate to the following steps in the following order: -Desalting while the pH is 2 to 4.9, preferably 2.5 to 4.7, more preferably 2.8 to 4.3, and even more preferably 3.2 to 4.0. -Optionally, concentration to a desired protein content while the pH is between 2 and 4.9, preferably 2.5 and 4.7, more preferably 2.8 and 4.3, and even more preferably 3.2 and 4.0, and - Physical reduction of microorganisms by heat treatment while the pH is 2 to 4.9, preferably 2.5 to 4.7, more preferably 2.8 to 4.3, and even more preferably 3.2 to 4.0.

[0450] In another preferred embodiment of the present invention, which is particularly suitable for providing a liquid BLG isolate having a pH in the range of 5.0 to 8.5, preferably in the range of 6.1 to 8.5, and more preferably in the range of 6.5 to 8.0, the provision of the liquid BLG isolate in step a) comprises subjecting the BLG concentrated composition to the following steps in the following order: - For example, adjusting the pH to 2-4.9 to dissolve BLG crystals in a BLG concentrated composition. -Optionally, concentrate to the desired protein content. - Physical reduction of microorganisms by heat treatment while the pH is 2 to 4.9, preferably 2.5 to 4.7, more preferably 2.8 to 4.3, and even more preferably 3.2 to 4.0. pH adjustment to a range of -5.0 to 8.5, preferably 6.1 to 8.5, and more preferably 6.5 to 8.0.

[0451] In another preferred embodiment of the present invention, which is particularly suitable for providing a liquid BLG isolate having a pH in the range of 6.1 to 8.5, preferably in the range of 6.3 to 8.0, and more preferably in the range of 6.5 to 7.5, the provision of the liquid BLG isolate in step a) comprises subjecting the BLG concentrated composition to the following steps in the following order: - For example, adjusting the pH to 2-4.9 to dissolve BLG crystals in a BLG concentrated composition. -Optionally, concentration to a desired protein content while the pH is between 2 and 4.9, preferably 2.5 and 4.7, more preferably 2.8 and 4.3, and even more preferably 3.2 and 4.0. - Physical reduction of microorganisms by heat treatment while the pH is 2 to 4.9, preferably 2.5 to 4.7, more preferably 2.8 to 4.3, and even more preferably 3.2 to 4.0. pH adjustment to a range of -6.1 to 8.5, preferably 6.3 to 8.0, more preferably 6.5 to 7.5, and Desalting at a pH in the range of -6.1 to 8.5, preferably in the range of 6.3 to 8.0, and more preferably in the range of 6.5 to 7.5.

[0452] In a more preferred embodiment of the present invention, which is particularly suitable for providing a liquid BLG isolate having a pH in the range of 6.1 to 8.5, preferably in the range of 6.1 to 8.5, and more preferably in the range of 6.5 to 8.0, the provision of the liquid BLG isolate in step a) includes subjecting the BLG concentrated composition to the following steps in the following order: - For example, adjusting the pH to at least pH 6.1 to dissolve the BLG crystals in the BLG concentrated composition. - Desalting while the pH is in the range of 6.1 to 8.5, preferably 6.5 to 8.0, more preferably 6.5 to 7.5 -Optionally, concentration of the desired protein while the pH is between 6.1 and 8.5, preferably 6.5 and 8.0, more preferably 6.5 and 7.5. -Optionally, physical reduction of microorganisms by heat treatment while the pH is in the range of 6.1 to 8.5, preferably 6.5 to 8.0, more preferably 6.5 to 7.5.

[0453] The temperature during the conversion of the BLG concentrated composition to a liquid BLG isolate is typically in the range of 0 to 82°C, and may involve higher temperatures for heat treatment. If the pH of the BLG stream during processing exceeds 4.9, the temperature is preferably in the range of 0 to 65°C, more preferably in the range of 0 to 15°C or 50 to 65°C, in order to reduce microbial growth.

[0454] When the pH of the liquid BLG isolate is up to 4.9, more preferably up to 4.1, the temperature is in the range of 0 to 82°C, advantageously in the range of 0 to 15°C, or preferably in the range of 50 to 80°C to reduce microbial growth. The inventors have found that it is particularly advantageous to carry out the process, or at least the concentration step as needed, at a temperature in the range of 50 to 80°C, more preferably in the range of 60 to 80°C, and even more preferably in the range of 65 to 78°C, as the higher temperature increases the efficiency of the concentration step and simultaneously contributes to the reduction of microorganisms. This embodiment makes it possible to produce a liquid BLG isolate having a high content of BLG and a very low microbial content while maintaining the nativeness of BLG. The resulting spray-dried powder has a high bulk density and a high level of nativeness, as well as a very favorable microbial profile.

[0455] In some preferred embodiments of the present invention, the process of converting the BLG concentrated composition into a liquid BLG isolate is carried out at a temperature in the range of 0 to 15°C, preferably in the range of 1 to 10°C.

[0456] In another preferred embodiment of the present invention, at least part, preferably the entire process, of the process of converting the BLG concentrated composition to a liquid BLG isolate is carried out at a temperature in the range of 50 to 82°C, preferably 55 to 80°C, and more preferably 60 to 78°C. This is particularly preferred when the pH of the protein stream during processing is up to 4.9, preferably up to 4.3, more preferably up to 3.7, and preferably in the range of 3.0 to 4.3.

[0457] In some preferred embodiments of the present invention, at least some processes, preferably the entire process, for converting a BLG concentrate composition into a liquid BLG isolate are carried out at a temperature in the range of 78 to 82°C while the pH is in the range of 3.0 to 3.7. In some preferred embodiments of the present invention, at least some processes, preferably the entire process, for converting a BLG concentrate composition into a liquid BLG isolate are carried out at a temperature in the range of 60 to 78°C while the pH is in the range of 3.7 to 4.3.

[0458] The BLG concentrate preferably has substantially the same protein composition as the liquid BLG isolate, and usually does not require the application of additional protein fractions when converting the BLG concentrate to a liquid BLG isolate.

[0459] In some preferred embodiments of the present invention, the BLG concentrated composition contains at least 85% by weight, preferably at least 88% by weight, more preferably at least 90% by weight, and even more preferably at least 92% of the total protein amount of BLG, most preferably at least 95% by weight of BLG relative to the total protein. It may be particularly preferred that the BLG concentrated composition contains at least 97% by weight, more preferably at least 99% by weight, for example, preferably about 100% by weight of BLG relative to the total protein.

[0460] In some preferred embodiments of the present invention, the BLG concentrated composition contains at least 5% by weight, preferably at least 10% by weight, more preferably at least 15% by weight, even more preferably at least 20% by weight of total protein, and most preferably at least 30% by weight of total protein, relative to the total protein.

[0461] In some preferred embodiments of the present invention, the BLG concentrated composition contains total protein in the range of 5 to 45% by weight, preferably in the range of 10 to 40% by weight, more preferably in the range of 15 to 38% by weight, and even more preferably in the range of 20 to 35% by weight.

[0462] In some preferred embodiments of the present invention, the total of alpha-lactalbumin (ALA) and caseinomacropeptide (CMP) constitutes at least 40% by weight of non-BLG proteins in the BLG concentrated composition, preferably at least 60% by weight, more preferably at least 70% by weight, and most preferably at least 90% by weight of non-BLG proteins in the BLG concentrated composition.

[0463] In another preferred embodiment of the present invention, each major non-BLG whey protein in the BLG concentrate is present as a weight percentage of the total protein, which is up to 25%, preferably up to 20%, more preferably up to 15%, even more preferably up to 10%, and most preferably up to 6% of its weight percentage of the total protein in a standard whey protein concentrate derived from sweet whey.

[0464] Even lower concentrations of major non-BLG whey proteins may be desirable. Therefore, in an additional preferred embodiment of the present invention, each major non-BLG whey protein in the BLG concentrate is present as a weight percentage of the total protein, which is up to 4%, preferably up to 3%, more preferably up to 2%, and even more preferably up to 1%, of its weight percentage of the total protein in a standard whey protein concentrate derived from sweet whey.

[0465] The inventors have observed that low levels of lactoferrin and / or lactoperoxidase are particularly advantageous in obtaining achromatic whey protein products.

[0466] Therefore, in some preferred embodiments of the present invention, lactoferrin is present in the BLG concentrate composition as a weight percentage of total protein, which is up to 25%, preferably up to 20%, more preferably up to 15%, even more preferably up to 10%, and most preferably up to 6% of its weight percentage of total protein in a standard whey protein concentrate derived from sweet whey. Even lower concentrations of lactoferrin may be desirable. Therefore, in an additional preferred embodiment of the present invention, lactoferrin is present as a weight percentage of total protein, which is up to 4%, preferably up to 3%, more preferably up to 2%, and even more preferably up to 1% of its weight percentage of total protein in a standard whey protein concentrate derived from sweet whey.

[0467] Similarly, in some preferred embodiments of the present invention, lactoperoxidase is present in the BLG concentrate composition as a weight percentage of total protein, which is up to 25%, preferably up to 20%, more preferably up to 15%, even more preferably up to 10%, and most preferably up to 6% of its weight percentage of total protein in a standard whey protein concentrate derived from sweet whey. Even lower concentrations of lactoperoxidase may be desirable. Therefore, in an additional preferred embodiment of the present invention, lactoperoxidase is present as a weight percentage of total protein, which is up to 4%, preferably up to 3%, more preferably up to 2%, and even more preferably up to 1% of its weight percentage of total protein in a standard whey protein concentrate derived from sweet whey.

[0468] In some preferred embodiments of the present invention, the BLG concentrated composition contains total solids in the range of 5 to 50% by weight, preferably 10 to 45% by weight, more preferably 15 to 40% by weight, and even more preferably 20 to 35% by weight.

[0469] In some preferred embodiments of the present invention, the BLG concentrated composition contains a water content in the range of 50-95% by weight, preferably 55-90% by weight, more preferably 60-85% by weight, and even more preferably 65-80% by weight.

[0470] In some preferred embodiments of the present invention, the BLG concentrate composition contains up to 60% by weight, preferably up to 50% by weight, more preferably up to 20% by weight, even more preferably up to 10% by weight, even more preferably up to 1% by weight, and most preferably up to 0.1% of carbohydrates.

[0471] In some preferred embodiments of the present invention, the BLG concentrate composition contains lipids in an amount of up to 10% by weight, preferably up to 5% by weight, more preferably up to 2% by weight, and even more preferably up to 0.1% by weight.

[0472] In some preferred embodiments of the present invention, the BLG concentrate is used directly as a liquid BLG isolate, for example, if it already has a desired pH and chemical composition.

[0473] Some embodiments of the present invention do not require the physical microbial reduction in step b) and therefore require only steps a) and c), while other preferred embodiments require the physical microbial reduction and therefore include all three steps a), b), and c).

[0474] Therefore, in some preferred embodiments of the present invention, the liquid BLG isolate is subjected to physical microbial reduction.

[0475] Useful examples of physical microbial reduction include one or more of the following: heating, bacterial filtration, UV irradiation, high-pressure treatment, pulsed electric field treatment, and ultrasound.

[0476] In some preferred embodiments of the present invention, physical microbial reduction includes, or further comprises, heat treatment.

[0477] Preferably, the heat treatment includes at least pasteurization.

[0478] In certain embodiments, the heat treatment includes heating the liquid BLG isolate to a temperature in the range of 70–82°C.

[0479] In some preferred embodiments of the present invention, the heat treatment temperature is in the range of 70 to 80°C, preferably 70 to 79°C, more preferably 71 to 78°C, even more preferably 72 to 77°C, most preferably 73 to 76°C, for example, about 75°C.

[0480] Preferably, the duration of the heat treatment is 1 second to 30 minutes when performed in a temperature range of 70 to 80 degrees Celsius. The longest exposure time is optimal at the lowest temperature in the temperature range, and vice versa.

[0481] In a particularly preferred embodiment of the present invention, the heat treatment is provided to be 70-78°C for 1 second to 30 minutes, more preferably 71-77°C for 1 minute to 25 minutes, and even more preferably 72-76°C for 2 minutes to 20 minutes.

[0482] In some embodiments, particularly when unfolding, higher temperatures may also be preferred, and optionally, aggregation of the BLG before drying is also required. For example, the heat treatment temperature may be at least 81°C, preferably at least 91°C, more preferably at least 100°C, even more preferably at least 120°C, and most preferably at least 140°C.

[0483] The heat treatment may include, for example, a temperature in the range of 90 to 130°C and a duration in the range of 5 seconds to 30 minutes. The heat treatment may include, for example, heating to a temperature in the range of 90 to 95°C for 1 to 30 minutes, for example, heating to approximately 120°C for approximately 20 seconds. Alternatively, the heat treatment may include heating to a temperature in the range of 115 to 125°C for 5 to 30 minutes, for example, heating to approximately 120°C for approximately 20 seconds.

[0484] Alternatively, the heat treatment may be, for example, a UHT type treatment, which typically involves a temperature in the range of 135–144°C and a duration in the range of 2–10 seconds.

[0485] Alternatively, however, the heat treatment may also include a temperature in the range of 145 to 180°C and a duration in the range of 0.01 to 2 seconds, and more preferably, a temperature in the range of 150 to 180°C and a duration in the range of 0.01 to 0.3 seconds.

[0486] The heat treatment may be carried out using conventional equipment such as plate or tubular heat exchangers, scrape heat exchangers, or retort systems. Alternatively, particularly preferably for heat treatments above 95°C, direct steam-based heating may be used, for example, using direct steam injection, direct steam infusion, or spray cooking. Furthermore, such direct steam-based heating is preferably used in combination with flash cooling. A good example of the implementation of spray cooking is found in International Publication No. 2009113858 A1, which is incorporated herein by reference for all purposes. A good example of the implementation of direct steam injection and direct steam infusion is found in International Publication No. 2009113858 A1 and International Publication No. 2010 / 085957 A3, which are incorporated herein by reference for all purposes. General aspects of high-temperature treatment are described, for example, in "Thermal technologies in food processing" ISBN 185573558 X, which is incorporated herein by reference for all purposes.

[0487] In some preferred embodiments of the present invention, the physical microbial reduction in step b) is sterilization resulting in a sterile liquid BLG isolate. Such sterilization can be achieved, for example, by combining bacterial filtration and pasteurization.

[0488] In some preferred embodiments of the present invention, a liquid BLG isolate having a pH preferably in the range of 2 to 4.9 is subjected to bacterial filtration and subsequently subjected to heat treatment using a temperature of up to 80°C, preferably up to 75°C. The combination of temperature and duration of this heat treatment is preferably selected to provide a liquid BLG isolate.

[0489] In another preferred embodiment of the present invention, the liquid BLG isolate is subjected to bacterial filtration and subsequently subjected to heat treatment for a period of up to 0.2 seconds, preferably up to 0.1 seconds, using a temperature of at least 150°C. The combination of temperature and duration of this heat treatment is preferably selected to provide a liquid BLG isolate.

[0490] Step c) of this method preferably includes spray drying or freeze-drying. Spray drying is particularly preferred.

[0491] The inventors have found it particularly advantageous to avoid exposing liquid BLG isolates to heat treatment regimes that unfold or denature a significant amount of BLG. Therefore, when preheating liquid BLG isolates before spraying, it is preferable to carefully control the heat load.

[0492] In some embodiments of the present invention, the liquid BLG isolate has a temperature of up to 70°C, preferably up to 60°C, and more preferably up to 50°C, when it reaches the outlet of a spraying device (e.g., a nozzle or atomizer). In some preferred embodiments of the present invention, the liquid BLG isolate has a temperature of up to 40°C, preferably up to 30°C, more preferably up to 20°C, preferably up to 10°C, and most preferably up to 5°C, when it reaches the outlet of a spraying device.

[0493] The spraying device of a spray dryer is a device that converts the solution or suspension to be dried into droplets that enter the drying chamber of the spray dryer, such as a nozzle or atomizer.

[0494] In some embodiments of the present invention, it is particularly preferable that the liquid BLG isolate has a temperature in the range of 0 to 60°C, preferably 2 to 40°C, more preferably 4 to 35°C, and most preferably 5 to 10°C, when it reaches the outlet of the spraying device.

[0495] The gas inlet temperature of the spray dryer is preferably in the range of 140 to 220°C, more preferably in the range of 160 to 200°C, and even more preferably in the range of 170 to 190°C, for example, preferably around 180°C. The gas outlet temperature from the spray dryer is preferably in the range of 50 to 95°C, more preferably in the range of 70 to 90°C, and even more preferably in the range of 80 to 88°C, for example, preferably around 85°C. As an empirical rule, it is said that the solid subjected to spray drying is heated to a temperature 10 to 15°C lower than the gas outlet temperature.

[0496] In some preferred embodiments of the present invention, the outlet temperature of the spray dryer is preferably in the range of 50 to 85°C, more preferably in the range of 60 to 80°C, and even more preferably in the range of 65 to 75°C, for example, preferably about 70°C.

[0497] The advantage of this method is that the liquid BLG isolate to be dried may have a very high solid content before the drying process, thus requiring less water removal and resulting in less energy consumption during the drying operation.

[0498] The inventors have found that the lower the degree of BLG unfolding, the higher the concentration of BLG that can be treated before spray drying.

[0499] In some preferred embodiments of the present invention, the liquid BLG isolate has a solid content of at least 10% by weight. Preferably, the liquid BLG isolate has a solid content of at least 20% by weight. More preferably, the liquid BLG isolate has a solid content of at least 25% by weight. Even more preferably, the liquid BLG isolate has a solid content of at least 30% by weight. Most preferably, the liquid BLG isolate has a solid content of at least 35% by weight.

[0500] In another preferred embodiment of the present invention, the liquid BLG isolate has a solid content in the range of 10 to 60% by weight. Preferably, the liquid BLG isolate has a solid content in the range of 15 to 50% by weight. More preferably, the liquid BLG isolate has a solid content in the range of 20 to 45% by weight. Even more preferably, the liquid BLG isolate has a solid content in the range of 25 to 40% by weight, for example, about 35% by weight.

[0501] In some preferred embodiments of the present invention, the liquid BLG isolate contains at least 10% by weight of total protein. Preferably, the liquid BLG isolate contains at least 20% by weight of total protein. More preferably, the liquid BLG isolate contains at least 25% by weight of total protein. Even more preferably, the liquid BLG isolate contains at least 30% by weight of total protein. Most preferably, the liquid BLG isolate contains at least 35% by weight of total protein.

[0502] In another preferred embodiment of the present invention, the liquid BLG isolate contains a total amount of protein in the range of 10 to 50% by weight. Preferably, the liquid BLG isolate contains a total amount of protein in the range of 15 to 45% by weight. More preferably, the liquid BLG isolate contains a total amount of protein in the range of 20 to 40% by weight. Even more preferably, the liquid BLG isolate contains a total amount of protein in the range of 25 to 38% by weight, for example, approximately 35% by weight.

[0503] The inventors have found that a higher protein content reduces the energy consumption required to convert liquid BLG isolates into powder, thereby increasing the BLG yield obtained from a drying unit of a given capacity.

[0504] The method of the present invention is preferably carried out using a mild temperature that does not impair the nutritional value of either non-aggregating BLG or other whey proteins in the whey protein solution.

[0505] In some preferred embodiments of the present invention, the non-aggregating BLG is not exposed to temperatures exceeding 90°C during the above method. Preferably, the non-aggregating BLG is not exposed to temperatures exceeding 80°C during the above method. Even more preferably, the non-aggregating BLG is not exposed to temperatures exceeding 75°C during the above method. It should be noted that although spray drying often uses temperatures exceeding 150°C, the short exposure time and simultaneous evaporation of water mean that the spray-dried protein does not experience temperatures exceeding 40-70°C.

[0506] The inventors have observed that prolonged heating during the drying process reduces the amount of BLG in its unmodified form. In some preferred embodiments of the present invention, the thermal exposure during the drying process is kept low enough to provide a degree of BLG modification of up to 5%, preferably up to 4%, more preferably up to 2%, even more preferably up to 0.5%, and even more preferably up to 0.1%. Most preferably, the drying process does not result in any detectable modification of BLG.

[0507] The drying process may further include fluidized bed drying, either integrated into a spray drying unit or as a separate unit operation performed after spray drying.

[0508] The combination of spray drying and fluid bed drying reduces the amount of water removed as the drying droplets move through the spray drying chamber, and instead, fluid bed drying removes residual water from the wet powder. This solution requires less energy to dry than drying by spray drying alone and further allows for modification of the powder, for example, by instantiation and / or agglomeration. Instantiation is preferably carried out by applying lecithin or another useful wetting agent to the surface of the powder. When instantiation is applied, the instantiation agent, for example, lecithin dissolved in edible oil, is typically added in the range of 0.5 to 2% by weight relative to the total weight of the final powder, preferably in the range of 1.0 to 1.5% by weight relative to the total weight of the final powder.

[0509] This method further preferably includes a step of packaging the BLG isolation powder. The packaged BLG isolation powder product includes a container containing the BLG isolation powder described herein, preferably a sealed container.

[0510] In some embodiments of the present invention, the BLG isolation powder is sealed in a container and optionally packaged with an inert gas.

[0511] Various containers can be used to store BLG isolation powder. Preferred containers include, for example, bags, barrels, pouches, boxes, cans, and sachets.

[0512] A particularly preferred embodiment of the present invention relates to a method for producing a dried BLG isolation powder containing BLG in an amount of at least 85% by weight, preferably at least 90% by weight, and more preferably at least 94% by weight, relative to total protein, the method being as follows: a)i) pH range of 2 to 4.9 ii) pH in the range of 6.1 to 8.5, iii) pH in the range of 5.0 to 6.0 A step of providing a liquid BLG isolate having, wherein the liquid BLG isolate contains an amount of BLG that is at least 85% by weight, preferably at least 90% by weight, and more preferably at least 94% by weight of total protein. b) Optionally, a step of subjecting the liquid BLG isolate to physical microbial reduction. c) A step of drying the liquid BLG isolate, preferably by spray drying, The provision of the liquid BLG isolate in step a) is as follows: -Next step, 1) A step of providing a whey protein solution comprising non-aggregating BLG and at least one additional whey protein, wherein the whey protein solution is supersaturated with respect to BLG and has a pH in the range of 5 to 6. 2) A step of crystallizing BLG in a supersaturated whey protein solution, 3) A step to separate BLG crystals from the remaining whey protein solution. 4) Optionally, a step of washing BLG crystals, for example, BLG crystals separated from step 3) or 5), and 5) optionally, preparing a BLG concentrated composition by a process including a step of recrystallizing BLG crystals, for example, BLG crystals obtained in step 3) or 4), and -The process involves treating the BLG concentrated composition to dissolve at least BLG crystals, thereby obtaining a liquid BLG isolate.

[0513] In the above embodiment, the BLG concentrated composition comprises BLG crystals separated from step 3), which are then dissolved by appropriate pH adjustment or by increasing conductivity and / or raising the temperature.

[0514] A particularly preferred embodiment of the present invention relates to a method for producing a dried BLG isolation powder containing BLG in an amount of at least 85% by weight, preferably at least 90% by weight, and more preferably at least 94% by weight, relative to total protein, the method being as follows: a) A step of providing a liquid BLG isolate having a pH in the range of 2.5 to 4.9, preferably 2.5 to 4.0, and more preferably 3.0 to 3.9, The liquid BLG isolate contains BLG in an amount of at least 85% by weight, preferably at least 90% by weight, and more preferably at least 94% by weight, relative to the total protein. b) Optionally, a step of subjecting the liquid BLG isolate to physical microbial reduction. c) A step of drying the liquid BLG isolate, preferably by spray drying, The provision of the liquid BLG isolate in step a) is as follows: -Next step, 1) A step of providing a whey protein solution comprising non-aggregating BLG and at least one additional whey protein, wherein the whey protein solution is supersaturated with respect to BLG and has a pH in the range of 5 to 6. 2) A step of crystallizing BLG in a supersaturated whey protein solution, 3) A step to separate BLG crystals from the remaining whey protein solution. 4) Optionally, a step of washing BLG crystals, for example, BLG crystals separated from step 3) or 5), and 5) optionally, preparing a BLG concentrated composition by a process including a step of recrystallizing BLG crystals, for example, BLG crystals obtained in step 3) or 4), and - This includes at least adjusting the pH of the BLG concentrated composition to a range of 2.5 to 4.9, preferably 2.5 to 4.0, and more preferably 3.0 to 3.9.

[0515] Another particularly preferred embodiment of the present invention relates to a method for producing a dried BLG isolation powder containing BLG in an amount of at least 85% by weight, preferably at least 90% by weight, and more preferably at least 94% by weight, relative to total protein, the method being as follows: a) A step of providing a liquid BLG isolate having a pH in the range of 6.1 to 8.5, preferably in the range of 6.3 to 8.0, and more preferably in the range of 6.5 to 7.5, The liquid BLG isolate contains BLG in an amount of at least 85% by weight, preferably at least 90% by weight, and more preferably at least 94% by weight, relative to the total protein. b) Optionally, a step of subjecting the liquid BLG isolate to physical microbial reduction. c) A step of drying the liquid BLG isolate, preferably by spray drying, The provision of the liquid BLG isolate in step a) is as follows: -Next step, 1) A step of providing a whey protein solution comprising non-aggregating BLG and at least one additional whey protein, wherein the whey protein solution is supersaturated with respect to BLG and has a pH in the range of 5 to 6. 2) A step of crystallizing BLG in a supersaturated whey protein solution, 3) A step to separate BLG crystals from the remaining whey protein solution. 4) Optionally, a step of washing BLG crystals, for example, BLG crystals separated from step 3) or 5), and 5) optionally, preparing a BLG concentrated composition by a process including a step of recrystallizing BLG crystals, for example, BLG crystals obtained in step 3) or 4), and - This includes at least adjusting the pH of the BLG concentrated composition to a range of 6.1 to 8.5, preferably 6.3 to 8.0, and more preferably 6.5 to 7.5.

[0516] An alternative, but also preferred, embodiment of the present invention relates to a method for producing a dried BLG isolation powder containing BLG in an amount of at least 85% by weight, preferably at least 90% by weight, and more preferably at least 94% by weight, relative to total protein, the method being as follows: a) A step of providing a liquid BLG isolate having a pH in the range of 5.0 to 8.5, preferably 6.1 to 8.5, more preferably 6.3 to 8.0, and even more preferably 6.5 to 7.5, The liquid BLG isolate contains BLG in an amount of at least 85% by weight, preferably at least 90% by weight, and more preferably at least 94% by weight, relative to the total protein. b) Optionally, a step of subjecting the liquid BLG isolate to physical microbial reduction. c) A step of drying the liquid BLG isolate, preferably by spray drying, The provision of the liquid BLG isolate in step a) is in the following order: -Next step, 1) A step of providing a whey protein solution comprising non-aggregating BLG and at least one additional whey protein, wherein the whey protein solution is supersaturated with respect to BLG and has a pH in the range of 5 to 6. 2) A step of crystallizing BLG in a supersaturated whey protein solution, 3) A step to separate BLG crystals from the remaining whey protein solution. 4) Optionally, a step of washing BLG crystals, for example, BLG crystals separated from step 3) or 5), and 5) optionally, preparing a BLG concentrated composition by a process including a step of recrystallizing BLG crystals, for example, BLG crystals obtained in step 3) or 4), and - A step of adjusting the pH of the BLG concentrated composition to a pH in the range of 2.5 to 4.9, preferably 2.5 to 4.0, and more preferably 3.0 to 3.9. -Includes pasteurization using a temperature of at least, preferably in the range of 70-82°C, more preferably in the range of 70-80°C, during which the pH is in the range of 2.5-4.9, preferably 2.5-4.0, more preferably in the range of 3.0-3.9, with physical microbial reduction. pH adjustment to a range of -5.0 to 8.5, preferably 6.1 to 8.5, more preferably 6.3 to 8.0, and even more preferably 6.5 to 7.5. -Optionally includes desalting.

[0517] In some preferred embodiments, the method of the present invention is carried out as a batch process. Alternatively, 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.

[0518] Furthermore, one aspect of the present invention relates to a liquid BLG isolate described herein. The liquid BLG isolate is particularly useful for obtaining spray-dried BLG isolate powder and can also be used as a liquid raw material in the manufacture of liquid or non-liquid food products. Alternatively, however, it is also preferable that the liquid BLG isolate be used as a beverage on its own.

[0519] In some preferred embodiments of the present invention, the BLG isolated powder of the present invention can be obtained by the method described herein.

[0520] In some preferred embodiments of the present invention, the liquid BLG isolate of the present invention can be obtained by the method described herein, except that the drying step is omitted.

[0521] One aspect of the present invention relates to the use of BLG isolated powder or liquid BLG isolated as defined herein as a raw material for the manufacture of food products. The food products are, for example, beverages or instant beverage powders.

[0522] The use of BLG isolated powder or liquid BLG isolate preferably provides one or more of the following effects: - Reduced level of dry mouthfeel - Improvement of the transparency of the resulting liquid containing BLG isolation powder or liquid BLG isolate. - Decrease in viscosity - Potential to increase the protein concentration of heat-treated food products. - Low color contribution (The inventors observed that the BLG isolate of the present invention provides less color to, for example, a protein beverage than the corresponding WPI solution).

[0523] In some preferred embodiments of the present invention, the use of BLG isolated powder is as a raw material for preparing a beverage having a protein content of at least 10-36% by weight, more preferably at least 15-35% by weight, even more preferably 20-34% by weight, and most preferably 25-33% by weight, where the BLG isolated powder contributes at least 90% by weight, more preferably at least 95% by weight, and most preferably the total protein of the beverage.

[0524] Since the BLG powder of the present invention contributes less to viscosity than conventional WPI, the BLG powder of the present invention is particularly suitable for high-protein beverages or shake powders for preparing high-protein beverages, and therefore provides a more drinkable beverage.

[0525] Food products, for example, have a pH in the range of 2 to 4.7, and furthermore, - A decrease in the level of dry mouthfeel. -Improved transparency, and / or - The beverage or instant beverage powder may have one or more of the above characteristics, and may preferably be a heat-treated beverage containing a protein content of at least 3-45% by weight, more preferably 11-40% by weight, even more preferably 15-38% by weight, and most preferably 20-36% by weight.

[0526] It should be noted that embodiments and features described in the context of one aspect of the present invention also apply to other aspects of the present invention.

[0527] All patent and non-patent references cited in this application are incorporated herein by reference in their entirety.

[0528] Next, the present invention will be described in more detail by the following non-limiting examples.

[0529] example Example 1: Method of Analysis Example 1.1: Determination of protein nativeness by endogenous tryptophan fluorescence Tryptophan (Trp) fluorescence spectroscopy is a well-known tool for monitoring protein folding and unfolding. Trp residues embedded in native proteins typically exhibit the highest fluorescence emission around 330 nm compared to those located in solvent-exposed positions, such as in unfolded proteins. In unfolded proteins, the wavelength of Trp fluorescence emission typically shifts to higher wavelengths, often measured around 350 nm. We hereby utilize this transition to monitor thermally induced unfolding by calculating the ratio of fluorescence emission at 330 nm and 350 nm and investigating the effect of heating temperature.

[0530] The analysis includes the following steps: The beverage composition was diluted to 0.6 mg / mL with MQ water. Transfer 300 μl of the sample to a white 96-well plate, avoiding air bubbles, or transfer 3 ml to a 10 mm quartz cuvette. The tryptophan fluorescence emission intensity in the 310-400 nm range was recorded from above by excitation at 295 using a 5 nm slit. Samples were measured at 22°C using a Cary Eclipse fluorescence spectrophotometer equipped with a plate reader accessory (G9810A) or a single cuvette holder. The emission intensity ratio was calculated by dividing the fluorescence emission intensity measured at 330 nm by the emission intensity at 350 nm, i.e., R = I330 / I350, and used as a measure of the protein's nativity. oAt least 1.11 R represents the dominant native BLG stereostructure, R values ​​less than o1.11 indicate at least partial unfolding and aggregation.

[0531] Example 1.2: Thermal stability at pH 3.9 Thermal stability at pH 3.9: Thermal stability at pH 3.9 is a measure of the protein composition's ability to maintain a clear state during prolonged pasteurization at pH 3.9.

[0532] Thermal stability at pH 3.9 is determined by forming an aqueous solution at pH 3.9, including 6.0% wt / wt protein by mixing the powder or liquid sample to be tested with water (or, alternatively, by concentrating by low-temperature evaporation in the case of dilute liquids), and adjusting the pH to 3.9 with a minimum amount of 0.1 M NaOH or 0.1 M HCl.

[0533] After allowing the pH-adjusted mixture to stand for 30 minutes, transfer 25 mL of the mixture to a 30 mL thin-walled glass test tube. Heat to 75.0°C for 300 seconds by immersing in a 75.0°C water bath. Immediately after heating, transfer the glass test tube to an ice bath to cool to 1–5°C, and measure the turbidity of the heat-treated sample according to Example 1.7.

[0534] Example 1.3: Determination of the degree of protein denaturation of a whey protein composition Since denatured whey protein is known to have lower solubility at pH 4.6 than at pH values ​​below or above pH 4.6, 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 the pH in which the protein in the solution is stable.

[0535] More specifically, in the case of whey protein, the whey protein composition to be analyzed (e.g., powder or aqueous solution) is converted as follows: - A first aqueous solution containing 5.0% total protein (by weight / by weight) and having a pH of 7.0 or 3.0, - A second aqueous solution containing 5.0% total protein (by weight) and with a pH of 4.6.

[0536] pH adjustment is performed using 3% (wt / wt) NaOH (aqueous solution) or 5% (wt / wt) HCl (aqueous solution).

[0537] Total protein content of the first aqueous solution (P pH7.0又は3.0 ) is determined according to Example 1.5.

[0538] The second aqueous solution is stored at room temperature for 2 hours, then centrifuged at 3000g for 5 minutes. The supernatant sample is collected and analyzed according to Example 1.5 to determine the protein concentration (S) in the supernatant. pH4.6 ) obtain.

[0539] The degree of protein denaturation, D, of the whey protein composition is calculated as follows: D=((P pH7.0又は3.0 -S pH4.6 ) / P pH7.0又は3.0 )*100%

[0540] Example 1.4 Determination of protein denaturation using reversed-phase ULC analysis (with pH 4.6 acid precipitation). BLG samples (unheated reference and heated BLG beverage compositions, etc.) were diluted to 2% with MQ water. 5 ml of protein solution, 10 ml of Milli-Q, 4 ml of 10% acetic acid, and 6 ml of 1.0 M NaOAc were mixed and stirred for 20 minutes to precipitate and aggregate denatured proteins at around pH 4.6. The solution was filtered through a 0.22 μm filter to remove aggregates and non-native proteins.

[0541] All samples were subjected to the same degree of dilution by adding polished water.

[0542] For each sample, the same volume was packed into a UPLC system equipped with a UPLC column (Protein BEH C4; 300 Å; 1.7 μm; 150 × 2.1 mm), and detection was performed at 214 nm.

[0543] The samples were run under the following conditions: Buffer A: Milli-Q water, 0.1% wt / wt TFA Buffer B: HPLC-grade acetonitrile, 0.1% wt / wt TFA Flow rate: 0.4ml / min Gradient: 0-6.00 min 24-45%B; 6.00-6.50 min 45-90%B; 6.50-7.00 min 90%B; 7.00-7.50 min 90-24%B, and 7.50-10.00 min 24%B.

[0544] The concentration of native BLG in the sample was determined using the area of ​​the BLG peak relative to the protein standard (Sigma L0130) (5-level calibration curve).

[0545] The sample was further diluted, and if it fell outside the linear range, it was reinjected.

[0546] Example 1.5: Determination of total protein The total protein content (true protein) of the sample is determined as follows: 1) Determination of the total nitrogen content of a sample in accordance with 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 non-protein nitrogen in a sample in accordance with ISO 8968-4|IDF 020-4 - Milk - Determination of nitrogen content - Part 4: Determination of non-protein nitrogen content. 3) The total amount of protein (m 総窒素 -m 非タンパク質-窒素 Calculate using * 6.38.

[0547] Example 1.6: Determination of non-aggregating BLG, ALA, and CMP The content of non-aggregating alpha-lactalbumin (ALA), beta-lactoglobulin (BLG), and caseinomacropeptide (CMP) was analyzed by HPLC at a rate of 0.4 ml / min. 25 microL of filtered sample was injected into two TSKgel3000PWxl (7.8 mm 30 cm, Tosohass, Japan) columns, which were attached to a pre-equilibrated PWxl column (6 mm × 4 cm, Tosohass, Japan) with an eluent (465 g Milli-Q water, 417.3 g acetonitrile, and 1 ml trifluoroacetic acid), and detected using a 210 nm UV detector.

[0548] Native alpha-lactalbumin (C アルファ ), beta-lactoglobulin (C ベータ ), and caseinomacropeptide (C CMP The quantitative determination of the content of ) was performed by comparing the peak area obtained for the corresponding standard protein with the peak area of ​​the sample.

[0549] The total amount of additional protein (non-BLG protein) was determined by subtracting the amount of BLG from the total amount of protein (determined according to Example 1.5).

[0550] Example 1.7: Measurement of turbidity Turbidity is the cloudiness or haze of a fluid caused by a large number of particles that are generally invisible to the naked eye, similar to smoke in the air.

[0551] Turbidity is measured in specific turbidity units (NTU).

[0552] 20 ml of beverage / sample was added to an NTU glass and placed in a Turbiquant® 3000 IR turbidimeter. The NTU value was measured after stabilization and repeated twice.

[0553] Example 1.8: Determination of viscosity The viscosity of the liquid was measured using a Gilson Viscoman viscometer or an equivalent viscometer, and the viscometer was set to 300s. -1The shear rate is reported. Unless otherwise specified, samples are equilibrated to 15°C before measurement and measured at that temperature.

[0554] Unless otherwise specified, viscosity is 300s -1 It is expressed in centipoise (cP) units at the shear rate. The higher the measured cP value, the higher the viscosity.

[0555] Example 1.9: Color determination Color was measured using a colorimeter (Konica Minolta, CR-400). 15g of the sample was added to a small Petri dish (55 × 14.2 mm, VWR catalog number 391-0895) to avoid air bubble formation. The protein content of the sample was standardized to 6.0 wt / wt% protein or less.

[0556] The chromatometer was calibrated to a white calibration plate (No. 19033177). The light source was set to D65, and the observer was set to 2 degrees. Color (CIELAB color space, a*, b*, L* values) was measured with the suspension covered by a lid, as the average of three individual readings at various locations in the petri dish.

[0557] The desalting water standard values ​​are as follows: L*39.97±0.3 a*0.00±0.06 b*-0.22±0.09

[0558] The measured values ​​were converted to delta / difference values ​​based on the desalted water measurements. Delta L* = L 6.0重量 / 重量%タンパク質に標準化したサンプル *-L 脱塩水 *Measured at room temperature. Delta a* = a 6.0重量 / 重量%タンパク質に標準化したサンプル *-a 脱塩水 *Measured at room temperature. Delta b* = b 6.0重量 / 重量%タンパク質に標準化したサンプル *-b 脱塩水 *Measured at room temperature.

[0559] The samples have been standardized to 6.0 wt / wt% protein or less.

[0560] The L*a*b* color space (also known as the CIELAB space) is one of the uniform color spaces defined by the International Commission on Illumination (CIE) in 1976 and used for quantitative reporting of lightness and hue (ISO 11664-4:2008(E) / CIE S 014-4 / E:2007).

[0561] In this space, L* represents brightness (a value from 0 to 100), with L*=0 representing the darkest black and L*=100 representing the brightest white.

[0562] The color channels a* and b* represent the true neutral gray values ​​at a*=0 and b*=0. The a* axis represents the green and red components, with green being negative and red being positive. The b* axis represents the blue and yellow components, with blue being negative and yellow being positive.

[0563] Example 1.10 Beverage Stability Test / Insoluble Protein Substances When centrifuged at 3000g for 5 minutes, if less than 15% of the total protein in the heated sample precipitated, the composition of the whey protein beverage was considered stable. Approximately 20g of the sample was added to a centrifuge tube and centrifuged at 3000g for 5 minutes. • Protein recovery was quantified using Kjeldahl analysis of the protein before centrifugation and the supernatant after centrifugation. See Example 1.5.

[0564] Calculate protein loss:

number

[0565] This parameter is sometimes referred to as the level of insoluble protein material and can be used for the analysis of both liquid and powder samples. If the sample is a powder, suspend 10 g of powder in 90 g of desalted water and hydrate at 22°C for 1 hour with gentle stirring. Centrifuge approximately 20 g of the sample (e.g., liquid sample or suspended powder sample) in a centrifuge tube at 3000 g for 5 minutes. Protein (P) before centrifugation total ) and the supernatant after centrifugation (P 3000×g Protein recovery was quantified according to Example 1.5 using Kjeldahl analysis.

[0566] Calculate the amount of insoluble protein:

number

[0567] Example 1.11: Sensory evaluation The heat-treated beverage preparations underwent descriptive sensory evaluation. The beverage preparations were heated using a plate heat exchanger. One volume of sample is mixed with one volume of water and compared to an unheated whey protein isolate. Lactic acid and citric acid are also used to create an attribute list before the final tasting session. [Table 1]

[0568] Participants' mouths were cleansed between each sample using crackers, white tea, melon, and water.

[0569] A 15 mL test sample at ambient temperature (20-25°C) was provided in a small cup.

[0570] The test samples were provided to 10 individuals in three different blocks in a randomized order, over three separate rounds.

[0571] The attributes (see the table above) were evaluated on a 15cm scale, with 0 = low intensity and 15 = high intensity.

[0572] Statistical analysis was performed using Panelcheck software with a three-way ANOVA test for multiple replicates. Samples were fixed, and panels were randomly assigned.

[0573] We assessed the significance of differences between samples using the Bonferroni correction, which represents the smallest statistically significant difference (pairwise comparison of groups associated with letters).

[0574] Example 1.12: Determination of transparency by imaging The beverage preparation was photographed by placing the sample in a turbidity NTU measuring vial that was touching a piece of paper with the text "lorem ipsum" written on it. The vial was photographed using a smartphone, and the inventors evaluated whether the text was clearly visible through the vial.

[0575] Example 1.13: Determination of Ash Content The ash content of food products is measured according to NMKL 173:2005, "Determination of the weight-based formula for ash content in food."

[0576] Example 1.14: Determination of conductivity The "electrical conductivity" (sometimes called "specific conductivity") of an aqueous solution is a measure of the solution's ability to conduct electricity. Conductivity is determined, for example, by measuring the AC resistance of the solution between two electrodes, and the result is typically expressed in millisiemens / cm (mS / cm). Conductivity can be measured, for example, according to EPA (U.S. Environmental Protection Agency) Method No. 120.1.

[0577] Unless otherwise specified, the conductivity values ​​described herein are normalized to 25°C.

[0578] Conductivity is measured using a conductivity meter (WTW Cond 3210 with Tetracon 325 electrodes).

[0579] The system is calibrated as described in the manual before use. The electrodes are thoroughly rinsed in the same type of medium in which the measurement will be performed to avoid localized dilution. The electrodes are lowered into the medium so that the area in which the measurement is performed is completely submerged. The electrodes are then agitated to remove any trapped air. The electrodes are then kept still until a stable value is obtained from the display and recorded.

[0580] Example 1.15: Determination of the total solids content of a solution The total solids content of a solution can be determined according to NMKL 110 2nd edition, 2005 (Total Solids (Water) - Gravimetric Analysis of Milk and Dairy Products). NMKL is an abbreviation for the Nordic Commission on Standard Analytical Methods and Food Analysis (Nordisk Metodikkomite for Naeringsmidler).

[0581] The water content of a solution can be calculated by subtracting the relative amount of total solids (% weight / weight) from 100%.

[0582] Example 1.16: Determining pH All pH values ​​were measured using a pH glass electrode and normalized to 25°C.

[0583] The pH glass electrode (with temperature compensation) should be thoroughly rinsed and calibrated before use.

[0584] If the sample is a liquid, the pH is measured directly in the solution at 25°C.

[0585] If the sample is in powder form, dissolve 10 grams of powder in 90 ml of demineralized water at room temperature while stirring vigorously. Then, measure the pH of the solution at 25°C.

[0586] Example 1.17: Determination of loosening density and bulk density The density of a dry powder is defined as the relationship between the weight and volume of the powder, analyzed under specific conditions using a special Stampf volumetric instrument (graduated cylinder). Density is typically expressed in g / mL or kg / L.

[0587] In this method, a sample of dry powder is packed into a graduated cylinder. After a specified number of taps, the volume of the product is read and its density is calculated.

[0588] This method allows us to define the following three types of densities: • Packing density: This is the mass of the powder divided by the volume of the powder after it has been transferred to the specified graduated cylinder. • Loosening density is the mass obtained by dividing the volume of powder after 100 taps according to the conditions specified in this standard. • Bulk density is the mass obtained by dividing the volume of the powder after 625 taps, according to the conditions specified in this standard.

[0589] This method uses a 250 ml graduated cylinder marked from 0 to 250 ml, a special graduated cylinder weighing 190 ± 15 g (J. Engelsmann AG67059 Ludwigshafen / Rh), and, for example, a Stampf volumetric instrument from J. Engelsmann AG.

[0590] The looseness density and bulk density of the dried product are determined by the following procedure.

[0591] Pre-processing: Store the sample to be measured at room temperature.

[0592] Next, rotate and invert the container repeatedly to thoroughly mix the sample (avoiding particle pulverization). The container should not be filled more than two-thirds of the way.

[0593] procedure: Measure 100.0 ± 0.1 grams of powder and transfer it to a graduated cylinder. Read the volume V0 in ml.

[0594] If 100g of powder does not fit in the cylinder, the amount needs to be reduced to 50 or 25 grams.

[0595] Secure the graduated cylinder to the Stampf volumetric gauge and tap it 100 times. Flatten the surface with a spatula and measure the volume V. 100 Read it in ml.

[0596] Change the number of tabs to 625 (including 100 taps). After lightly tapping, level the surface and adjust the capacitance V. 625 Read it in ml.

[0597] Density calculation: The loosening density and bulk density, expressed in g / mL, are calculated according to the following formula: Bulk density = M / V In the formula, M represents the weight of the sample in grams, and V represents the volume in ml after 625 taps.

[0598] Example 1.18: Determining the water content of powder The water content of food products is measured according to ISO 5537:2004 (powdered milk - measurement of water content (standard method)). NMKL is an abbreviation for the Nordic Commission on Standard Analytical Methods and Food Analysis (Nordisk Metodikkomite for Naeringsmidler).

[0599] Example 1.19: Measurement of calcium, magnesium, sodium, potassium, and phosphorus levels (ICP-MS method) The total amounts of calcium, magnesium, sodium, potassium, and phosphorus are determined using a procedure in which the sample is first decomposed using microwave decomposition, and then the total amount of minerals (which may be multiple) is determined using an ICP instrument.

[0600] Device: The microwave is manufactured by Anton Paar, and the ICP is an Optima 2000DV manufactured by PerkinElmer Inc.

[0601] material: 1M HNO3 Yttrium in 2% HNO3 Appropriate standards for calcium, magnesium, sodium, potassium, and phosphorus in 5% HNO3

[0602] Pre-processing: Measure out a certain amount of powder and transfer it to a microwave decomposition tube. Add 5 ml of 1 M HNO3. Decompose the sample in the microwave according to the microwave instructions. Place the decomposed tube in a fume hood, remove the lid, and allow the volatile fumes to evaporate.

[0603] Measurement procedure: Transfer the pre-treated sample to the DigiTUBE using a known amount of Milli-Q water. Add the yttrium solution in 2% HNO3 to the decomposition tube (approximately 0.25 ml per 50 ml of diluted sample) and dilute to a known volume using Milli-Q water. Analyze the sample by ICP using the procedure described by the manufacturer.

[0604] Blind samples are prepared by diluting a mixture of 10 ml of 1 M HNO3 and 0.5 mL of yttrium 2% HNO3 solution to a final volume of 100 mL using Milli-Q water.

[0605] Prepare at least three standard samples with concentrations that fall between the expected sample concentration.

[0606] Example 1.20: Determination of furosen levels: Furosine levels 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 the total protein amount is determined according to Example 1.5. Furosine levels are reported in mg of furosine per 100 g of protein.

[0607] Example 1.21: Determination of the crystallinity of BLG in liquid The degree of crystallinity of BLG in a liquid with a pH in the range of 5-6 is determined using the following method. a) Transfer a 10 mL sample of the liquid in question to a Maxi-Spin filter equipped with a CA membrane with a pore size of 0.45 microns. b) Keep the centrifuge at 2°C and immediately rotate the filter at 1500g for 5 minutes. c) Add 2 ml of cold Milli-Q water (2°C) to the material-holding side of the spin filter, immediately spin the filter at 1500 g for 5 minutes while keeping the centrifuge cooled to 2°C, collect the permeate (permeate A), measure the volume, and determine the BLG concentration by HPLC using the method outlined in Example 1.6. d) Add 4 mL of 2 M NaCl to the material-holding side of the filter, stir quickly, and let the mixture stand at 25°C for 15 minutes. e) Immediately rotate the filter at 1500g 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 1.6, and convert the results to the total weight of BLG rather than the weight percentage. The weight of BLG in permeate A is m 透過物A It is called that, and the weight of BLG in permeate solution B is m 透過物B It is called that. g) The degree of crystallinity of the liquid for BLG is determined as follows: Crystallinity=m 透過物B / (m 透過物A +m 透過物B )*100%

[0608] Example 1.22: Determination of the degree of crystallinity of BLG in dry powder This method is used to determine the degree of crystallinity of BLG in a dry powder. a) Mix 5.0 grams of powder sample with 20.0 grams of cold Milli-Q water (2°C) and let stand at 2°C for 5 minutes. b) Transfer the liquid sample to a Maxi-Spin filter with a 0.45 micron CA membrane. c) Keep the centrifuge at 2°C and immediately rotate the filter at 1500g for 5 minutes. d) Add 2 mL of cold Milli-Q water (2°C) to the material-holding side of the spin filter, immediately rotate the filter at 1500 g for 5 minutes, collect the permeate (permeate A), measure the volume to determine the BLG. Concentrate via HPLC using the method outlined in Example 1.6, and convert the results to the total weight of BLG rather than weight percentage. The weight of BLG in permeate A is m 透過液A It is called that. f) Next, the degree of crystallinity of BLG in the powder is calculated using the following formula:

number

[0609] If the total amount of BLG in a powder sample is unknown, it may be determined by suspending another 5 g of powder sample (from the same powder source) in 20.0 g of Milli-Q water, adding aqueous NaOH solution to adjust the pH to 7.0, letting the mixture stand at 25°C for 1 hour with stirring, and finally determining the total amount of BLG in the powder sample using Example 1.6.

[0610] Example 1.23: Determination of UF transmission conductivity Transfer a 15 mL sample to an Amicon Ultra-15 centrifugal filter unit with a 3 kDa cutoff (3000 NMWL) and centrifuge at 4000 g for 20–30 minutes, or until a sufficient volume of UF permeate to measure conductivity accumulates at the bottom of the filter unit. Measure conductivity immediately after centrifugation. Sample handling and centrifugation should be performed at the temperature of the sample source.

[0611] Example 1.24: Detection of dried BLG crystals in powder The presence of dried BLG crystals in the powder can be determined by the following method:

[0612] Resuspend the powder sample to be analyzed and gently mix it with demineralized water at 4°C in a weight ratio of 2 parts water to 1 part powder, then rehydrate at 4°C for 1 hour.

[0613] The rehydrated sample is examined by microscopy to identify the presence of crystals, preferably using plane-polarized light to detect birefringence.

[0614] The crystalline material is separated and subjected to X-ray crystallography to confirm the existence of a crystalline structure. Preferably, it is also confirmed that the crystal lattice (space group and unit cell dimensions) corresponds to the lattice of a BLG crystal.

[0615] The chemical composition of the separated crystalline material is analyzed to confirm that the solid is mainly composed of BLG.

[0616] Example 1.25: Determining the total amount of lactose The total amount of lactose is measured according to ISO 5765-2:2002 (IDF 79-2:2002) "Powdered milk, dried ice mix, and processed cheese - Measurement of lactose content - Part 2: Enzymatic method using the galactose portion of lactose".

[0617] Example 1.26: Determining the total amount of carbohydrates: The amount of carbohydrates is determined using the Sigma Aldrich Total Carbohydrate Assay Kit (Cat MAK104-1KT), in which carbohydrates are hydrolyzed and converted to furfural and hydroxyfurfural, which are then converted into chromatogens that are spectrophotometrically monitored at 490 nm.

[0618] Example 1.27: Determination of total lipid content The amount of lipids is determined according to ISO 1211:2010 (Determination of fat content - Rose-Gottlieb gravimetric method).

[0619] Example 1.28: Brix's decision Brix measurements were performed using a PAL-α digital handheld refractometer (Atago) calibrated to polishing water (water filtered by reverse osmosis, which yields a maximum conductivity of 0.05 mS / cm).

[0620] Approximately 500 μl of sample was transferred to the prism surface of the instrument, and the measurement was started. The measured values ​​were read and recorded.

[0621] The Brix value of whey protein solution is proportional to the total solids (TS) content, and TS (% weight / weight) is approximately Brix*0.85.

[0622] Brix is ​​sometimes referred to as Brix degree or simply Brix.

[0623] Example 1.29 Measurement of lactoferrin and lactoperoxidase Lactoferrin concentration is determined by an ELISA immunoassay, as outlined in Soyeurt 2012 (Soyeurt et al; Mid-infrared prediction of lactoferrin content in bovine milk: potential indicator of mastitis; Animal (2012), 6:11, pp1830-1838).

[0624] The lactoperoxidase concentration is determined using a commercially available bovine lactoperoxidase kit.

[0625] Example 1.30: Determining the number of colony-forming units The determination of the number of colony-forming units per gram of sample is performed according to ISO 4833-1:2013(E): Microbiology of food products and animal feed ingredients - Horizontal method for enumeration of microorganisms - Colony counting method at 30°C.

[0626] Example 1.31: Determination of total amounts of BLG, ALA, and CMP This procedure is a liquid chromatography (HPLC) method for the quantitative analysis of proteins such as ALA, BLG, and CMP, and optionally other protein species in the composition. In contrast to the method in Example 1.6, this method also measures the proteins present in aggregates and thus provides an estimate of the total amount of protein species in the composition in question.

[0627] The separation mode is size exclusion chromatography (SEC), which uses 6M guanidine HCl buffer as both the sample solvent and the HPLC mobile phase. Mercaptoethanol is used as a reducing agent to reduce the disulfide (SS) of proteins or protein aggregates to create unfolded monomeric structures.

[0628] Sample preparation can be easily achieved by dissolving 10 mg of protein in the mobile phase.

[0629] Two TSK-GEL G3000SWXL (7.7mm x 30.0cm) columns (GPC columns) and a guard column are arranged in series to achieve proper separation of major proteins in the raw material.

[0630] The eluted analytes are detected and quantified by UV detection (280 nm).

[0631] Equipment / Materials: 1. HPLC pump 515 (Waters) with manual sealing washer 2. HPLC Pump Controller Module II (Waters) 3. Autosampler 717 (Waters) 4. Dual absorbance detector 2487 (Waters) 5. Computer software capable of generating quantitative reports (Empower 3, Waters) 6. Analytical columns: Two TSK-GEL G3000SWXL (7.8 × 300 mm, P / N: 08541). Guard column: TSK-Guard Column SWxL (6.0 x 40 mm, P / N: 08543). 7. Ultrasonic bath (Branson 5200) 8.25 mm syringe filter with 0.2 μm cellulose acetate membrane (514-0060, VWR)

[0632] procedure: Mobile phase: A. Stock buffer solution. 1. Weigh 56.6 g of Na2HPO4, 3.5 g of NaH2PO4, and 2.9 g of EDTA into a 1000 ml beaker. Dissolve in 800ml of water. 2. Measure the pH and adjust it to 7.5 ± 0.1 if necessary using HCl (to lower the pH) or NaOH (to raise the pH). Transfer to a 1000 ml volumetric flask and dilute with water to adjust the volume.

[0633] B. 6M guanidine HCl mobile phase. 1. Weigh 1146 g of guanidine HCl into a 2000 mL beaker and add 200 ml of stock buffer (A). 2. Dilute this solution with water to approximately 1600 ml while stirring with a magnetic stirrer (50°C). 3. Adjust the pH to 7.5 ± 0.1 with NaOH. 4. Transfer to a 2000 ml volumetric flask and dilute with water to adjust the volume. 5. Filter using a solvent filtration apparatus equipped with a 0.22 μm membrane filter.

[0634] Calibration standard. Calibration standards for each protein to be quantified are prepared using the following method: 1. Accurately weigh approximately 25 mg of protein reference standard into 10 ml (to the nearest 0.01 mg), Place in a volumetric flask and dissolve in 10 ml of water. This is a protein stock standard solution (S1) for proteins. 2,200 μl of S1 is pipetted into a 20 ml volumetric flask and diluted with the mobile phase to adjust the volume. This is the low-dilution standard solution WS1. 3,500 μL of S1 is pipetted into a 10 ml volumetric flask, and the volume is adjusted by diluting with the mobile phase. This is standard solution WS2. Pipette 4,500 μL of S1 into a 5 ml volumetric flask and dilute with the mobile phase to adjust the volume. This is standard solution WS3. Pipette 5.750 μL of S1 into a 5 ml volumetric flask and dilute with the mobile phase to adjust the volume. This is the standard solution WS4. 6.1.0 mL of S1 is pipetted into a 5 mL volumetric flask and diluted to the target volume with the mobile phase. This is the highly diluted standard solution WS5. 7. Using a graduated disposable pipette, transfer 1.5 mL of WS1-5 into separate vials. Add 10 μL of 2-mercaptoethanol to each vial and cap. Vortex the solution for 10 seconds. Allow the standard solution to stand at ambient temperature for approximately one hour. Filter the standard solution using an 8.0.22 μm cellulose acetate syringe filter.

[0635] Protein purity is measured using Kjeldahl (N × 6.38) and the area percentage from standard solution WS5 is measured using HPLC. Protein (mg) = "Standard protein weight" (mg) × P1 × P2 P1 = P% (Kjeldahl) P2 = Protein Area % (HPLC)

[0636] Sample preparation 1. Measure an amount equivalent to 25 mg of the original sample protein into a 25 mL volumetric flask. 2. Add approximately 20 mL of mobile phase and allow the sample to dissolve for about 30 minutes. 3. Add the mobile phase to the volume, and add 167 μL of 2-mercaptoethanol to 25 ml of the sample solution. 4. Sonicate the sample for approximately 30 minutes, then leave it at ambient temperature for approximately 1.5 hours. 5. Mix the solutions and filter them using a 0.22 μl cellulose acetate syringe filter.

[0637] HPLC system / column Column equilibration 1. Connect the GPC Guard column and the two GPC analysis columns in series. New columns are typically shipped in phosphate buffer. 2. Gradually flow water through a new column at a rate of 0.1 mL / min to 0.5 mL / min for 30 to 60 minutes. Continue flashing for about an hour. 3. Gradually reduce the flow rate from 0.5 mL / min to 0.1 mL / min. The mobile phase is replaced in the reservoir. 4. To avoid pressure shock, gradually increase the pump flow rate from 0.1 mL / min to 0.5 mL / min over 30-60 minutes, and then maintain it at 0.5 mL / min. 5. Inject 10 different samples into the column to saturate it, and wait for the peaks to elute. This helps with column conditioning. This process can be carried out without waiting for each injection to complete before the next injection. 6. Equilibrium the mobile phase for at least one hour.

[0638] Calculation of the result The quantitative determination of the content of the proteins to be quantified, such as alpha-lactalbumin, beta-lactoglobulin, and caseinomacropeptides, was performed by comparing the peak area obtained for the corresponding standard protein with the peak area of ​​the sample. The results are reported as the specific protein / 100g of the original sample, or as the weight percentage of the specific protein relative to the weight of the original sample.

[0639] Example 2: Preparation of spray-dried acidic BLG isolated powder Whey protein feed Lactose-depleted UF holding liquid derived from sweet whey from a standard cheese-making process was filtered through a 1.2-micron filter and fat was reduced using a Synder FR membrane before being used as feed for the BLG crystallization process. The chemical composition of the feed is shown in Table 1. The inventors recognize that all weight percentages of specific proteins such as BLG and ALA mentioned in this example relate to the weight percentage of non-aggregating proteins relative to total protein.

[0640] conditioning Sweet whey feed is fed to a feed concentration of 21% (TS) ± 5 total solids using a 46 mil spacer and a feed pressure of 1.5-3.0 bar, with a Koch HFK-328 type membrane (70m³). 2 The samples were conditioned on an ultrafiltration setup at 20°C using a membrane and polishing water (water filtered by reverse osmosis, yielding a conductivity of up to 0.05 mS / cm) as the diafiltration medium. The pH was then adjusted by adding HCl to approximately 5.5. Diafiltration was continued until the decrease in conductivity of the retained solution fell below 0.1 mS / cm over 20 minutes, after which the permeate flow was 1.43 L / hour / m 2 The holding solution was concentrated until it fell below a certain level. A first sample of the concentrated holding solution was taken and centrifuged at 3000g for 5 minutes. The supernatant of the first sample was used to measure the BLG yield.

[0641] crystallization The concentrated holding solution was transferred to a 300L crystallization tank, where it was seeded with pure BLG crystal material made from BLG crystals that had been rehydrated and spray-dried. Subsequently, the seeded whey protein solution was cooled from 20°C to approximately 6°C over approximately 10 hours to form and grow BLG crystals.

[0642] After cooling, a sample of the whey protein solution containing crystals (second sample) was taken, and the BLG crystals were separated by centrifugation at 3000g for 5 minutes. The supernatant and crystal pellet from the second sample were subjected to HPLC analysis as described below. The crystallization yield was calculated as 57%, as outlined below. [Table 2]

[0643] Determination of BLG yield using HPLC: The supernatants of the first and second samples were diluted to the same extent by adding polishing water, and the diluted supernatants were filtered through a 0.22 μm filter. For each filtered and diluted supernatant, the same volume was loaded into an HPLC system equipped with a Phenomenex Jupiter® 5 μm C4 300 Å, LC column 250 × 4.6 mm, Ea., and detected at 214 nm.

[0644] The samples were run under the following conditions: Buffer A: MilliQ water, 0.1% wt / wt TFA Buffer B: HPLC-grade acetonitrile, 0.085% wt / mTFA Flow rate: 1ml / min Column temperature: 40℃ Gradient: 0-30 minutes 82-55%A and 18-45%B; 30-32 minutes 55-10%A and 45-90%B; 32.5-37.5 minutes 10%A and 90%B; 38-48 minutes 10-82%A and 90-18%B.

[0645] Data processing: Since both supernatants were processed in the same way, the relative yield can be calculated by directly comparing the area of ​​the BLG peak. Because the crystals contain only BLG and all samples were processed in the same way, the concentration of alpha-lactalbumin (ALA), and therefore the ALA area, should be the same in all samples. Thus, the ALA area before and after crystallization is used as a correction factor (cf) when calculating the relative yield.

number

[0646] The relative yield is calculated using the following formula.

number

[0647] Acid dissolution of BLC crystals The remaining material from the crystallization tank was separated using a decanter at 350g, 2750 RPM, with a 150 RPM difference due to 64 spacers, and a feed flow of 75 L / hour. The feed was then mixed with polishing water in a 1:2 ratio. Next, the BLG crystals / solid phase from the decanter was mixed with polishing water to form a thinner slurry before adding phosphoric acid to lower the pH to approximately 3.0 in order to rapidly dissolve the crystals.

[0648] After dissolving the BLG crystals, the pure BLG protein solution was concentrated to 15 Brix at the same UF setting used to prepare the feed for crystallization, and the pH was adjusted to a final pH of approximately 3.8. The liquid BLG isolate was then heated to 75°C for 5 minutes and subsequently cooled to 10°C. The heat treatment was found to reduce the microbial load from 137,000 CFU / g before heat treatment to less than 1,000 CFU / g after heat treatment. The heat treatment did not cause protein denaturation, and the intrinsic tryptophan fluorescence emission ratio (330nm / 350nm) was determined to be 1.20, indicating the native three-dimensional structure of the BLG molecule.

[0649] BLG was dried in a pilot plant spray dryer with an inlet temperature of 180°C and an outlet temperature of 75°C. The resulting powder sampled at the outlet had a water content of approximately 4% by weight / weight. The chemical composition of the powder is shown in Table 2. The dried powder sample was dissolved, the degree of protein denaturation was determined to be 1.5%, and the intrinsic tryptophan fluorescence emission ratio (I330 / I350) was measured to be 1.20. [Table 3]

[0650] The bulk density (625 taps) of the spray-dried powder is 0.2-0.3 g / cm³. 3 It was estimated that...

[0651] Conclusion: By using the process described above, the inventors were able to produce a high-purity BLG product that could be heat-treated without substantially causing protein denaturation or unfolding during processing. The heat treatment significantly reduced bacterial levels without damaging the protein product.

[0652] The inventors have observed that increasing the protein content before spray drying can result in an even higher bulk density. They have also observed that lower levels of denaturation can be achieved when the inlet and / or outlet temperatures used in spray drying are reduced.

[0653] Example 3: Preparation of spray-dried pH-neutral BLG isolated powder Using the same protocol and experimental setup as in Example 2, the lactose-reduced whey protein isolate shown in Table 3 was conditioned and used as feed for crystallization. The crystallization yield was calculated to be 68%.

[0654] The inventors recognize that all weight percentages of specific proteins such as BLG and ALA mentioned in this example are related to the weight percentage of non-aggregating proteins relative to total protein. [Table 4]

[0655] The remaining material from the crystallization tank was separated on a decanter at 350 g, 2750 RPM, a 150 RPM difference using 64 spacers, and a feed rate of 75 L / hour. The feed was then mixed with polishing water in a 1:2 ratio. Next, to rapidly dissolve the crystals, 0.1 M potassium hydroxide was added, and the BLG crystals / solid phase from the decanter was mixed with polishing water to form a thinner slurry before adjusting the pH to approximately 7.

[0656] After dissolving the crystals, the pure BLG protein solution was concentrated to 15 Brix at the same UF setting used to prepare the whey protein solution for crystallization, and the pH was adjusted to a final pH of 7.0. The BLG was dried in a pilot plant spray dryer with an inlet temperature of 180°C and an outlet temperature of 75°C. The powder obtained by sampling at the outlet had a water content of approximately 4% wt / kg. The composition of the powder is shown in Table 4. After drying, a portion of the powder was dissolved in desalted water, and the degree of protein denaturation was determined to be 9.0%, with an intrinsic tryptophan fluorescence emission ratio (330nm / 350nm) of 1.16. [Table 5]

[0657] The bulk density (625 taps) of the spray-dried powder is 0.2-0.3 g / cm³. 3 It was estimated that...

[0658] Conclusion: By using the process described above, the inventors can produce a pH-neutral, high-purity BLG product with minimal or no protein denaturation during processing. The inventors have observed that even higher bulk density can be obtained by increasing the protein content before spray drying. The inventors have also observed that even lower levels of denaturation can be obtained when the inlet and / or outlet temperatures used for spray drying are reduced. The level of denaturation can be further reduced by reducing the mineral content before spray drying.

[0659] Example 4: Wettability of spray-dried isolated BLG powder The wettability of acidic or pH-neutral spray-dried BLG isolate powders prepared according to Examples 2 and 3 was compared to that of conventional spray-dried whey protein isolates (WPI). Wettability was measured as the time it took for the entire powder sample to become wet. 0.5 grams of powder were weighed and placed on the surface of 100 g of demineralized water (10°C) in a 5 cm diameter cylindrical container. The time from when the powder was placed on the water surface until it dissolved or passed through the water surface was measured. The results are shown below. [Table 6]

[0660] Conclusion: It was surprising that the uncoated BLG isolate powder (Samples 2 and 3) wetted far better than standard WPI (Sample 1). This indicates that the BLG isolate powder of the present invention is a useful ingredient for instant beverage powders where rapid wettability and dissolution are important. The BLG isolate powder further offers improved utility in the production of protein beverages compared to conventional WPI, as it wets and dissolves protein beverages much faster, and therefore dispersion and dissolution during production are easier. Ultimately, this could reduce the time required to produce high-protein beverages and potentially increase the hourly production capacity of beverage manufacturing plants.

[0661] Example 5: Acidic BLG isolate with reduced dry mouthfeel level Two protein beverages, A and B, containing an amount of acidic spray-dried BLG isolated powder prepared in Example 2 sufficient to provide 6.0% total protein, were pH-adjusted to 3.7 and subjected to heat treatment at 75°C for 15 seconds for A, or at 120°C for 20 seconds for B. Both beverages were immediately cooled and stored in a refrigerator at 5°C. By measuring the intrinsic fluorescence emission ratio (I330 / I350), it was confirmed that the protein in beverage A still maintained its native three-dimensional structure, while significant unfolding and denaturation had occurred in beverage B.

[0662] The turbidity of beverages A and B was measured according to Example 1.7, and both beverages had a turbidity of less than 40 NTU, while an equivalent standard WPI had a turbidity of more than 200 NTU. Therefore, the acidic BLG isolate of the present invention is clearly very suitable for the production of clear, acidic, high-protein beverages.

[0663] Less than a week after production, both beverages were subjected to sensory testing by a trained sensory testing panel, and it was found that the dry mouthfeel characteristic of heat-treated acidic whey protein beverages was more than 100% higher in beverage B (dry mouthfeel score: approximately 10.5 on a scale of 0-12) than in beverage A (dry mouthfeel score: approximately 5.0 on a scale of 0-12).

[0664] These findings indicate that protein denaturation contributes to the dry mouthfeel of acidic protein beverages, and that this dry mouthfeel can be significantly reduced by limiting or avoiding protein denaturation.

[0665] Example 6: Preparation of neutral BLG isolate with very low microbial content The inventors have found that the present invention makes it possible to obtain a pH-neutral BLG powder with a very low bacterial content and high protein nativeity. This is demonstrated in an example of the present invention where the total process time was extended by inserting a 6-day storage period at 10°C during the final step of the conditioning process to challenge the microbial quality of the product.

[0666] Whey protein feed: Lactose-depleted UF holding liquid derived from sweet whey from a standard cheese-making process was filtered through a 1.2-micron filter and fat was reduced using a Synder FR membrane before being used as feed for the BLG crystallization process. The chemical composition of the feed is shown in Table 5. The inventors recognize that all weight percentages of specific proteins such as BLG and ALA mentioned in this example relate to the weight percentage of non-aggregating proteins relative to total protein. [Table 7]

[0667] conditioning: Sweet whey feed was conditioned on an ultrafiltration setup at approximately 10°C using an Alfa Laval GR82PE type membrane with a feed concentration of 21% (TS) ± 5 total solids, a 30 mil spacer, and a feed pressure of 1.5–3.0 bar, and polishing water (water filtered by reverse osmosis, achieving a conductivity of up to 0.05 mS / cm) as the diafiltration medium. The pH was adjusted to approximately 5.9 using diluted hydrochloric acid, and diafiltration was performed on a batch ultrafiltration setup. Diafiltration was continued until the decrease in conductivity of the retained solution fell below 0.1 mS / cm over a 20-minute period.

[0668] Next, the diafiltration-filtered whey protein feed was stored at 10°C for 6 days to test the microbial quality of the resulting product.

[0669] After storage, the whey protein feed was heated to 20°C, then HCl was added to adjust the pH, and dilute hydrochloric acid was used to bring the pH to approximately 5.5.

[0670] Whey protein feed was conditioned on an ultrafiltration setup at 20°C using an Alfa Laval GR82PE type membrane with a feed concentration of 21% (TS) ± 5 total solids, a 30 mil spacer, and a feed pressure of 1.5–3.0 bar, with polished water as the diafiltration medium. Ultrafiltration was performed as a continuous ultrafiltration setup, with diafiltration added so that the end-retaining solution had a conductivity of 1.9–2.2 mS / cm and a TS of 22 ± 5. The retaining solution was collected in an 800 L tank equipped with a mantle and agitator. Upon filling the tank, a first sample of the concentrated retaining solution was taken for HPLC analysis. The HPLC analysis of this example was performed as described in Example 2.

[0671] Crystallization: The concentrated holding solution was transferred to an 800L crystallization tank, where it was seeded with pure BLG crystal material made from rehydrated and spray-dried BLG crystals. The crystals were added to 1L of conditioned WPI and rapidly cooled to below 5°C on ice. Subsequently, the seeded whey protein solution was cooled from 20°C to approximately 6°C over approximately 4 hours to form and grow BLG crystals.

[0672] After cooling, a sample of the whey protein solution containing crystals (second sample) was taken, and the BLG crystals were separated by centrifugation at 3000g for 5 minutes. The supernatant and crystal pellet from the second sample were subjected to HPLC analysis as described below. The crystallization yield was calculated and determined as a percentage, as outlined below.

[0673] Decanter separation: The crystals produced in an 800L tank were separated from the mother liquor on a decanter (LEMITECH MD80) at a difference of 2750 RPM using a 600g, 64 mil spacer (mil means 1 / 1000 inch), with a feed flow of 150 L / hour and a mixture of polishing water and tank feed in a ratio of 1 volume tank feed to 2 volumes polishing water.

[0674] Dissolution of crystals: The crystals collected from the decanter were diluted with polishing water to a total solids concentration of approximately 10%, and then the pH was adjusted to pH 3 using dilute hydrochloric acid. The acidified BLG solution was maintained at pH 3 at approximately 6°C for approximately 16 to 48 hours. pH adjustment of acidified BLG solution:

[0675] Next, the acidified BLG solution was adjusted to pH 7 using a diluted mixture of potassium hydroxide and sodium hydroxide, and then conditioned to a concentration of approximately 16% TS in an ultrafiltration setup at approximately 10°C using an Alfa Laval GR82PE type membrane with a 30 mil spacer and a feed pressure of 1.5–3.0 bar. Precision filtration:

[0676] A portion of the concentrated BLG solution was subjected to microfiltration using a Membralox EP-1940-GL-UTP membrane with a nominal pore size of 0.8 microns. Microfiltration was performed at approximately 10°C with a feed pressure of 3.5. The MF permeate was collected and prepared for drying.

[0677] Analysis of MF permeate samples for their chemical composition (see results in Table 6) and microbiology (Example 1.30) revealed, surprisingly, a level of less than 10 CFU / g (essentially, no colonies were identifiable, and the tested samples appeared nearly sterile). Sterility can be achieved, for example, by using a more robust microfiltration membrane and ensuring sterility on the permeate side of the membrane.

[0678] In an equivalent process without acidification and microfiltration, BLG solutions with concentrations exceeding 1,000,000 cfu / g were readily obtained. [Table 8]

[0679] Conclusion: Even under heavy microbiological loads, the present invention makes it possible to provide sterile or nearly sterile, pH-neutral BLG products with a high degree of protein-native properties.

[0680] Example 7: Compare the reverse osmosis concentration and powder bulk density of acidic and neutral BLG with that of standard neutral WPI. The inventors have found that a spray-dried preparation of purified native BLG can provide a powder with a higher bulk density than an equivalent whey protein isolate spray-dried under the same conditions.

[0681] The inventors further found that the purified native BLG liquid high-protein preparation has a lower viscosity than the equivalent whey protein isolate. This finding makes it possible to concentrate the BLG isolate to a higher total protein concentration (and lower water content) before drying, thus reducing water removal compared to the equivalent WPI powder and thus reducing energy consumption per kg of dry protein. Furthermore, as viscosity decreases, the energy required for membrane filtration also decreases, allowing for energy-saving membrane filtration, such as microfiltration.

[0682] The above findings were confirmed in the following experiment.

[0683] Raw materials for reverse osmosis and drying: Neutral BLG: The MF holding solution produced in Example 6 was concentrated on an Alfa Laval RO98pHt membrane at a feed pressure of up to 52 bar, and the temperature was kept below 15°C during conditioning. Concentration continued until Brix reached 32.2. During concentration, samples were taken for viscosity and Brix measurements as described in Examples 1.8B and 1.28. The Brix of each sample was converted to protein (weight / weight %) using the following formula.

number

[0684] The conversion of 0.85 was obtained empirically, and there is a good relationship between the Brix degree and the total solid. The results are shown in Figure 6.

[0685] The total solid content of the whey protein solution is approximately Brix* 0.85.

[0686] Acidic BLG: The acidic BLG solution was prepared as in Example 6 (except that ultrafiltration at pH 5.92 was not performed before continuous ultrafiltration at pH 5.5, and that ultrafiltration was performed at pH 3). The feed composition is shown in Table 7. The acidic BLG permeate subjected to the MF process described in Example 6 was then concentrated on an Alfa Laval RO98pHt reverse osmosis (RO) membrane at a maximum feed pressure of 52 bar, and the temperature was kept below 15°C during conditioning. Concentration was continued until a Brix of 40.0 was obtained. During concentration, samples were taken for viscosity and Brix measurements as described in Examples 1.8B and 1.28. The Brix values ​​were converted to protein concentrations, and the results are shown in Figure 6. Before drying, the BLG was diluted to a Brix of 35.5 with polishing water. [Table 9]

[0687] WPI reference: To compare acidic BLG products and pH-neutral BLG products with conventional pH-neutral WPI products, a standard liquid-concentrated WPI based on sweet whey was collected from the production of Arla Foods Danmark Protein and dried in the same pilot plant spray dryer under the same conditions as the acidic and pH-neutral BLG samples. The composition of the liquid-concentrated WPI is shown in Table 8.

[0688] The viscosity of concentrated WPI was measured first, and then the concentrated WPI was gradually diluted with polishing water to show the correlation between WPI Brix and viscosity for comparison with BLG measurements.

[0689] Viscosity curve: The temperature of the sample taken in RO and the diluted WPI reference were adjusted to 15°C before viscosity measurements (performed three times), as described in Example 1.8B. Brix was measured as described in Example 1.28.

[0690] The concentrated liquid WPI was dried at 36.0 Brix. [Table 10]

[0691] Spray drying: After concentration by reverse osmosis (RO), all samples were dried without preheating in a pilot plant spray dryer with an inlet temperature of 180°C and an outlet temperature of 85°C. The temperature of all samples was maintained below 12°C until spray drying. The bulk density of the spray-dried powders was then measured without stomping, and with 100 and 625 stomps, as described in Example 1.17. Surprisingly, both the acidic and pH-neutral BLG powders had significantly and consistently higher bulk densities than the WPI reference. On average, the bulk density of pH-neutral BLG was 18.4 percent higher than the WPI reference powder, and the acidic BLG was 22.2 percent higher. The results of the bulk density measurements are shown in Table 9 and illustrated in Figure 5. [Table 11]

[0692] Conclusion: Both acidic and pH-neutral BLG isolates exhibited lower viscosity compared to the WPI reference, making them easier to concentrate under RO. This finding allows for the concentration of BLG isolates to higher total protein concentrations (and lower water content) before drying, resulting in less water removal and therefore lower energy consumption per kg of dry protein compared to the equivalent WPI powder. Furthermore, the reduced viscosity decreases the energy required for membrane filtration, such as microfiltration, thus reducing energy consumption.

[0693] Since the BLG powder of the present invention contributes less to viscosity than conventional WPI, the BLG powder of the present invention is particularly suitable for high-protein beverages or shake powders for preparing high-protein beverages, and therefore provides a more drinkable beverage.

[0694] Surprisingly, the spray-dried BLG powder also had a higher bulk density compared to the WPI reference, even when the BLG was dried with a slightly lower amount of total solids, as explained by the Brix value. Higher bulk density of powder tends to result in less transport volume per unit weight during transport, and higher particle density tends to result in less dust during handling.

[0695] [Claim 1] BLG isolated powder, preferably prepared by spray drying, having a pH in the range of i) 2.5 to 4.9, ii) 6.1 to 8.5, or iii) 5.0 to 6.0, and containing the following: At least 30% by weight / total protein, Beta-lactoglobulin (BLG) in an amount of at least 85% by weight relative to total protein. Up to 10% by weight / amount of water, The BLG isolated powder is At least 0.2 g / cm³ 3 bulk density, An intrinsic tryptophan fluorescence emission ratio of at least 1.11 (I330 / I350) Up to 10% protein denaturation, Thermal stability of up to 200 NTU at pH 3.9, and It has one or more colony-forming units of up to 15,000 per gram. [Claim 2] The BLG isolation powder according to claim 1, having a pH in the range of 2.5 to 4.9. [Claim 3] The BLG isolation powder according to claim 1, having a pH in the range of 6.1 to 8.5. [Claim 4] The BLG isolation powder according to claim 1, having a pH in the range of 5.0 to 6.0. [Claim 5] BLG isolation powder according to any one of claims 1 to 4, comprising at least 40% by weight / weight, preferably at least 50% by weight / weight, at least 60% by weight / weight, more preferably at least 70% by weight / weight, even more preferably at least 80% by weight / weight, even more preferably at least 90% by weight / weight, and most preferably at least 92% by weight / weight of total protein. [Claim 6] BLG isolation powder according to any one of claims 1 to 5, comprising BLG in an amount of at least 88% by weight, preferably at least 90% by weight, relative to total protein. [Claim 7] BLG isolated powder according to any one of claims 1 to 6, comprising lipids in an amount of up to 10% by weight, preferably up to 5% by weight, more preferably up to 2% by weight, and even more preferably up to 0.1% by weight. [Claim 8] At least 0.20 g / cm³ 3 Preferably at least 0.30 g / cm³ 3 BLG isolation powder according to any one of claims 1 to 7, having a bulk density. [Claim 9] BLG isolation powder according to any one of claims 1 to 8, having an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.11. [Claim 10] BLG isolation powder according to any one of claims 1 to 9, having a maximum protein denaturation degree of 10%. [Claim 11] Liquid BLG isolate having a pH in the range of i) 2-4.9, ii) 6.1-8.5, or iii) 5.0-6.0, and containing the following: At least 10% by weight / weight of total protein, Beta-lactoglobulin (BLG) in an amount of at least 85% by weight relative to total protein, At least an intrinsic tryptophan fluorescence emission ratio of 1.11 (I330 / I350), Up to 10% protein denaturation, Thermal stability of up to 200 NTU at pH 3.9, and It has one or more colony-forming units, preferably up to 15,000 per gram, and more preferably up to 1,000 per gram. [Claim 12] A method for producing a dried BLG isolation powder according to claim 1, comprising the following steps: a) To provide a liquid BLG isolate having the following: i) pH in the range of 2 to 4.9 ii) pH in the range of 6.1 to 8.5, iii) pH in the range of 5.0 to 6.0 The liquid BLG isolate contains BLG in an amount of at least 85% by weight relative to the total protein. b) Optionally, subject the liquid BLG isolate to physical microbial reduction. c) Dry the liquid BLG isolate by spray drying. [Claim 13] The method according to claim 12, wherein the liquid BLG isolate contains a total solids content in the range of 5 to 50% by weight. [Claim 14] The method according to claim 12 or 13, wherein the protein fraction of the liquid BLG isolate has an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.11. [Claim 15] The method according to any one of claims 12 to 14, wherein the protein of the liquid BLG isolate has a protein denaturation degree of up to 10% by weight / weight. [Claim 16] The provision of liquid BLG isolates BLG from whey protein feed, and optionally, the obtained BLG concentrated composition. Desalting, Mineral supplementation, Dilution, Physical microbial reduction, Concentration, and pH adjustment, The method according to any one of claims 12 to 15, comprising subjecting to one or more steps selected from the group. [Claim 17] The method according to any one of claims 12 to 16, wherein the liquid BLG isolate is preferably subjected to a physical microbial reduction comprising or further comprising a heat treatment. [Claim 18] Use as a raw material for the manufacture of heat-treated beverages, in a food product, e.g., a beverage or instant beverage powder, of the BLG isolated powder according to any one of claims 1 to 10 or the liquid BLG isolate according to claim 11, having a pH in the range of 2 to 4.7 and further comprising: A decrease in the level of dry mouthfeel. Improved transparency, and / or The protein content is increased, and the protein content is one or more of the above, preferably at least 3-45% by weight, more preferably 11-40% by weight, even more preferably 15-38% by weight, and most preferably 20-36% by weight.

Claims

1. BLG isolated powder, prepared by spray drying, having a pH in the range of 6.1 to 8.5, and containing the following: - At least 30% by weight / weight of total protein, - Beta-lactoglobulin (BLG) in an amount of at least 90% by weight relative to total protein. - Up to 10% by weight / amount of water by weight, The BLG isolated powder is It has a maximum protein denaturation degree of 10% and a maximum of 15,000 colony-forming units / g; The pH of the aforementioned powder is defined as the pH of a liquid prepared by dissolving 10 g of the powder in 90 ml of demineralized water at room temperature, measured at 25°C.

2. At least 0.2 g / cm³ ３ bulk density, At least an intrinsic tryptophan fluorescence emission ratio of 1.11 (I330 / I350), Thermal stability of up to 200 NTU at pH 3.9, Having one or more of the following: BLG isolated powder according to claim 1.

3. BLG isolated powder according to claim 1 or 2, comprising at least 40% by weight of total protein.

4. Claim 1, comprising BLG in an amount of at least 60% by weight relative to total protein. BLG isolated powder as described in any of the following three items.

5. Claim 1, comprising BLG in an amount of at least 80% by weight relative to total protein. BLG isolated powder as described in any of the following four:

6. Claim 1, comprising BLG in an amount of at least 92% by weight relative to total protein. BLG isolated powder as described in any of the following five items.

7. BLG isolation powder according to any one of claims 1 to 6, comprising BLG in an amount of at least 94% by weight relative to total protein.

8. BLG isolation powder according to any one of claims 1 to 7, having a maximum of 10,000 colony-forming units / g.

9. The BLG isolation powder according to any one of claims 1 to 8, having a maximum of 5,000 colony-forming units / g.

10. BLG isolated powder according to any one of claims 1 to 9, comprising up to 10% by weight of lipids.

11. At least 0.20 g / cm³ ３ BLG isolated powder according to any one of claims 1 to 10, having a bulk density.

12. BLG isolation powder according to any one of claims 1 to 11, having an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.

11.

13. A liquid BLG isolate having a pH in the range of 6.1 to 8.5, comprising the following: At least 10% by weight / weight of total protein, Beta-lactoglobulin (BLG) in an amount of at least 92% by weight relative to total protein. The liquid BLG isolate is Maximum protein denaturation level of 10% and maximum colony-forming units / g It has.

14. At least 0.2 g / cm³ ３ bulk density, An intrinsic tryptophan fluorescence emission ratio of at least 1.11 (I330 / I350) Thermal stability of up to 200 NTU at pH 3.9, and Having one or more of the following: The liquid BLG isolate according to claim 13.

15. The liquid BLG isolation powder according to claim 13 or 14, comprising at least 15% by weight of total protein.

16. A liquid BLG isolation powder according to any one of claims 13 to 15, comprising BLG at a weight of at least 95% of total protein.

17. A liquid BLG isolation powder according to any one of claims 13 to 16, having a maximum of 10,000 colony-forming units / g.

18. A method for producing a dried BLG isolation powder according to claim 1, comprising the following steps: a) To provide a liquid BLG isolate having the following: ii) pH in the range of 6.1 to 8.5, or The liquid BLG isolate contains BLG in an amount of at least 90% by weight relative to the total protein. b) Optionally, subject the liquid BLG isolate to physical microbial reduction. c) Dry the liquid BLG isolate by spray drying.

19. The method according to claim 18, wherein the liquid BLG isolate contains a total solids content in the range of 5 to 50% by weight.

20. The method according to claim 18 or 19, wherein the protein fraction of the liquid BLG isolate has an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.

11.

21. The provision of liquid BLG isolate involves isolating BLG from whey protein feed, and optionally, using the resulting BLG concentrated composition. Desalting, Mineral supplementation, Dilution, physical microbial reduction, Concentration, and pH adjustment, The method according to any one of claims 18 to 20, comprising subjecting to one or more steps selected from the group.

22. The method according to any one of claims 18 to 21, wherein the liquid BLG isolate is subjected to physical microbial reduction.

23. A food product of the BLG isolated powder according to any one of claims 1 to 12 or the liquid BLG isolate according to any one of claims 13 to 17, having the following use as a raw material for the manufacture of a heat-treated beverage: A decrease in the level of dry mouthfeel. Improved transparency, and / or Increased protein content.

24. The use of claim 23, wherein the food product is a beverage or instant beverage powder.

25. Use of claim 23 or 24, wherein the food product has a pH in the range of 2 to 4.7.

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