Process for preparing beta-lactoglobulin isolate and related methods and uses

Abstract

The present invention relates to novel beta-lactoglobulin (BLG) isolates, methods for producing such isolates and the use of the powders, e.g. in beverage applications.

Description

Technical Field

[0001] The present invention relates to novel beta-lactoglobulin (BLG) isolates and methods for producing such isolates, as well as the use of the powders in, for example, beverage applications. Background Art

[0002] BLG is known as the gel-forming component of whey protein and is prone to undesirable aggregation and gel formation even at pasteurization temperatures. BLG isolate is considered to be more heat-sensitive than traditional whey protein isolate, which contains more heat-resistant proteins in addition to BLG: alpha-lactalbumin (ALA) and caseinomacropeptide (CMP). Previously, the industrial food use of BLG isolate was limited due to the high price of BLG isolate and problems with the handling of freeze-dried BLG isolate.

[0003] The isolation of beta-lactoglobulin (BLG) from milk serum or whey is the subject of numerous publications, usually involving multiple separation steps and generally 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. 2001, 84 (3): 562-571) describe the purification of BLG from freshly squeezed milk by low temperature acid coagulation of casein and by a combination of affinity chromatography (DEAE Sepharose) and gel permeation chromatography of the resulting acid whey. The resulting BLG composition is said to contain 0.985 g of β-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. 2002, 85: 1639-1645) disclosed a method for scaling up the production of native BLG based on affinity chromatography. The BLG composition was dried by freeze drying.

[0006] US2790790A1 describes a method for separating BLG from whey by adding NaCl at pH 3.6-4.0, but does not describe drying the separated BLG.

[0007] Palmer (Crystalline Globulin from Cow's Milk, J. Biol. Chem. 1934, 104: 359-372) reported a laborious and time-consuming method for producing protein crystals based on acid whey, using the following steps: precipitation of undesirable protein salts, pH adjustment and dialysis to remove other unwanted proteins. Finally, when a high-purity BLG solution is obtained, BLG is crystallized. The method lasts for more than 12 days and requires the addition of toluene. Therefore, the procedure disclosed by Palmer is incompatible with food production safety, and the product it provides is obviously inedible. Palmer reported that BLG crystals can be dried by ethanol and ether.

[0008] EP 0604684 A1 discloses a method for recovering a whey protein concentrate rich in α-lactalbumin and / or β-lactoglobulin from a whey protein product. The method comprises: a) incubating a solution containing the whey protein product with a calcium-binding ion exchange resin in acid form to cause destabilization of α-lactalbumin; b) adjusting the pH of the treated protein product solution to 4.3-4.8 after separating the resin; c) incubating the protein product solution at a temperature of 10-50°C to promote α-lactalbumin flocculation; d) fractionating the protein in the protein product solution at pH 4.3-4.8 to provide a fraction rich in α-lactalbumin and a fraction rich in β-lactoglobulin; e) sufficiently increasing the pH of the fraction rich in α-lactalbumin to dissolve the α-lactalbumin fraction; and f) optionally, sufficiently increasing the pH of the fraction rich in β-lactoglobulin to neutralize the β-lactoglobulin fraction.

[0009] WO2010 / 037736 A1 discloses the separation of whey protein and the preparation of whey products and whey isolates, in particular, the separation of β-lactoglobulin products and the separation of α-lactalbumin-rich whey protein isolates from animal whey. The α-lactalbumin-rich whey protein isolate provided by the present invention has not only a low β-lactoglobulin content, but also a high α-lactalbumin and immunoglobulin G content.

[0010] WO2011 / 112695 A1 discloses a nutritional composition containing whey protein micelles and leucine, which provides a sufficient amount of leucine to improve protein synthesis in humans while maintaining a low-viscosity fluid matrix and acceptable sensory properties. Summary of the invention

[0011] The present inventors have found that a high-functionality BLG isolate powder can be obtained by drying (preferably spray drying) a high-BLG liquid BLG isolate having a pH of: i) 2 to 4.9; or ii) 6.1 to 8.5; or iii) 5.0 to 6.0.

[0012] Thus, one aspect of the present invention relates to a BLG isolate powder, preferably prepared by spray-drying, having a pH of i) 2.5 to 4.9, ii) 6.1 to 8.5 or iii) 5.0 to 6.0, comprising:

[0013] - at least 30% w / w total protein;

[0014] - at least 85% w / w beta-lactoglobulin (BLG) relative to the total amount of protein;

[0015] - up to 10% w / w water;

[0016] The BLG isolate powder has one or more of the following:

[0017] - Bulk density of at least 0.2 g / cm 3 ;

[0018] - an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.11;

[0019] - protein denaturation of up to 10%;

[0020] - a thermal stability of at most 200 NTU at pH 3.9; and

[0021] - Up to 15000 colony forming units / g.

[0022] Another aspect of the present invention relates to a liquid BLG isolate having a pH of i) 2 to 4.9, ii) 6.1 to 8.5 or iii) 5.0 to 6.0, the liquid BLG isolate comprising:

[0023] - at least 10% w / w total protein;

[0024] - at least 85% w / w beta-lactoglobulin (BLG) relative to the total amount of protein;

[0025] The BLG isolate powder has one or more of the following:

[0026] - an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.11;

[0027] - protein denaturation of up to 10%;

[0028] - a thermal stability of at most 200 NTU at pH 3.9; and

[0029] - Up to 1000 colony forming units / g.

[0030] Yet another aspect of the present invention relates to a method for producing a dry BLG isolate powder having a BLG content of at least 85% w / w relative to the total amount of protein, the method comprising the steps of:

[0031] a) providing a liquid BLG isolate having the following characteristics:

[0032] i) pH 2 to 4.9;

[0033] ii) pH 6.1 to 8.5; or

[0034] iii) pH 5.0 to 6.0;

[0035] The liquid BLG isolate has a BLG content of at least 85% w / w relative to the total amount of protein;

[0036] b) optionally, subjecting the liquid BLG isolate to physical microbial reduction;

[0037] c) drying the liquid BLG isolate, preferably spray drying.

[0038] Another aspect of the present invention relates to the use of a BLG isolate powder or liquid BLG isolate as described herein as an ingredient for producing a food (e.g., a beverage or instant beverage powder) having a pH of 2 to 4.7 and further having one or more of the following:

[0039] - Reduced dry mouthfeel;

[0040] - improved transparency; and / or

[0041] - Increased protein content, the protein content of the heat treated beverage is preferably at least 3-45% w / w, more preferably 11-40% w / w, even more preferably 15-38% w / w, most preferably 20-36% w / w. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 A schematic diagram of the method of producing BLG isolate powder of the present invention is provided.

[0043] Figure 2 A schematic diagram of the process of the present invention for producing BLG isolate powder starting from a whey protein raw material is provided, and the terms used in this patent specification are explained.

[0044] Figure 3 This is a microscope photo of BLG crystals.

[0045] Figure 4 Microscope photographs of intact BLG crystals and fragmented BLG crystals are shown.

[0046] Figure 5 It was shown that the bulk density of the spray-dried BLG isolate was higher compared to the comparable WPI dried under the same conditions.

[0047] Figure 6 It was shown that the high protein liquid BLG isolate had a lower viscosity compared to the equivalent WPI solution. DETAILED DESCRIPTION

[0048] definition

[0049] In the context of the present invention, the term "β-lactoglobulin" or "BLG" relates to β-lactoglobulin from a mammalian species, e.g. in native, unfolded and / or glycosylated form, including naturally occurring gene variants. The term also includes aggregated BLG, precipitated BLG and crystallized BLG. When referring to the amount of BLG, it refers to the total amount of BLG (including aggregated BLG). The total amount of BLG is determined according to Example 1.31. The term "aggregated BLG" refers to BLG that is at least partially unfolded and aggregated with other denatured BLG molecules and / or other denatured whey proteins, typically by hydrophobic interactions and / or covalent bonds.

[0050] BLG is the most predominant protein in bovine whey and cow serum, and there are several genetic variants, with the major variants in bovine milk being labeled A and B. BLG is a lipoprotein that binds a variety of hydrophobic molecules, suggesting a role in their transport. BLG has also been shown to bind iron via siderophores, possibly playing a role in combating pathogens. The homologue of BLG is missing in human breast milk.

[0051] Bovine BLG is a relatively small protein with about 162 amino acid residues and a molecular weight of about 18.3 kDa to 18.4 kDa. As measured using nuclear magnetic resonance spectroscopy, it is primarily a dimer under physiological conditions, but dissociates into monomers at a pH below about 3, retaining its native state. However, BLG also exists as a tetramer, octamer, and other multimers under various natural conditions.

[0052] In the context of the present invention, the term "non-aggregated β-lactoglobulin" or "non-aggregated BLG" also relates to β-lactoglobulin from mammalian species, e.g. in native, unfolded and / or glycosylated form, including naturally occurring gene variants. However, the term does not include aggregated BLG, precipitated BLG or crystallized BLG. The amount or concentration of non-aggregated BLG is measured according to Example 1.6.

[0053] By calculating (m 总BLG -m 非聚集BLG ) / m 总BLG *100%, to determine the percentage of non-aggregated BLG relative to total BLG. m 总BLG is the concentration or amount of BLG measured according to Example 1.31, m 非聚集BLG is the concentration or amount of non-aggregated BLG measured according to Example 1.6.

[0054] In the context of the present invention, the term "crystalline" relates to a solid material whose constituents (eg atoms, molecules or ions) are arranged in a highly ordered microstructure, forming a crystalline lattice extending in all directions.

[0055] In the context of the present invention, the term "BLG crystals" relates to protein crystals that primarily comprise non-aggregated BLG (preferably native BLG) arranged in a highly ordered microstructure, forming a lattice extending in all directions. For example, BLG crystals may be monolithic or polycrystalline, and may be, for example, complete crystals, crystal fragments, or a combination thereof. For example, crystal fragments are formed when complete crystals are mechanically sheared during processing. Crystal fragments also have the highly ordered microstructure of the crystal, but may lack the uniform surface and / or uniform edges or corners of the complete crystal. See, for example, Figure 3 As examples of many complete BLG crystals, Figure 4 are examples of BLG crystal fragments. In both cases, BLG crystals or crystal fragments can be visually identified as well-defined, compact and co-ordered structures using an optical microscope. Typically, BLG crystals or crystal fragments are at least partially transparent. In addition, it is known that protein crystals are birefringent, and this optical property can be used to identify unknown particles with a crystalline structure. On the other hand, non-crystalline BLG aggregates usually appear as open or porous masses with unclear boundaries, opacity and irregular size.

[0056] In the context of the present invention, the term "crystallization" relates to the formation of protein crystals. For example, crystallization occurs spontaneously or is initiated by the addition of seed crystals.

[0057] “Mother liquor”

[0058] In the context of the present invention, the term "mother liquor" relates to the whey protein solution remaining after BLG has been crystallized and the BLG crystals have been at least partially removed. The mother liquor may still contain some BLG crystals, but typically only small BLG crystals that escaped separation.

[0059] In the context of the present invention, the term "edible composition" relates to a composition that is safe for human consumption and use as a food ingredient and does not contain problematic toxic components such as toluene or other hazardous organic solvents.

[0060] In the context of the present invention, the term "ALA" or "α-lactalbumin" relates to α-lactalbumin from mammalian species, e.g. in native and / or glycosylated form, including naturally occurring gene variants. The term also includes aggregated ALA and precipitated BLG. When referring to the amount of ALA, it refers to the total amount of ALA (including e.g. aggregated ALA). The total amount of ALA is determined according to Example 1.31. The term "aggregated ALA" relates to ALA that is typically at least partially unfolded and aggregated with other denatured ALA molecules and / or other denatured whey proteins, typically by hydrophobic interactions and / or covalent bonds.

[0061] Alpha-lactalbumin (ALA) is a protein present in the milk of almost all mammalian species. ALA forms the regulatory subunit of the lactose synthase (LS) heterodimer, while β-1,4-galactosyltransferase (β4Gal-T1) forms the catalytic component. Together, these proteins allow LS to produce lactose by transferring the galactose moiety to glucose. One of the main structural differences of β-lactoglobulin is that ALA does not have any free thiol groups that could serve as a starting point for covalent aggregation reactions.

[0062] In the context of the present invention, the term "non-aggregated ALA" also relates to ALA from mammalian species, e.g. in native, unfolded and / or glycosylated form, including naturally occurring gene 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.

[0063] By calculating (m 总ALA -m 非聚集ALA ) / m 总ALA *100%, to determine the percentage of non-aggregated ALA relative to total ALA. m 总ALA is the concentration or amount of ALA measured according to Example 1.31, m 非聚集ALA is the concentration or amount of non-aggregated ALA measured according to Example 1.6.

[0064] In the context of the present invention, the term "casein macropeptide" or "CMP" relates to a hydrophilic peptide, residues 106-169, derived from "κ-CN" or "κ-casein" of mammalian species by hydrolysis of aspartic proteases (e.g. chymosin), e.g. in native and / or glycosylated form, including naturally occurring genetic variants.

[0065] In the context of the present invention, the term "BLG isolate" refers to a composition having a BLG content of at least 85% w / w relative to the total amount of protein. Preferably, the total amount of protein of the BLG isolate is at least 30% w / w, preferably at least 80% w / w relative to the total amount of solids.

[0066] In the context of the present invention, the term "BLG isolate powder" relates to BLG isolate in powder form, which is preferably a free-flowing powder.

[0067] In the context of the present invention, the term "BLG isolate liquid" relates to a BLG isolate in liquid form, which is preferably an aqueous solution.

[0068] The term "whey" relates to the liquid phase remaining after casein in milk has been precipitated and removed. For example, casein is precipitated by acidifying milk and / or using rennet. There are several types of whey, such as "sweet whey", which is a whey product produced upon precipitation of casein by rennet, and "acid whey", which is a whey product produced upon precipitation of casein by acid. For example, casein is precipitated by acid by adding edible acids or by bacterial cultures.

[0069] The term "serum" relates to the liquid that remains when casein and milk fat globules are removed from milk, for example by microfiltration or macroporous ultrafiltration. Serum may also be referred to as "ideal whey".

[0070] The term "whey proteins" or "serum proteins" relates to proteins present in whey.

[0071] In the context of the present invention, the term "whey protein" relates to proteins found in whey or milk hey. The whey protein may be a subset of the protein species found in whey or milk hey, even a single whey protein species, or it may be a complete set of protein species found in whey or / and milk hey.

[0072] In the context of the present invention, the major non-BLG proteins of the standard whey protein concentrate from sweet whey are ALA, CMP, bovine serum albumin, immunoglobulins, osteopontin, lactoferrin and lactoperoxidase. In the context of the present invention, the major non-BLG whey proteins of the standard whey protein concentrate from sweet whey are in the weight percentages:

[0073] 18% w / w ALA relative to total protein;

[0074] 18% w / w CMP relative to the total protein;

[0075] 4% w / w BSA relative to the total protein;

[0076] 5% w / w casein relative to the total protein;

[0077] 6% w / w immunoglobulin relative to the total protein;

[0078] 0.5% w / w osteopontin relative to the total protein;

[0079] 0.1% w / w lactoferrin relative to the total protein; and

[0080] 0.1% w / w lactoperoxidase relative to the total protein.

[0081] The term "casein" refers to casein found in milk, including the two native micellar caseins found in raw milk, individual casein species and caseinates.

[0082] In the context of the present invention, a "supersaturated" or "BLG supersaturated" liquid contains a concentration of dissolved non-aggregated BLG that is higher than the saturation point of non-aggregated BLG in the liquid under given physical and chemical conditions. The term "supersaturation" is well known in the art of crystallization (see, for example, Gérard Coquerela, Crystallization of molecular systems from solution: phase diagrams, supersaturation and other basic concepts, Chemical Society Reviews 2014, pp. 2286-2300, No. 7), and supersaturation can be determined by a variety of different measurement techniques (e.g., by spectroscopy or particle size analysis). In the context of the present invention, the supersaturation of BLG is determined by the following procedure.

[0083] Procedure for detecting whether a liquid is supersaturated with BLG under specific conditions:

[0084] a) Transfer 50 mL of the liquid sample to be tested into a centrifuge tube (VWR catalog number 525-0402) with a height of 115 mm, an inner diameter of 25 mm, and a capacity of 50 mL. During steps a) to h), care should be taken to maintain the sample and its subsequent fractions in the original physical and chemical conditions of the liquid;

[0085] b) immediately centrifuging the sample at 3000 g for 3.0 minutes with an acceleration of up to 30 seconds and a deceleration of up to 30 seconds;

[0086] c) After centrifugation, immediately transfer as much supernatant as possible to a second centrifuge tube (of the same type as in step a) (if a precipitate has formed, do not disturb the precipitate);

[0087] d) taking a subsample of 0.05 mL of the supernatant (subsample A);

[0088] e) adding 10 mg of BLG crystals having a particle size of at most 200 μm (non-aggregated BLG having a purity of at least 98% relative to the total amount of solids) to a second centrifuge tube and stirring the mixture;

[0089] f) allowing the second centrifuge tube to stand at the original temperature for 60 minutes;

[0090] g) immediately after step f), centrifuge the second centrifuge tube at 500 g for 10 minutes and take another 0.05 mL subsample of the supernatant (subsample B);

[0091] h) recovering the centrifugal precipitate (if any) from step g), resuspending it in MilliQ water, and immediately checking the suspension for the presence of crystals observable under a microscope;

[0092] i) The concentration of non-aggregated BLG in sub-samples A and B was determined using the method outlined in Example 1.6 - the results are expressed as % w / w of BLG relative to the total weight of the sub-sample. The concentration of non-aggregated BLG in sub-sample A is referred to as C BLG，A , the concentration of non-aggregated BLG in subsample B is called C BLG，B ;

[0093] j) If C BLG，B Below C BLG，A and crystals are observed in step i), then the liquid from which the sample of step a) was taken is (under the specific conditions) supersaturated.

[0094] In the context of the present invention, the terms "liquid" and "solution" include compositions that do not contain particulate matter, as well as compositions that contain a liquid in combination with solid and / or semi-solid particles (e.g., protein crystals or other protein particles). Thus, a "liquid" or "solution" may be a suspension or even a slurry. However, "liquids" and "solutions" are preferably pumpable.

[0095] In the context of the present invention, the terms "whey protein concentrate" (WPC) and "serum protein concentrate" (SPC) relate to dry or aqueous compositions comprising a total amount of 20-89% w / w protein relative to the total amount of solids.

[0096] Preferably, the WPC or SPC comprises:

[0097] 20-89% w / w protein relative to total solids;

[0098] 15-70% w / w BLG relative to the total protein;

[0099] 8-50% w / w ALA relative to the total amount of protein; and

[0100] 0-40% w / w CMP relative to protein.

[0101] Alternatively, but also preferably, the WPC or SPC may comprise:

[0102] 20-89% w / w protein relative to total solids;

[0103] 15-90% w / w BLG relative to the total protein;

[0104] 4-50% w / w ALA relative to the total amount of protein; and

[0105] 0-40% w / w CMP relative to protein.

[0106] Preferably, the WPC or SPC comprises:

[0107] 20-89% w / w protein relative to total solids;

[0108] 15-80% w / w BLG relative to the total protein;

[0109] 4-50% w / w ALA relative to the total amount of protein; and

[0110] 0-40% w / w CMP relative to protein.

[0111] More preferably, the WPC or SPC comprises:

[0112] 70-89% w / w protein relative to total solids;

[0113] 30-90% w / w BLG relative to the total protein;

[0114] 4-35% w / w ALA relative to the total amount of protein; and

[0115] 0-25% w / w CMP relative to protein.

[0116] Typically, SPC contains no CMP or only trace amounts of CMP.

[0117] The terms "whey protein isolate" (WPI) and "serum protein isolate" (SPI) relate to dry or aqueous compositions comprising a total amount of protein of 90-100% w / w relative to the total amount of solids.

[0118] Preferably, the WPI or SPI comprises:

[0119] 90-100% w / w protein relative to total solids;

[0120] 15-70% w / w BLG relative to the total protein;

[0121] 8-50% w / w ALA relative to the total amount of protein; and

[0122] 0-40% w / w CMP relative to the total amount of protein.

[0123] Alternatively, but preferably, the WPI or SPI may comprise:

[0124] 90-100% w / w protein relative to total solids;

[0125] 30-95% w / w BLG relative to the total protein;

[0126] 4-35% w / w ALA relative to the total amount of protein; and

[0127] 0-25% w / w CMP relative to the total amount of protein.

[0128] Preferably, the WPI or SPI comprises:

[0129] 90-100% w / w protein relative to total solids;

[0130] 30-90% w / w BLG relative to the total protein;

[0131] 4-35% w / w ALA relative to the total amount of protein; and

[0132] 0-25% w / w CMP relative to the total amount of protein.

[0133] Typically, SPI contains no CMP or only trace amounts of CMP.

[0134] In the context of the present invention, the term "other proteins" refers to proteins other than BLG. Typically, the other proteins present in the whey protein solution comprise one or more non-BLG proteins found in milk serum or whey. Non-limiting examples of such proteins are α-lactalbumin, bovine serum albumin, immunoglobulins, casein macropeptide (CMP), osteopontin, lactoferrin and milk fat globule membrane protein.

[0135] The terms “consisting essentially of” and “consisting essentially of” mean that the claim or feature covers the specified materials or steps as well as those that do not materially affect the basic and novel characteristics of the claimed invention.

[0136] In the context of the present invention, the phrase "Y and / or X" means "Y" or "X" or "Y and X". Following the same logic, the phrase "n1, n2, ..., n i-1 and / or i " means "n1" or "n2" or ... or "n i-1 " or "n i " or components n1, n2, ..., n i-1 and n i Any combination of .

[0137] In the context of the present invention, the term "dry" means that the composition or product in question comprises at most 10% w / w, preferably at most 6% w / w, more preferably even less water.

[0138] In the context of the present invention, the term "physical reduction of microorganisms" relates to a physical interaction with a composition that results in a reduction in the total amount of viable microorganisms of the composition. The term does not include the addition of chemicals that kill microorganisms. Furthermore, the term does not include thermal exposure of the atomized droplets of the liquid during spray drying, but includes possible preheating prior to spray drying.

[0139] In the context of the present invention, the pH of a powder refers to the pH of 10 g of powder mixed into 90 g of demineralised water, measured according to Example 1.16.

[0140] In the context of the present invention, the weight percentage (% w / w) of a component of a composition, product or material refers to the weight percentage of that component relative to the particular composition, product or material, unless another reference (e.g., total solids or total protein) is specifically mentioned.

[0141] In the context of the present invention, the process step "concentrate" and the verb "concentrate" refer to the concentration of protein, including the concentration of protein based on the total solids and the concentration of protein based on the total weight. For example, this means that concentration does not necessarily require that the absolute protein concentration w / w of the composition is increased, as long as the protein content is increased relative to the total solids.

[0142] In the context of the present invention, the term "weight ratio" between component X and component Y refers to the weight ratio calculated by m X / m Y The value obtained, where m Xis the amount (weight) of component X, m Y is the amount of component Y (by weight).

[0143] In the context of the present invention, the term "at least pasteurized" relates to a heat treatment with a microbial killing effect equal to or higher than 70°C for 10 seconds. The reference for determining the bacterial killing effect is Escherichia coli O157:H7.

[0144] In the context of the present invention, the term "whey protein raw material" refers to the whey protein source of the liquid BLG isolate. Compared with the liquid BLG isolate, the whey protein raw material has a lower content of BLG relative to the total amount of protein, and is typically WPC, WPI, SPC or SPI.

[0145] In the context of the present invention, the term "BLG-enriched composition" relates to a BLG-enriched composition obtained by separating BLG from a whey protein raw material. Typically, a BLG-enriched composition comprises the same whey protein as the whey protein raw material; however, BLG is present in the composition at a significantly higher concentration (relative to the total amount of protein) than in the whey protein raw material. For example, a BLG-enriched composition can be prepared from a whey protein raw material by chromatography, protein crystallization and / or membrane-based protein fractionation. Relative to the total amount of protein, the BLG-enriched composition comprises at least 85% w / w, preferably at least 90% w / w of BLG. In some cases, a BLG-enriched composition can be used directly as a liquid BLG isolate. However, additional processing is generally required to convert the BLG-enriched composition into a liquid BLG isolate.

[0146] In the context of the present invention, the term "whey protein solution" is used to describe a specific aqueous whey protein composition which is supersaturated with BLG in salting-in mode and which can be used to prepare BLG crystals.

[0147] In the context of the present invention, the term "sterile" means that the sterile composition or product in question does not contain any living microorganisms and therefore there is no microbial growth during storage at room temperature. A sterilized composition is sterile.

[0148] When a liquid, such as a beverage formulation, is sterilized and aseptically packaged in a sterile container, the liquid typically has a shelf life of at least six months at room temperature. The sterilization process kills spores and microorganisms that may cause the liquid to spoil.

[0149] In the context of the present invention, the term "protein fraction" relates to the protein of the composition under investigation, for example the protein in a powder or beverage formulation.

[0150] In the context of the present invention, the term "dry mouthfeel" relates to a sensation in the oral cavity which feels like dryness of the mouth and teeth and leads to a minimization of saliva production.

[0151] Therefore, dry mouthfeel is not a taste in itself, but rather a physical texture and time-related sensation in the mouth.

[0152] In the context of the present invention, unless otherwise specified, the term "mineral" as used herein refers to any of macrominerals, trace minerals or trace minerals, other minerals and combinations thereof. Major minerals include: calcium, phosphorus, potassium, sulfur, sodium, chlorine, magnesium. Trace minerals or trace minerals include: iron, cobalt, copper, zinc, molybdenum, iodine, selenium, manganese. Other minerals include: chromium, fluorine, boron, lithium and strontium.

[0153] In the context of the present invention, unless otherwise specified, the terms "lipid", "fat" and "oil" used herein are used interchangeably to refer to lipid materials derived from plants or animals or obtained by processing plants or animals. These terms also include synthetic lipid materials, as long as such synthetic materials are suitable for human consumption.

[0154] In the context of the present invention, the term "transparent" includes beverage formulations that have a visually clear appearance and allow light to pass through and appear different images therethrough. A clear beverage has a turbidity of at most 200 NTU.

[0155] In the context of the present invention, the term "opaque" includes beverage formulations having a visually unclear appearance and a turbidity greater than 200 NTU.

[0156] One aspect of the present invention relates to a β-lactoglobulin (BLG) isolate powder, preferably, the isolate powder is prepared by spray drying, the pH of the isolate powder is i) 2 to 4.9, ii) 6.1 to 8.5 or iii) 5.0 to 6.0, and the isolate powder comprises:

[0157] - at least 30% w / w total protein;

[0158] - at least 85% w / w beta-lactoglobulin (BLG) relative to the total amount of protein; and

[0159] - Up to 10% w / w water.

[0160] Preferably, the BLG isolate powder has one or more of the following:

[0161] - Bulk density of at least 0.2 g / cm 3 ;

[0162] - an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.11;

[0163] - protein denaturation of up to 10%;

[0164] - a thermal stability of at most 200 NTU at pH 3.9; and

[0165] - Up to 1000 colony forming units / g.

[0166] Preferably, the BLG isolate powder is an edible composition.

[0167] In some preferred embodiments of the present invention, the pH of the BLG isolate powder is 2 to 4.9. Such powder is particularly useful for acidic foods (especially acidic beverages).

[0168] In another preferred embodiment of the present invention, the pH of the BLG isolate powder is 6.1 to 8.5.

[0169] In some preferred embodiments of the present invention, the total amount of protein in the BLG isolate powder is at least 40% w / w, preferably at least 50% w / w, at least 60% w / w, more preferably at least 70% w / w, even more preferably at least 80% w / w.

[0170] Even higher protein contents may be desired; in some preferred embodiments of the invention, the total protein content of the BLG isolate powder is at least 85% w / w, preferably at least 90% w / w, at least 92% w / w, more preferably at least 94% w / w, even more preferably at least 95% w / w.

[0171] The total amount of protein was measured according to Example 1.5.

[0172] In some preferred embodiments of the present invention, the BLG isolate powder comprises at least 96% w / w, preferably at least 96.5% w / w, more preferably at least 97% w / w, even more preferably at least 98%, most preferably at least 99.5% w / w BLG relative to the total amount of protein.

[0173] In some preferred embodiments of the present invention, the BLG isolate powder comprises 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 (e.g. 100.0% w / w) BLG relative to the total amount of protein.

[0174] In some preferred embodiments of the present invention, the sum of α-lactalbumin (ALA) and casein macropeptide (CMP) accounts for at least 40% w / w, preferably at least 60% w / w, even more preferably at least 70% w / w, most preferably at least 90% w / w of the non-BLG protein of the powder.

[0175] In other preferred embodiments of the present invention, each major non-BLG whey protein, expressed as a weight percentage relative to the total amount of protein, is at most 25%, preferably at most 20%, more preferably at most 15%, even more preferably at most 10%, most preferably at most 6% by weight relative to the total amount of protein in a standard whey protein concentrate from sweet whey.

[0176] Even lower concentrations of the major non-BLG whey proteins may be desired. Therefore, in other preferred embodiments of the invention, each major non-BLG whey protein, expressed as a weight percentage relative to the total amount of protein, is at most 4%, preferably at most 3%, more preferably at most 2%, even more preferably at most 1% by weight relative to the total amount of protein in a standard whey protein concentrate from sweet whey.

[0177] In some preferred embodiments of the invention, ALA comprises at most 80% w / w, preferably at most 60% w / w, even more preferably at most 40% w / w, most preferably at most 30% w / w of the non-BLG proteins in the BLG isolate powder.

[0178] Even lower levels of ALA may be preferred; thus, in some preferred embodiments of the invention, ALA comprises at most 20% w / w, preferably at most 15% w / w, even more preferably at most 10% w / w, most preferably at most 5% w / w of the non-BLG protein in the BLG isolate powder.

[0179] The inventors have observed that reducing lactoferrin and / or lactoperoxidase is particularly advantageous for obtaining a whey protein product that is neutral in colour.

[0180] Therefore, in some preferred embodiments of the present invention, the lactoferrin, expressed as a percentage by weight relative to the total amount of protein, is at most 25%, preferably at most 20%, more preferably at most 15%, even more preferably at most 10%, most preferably at most 6%, relative to the total amount of protein of a standard whey protein concentrate from sweet whey. Even lower concentrations of lactoferrin may be desired. Therefore, in other preferred embodiments of the present invention, the lactoferrin, expressed as a percentage by weight relative to the total amount of protein, is at most 4%, preferably at most 3%, more preferably at most 2%, even more preferably at most 1%, relative to the total amount of protein of a standard whey protein concentrate from sweet whey.

[0181] Similarly, in some preferred embodiments of the invention, the lactoperoxidase is present in a weight percentage relative to the total amount of protein of at most 25%, preferably at most 20%, more preferably at most 15%, even more preferably at most 10%, most preferably at most 6%, relative to the total amount of protein of the standard whey protein concentrate from sweet whey. Even lower concentrations of lactoperoxidase may be required. Therefore, in other preferred embodiments of the invention, the lactoperoxidase is present in a weight percentage relative to the total amount of protein of at most 4%, preferably at most 3%, more preferably at most 2%, even more preferably at most 1%, relative to the total amount of protein of the standard whey protein concentrate from sweet whey.

[0182] Lactoferrin and lactoperoxidase were quantified according to Example 1.29.

[0183] In some preferred embodiments of the present invention, the water content of the BLG isolate powder is at most 10% w / w, preferably at most 7% w / w, more preferably at most 6% w / w, even more preferably at most 4% w / w, most preferably at most 2% w / w.

[0184] In some preferred embodiments of the present invention, the carbohydrate content of the BLG isolate powder is at most 60% w / w, preferably at most 50% w / w, more preferably at most 20% w / w, even more preferably at most 10% w / w, even more preferably at most 1% w / w, most preferably at most 0.1%. For example, the BLG isolate powder may contain carbohydrates such as lactose, oligosaccharides and / or hydrolysates of lactose (i.e., glucose and galactose), sucrose and / or maltodextrin.

[0185] In some preferred embodiments of the present invention, the lipid content of the BLG isolate powder is at most 10% w / w, preferably at most 5% w / w, more preferably at most 2% w / w, even more preferably at most 0.1% w / w.

[0186] Lipid content according to ISO 1211:2010 (Determination of fat content – -Gottlieb weight method, Determination of Fat Content– -Gottlieb Gravimetric Method).

[0187] The present inventors have discovered that it may be advantageous to control the mineral content to achieve certain desired properties of the BLG isolate powder.

[0188] In some preferred embodiments of the present invention, the total amount of Na, K, Mg and Ca in the BLG isolate powder is at most 10 mmol / g protein. Preferably, the total amount of Na, K, Mg and Ca in the BLG isolate powder is at most 6 mmol / g protein, more preferably at most 4 mmol / g protein, even more preferably at most 2 mmol / g protein.

[0189] In other preferred embodiments of the present invention, the total amount of Na, K, Mg and Ca in the BLG isolate powder is at most 1 mmol / g protein. Preferably, the total amount of Na, K, Mg and Ca in the BLG isolate powder is at most 0.6 mmol / g protein, more preferably at most 0.4 mmol / g protein, even more preferably at most 0.2 mmol / g protein, most preferably at most 0.1 mmol / g protein.

[0190] In other preferred embodiments of the present invention, the total amount of Mg and Ca in the BLG isolate powder is at most 5 mmol / g protein. Preferably, the total amount of Mg and Ca in the BLG isolate powder 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.

[0191] In other preferred embodiments of the present invention, the total amount of Mg and Ca in the BLG isolate powder is at most 0.3 mmol / g protein. Preferably, the total amount of Mg and Ca in the BLG isolate powder 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, most preferably 0.01 mmol / g protein.

[0192] The inventors have found that a low phosphorus / low potassium variant of BLG isolate powder can be prepared which is particularly useful for patients with kidney disease. To produce such a product, the BLG isolate powder must have equally low amounts of phosphorus and potassium.

[0193] Therefore, in some preferred embodiments of the present invention, the total phosphorus content of the BLG isolate powder is at most 100 mg phosphorus / 100 g protein. Preferably, the total phosphorus content of the BLG isolate powder is at most 80 mg phosphorus / 100 g protein. More preferably, the total phosphorus content of the BLG isolate powder is at most 50 mg phosphorus / 100 g protein. Even more preferably, the total phosphorus content of the BLG isolate powder is at most 20 mg phosphorus / 100 g protein. The total phosphorus content of the BLG isolate powder is at most 5 mg phosphorus / 100 g protein.

[0194] In some preferred embodiments of the present invention, the BLG isolate powder comprises at most 600 mg potassium / 100 g protein. More preferably, the BLG isolate powder comprises at most 500 mg potassium / 100 g protein. More preferably, the BLG isolate powder comprises at most 400 mg potassium / 100 g protein. More preferably, the BLG isolate powder comprises at most 300 mg potassium / 100 g protein. Even more preferably, the BLG isolate powder comprises at most 200 mg potassium / 100 g protein. Even more preferably, the BLG isolate powder comprises at most 100 mg potassium / 100 g protein. Even more preferably, the BLG isolate powder comprises at most 50 mg potassium / 100 g protein. Even more preferably, the BLG isolate powder comprises at most 10 mg potassium / 100 g protein.

[0195] The phosphorus content relates to the total amount of elemental phosphorus in the composition under investigation, measured according to Example 1.19. Similarly, the potassium content relates to the total amount of elemental potassium in the composition under investigation, measured according to Example 1.19.

[0196] In some preferred embodiments of the present invention, the BLG isolate powder comprises at most 100 mg phosphorus / 100 g protein and at most 700 mg potassium / 100 g protein, preferably at most 80 mg phosphorus / 100 g protein and at most 600 mg potassium / 100 g protein, more preferably at most 60 mg phosphorus / 100 g protein and at most 500 mg potassium / 100 g protein, more preferably at most 50 mg phosphorus / 100 g protein and at most 400 mg potassium / 100 g protein, or more preferably at most 20 mg phosphorus / 100 g protein and at most 200 mg potassium / 100 g protein, or even more preferably at most 10 mg phosphorus / 100 g protein and at most 50 mg potassium / 100 g protein. In some preferred embodiments of the present invention, the BLG isolate powder comprises at most 100 mg phosphorus / 100 g protein and at most 340 mg potassium / 100 g protein.

[0197] The low phosphorus and / or low potassium composition of the present invention can be used as a food ingredient for producing food for a patient population with reduced renal function.

[0198] The present inventors have found that for certain applications (e.g. acidic foods, especially acidic beverages) it is particularly advantageous for the acidic BLG isolate powder to have a pH of at most 4.9, even more preferably at most 4.3. This is particularly true for high protein, clear acidic beverages.

[0199] In the context of the present invention, the turbidity of a clear liquid is at most 200 NTU, measured according to Example 1.7.

[0200] Therefore, in some preferred embodiments of the present invention, the pH of the BLG isolate powder is 2 to 4.9. Preferably, the pH of the BLG isolate powder is 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, but also preferably, the pH of the BLG isolate powder can be 3.6 to 4.3.

[0201] The present inventors have found that for certain applications (e.g., pH-neutral foods, particularly pH-neutral beverages), pH-neutral BLG isolate powders are particularly advantageous. This is especially true for high-protein, clear or opaque pH-neutral beverages.

[0202] Therefore, in some preferred embodiments of the present invention, the pH of the BLG isolate powder is 6.1 to 8.5. Preferably, the pH of the powder is 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.

[0203] In other preferred embodiments of the present invention, the pH of the BLG isolate powder is 5.0 to 6.0. Preferably, the pH of the powder is 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.

[0204] Advantageously, the bulk density of the BLG isolate powder of the present invention may be at least 0.20 g / cm 3 , preferably at least 0.30 g / cm 3 , more preferably at least 0.40 g / cm 3 , even more preferably at least 0.45 g / cm 3 , even more preferably at least 0.50 g / cm 3 , most preferably at least 0.6 g / cm 3 .

[0205] Low density powders (such as freeze dried BLG isolate) are fluffy and easily enter the air at the production site during use. This is problematic because it increases the risk of cross contamination of freeze dried powders with other foods, and dusty environments are known to cause hygiene problems. In extreme cases, dusty environments can also increase the risk of dust explosions.

[0206] The high density versions of the present invention are easier to handle and less prone to leakage into the surrounding air.

[0207] Another advantage of the high density variants of the present invention is that they take up less space during transportation, thereby increasing the weight of BLG isolate powder that can be transported per unit volume.

[0208] Another advantage of the high density variants of the present invention is that they are less susceptible to segregation when used in powdered mixtures with other powdered food ingredients, such as powdered sugar (bulk density of about 0.56 g / cm 3 ), sugar (bulk density about 0.71g / cm 3 ), citric acid powder (bulk density about 0.77g / cm 3 ).

[0209] For example, the bulk density of the BLG isolate powder of the present invention may be 0.2 to 1.0 g / cm 3 , preferably 0.30 to 0.9 g / cm 3 , more preferably 0.40 to 0.8 g / cm 3 , even more preferably 0.45 to 0.75 g / cm 3 , even more preferably 0.50 to 0.75 g / cm 3 , and most preferably 0.6 to 0.75 g / cm 3 .

[0210] In some preferred embodiments of the present invention, the bulk density of the BLG isolate powder is 0.45 to 1.2 g / cm 3 , preferably 0.46 to 1.0 g / cm 3 , more preferably 0.47 to 0.8 g / cm 3 , even more preferably 0.48 to 0.75 g / cm 3 , even more preferably 0.48 to 0.6 g / cm 3 , and most preferably 0.50 to 0.6 g / cm 3 .

[0211] The bulk density of the powders was measured according to Example 1.17.

[0212] The present inventors have found that maintaining the native conformation of BLG is advantageous when BLG is used in acidic beverages, and the present inventors have observed that increased unfolding of BLG results in an increased dry mouthfeel.

[0213] The intrinsic tryptophan fluorescence emission ratio (I330 / I350) is a measure of the degree of BLG expansion; the inventors have found that at high intrinsic tryptophan fluorescence emission ratios (associated with low BLG expansion or no BLG expansion), a lower dry mouthfeel is observed. The intrinsic tryptophan fluorescence emission ratio (I330 / I350) is measured according to Example 1.1.

[0214] In some preferred embodiments of the present invention, the intrinsic tryptophan fluorescence emission ratio (I330 / I350) of the BLG isolate powder is at least 1.11.

[0215] In some preferred embodiments of the present invention, the intrinsic tryptophan fluorescence emission ratio (I330 / I350) of the BLG isolate powder is at least 1.12, preferably at least 1.13, more preferably at least 1.15, even more preferably at least 1.17, most preferably at least 1.19.

[0216] If the BLG isolate powder contains a large amount of non-protein substances, it is preferred to separate the protein fraction before measuring the intrinsic tryptophan fluorescence emission ratio. Therefore, in some preferred embodiments of the present invention, the intrinsic tryptophan fluorescence emission ratio of the protein fraction of the BLG isolate powder is at least 1.11.

[0217] In some preferred embodiments of the present invention, the intrinsic tryptophan fluorescence emission ratio (I330 / I350) of the protein fraction of the BLG isolate powder is 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.

[0218] For example, the protein fraction can be separated from the BLG isolate powder by dissolving the BLG isolate powder in demineralized water and subjecting the solution to dialysis or ultrafiltration-based diafiltration using a protein-retaining filter. If the BLG isolate powder contains interfering levels of lipids, the lipids can be removed by, for example, microfiltration. The microfiltration and ultrafiltration / diafiltration steps can be combined to remove lipids and small molecules from the protein fraction.

[0219] It is generally preferred that a majority of the BLG in the BLG isolate powder is non-aggregated BLG. Preferably, at least 50% of the BLG is non-aggregated BLG. More preferably, at least 80% of the BLG is non-aggregated BLG. Even more preferably, at least 90% of the BLG is non-aggregated BLG. Most preferably, at least 95% of the BLG is non-aggregated BLG. Even more preferably, about 100% of the BLG in the BLG isolate powder is non-aggregated BLG.

[0220] In some preferred embodiments of the present invention, the protein denaturation degree of the BLG isolate powder is at most 10%, preferably at most 8%, more preferably at most 6%, even more preferably at most 3%, even more preferably at most 1%, and most preferably at most 0.2%.

[0221] However, for example, if an opaque beverage is desired, it may also be preferred that the BLG isolate powder has a significant level of protein denaturation. Therefore, in other preferred embodiments of the present invention, the protein denaturation of the BLG isolate powder is at least 11%, preferably at least 20%, more preferably at least 40%, even more preferably at least 50%, even more preferably at least 75%, most preferably at least 90%.

[0222] If the BLG isolate powder has a significant level of protein denaturation, it is generally preferred to maintain low levels of insoluble protein material (ie precipitated protein material) which will precipitate in the beverage during storage.The level of insoluble material is determined according to Example 1.10.

[0223] In some preferred embodiments of the present invention, the BLG isolate powder contains at most 20% w / w insoluble protein matter, preferably at most 10% w / w insoluble protein matter, more preferably at most 5% w / w insoluble protein matter, even more preferably at most 3% w / w insoluble protein matter, most preferably at most 1% w / w insoluble protein matter. Even preferably, the BLG isolate powder does not contain any insoluble protein matter at all.

[0224] The inventors have found that the heat stability of the BLG isolate powder at pH 3.9 is a good indicator of its usefulness for a clear high protein beverage.The heat stability at pH 3.9 was measured according to Example 1.2.

[0225] Particularly preferably, the thermal stability of the BLG isolate powder at pH 3.9 is at most 200 NTU, preferably at most 100 NTU, more preferably at most 60 NTU, even more preferably at most 40 NTU, most preferably at most 20 NTU. Even better thermal stability is possible; the thermal stability of the BLG isolate powder at pH 3.9 is preferably at most 10 NTU, preferably at most 8 NTU, more preferably at most 4 NTU, even more preferably at most 2 NTU.

[0226] The microbial content of the BLG isolate powder is preferably kept to a minimum. However, it is challenging to simultaneously obtain high protein nativeness and low levels of microorganisms, as the sterilization process often results in protein unfolding and denaturation. The present invention makes it possible to obtain very low levels of microorganisms while maintaining a high level of BLG nativeness.

[0227] In some embodiments of the invention, the liquid BLG isolate comprises at most 500.000 CFU / g, preferably at most 100.000 CFU / g, more preferably at most 50.000 CFU / g, even more preferably at most 25.000 CFU / g.

[0228] Even lower levels of microorganisms are also preferred, and therefore, in some preferred embodiments of the invention, the BLG isolate powder comprises at most 15,000 colony forming units (CFU) / g. Preferably, the BLG isolate powder comprises at most 10,000 CFU / g. More preferably, the BLG isolate powder comprises at most 5,000 CFU / g. Even more preferably, the BLG isolate powder comprises at most 1,000 CFU / g. Even more preferably, the BLG isolate powder comprises at most 300 CFU / g. Most preferably, the BLG isolate powder comprises at most 100 CFU / g, such as at most 10 CFU / g. In particularly preferred embodiments, the powder is sterile. For example, sterile BLG isolate powder can be prepared by combining several physical microbial reduction methods (e.g., microfiltration and heat treatment at acidic pH) during the production of the BLG isolate powder. Preferably, drying is carried out in an aseptic drying system (e.g., an aseptic spray dryer).

[0229] In some preferred embodiments of the present invention, the pH of the BLG isolate powder is i) 2 to 4.9, ii) 6.1 to 8.5 or iii) 5.0 to 6.0, and the BLG isolate powder comprises:

[0230] - a total amount of protein of at least 30% w / w, preferably at least 80% w / w, even more preferably at least 90% w / w;

[0231] - at least 85% w / w, preferably at least 90% w / w beta-lactoglobulin (BLG) relative to the total amount of protein;

[0232] - up to 6% w / w water;

[0233] - up to 2% w / w, preferably up to 0.5% w / w, lipids;

[0234] The BLG isolate powder has:

[0235] - an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.11;

[0236] - the degree of protein denaturation is at most 10%; and

[0237] - Thermostability at pH 3.9 is up to 200 NTU.

[0238] In some preferred embodiments of the present invention, the pH of the BLG isolate powder is i) 2 to 4.9 or ii) 6.1 to 8.5, and the BLG isolate powder comprises:

[0239] - a total amount of protein of at least 30% w / w, preferably at least 80% w / w, even more preferably at least 90% w / w;

[0240] - at least 85% w / w, preferably at least 90% w / w, more preferably at least 94% w / w beta-lactoglobulin (BLG) relative to the total amount of protein;

[0241] - up to 6% w / w water;

[0242] - up to 2% w / w, preferably up to 0.5% w / w, lipids;

[0243] The BLG isolate powder has:

[0244] - an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.11;

[0245] - a degree of protein denaturation of at most 10%, preferably at most 5%; and

[0246] - A thermal stability at pH 3.9 of at most 70 NTU, preferably at most 50 NTU, even more preferably at most 40 NTU.

[0247] In some preferred embodiments of the present invention, the pH of the BLG isolate powder is i) 2 to 4.9 or ii) 6.1 to 8.5, and the BLG isolate powder comprises:

[0248] - at least 30% w / w total protein;

[0249] - at least 85% w / w, preferably at least 90% w / w beta-lactoglobulin (BLG) relative to the total amount of protein;

[0250] - up to 6% w / w water;

[0251] The BLG isolate powder has:

[0252] - At least 0.2g / cm 3 The bulk density;

[0253] - an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.11;

[0254] - the degree of protein denaturation is at most 10%; and

[0255] - Thermostability at pH 3.9 is up to 200 NTU.

[0256] In another preferred embodiment of the present invention, the pH of the BLG isolate powder is 2 to 4.9, and the BLG isolate powder comprises:

[0257] - a total amount of protein of at least 80% w / w, preferably at least 90% w / w, even more preferably at least 94% w / w;

[0258] - at least 85% w / w, preferably at least 90% w / w, more preferably at least 94% w / w beta-lactoglobulin (BLG) relative to the total amount of protein;

[0259] - up to 6% w / w water;

[0260] - up to 2% w / w, preferably up to 0.5% w / w, lipids;

[0261] The BLG isolate powder has:

[0262] - At least 0.2g / cm 3 , preferably at least 0.3 g / cm 3 , more preferably at least 0.4 g / cm 3 The bulk density;

[0263] - an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.11;

[0264] - a degree of protein denaturation of at most 10%, preferably at most 5%, more preferably at most 2%; and

[0265] - A thermal stability at pH 3.9 of at most 50 NTU, preferably at most 30 NTU, even more preferably at most 10 NTU.

[0266] In another preferred embodiment of the present invention, the pH of the BLG isolate powder is 3.0 to 4.3, preferably 3.6 to 4.1, and the BLG isolate powder comprises:

[0267] - a total amount of protein of at least 80% w / w, preferably at least 90% w / w, even more preferably at least 94% w / w;

[0268] - at least 90% w / w, preferably at least 92% w / w, even more preferably at least 94% w / w beta-lactoglobulin (BLG) relative to the total amount of protein;

[0269] - up to 6% w / w water;

[0270] - up to 2% w / w, preferably up to 0.5% w / w, lipids;

[0271] The BLG isolate powder has:

[0272] - At least 0.2g / cm 3 , preferably at least 0.3 g / cm 3 , more preferably at least 0.4 g / cm 3 The bulk density;

[0273] - the intrinsic tryptophan fluorescence emission ratio (I330 / I350) is at least 1.11, preferably at least 1.13;

[0274] - a degree of protein denaturation of at most 10%, preferably at most 5%, more preferably at most 2%; and

[0275] - A thermal stability at pH 3.9 of at most 50 NTU, preferably at most 30 NTU, even more preferably at most 10 NTU.

[0276] In another preferred embodiment of the present invention, the pH of the BLG isolate powder is 6.1 to 8.5, and the BLG isolate powder comprises:

[0277] - a total amount of protein of at least 80% w / w, preferably at least 90% w / w, even more preferably at least 94% w / w;

[0278] - at least 85% w / w, preferably at least 90% w / w, more preferably at least 94% w / w beta-lactoglobulin (BLG) relative to the total amount of protein;

[0279] - up to 6% w / w water;

[0280] - up to 2% w / w, preferably up to 0.5% w / w, lipids;

[0281] The BLG isolate powder has:

[0282] - At least 0.2g / cm 3 , preferably at least 0.3 g / cm 3 , more preferably at least 0.4 g / cm 3 The bulk density;

[0283] - a degree of protein denaturation of at most 10%, preferably at most 5%, more preferably at most 2%; and

[0284] - A thermal stability at pH 3.9 of at most 50 NTU, preferably at most 30 NTU, even more preferably at most 10 NTU.

[0285] In another preferred embodiment of the present invention, the pH of the BLG isolate powder is 6.1 to 8.5, and the BLG isolate powder comprises:

[0286] - a total amount of protein of at least 80% w / w, preferably at least 90% w / w, even more preferably at least 94% w / w;

[0287] - at least 85% w / w, preferably at least 90% w / w, more preferably at least 94% w / w beta-lactoglobulin (BLG) relative to the total amount of protein;

[0288] - up to 6% w / w water;

[0289] - up to 2% w / w, preferably up to 0.5% w / w, lipids;

[0290] The BLG isolate powder has:

[0291] - At least 0.2g / cm 3 , preferably at least 0.3 g / cm 3 , more preferably at least 0.4 g / cm 3 The bulk density;

[0292] - a degree of protein denaturation of at most 10%, preferably at most 5%, more preferably at most 2%; and

[0293] - A thermal stability at pH 3.9 of at most 50 NTU, preferably at most 30 NTU, even more preferably at most 10 NTU.

[0294] In another preferred embodiment of the present invention, the pH of the BLG isolate powder is 5.0-6.0, and the BLG isolate powder comprises:

[0295] - a total amount of protein of at least 80% w / w, preferably at least 90% w / w, even more preferably at least 94% w / w;

[0296] - at least 85% w / w, preferably at least 90% w / w, more preferably at least 94% w / w beta-lactoglobulin (BLG) relative to the total amount of protein;

[0297] - up to 6% w / w water;

[0298] - up to 2% w / w, preferably up to 0.5% w / w, lipids;

[0299] The BLG isolate powder has:

[0300] - At least 0.2g / cm 3 , preferably at least 0.3 g / cm 3 , more preferably at least 0.4 g / cm 3 The bulk density;

[0301] - the degree of protein denaturation is at most 10%, preferably at most 5%, more preferably at most 2%;

[0302] - a thermal stability at pH 3.9 of at most 50 NTU, preferably at most 30 NTU, even more preferably at most 10 NTU; and

[0303] - Preferably, the BLG crystallinity is less than 10%.

[0304] In some preferred embodiments of the present invention, the pH of the BLG isolate powder is i) 3.0-4.3, ii) 6.5-7.5 or iii) 5.0-6.0, and the BLG isolate powder comprises:

[0305] - a total amount of protein of at least 90% w / w, preferably at least 92% w / w, even more preferably at least 94% w / w;

[0306] - at least 92% w / w, preferably at least 94% w / w beta-lactoglobulin (BLG) relative to the total amount of protein;

[0307] - up to 6% w / w water;

[0308] - up to 0.5% w / w, preferably up to 0.1% w / w, lipids;

[0309] The BLG isolate powder has:

[0310] - an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.15;

[0311] - The degree of protein denaturation is at most 5%;

[0312] - a thermal stability of at most 40 NTU at pH 3.9; and

[0313] - at most 15000 colony forming units / g, preferably at most 1000 colony forming units / g, more preferably at most 100 colony forming units / g, most preferably said BLG isolate powder is sterile.

[0314] In some preferred embodiments of the present invention, the pH of the BLG isolate powder is i) 3.0-4.3, ii) 6.5-7.5 or iii) 5.0-6.0, and the BLG isolate powder comprises:

[0315] - a total amount of protein of at least 90% w / w, preferably at least 92% w / w, even more preferably at least 94% w / w;

[0316] - at least 92% w / w, preferably at least 94% w / w beta-lactoglobulin (BLG) relative to the total amount of protein;

[0317] - up to 6% w / w water;

[0318] - up to 0.5% w / w, preferably up to 0.1% w / w, lipids;

[0319] The BLG isolate powder has:

[0320] - The degree of protein denaturation is at most 5%;

[0321] - a thermal stability of at most 40 NTU at pH 3.9; and

[0322] - at most 15000 colony forming units / g, preferably at most 1000 colony forming units / g, more preferably at most 100 colony forming units / g, most preferably said BLG isolate powder is sterile.

[0323] In some preferred embodiments of the present invention, the pH of the BLG isolate powder is i) 3.0-4.3, ii) 6.5-7.5 or iii) 5.0-6.0, and the BLG isolate powder comprises:

[0324] - a total amount of protein of at least 90% w / w, preferably at least 92% w / w, even more preferably at least 94% w / w;

[0325] - at least 92% w / w, preferably at least 94% w / w beta-lactoglobulin (BLG) relative to the total amount of protein;

[0326] - up to 6% w / w water;

[0327] - up to 0.5% w / w, preferably up to 0.1% w / w, lipids;

[0328] The BLG isolate powder has:

[0329] - an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.15;

[0330] - a thermal stability of at most 40 NTU at pH 3.9; and

[0331] - at most 15000 colony forming units / g, preferably at most 1000 colony forming units / g, more preferably at most 100 colony forming units / g, most preferably said BLG isolate powder is sterile.

[0332] In some preferred embodiments of the present invention, the pH of the BLG isolate powder is i) 3.0-4.3, ii) 6.5-7.5 or iii) 5.0-6.0, and the BLG isolate powder comprises:

[0333] - a total amount of protein of at least 90% w / w, preferably at least 92% w / w, even more preferably at least 94% w / w;

[0334] - at least 92% w / w, preferably at least 94% w / w beta-lactoglobulin (BLG) relative to the total amount of protein;

[0335] - up to 6% w / w water;

[0336] - up to 0.5% w / w, preferably up to 0.1% w / w, lipids;

[0337] The BLG isolate powder has:

[0338] - an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.15;

[0339] - the degree of protein denaturation is at most 5%; and

[0340] - at most 15000 colony forming units / g, preferably at most 1000 colony forming units / g, more preferably at most 100 colony forming units / g, most preferably said BLG isolate powder is sterile.

[0341] In some preferred embodiments of the present invention, the pH of the BLG isolate powder is i) 3.0-4.3, ii) 6.5-7.5 or iii) 5.0-6.0, and the BLG isolate powder comprises:

[0342] - a total amount of protein of at least 90% w / w, preferably at least 92% w / w, even more preferably at least 94% w / w;

[0343] - at least 92% w / w, preferably at least 94% w / w beta-lactoglobulin (BLG) relative to the total amount of protein;

[0344] - up to 6% w / w water;

[0345] - up to 0.5% w / w, preferably up to 0.1% w / w, lipids;

[0346] The BLG isolate powder has:

[0347] - an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.15;

[0348] - the degree of protein denaturation is at most 5%; and

[0349] - Thermostability at pH 3.9 is up to 40 NTU.

[0350] In some preferred embodiments of the present invention, the pH of the BLG isolate powder is i) 3.0-4.3, ii) 6.5-7.5 or iii) 5.0-6.0, and the BLG isolate powder comprises:

[0351] - a total amount of protein of at least 90% w / w, preferably at least 92% w / w, even more preferably at least 94% w / w;

[0352] - at least 92% w / w, preferably at least 94% w / w beta-lactoglobulin (BLG) relative to the total amount of protein;

[0353] - up to 6% w / w water;

[0354] - up to 0.5% w / w, preferably up to 0.1% w / w, lipids;

[0355] The BLG isolate powder has:

[0356] - an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.15;

[0357] - a thermal stability of at most 40 NTU at pH 3.9; and

[0358] - at most 15000 colony forming units / g, preferably at most 1000 colony forming units / g, more preferably at most 100 colony forming units / g, most preferably said BLG isolate powder is sterile.

[0359] In some preferred embodiments of the present invention, the pH of the BLG isolate powder is i) 3.0-4.3 or ii) 6.3-7.5, and the BLG isolate powder comprises:

[0360] - a total amount of protein of at least 30% w / w, preferably at least 50% w / w, even more preferably at least 80% w / w;

[0361] - at least 90% w / w, more preferably at least 94% w / w beta-lactoglobulin (BLG) relative to the total amount of protein

[0362] - up to 6% w / w water;

[0363] - up to 0.5% w / w, preferably up to 0.1% w / w, lipids;

[0364] The BLG isolate powder has:

[0365] - an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.15;

[0366] - a protein denaturation degree of at most 5%, preferably at most 2%; and

[0367] - A thermal stability at pH 3.9 of at most 40 NTU, preferably at most 20 NTU, even more preferably at most 10 NTU.

[0368] In some preferred embodiments of the present invention, the pH of the BLG isolate powder is i) 3.0 to 4.3 or ii) 6.3 to 7.5, and comprises:

[0369] - at least 30% w / w total protein;

[0370] - at least 85% w / w, preferably at least 90% w / w beta-lactoglobulin (BLG) relative to the total amount of protein;

[0371] - up to 6% w / w water;

[0372] The BLG isolate powder has:

[0373] - an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.15;

[0374] - the degree of protein denaturation is at most 5%; and

[0375] - Thermostability at pH 3.9 is up to 40 NTU.

[0376] In another preferred embodiment of the present invention, the pH of the BLG isolate powder is 3.0 to 4.3, and the BLG isolate powder comprises:

[0377] - a total amount of protein of at least 90% w / w, preferably at least 94% w / w;

[0378] - at least 94% w / w, preferably at least 96% w / w beta-lactoglobulin (BLG) relative to the total amount of protein;

[0379] - up to 6% w / w water;

[0380] - up to 0.5% w / w, preferably up to 0.1% w / w, lipids;

[0381] The BLG isolate powder has:

[0382] - Bulk density is 0.45~0.8g / cm 3 , preferably 0.50~0.6g / cm 3 ;

[0383] - an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.15;

[0384] - a degree of protein denaturation of at most 5%, more preferably at most 2%; and

[0385] - Thermostability at pH 3.9 is at most 30 NTU, preferably at most 10 NTU.

[0386] In another preferred embodiment of the present invention, the pH of the BLG isolate powder is 6.3-7.5, and the BLG isolate powder comprises:

[0387] - a total amount of protein of at least 90% w / w, even more preferably at least 94% w / w;

[0388] - at least 94% w / w, even more preferably at least 96% w / w beta-lactoglobulin (BLG), relative to the total amount of protein;

[0389] - up to 6% w / w water;

[0390] - up to 0.5% w / w, preferably up to 0.1% w / w, lipids;

[0391] The BLG isolate powder has:

[0392] - Bulk density is 0.45~0.8g / cm 3 , preferably 0.50~0.6g / cm 3 ;

[0393] - a degree of protein denaturation of at most 10%, preferably at most 5%, more preferably at most 2%; and

[0394] - A thermal stability at pH 3.9 of at most 50 NTU, preferably at most 30 NTU, even more preferably at most 10 NTU.

[0395] In another preferred embodiment of the present invention, the pH of the BLG isolate powder is 5.0-6.0, and the BLG isolate powder comprises:

[0396] - a total amount of protein of at least 90% w / w, even more preferably at least 94% w / w;

[0397] - at least 90% w / w, even more preferably at least 94% w / w beta-lactoglobulin (BLG), relative to the total amount of proteins;

[0398] - up to 6% w / w water;

[0399] - up to 0.5% w / w, preferably up to 0.1% w / w, lipids;

[0400] The BLG isolate powder has:

[0401] - Bulk density is 0.45~0.8g / cm 3 , preferably 0.50~0.6g / cm 3 ;

[0402] - the degree of protein denaturation is at most 10%, preferably at most 5%, more preferably at most 2%;

[0403] - a thermal stability at pH 3.9 of at most 50 NTU, preferably at most 30 NTU, even more preferably at most 10 NTU; and

[0404] - Preferably, the crystallinity of BLG is less than 10%, more preferably at most 1%.

[0405] Yet another aspect of the present invention relates to a method for producing a dry BLG isolate powder having a BLG content of at least 85% w / w relative to the total amount of protein, the method comprising the steps of:

[0406] a) providing a liquid BLG isolate having the following characteristics:

[0407] i) pH 2 to 4.9;

[0408] ii) pH 6.1 to 8.5; or

[0409] iii) pH 5.0 to 6.0;

[0410] The liquid BLG isolate has a BLG content of at least 85% w / w relative to the total amount of protein;

[0411] b) optionally, subjecting the liquid BLG isolate to physical microbial reduction;

[0412] c) drying the liquid BLG isolate, preferably spray drying.

[0413] Preferably, the liquid BLG isolate is an edible composition.

[0414] Preferably, the liquid BLG isolate is obtained from the milk of a mammal, preferably a ruminant, such as a cow, sheep, goat, buffalo, camel, llama, horse and / or deer. Proteins derived from bovine milk are particularly preferred. Thus, the BLG is preferably bovine BLG.

[0415] In some preferred embodiments of the present invention, the BLG content of the liquid BLG isolate is 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, relative to the total amount of protein.

[0416] In some preferred embodiments of the present invention, the BLG content of the liquid BLG isolate is 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, relative to the total amount of protein.

[0417] In some preferred embodiments of the present invention, the total amount of protein in the liquid BLG isolate is at least 5% w / w, preferably at least 10% w / w, more preferably at least 15% w / w, even more preferably at least 20% w / w, most preferably at least 30% w / w.

[0418] In some preferred embodiments of the present invention, the total protein content of the liquid BLG isolate is 5-40% w / w, preferably 10-35% w / w, more preferably 15-30% w / w, even more preferably 20-25% w / w.

[0419] The present inventors have observed that increasing the BLG concentration in the liquid BLG isolate results in a spray-dried powder with a higher bulk density; therefore preferably, the liquid BLG isolate has a relatively high BLG concentration.

[0420] Therefore, in other preferred embodiments of the present invention, the total protein content of the liquid BLG isolate is 10-40% w / w, preferably 20-38% w / w, more preferably 24-36% w / w, even more preferably 28-34% w / w.

[0421] In some preferred embodiments of the present invention, the sum of α-lactalbumin (ALA) and casein macropeptide (CMP) accounts for at least 40% w / w, preferably at least 60% w / w, even more preferably at least 70% w / w, and most preferably at least 90% w / w of the non-BLG proteins in the liquid BLG isolate.

[0422] In some preferred embodiments of the invention, ALA comprises at most 80% w / w, preferably at most 60% w / w, even more preferably at most 40% w / w, most preferably at most 30% w / w of the non-BLG proteins in the liquid BLG isolate.

[0423] Even lower amounts of ALA may be preferred; thus, in some preferred embodiments of the invention, ALA comprises at most 20% w / w, preferably at most 15% w / w, even more preferably 10% w / w, most preferably at most 5% w / w of the non-BLG protein in the liquid BLG isolate.

[0424] In other preferred embodiments of the present invention, each major non-BLG whey protein in the liquid BLG isolate, expressed as a weight percentage relative to the total amount of protein, is at most 25%, preferably at most 20%, more preferably at most 15%, even more preferably at most 10%, most preferably at most 6% by weight relative to the total amount of protein in a standard whey protein concentrate from sweet whey.

[0425] Even lower concentrations of major non-BLG whey proteins are desired. Therefore, in other preferred embodiments of the invention, each major non-BLG whey protein in the liquid BLG isolate, expressed as a weight percentage relative to the total amount of protein, is at most 4%, preferably at most 3%, more preferably at most 2%, even more preferably at most 1% by weight relative to the total amount of protein in a standard whey protein concentrate from sweet whey.

[0426] The inventors have found that low levels of lactoferrin and / or lactoperoxidase are particularly advantageous for obtaining a whey protein product that is neutral in colour.

[0427] Therefore, in some preferred embodiments of the present invention, the lactoferrin in the liquid BLG isolate, expressed as a percentage by weight relative to the total amount of protein, is at most 25%, preferably at most 20%, more preferably at most 15%, even more preferably at most 10%, most preferably at most 6%, relative to the total amount of protein of a standard whey protein concentrate from sweet whey. Even lower concentrations of lactoferrin may be desired. Therefore, in other preferred embodiments of the present invention, the lactoferrin, expressed as a percentage by weight relative to the total amount of protein, is at most 4%, preferably at most 3%, more preferably at most 2%, even more preferably at most 1%, relative to the total amount of protein of a standard whey protein concentrate from sweet whey.

[0428] Similarly, in some preferred embodiments of the invention, the lactoperoxidase in the liquid BLG isolate, expressed as a percentage by weight relative to the total amount of protein, is at most 25%, preferably at most 20%, more preferably at most 15%, even more preferably at most 10%, most preferably at most 6%, relative to the total amount of protein of a standard whey protein concentrate from sweet whey. Even lower concentrations of lactoperoxidase may be required. Therefore, in other preferred embodiments of the invention, the lactoperoxidase, expressed as a percentage by weight relative to the total amount of protein, is at most 4%, preferably at most 3%, more preferably at most 2%, even more preferably at most 1%, relative to the total amount of protein of a standard whey protein concentrate from sweet whey.

[0429] In some preferred embodiments of the present invention, the liquid BLG isolate comprises a total solid content of 5-50% w / w, preferably 10-40% w / w, more preferably 15-35% w / w, even more preferably 20-30% w / w.

[0430] Preferably, the fraction of the liquid BLG isolate not counted as part of the total solids consists essentially of water or even consists of water.

[0431] In some preferred embodiments of the present invention, the liquid BLG isolate comprises water in an amount of 50-95% w / w, preferably 60-90% w / w, more preferably 65-85% w / w, even more preferably 70-80% w / w.

[0432] In some preferred embodiments of the present invention, the carbohydrate content of the liquid BLG isolate is at most 60% w / w, preferably at most 50% w / w, more preferably at most 20% w / w, even more preferably at most 10% w / w, even more preferably at most 1% w / w, most preferably at most 0.1%. For example, the liquid BLG isolate may contain carbohydrates such as lactose, oligosaccharides and / or hydrolysates of lactose (i.e., glucose and galactose), sucrose and / or maltodextrin.

[0433] In some preferred embodiments of the present invention, the lipid content of the liquid BLG isolate is at most 10% w / w, preferably at most 5% w / w, more preferably at most 2% w / w, even more preferably at most 0.1% w / w.

[0434] The present inventors have discovered that it may be advantageous to control the mineral content to achieve certain desired properties of the liquid BLG isolate.

[0435] In some preferred embodiments of the present invention, the total amount of Na, K, Mg and Ca of the liquid BLG isolate is at most 10 mmol / g protein. Preferably, the total amount of Na, K, Mg and Ca of the liquid BLG isolate is at most 6 mmol / g protein, more preferably at most 4 mmol / g protein, even more preferably at most 2 mmol / g protein.

[0436] In other preferred embodiments of the present invention, the total amount of Na, K, Mg and Ca of the liquid BLG isolate is at most 1.0 mmol / g protein. Preferably, the total amount of Na, K, Mg and Ca of the liquid BLG isolate is at most 0.6 mmol / g protein, more preferably at most 0.4 mmol / g protein, even more preferably at most 0.2 mmol / g protein, most preferably at most 0.1 mmol / g protein.

[0437] In other preferred embodiments of the present invention, the total amount of Mg and Ca of the liquid BLG isolate is at most 5 mmol / g protein. Preferably, the total amount of Mg and Ca of 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.

[0438] In other preferred embodiments of the present invention, the total amount of Mg and Ca of the liquid BLG isolate is at most 0.3 mmol / g protein. Preferably, the total amount of Mg and Ca of 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, most preferably at most 0.01 mmol / g protein.

[0439] The inventors have discovered that it is possible to prepare 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 liquid BLG isolate must have equally low levels of phosphorus and potassium.

[0440] Therefore, in some preferred embodiments of the present invention, the total phosphorus content of the liquid BLG isolate is at most 100 mg phosphorus / 100 g protein. Preferably, the total phosphorus content of the liquid BLG isolate is at most 80 mg phosphorus / 100 g protein. More preferably, the total phosphorus content of the liquid BLG isolate is at most 50 mg phosphorus / 100 g protein. Even more preferably, the total phosphorus content of the liquid BLG isolate is at most 20 mg phosphorus / 100 g protein. The total phosphorus content of the liquid BLG isolate is at most 5 mg phosphorus / 100 g protein.

[0441] In some preferred embodiments of the present invention, the liquid BLG isolate comprises at most 600 mg potassium / 100 g protein. More preferably, the liquid BLG isolate comprises at most 500 mg potassium / 100 g protein. More preferably, the liquid BLG isolate comprises at most 400 mg potassium / 100 g protein. More preferably, the liquid BLG isolate comprises at most 300 mg potassium / 100 g protein. Even more preferably, the liquid BLG isolate comprises at most 200 mg potassium / 100 g protein. Even more preferably, the liquid BLG isolate comprises at most 100 mg potassium / 100 g protein. Even more preferably, the liquid BLG isolate comprises at most 50 mg potassium / 100 g protein. Even more preferably, the liquid BLG isolate comprises at most 10 mg potassium / 100 g protein.

[0442] The phosphorus content relates to the total amount of elemental phosphorus in the composition under investigation, measured according to Example 1.19. Similarly, the potassium content relates to the total amount of elemental potassium in the composition under investigation, measured according to Example 1.19.

[0443] In some preferred embodiments of the present invention, the liquid BLG isolate comprises at most 100 mg phosphorus / 100 g protein and at most 700 mg potassium / 100 g protein, preferably at most 80 mg phosphorus / 100 g protein and at most 600 mg potassium / 100 g protein, more preferably at most 60 mg phosphorus / 100 g protein and at most 500 mg potassium / 100 g protein, more preferably at most 50 mg phosphorus / 100 g protein and at most 400 mg potassium / 100 g protein, or more preferably at most 20 mg phosphorus / 100 g protein and at most 200 mg potassium / 100 g protein, or more preferably at most 10 mg phosphorus / 100 g protein and at most 50 mg potassium / 100 g protein. In some preferred embodiments of the present invention, the liquid BLG isolate comprises at most 100 mg phosphorus / 100 g protein and at most 340 mg potassium / 100 g protein.

[0444] The low phosphorus and / or low potassium composition of the present invention can be used as a food ingredient for producing food for a patient population with reduced renal function.

[0445] In some preferred embodiments of the present invention, the pH of the liquid BLG isolate is 2-4.9, preferably 2.5-4.7, more preferably 2.8-4.4, even more preferably 3.0-4.0, most preferably 3.4-3.9.

[0446] In other preferred embodiments of the present invention, the pH of the liquid BLG isolate is 6.1 to 8.5, preferably 6.2 to 8.0, more preferably 6.3 to 7.7, even more preferably 6.5 to 7.5.

[0447] In other preferred embodiments of the present invention, the pH of the liquid BLG isolate is 5.0-6.0, preferably 5.1-5.9, more preferably 5.2-5.8, and even more preferably 5.3-5.7. When the pH of the liquid BLG isolate is 5.0-6.0, it is generally preferred that the liquid BLG isolate does not contain any BLG crystals. This can be achieved by ensuring that the liquid BLG isolate is below the saturation point of BLG, for example by increasing the temperature and / or adding salt. Alternatively, even if BLG is supersaturated, the liquid BLG isolate can be kept free of crystals as long as it is kept in the metastable region and the crystallization promoter is not brought into contact with the liquid BLG isolate.

[0448] Preferably, the liquid BLG isolate has a low microbial content; this is particularly possible if the BLG-enriched composition already has a low microbial content.

[0449] In some embodiments of the invention, the liquid BLG isolate comprises at most 500.000 CFU / g, preferably at most 100.000 CFU / g, more preferably at most 50.000 CFU / g, even more preferably at most 10.000 CFU / g.

[0450] Thus, in some preferred embodiments of the invention, the liquid BLG isolate comprises at most 1000 colony forming units (CFU) / g. Preferably, the liquid BLG isolate comprises at most 600 CFU / g. More preferably, the liquid BLG isolate comprises at most 300 CFU / g. Even more preferably, the liquid BLG isolate comprises at most 100 CFU / g. Even more preferably, the liquid BLG isolate comprises at most 50 CFU / g. Most preferably, the liquid BLG isolate comprises at most 20 CFU / g, such as at most 10 CFU / g. In particularly preferred embodiments, the powder is sterile. For example, a sterile liquid BLG isolate can be prepared by combining several physical microbial reduction processes during the production of the BLG isolate powder, such as microfiltration and heat treatment at low pH (up to pH 4.0).

[0451] The preparation of BLG isolate powder with low protein unfolding requires that the liquid BLG isolate already has a low protein unfolding degree, since the unfolding of BLG appears to be an irreversible process.

[0452] If a BLG isolate powder or a liquid BLG isolate having a low degree of BLG development is desired, it is preferred that the liquid BLG isolate have an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.11.

[0453] In some preferred embodiments of the present invention, the intrinsic tryptophan fluorescence emission ratio (I330 / I350) of the liquid BLG isolate is at least 1.12, preferably at least 1.13, more preferably at least 1.15, even more preferably at least 1.17, most preferably at least 1.19.

[0454] If the liquid BLG isolate contains a large amount of non-protein substances, it is preferred to separate the protein fraction before measuring the intrinsic tryptophan fluorescence emission ratio. Therefore, in some preferred embodiments of the present invention, the intrinsic tryptophan fluorescence emission ratio (I330 / I350) of the protein fraction of the liquid BLG isolate is at least 1.11.

[0455] Preferably, the intrinsic tryptophan fluorescence emission ratio (I330 / I350) of the protein fraction of the liquid BLG isolate may be at least 1.12, more preferably at least 1.13, even more preferably at least 1.15, even more preferably at least 1.17, most preferably at least 1.19.

[0456] For example, the protein fraction can be separated from the BLG isolate powder by dialysis using a protein retaining filter or ultrafiltration based diafiltration.

[0457] Preparation of BLG isolate powder with low protein development requires that the liquid BLG isolate already has a low degree of protein denaturation, since protein denaturation of BLG appears to be an irreversible process. Therefore, in some preferred embodiments of the present invention, the degree of protein denaturation of the liquid BLG isolate is at most 10% w / w, preferably at most 6% w / w, more preferably at most 4% w / w, even more preferably at most 2% w / w, and most preferably at most 1% w / w.

[0458] It is generally preferred that a large amount of BLG in the liquid BLG isolate is non-aggregated BLG. Preferably, at least 50% of the BLG is non-aggregated BLG. More preferably, at least 80% of the BLG is non-aggregated BLG. Even more preferably, at least 90% of the BLG is non-aggregated BLG. Most preferably, at least 95% of the BLG is non-aggregated BLG. Even more preferably, about 100% of the BLG in the liquid BLG isolate is non-aggregated BLG.

[0459] However, it may also be preferred that the level of protein denaturation of the liquid BLG isolate is significant, for example if an opaque beverage is desired. Therefore, in other preferred embodiments of the present invention, the protein denaturation of the BLG isolate powder is at least 11%, preferably at least 20%, more preferably at least 40%, even more preferably at least 50%, even more preferably at least 75%, most preferably at least 90%.

[0460] If the level of protein denaturation of the liquid BLG isolate is significant, it is generally preferred to keep the level of insoluble protein material, ie precipitated protein material which will settle in the beverage during storage, low. The level of insoluble material is determined according to Example 1.10.

[0461] In some preferred embodiments of the present invention, the liquid BLG isolate contains at most 20% w / w insoluble protein matter, preferably at most 10% w / w insoluble protein matter, more preferably at most 5% w / w insoluble protein matter, even more preferably at most 3% w / w insoluble protein matter, most preferably at most 1% w / w insoluble protein matter. Even preferably, the liquid BLG isolate does not contain any insoluble protein matter at all.

[0462] As mentioned above, the inventors have found that the heat stability of a liquid BLG isolate at pH 3.9 is a good indicator of its usefulness for a clear high protein beverage.The heat stability at pH 3.9 was measured according to Example 1.2.

[0463] Particularly preferably, the liquid BLG isolate has a thermal stability of at most 200 NTU, preferably at most 100 NTU, more preferably at most 60 NTU, even more preferably at most 40 NTU, and most preferably at most 20 NTU at pH 3.9. Even better thermal stability is possible, the thermal stability of the liquid BLG isolate at pH 3.9 is preferably at most 10 NTU, preferably at most 8 NTU, more preferably at most 4 NTU, and even more preferably at most 2 NTU.

[0464] In some preferred embodiments of the present invention, the turbidity of the liquid BLG isolate is at most 200 NTU, preferably at most 100 NTU, more preferably at most 50 NTU, even more preferably at most 20 NTU, even more preferably at most 10 NTU, most preferably at most 2 NTU.

[0465] In other preferred embodiments of the present invention, the turbidity of the liquid BLG isolate is more than 200NTU, preferably at least 400NTU, more preferably at least 800NTU, even more preferably at least 1000NTU, even more preferably at least 2000NTU, most preferably at least 5000NTU. Particularly preferably, such liquid BLG isolate is used to produce opaque beverages.

[0466] The inventors have observed that the liquid BLG isolate of the present invention unexpectedly has a lower viscosity than a comparable liquid WPI. The inventors have found that this makes the liquid BLG isolate particularly suitable for use as a high protein beverage as it allows high protein content to be obtained without resulting in undesirably high viscosity.

[0467] In some preferred embodiments of the present invention, the liquid BLG isolate is heated at 15°C for 300 s -1 The viscosity at the shear rate is visc(p)±50%, more preferably visc(p)±40%, even more preferably visc(p)±30%, most preferably visc(p)±25%.

[0468] visc(p) is defined as:

[0469] visc(p)=0.3556e 0.1262*p (for p≤23%) or 0.0254*e 0.24*p (for p>23%).

[0470] p is the total amount of protein (expressed as % w / w) of the liquid BLG isolate; thus, for example, if the protein content is 31% w / w, then p is 31.

[0471] This means, for example, that if a liquid BLG isolate (protein content p) is heated at 15°C and 300 s -1The viscosity at the shear rate is visc(p) ± 25%, then the viscosity of the liquid BLG isolate is at least visc(p) – 25% and at most visc(p) + 25%. For example, if the protein content p of the liquid BLG isolate is 31% w / w, the minimum and maximum viscosities for this example are:

[0472] Minimum viscosity (in cP): 0.0254*e 0.24*31 –25%=43cP –25%=32cP;

[0473] Maximum viscosity (in cP): 0.0254*e 0.24*31 +25%=43cP+25%=54cP.

[0474] In other preferred embodiments of the present invention, the liquid BLG isolate is heated at 15°C for 300 s -1 The viscosity at the shear rate is visc(p)±20%, more preferably visc(p)±15%, even more preferably visc(p)±10%, most preferably visc(p)±5%.

[0475] According to Example 1.8 but using a temperature of 15°C and 300 s -1 The viscosity of the liquid BLG isolate was measured at a shear rate of 1.54°.

[0476] In some preferred embodiments of the present invention, the liquid BLG isolate is heated at 15°C for 300 s -1 The viscosity at shear rate is:

[0477] - at least visc(p) – 20%; and

[0478] - at most visc 最大值 (p)–20%.

[0479] In other preferred embodiments of the present invention, the liquid BLG isolate is heated at 15°C for 300 s -1 The viscosity at shear rate is:

[0480] - at least visc(p) – 10%; and

[0481] - at most visc 最大值 (p)–40%.

[0482] In other preferred embodiments of the present invention, the liquid BLG isolate is heated at 15°C for 300 s -1 The viscosity at shear rate is:

[0483] - at least visc(p) – 10%; and

[0484] - at most visc最大值 (p)–50%.

[0485] visc 最大值 (p) is defined as: visc 最大值 (p)≤0.611*e (0.1494*p) cP.

[0486] In other preferred embodiments of the present invention, the liquid BLG isolate is heated at 15°C for 300 s -1 The viscosity at a shear rate of up to visc 最大值 (p) - 10%, more preferably up to visc 最大值 (p) - 20%, even more preferably visc 最大值 (p) – 30%, most preferably visc 最大值 (p)–50%.

[0487] In some preferred embodiments of the present invention, the pH of the liquid BLG isolate is 2.8 to 4.3, preferably 3.0 to 4.0, and the liquid BLG isolate comprises:

[0488] - 20-34% w / w, more preferably 24-32% w / w, even more preferably 28-32% w / w of total protein;

[0489] - at least 90% w / w, more preferably at least 94% w / w beta-lactoglobulin (BLG) relative to the total amount of protein;

[0490] Preferably, the BLG isolate powder has one or more of the following:

[0491] - an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.15;

[0492] - The degree of protein denaturation is at most 2%;

[0493] - Thermostability at pH 3.9 is at most 20 NTU;

[0494] - Sterility; and

[0495] - The viscosity at 15°C is visc(p) ± 25%; where p is the protein content (in % w / w), visc(p) = 0.3556e 0.1262*p (for p≤23%) or 0.0254*e 0.24*p (for p>23%).

[0496] In other preferred embodiments of the present invention, the pH of the liquid BLG isolate is 6.3 to 8.0, more preferably 6.5 to 7.5, and the liquid BLG isolate comprises:

[0497] - 20-34% w / w, more preferably 24-32% w / w, even more preferably 28-30% w / w of total protein;

[0498] - at least 90% w / w, more preferably at least 94% w / w beta-lactoglobulin (BLG) relative to the total amount of protein;

[0499] Preferably, the BLG isolate powder has one or more of the following:

[0500] - an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.15;

[0501] - The degree of protein denaturation is at most 5%;

[0502] - Thermostability at pH 3.9 is up to 40 NTU;

[0503] - Sterility; and

[0504] - The viscosity at 15°C is visc(p) ± 25%; where p is the protein content (in % w / w), and visc(p) = 0.3556e 0.1262*p (for p≤23%) or 0.0254*e 0.24*p (for p>23%).

[0505] Such acidic high protein liquid BLG isolates are particularly suitable for producing high quality BLG isolate powders and have unexpectedly low viscosity compared to comparable WPIs, which results in lower energy consumption for processing (e.g., microfiltration). In addition, acidic high protein liquid BLG isolates can produce acidic whey protein powders with much higher bulk densities than can be achieved with conventional WPIs of the same protein content (see, e.g., Example 7).

[0506] Liquid BLG isolate can be provided in a number of different ways.

[0507] Typically, providing a liquid BLG isolate involves or even consists of isolating BLG from a whey protein material to provide a BLG-enriched composition by one or more of the following methods:

[0508] - Crystallization or precipitation of BLG by salting;

[0509] - crystallization or precipitation of BLG by salting-out;

[0510] - ion exchange chromatography; and

[0511] - Fractionation of whey protein by ultrafiltration.

[0512] A particularly preferred way of providing a BLG-enriched composition is by crystallization of BLG, preferably by salting-out or salting-out.

[0513] Preferably, the whey protein raw material is WPC, WPI, SPC, SPI or a combination thereof.

[0514] The term "whey protein raw material" refers to a composition from which a BLG-enriched composition and subsequent liquid BLG isolate can be obtained. The chemical features and embodiments described for the whey protein solution also apply to the whey protein raw material, except that: generally, the BLG of the whey protein raw material is not supersaturated, and the pH of the raw material is not limited to 5-6.

[0515] In some embodiments of the present invention, the preparation of the BLG-enriched composition comprises or even consists of: crystallization of high-salt BLG at a pH of 3.6-4.0 according to US 2,790,790A1.

[0516] In other embodiments of the present invention, the preparation of a BLG-enriched composition comprises or even consists of the method described by de Jongh et al. (Mild Isolation Procedure Discloses New Protein Structural Properties of β-Lactoglobulin, J. Dairy Sci. 2001, 84(3): 562-571) or Vyas et al. (Scale-Up of Native β-Lactoglobulin Affinity Separation Process, J. Dairy Sci. 2002, 85: 1639-1645).

[0517] However, in a particularly preferred embodiment of the present invention, the BLG-enriched composition is prepared by crystallization under saline conditions at pH 5-6 as described in PCT Application No. PCT / EP2017 / 084553 (the contents of which are incorporated herein by reference for all purposes).

[0518] In some preferred embodiments of the present invention, the BLG-enriched composition is an edible BLG composition according to PCT / EP2017 / 084553, which comprises at least 90% BLG relative to the total amount of protein and preferably comprises BLG crystals.

[0519] Preferably, the BLG-enriched composition is prepared by a method comprising the following steps:

[0520] 1) providing a whey protein solution, wherein the whey protein solution comprises non-aggregated BLG and at least one other whey protein, wherein the whey protein solution is supersaturated with BLG and has a pH of 5 to 6;

[0521] 2) crystallization of non-aggregated BLG in a supersaturated whey protein solution;

[0522] 3) separating BLG crystals from the remaining whey protein solution;

[0523] 4) optionally, washing the BLG crystals, such as the separated BLG crystals obtained from step 3) or 5); and

[0524] 5) Optionally, recrystallize BLG crystals, such as the BLG crystals obtained from step 3) or 4).

[0525] This method for preparing a BLG-enriched composition comprises the mandatory steps 1), 2) and 3) and is performed in this order, and may optionally include steps 4) and / or 5) in any order and number of iterations. However, steps 4) and 5) are usually after step 3). Alternatively or additionally, wash water may be added to the whey protein solution containing the crystals before separation.

[0526] Furthermore, the method may comprise the step of drying the BLG-enriched composition. However, it is currently preferred to use the BLG-enriched composition without drying it to avoid the risk of damaging the protein during drying.

[0527] As described above, step 1) of the crystallization process involves providing a whey protein solution comprising non-aggregated BLG and at least one other whey protein.

[0528] Preferably, the whey protein solution comprises at least one other non-aggregated whey protein selected from the group consisting of α-lactalbumin, bovine serum albumin, immunoglobulins, casein macropeptide (CMP), osteopontin, lactoferrin, lactoperoxidase, milk fat globule membrane protein and combinations thereof.

[0529] In some embodiments of the invention, the whey protein solution comprises at most 10% w / w, preferably at most 5% w / w, more preferably at most 1% w / w, even preferably at most 0.5% w / w casein relative to the total amount of protein. In some preferred embodiments of the invention, the whey protein solution does not comprise any detectable amount of casein.

[0530] In some preferred embodiments of the present invention, the whey protein solution of step 1) contains at least 5% w / w of other whey proteins relative to the total amount of protein. Preferably, the whey protein solution of step 1) contains at least 10% w / w of other whey proteins relative to the total amount of protein. More preferably, the whey protein solution of step 1) contains at least 15% w / w of other whey proteins relative to the total amount of protein. Even more preferably, the whey protein solution of step 1) contains at least 20% w / w of other whey proteins relative to the total amount of protein. Most preferably, the whey protein solution of step 1) may contain at least 30% w / w of other whey proteins relative to the total amount of protein.

[0531] In other preferred embodiments of the present invention, the whey protein solution of step 1) comprises at least 1% w / w of other whey proteins relative to the total amount of protein. Preferably, the whey protein solution of step 1) comprises at least 2% w / w of other whey proteins relative to the total amount of protein. Even more preferably, the whey protein solution of step 1) comprises at least 3% w / w of other whey proteins relative to the total amount of protein. Most preferably, the whey protein solution of step 1) may comprise at least 4% w / w of other whey proteins relative to the total amount of protein.

[0532] In other preferred embodiments of the present invention, the whey protein solution of step 1) contains at least 35% w / w of other whey proteins relative to the total amount of protein. Preferably, the whey protein solution of step 1) may contain at least 40% w / w of other whey proteins relative to the total amount of protein. More preferably, for example, the whey protein solution of step 1) may contain at least 45% w / w of other whey proteins relative to the total amount of protein. Even more preferably, the whey protein solution of step 1) may contain at least 50% w / w of other whey proteins relative to the total amount of protein.

[0533] In some preferred embodiments of the present invention, the whey protein solution of step 1) contains 5 to 90% w / w of other whey proteins relative to the total amount of protein. Preferably, the whey protein solution of step 1) may contain 10 to 80% w / w of other whey proteins relative to the total amount of protein. For example, the whey protein solution of step 1) may contain whey protein containing 20 to 70% w / w of other whey proteins relative to the total amount of protein. Preferably, the whey protein solution of step 1) contains 30 to 70% w / w of other whey proteins relative to the total amount of protein.

[0534] As described above, the inventors have discovered that non-aggregated BLG can be crystallized without the use of an organic solvent. This purification method can also be used to refine preparations containing whey proteins that have undergone some BLG purification, which provides a simple method for further improving the purity of non-aggregated BLG. Therefore, in some preferred embodiments of the present invention, the whey protein solution of step 1) contains 1 to 20% w / w of other whey proteins relative to the total amount of protein. Preferably, the whey protein solution of step 1) may contain 2 to 15% w / w of other whey proteins relative to the total amount of protein. Even more preferably, for example, the whey protein solution of step 1) may contain 3 to 10% w / w of other whey proteins relative to the total amount of protein.

[0535] In some embodiments of the present invention, the whey protein solution of step 1) comprises at least 5% w / w of non-aggregated ALA relative to the total amount of protein. Preferably, the whey protein solution of step 1) comprises at least 10% w / w of non-aggregated ALA relative to the total amount of protein. Even more preferably, the whey protein solution of step 1) comprises at least 15% w / w of non-aggregated ALA relative to the total amount of protein. Alternatively, the whey protein solution of step 1) may comprise at least 20% w / w of non-aggregated ALA relative to the total amount of protein.

[0536] In some preferred embodiments of the present invention, the whey protein solution of step 1) contains at least 25% w / w of non-aggregated ALA relative to the total amount of protein. Preferably, the whey protein solution of step 1) contains at least 30% w / w of non-aggregated ALA relative to the total amount of protein. The whey protein solution of step 1) preferably contains at least 35% w / w of non-aggregated ALA relative to the total amount of protein. Even more preferably, the whey protein solution of step 1) may contain at least 40% w / w of non-aggregated ALA relative to the total amount of protein.

[0537] In some preferred embodiments of the present invention, the whey protein solution of step 1) contains 5-95% w / w non-aggregated ALA relative to the total amount of protein. Preferably, the whey protein solution of step 1) contains 5-70% w / w non-aggregated ALA relative to the total amount of protein. Even more preferably, the whey protein solution of step 1) may contain 10-60% w / w non-aggregated ALA relative to the total amount of protein. The whey protein solution of step 1) preferably contains 12-50% w / w non-aggregated ALA relative to the total amount of protein. Even more preferably, the whey protein solution of step 1) may contain 20-45% w / w non-aggregated ALA relative to the total amount of protein.

[0538] In some preferred embodiments of the present invention, the weight ratio between non-aggregated BLG and non-aggregated ALA in the whey protein solution of step 1) is at least 0.01. Preferably, the weight ratio between non-aggregated BLG and non-aggregated ALA in the whey protein solution of step 1) is at least 0.5. Even more preferably, the weight ratio between non-aggregated BLG and non-aggregated ALA in the whey protein solution of step 1) is at least 1, such as at least 2. For example, the weight ratio between non-aggregated BLG and non-aggregated ALA in the whey protein solution of step 1) can be at least 3.

[0539] In some preferred embodiments of the present invention, the weight ratio between non-aggregated BLG and non-aggregated ALA in the whey protein solution of step 1) is 0.01 to 20. Preferably, the weight ratio between non-aggregated BLG and non-aggregated ALA in the whey protein solution of step 1) is 0.2 to 10. Even more preferably, the weight ratio between non-aggregated BLG and non-aggregated ALA in the whey protein solution of step 1) is 0.5 to 4. For example, the weight ratio between non-aggregated BLG and non-aggregated ALA in the whey protein solution of step 1) may be 1 to 3.

[0540] In some preferred embodiments of the present invention, the whey protein solution of step 1) contains at least 1% w / w of non-aggregated BLG relative to the total amount of protein. Preferably, the whey protein solution of step 1) contains at least 2% w / w of non-aggregated BLG relative to the total amount of protein. Even more preferably, the whey protein solution of step 1) contains at least 5% w / w of non-aggregated BLG relative to the total amount of protein. Preferably, the whey protein solution of step 1) may contain at least 10% w / w of non-aggregated BLG relative to the total amount of protein.

[0541] In some preferred embodiments of the present invention, the whey protein solution of step 1) contains at least 12% w / w of non-aggregated BLG relative to the total amount of protein. For example, the whey protein solution of step 1) may contain at least 15% w / w of non-aggregated BLG relative to the total amount of protein. For example, the whey protein solution of step 1) may contain at least 20% w / w of non-aggregated BLG relative to the total amount of protein. Alternatively, the whey protein solution of step 1) may contain at least 30% w / w of non-aggregated BLG relative to the total amount of protein.

[0542] In some particularly preferred embodiments of the present invention, the whey protein solution of step 1) contains up to 95% w / w of non-aggregated BLG relative to the total amount of protein. Preferably, the whey protein solution of step 1) may contain up to 90% w / w of non-aggregated BLG relative to the total amount of protein. More preferably, for example, the whey protein solution of step 1) may contain up to 85% w / w of non-aggregated BLG relative to the total amount of protein. Even more preferably, for example, the whey protein solution of step 1) may contain up to 80% w / w of non-aggregated BLG. Preferably, the whey protein solution of step 1) may contain up to 78% w / w of non-aggregated BLG relative to the total amount of protein. Preferably, the whey protein solution of step 1) may contain up to 75% w / w of non-aggregated BLG relative to the total amount of protein.

[0543] In some preferred embodiments of the present invention, the whey protein solution of step 1) contains 1 to 95% w / w of non-aggregated BLG relative to the total amount of protein. Preferably, the whey protein solution of step 1) may contain 5 to 90% w / w of non-aggregated BLG relative to the total amount of protein. More preferably, the whey protein solution of step 1) contains 10 to 85% w / w of non-aggregated BLG relative to the total amount of protein. Even more preferably, the whey protein solution of step 1) contains 10 to 80% w / w of non-aggregated BLG relative to the total amount of protein. Most preferably, the whey protein solution of step 1) may contain 20 to 70% w / w of non-aggregated BLG relative to the total amount of protein.

[0544] In other preferred embodiments of the present invention, the whey protein solution of step 1) contains 10-95% w / w non-aggregated BLG relative to the total amount of protein. Preferably, the whey protein solution of step 1) may contain 12-90% w / w non-aggregated BLG relative to the total amount of protein. More preferably, the whey protein solution of step 1) contains 15-85% w / w non-aggregated BLG relative to the total amount of protein. Even more preferably, the whey protein solution of step 1) contains 15-80% w / w non-aggregated BLG relative to the total amount of protein. Most preferably, the whey protein solution of step 1) may contain 30-70% w / w non-aggregated BLG relative to the total amount of protein.

[0545] In some preferred embodiments of the present invention, the whey protein solution of step 1) comprises at least 0.4% w / w non-aggregated BLG relative to the weight of the whey protein solution. Preferably, the whey protein solution comprises at least 1.0% w / w non-aggregated BLG. More preferably, the whey protein solution comprises at least 2.0% w / w non-aggregated BLG. Even more preferably, the whey protein solution comprises at least 4% w / w non-aggregated BLG.

[0546] Higher concentrations of non-aggregated BLG are even more preferred. Preferably, the whey protein solution comprises at least 6% w / w of non-aggregated BLG. More preferably, the whey protein solution comprises at least 10% w / w of non-aggregated BLG. Even more preferably, the whey protein solution comprises at least 15% w / w of non-aggregated BLG.

[0547] In some preferred embodiments of the present invention, the whey protein solution of step 1) contains 0.4-45% w / w non-aggregated BLG relative to the weight of the whey protein solution. Preferably, the whey protein solution contains 1-35% w / w non-aggregated BLG. More preferably, the whey protein solution contains 4-30% w / w non-aggregated BLG. Even more preferably, the whey protein solution contains 10-25% w / w non-aggregated BLG.

[0548] Higher contents of BLG are particularly preferred; therefore, in some preferred embodiments of the present invention, the whey protein solution of step 1) comprises 10-45% w / w non-aggregated BLG relative to the weight of the whey protein solution. Preferably, the whey protein solution preferably comprises 15-40% w / w non-aggregated BLG. More preferably, the whey protein solution comprises 20-39% w / w non-aggregated BLG. Even more preferably, the whey protein solution comprises 25-38% w / w non-aggregated BLG.

[0549] It is generally preferred that a substantial amount of the BLG in the whey protein solution is non-aggregated BLG. Preferably, at least 50% of the BLG is non-aggregated BLG. More preferably, at least 80% of the BLG is non-aggregated BLG. Even more preferably, at least 90% of the BLG is non-aggregated BLG. Most preferably, at least 95% of the BLG is non-aggregated BLG. Even more preferably, about 100% of the BLG in the whey protein solution is non-aggregated BLG.

[0550] Any suitable whey protein source can be used to prepare the whey protein solution.In some preferred embodiments of the present invention, the whey protein solution comprises whey protein concentrate, whey protein concentrate, whey protein isolate, whey protein isolate or a combination thereof, or even consists thereof.

[0551] Preferably, the whey protein solution is a demineralised whey protein solution.

[0552] In the present context, the term "demineralized" means that the conductivity of the whey protein solution is at most 15 mS / cm, preferably at most 10 mS / cm, even more preferably at most 8 mS / cm. The UF permeate conductivity of the demineralized whey protein solution is preferably at most 7 mS / cm, more preferably at most 4 mS / cm, even more preferably at most 1 mS / cm.

[0553] Particularly preferably, the whey protein solution is a demineralized whey protein concentrate, a demineralized whey protein isolate, a demineralized whey protein concentrate or a demineralized whey protein isolate.

[0554] In some particularly preferred embodiments of the present invention, the whey protein solution comprises or even consists of the following components: demineralized and pH adjusted whey protein concentrate, whey protein concentrate, whey protein isolate, whey protein isolate or a combination thereof.

[0555] For example, the whey protein solution may comprise, or even consist of, a demineralized whey protein concentrate. Alternatively, the whey protein solution may comprise, or even consist of, a demineralized whey protein concentrate. Alternatively, the whey protein solution may comprise, or even consist of, a demineralized whey protein isolate. Alternatively, the whey protein solution may comprise, or even consist of, a demineralized whey protein isolate.

[0556] The BLG-enriched composition is preferably prepared from the milk of a mammal, preferably from the milk of a ruminant, such as a cow, sheep, goat, buffalo, camel, llama, mare and / or deer. Proteins derived from bovine milk are particularly preferred.

[0557] Preferably, the protein in the whey protein solution is as close to its native state as possible and preferably has been subjected to only mild heat treatment, if any.

[0558] In some preferred embodiments of the invention, the whey protein solution has a furosine value of at most 80 mg / 100 g protein. Preferably, the whey protein solution has a furosine value of at most 40 mg / 100 g protein. More preferably, the whey protein solution has a furosine value of at most 20 mg / 100 g protein. Even more preferably, the whey protein solution has a furosine value of at most 10 mg / 100 g protein. Most preferably, the whey protein solution has a furosine value of at most 5 mg / 100 g protein, for example, the furosine value is preferably 0 mg / 100 g protein.

[0559] Typically, in addition to protein, a whey protein solution also comprises other ingredients. A whey protein solution may comprise other ingredients commonly found in whey or milk whey, such as minerals, carbohydrates and / or lipids. Alternatively or additionally, a whey protein solution may comprise ingredients that are non-natural to whey or milk whey. However, preferably, such non-natural ingredients should be safe for use in food production, preferably also safe for human consumption.

[0560] The process of the present invention is particularly advantageous for separating BLG from a crude whey protein solution which contains other solids besides BLG.

[0561] For example, the whey protein solution may contain carbohydrates, such as lactose, oligosaccharides and / or hydrolysates of lactose (i.e. glucose and galactose). For example, the whey protein solution may contain 0-40% w / w (e.g. 1-30% w / w or 2-20% w / w) carbohydrates.

[0562] In some preferred embodiments of the invention, the whey protein solution comprises at most 20% w / w carbohydrates, preferably at most 10% w / w carbohydrates, more preferably at most 5% w / w carbohydrates, even more preferably at most 2% w / w carbohydrates.

[0563] The whey protein solution may further comprise lipids, for example in the form of triglycerides and / or other lipid types (eg phospholipids).

[0564] In some embodiments of the invention, the whey protein solution of step 1) comprises a total amount of lipids of at most 15% w / w relative to the total amount of solids. Preferably, the whey protein solution of step 1) comprises a total amount of lipids of at most 10% w / w relative to the total amount of solids. More preferably, the whey protein solution of step 1) comprises a total amount of lipids of at most 6% w / w relative to the total amount of solids. Even more preferably, the whey protein solution of step 1) comprises a total amount of lipids of at most 1.0% w / w relative to the total amount of solids. Most preferably, the whey protein solution of step 1) comprises a total amount of lipids of at most 0.5% w / w relative to the total amount of solids.

[0565] Typically, the total amount of protein in the whey protein solution is at least 1% w / w relative to the weight of the whey protein solution. Preferably, the total amount of protein in the whey protein solution is at least 5% w / w. More preferably, the total amount of protein in the whey protein solution is at least 10% w / w. Even more preferably, the total amount of protein in the whey protein solution is at least 15% w / w.

[0566] In some preferred embodiments of the present invention, the total amount of protein in the whey protein solution is 1-50% w / w. Preferably, the total amount of protein in the whey protein solution is 5-40% w / w. More preferably, the total amount of protein in the whey protein solution is 10-30% w / w. Even more preferably, the total amount of protein in the whey protein solution is 15-25% w / w.

[0567] The total amount of protein in the whey protein solution was determined according to Example 1.5.

[0568] Typically, the whey protein solution is prepared by subjecting the whey protein raw material to one or more adjustments that result in a whey protein solution that is supersaturated with BLG.

[0569] Preferably, the whey protein raw material is WPC, WPI, SPC, SPI or a combination thereof.

[0570] The term "whey protein raw material" relates to a composition from which a BLG-enriched composition and a subsequent liquid BLG isolate is obtained. For example, a whey protein raw material can be converted into a whey protein solution supersaturated with BLG. Typically, a whey protein raw material is an aqueous solution comprising BLG and at least one other whey protein, but typically unsaturated with BLG.

[0571] The embodiments regarding the chemical composition of the whey protein solution are equally applicable to the whey protein raw material. However, typically, at least one parameter of the whey protein raw material is set to avoid supersaturation or at least spontaneous crystallization.

[0572] In some preferred embodiments of the present invention, the supersaturated whey protein solution is prepared by performing one or more of the following adjustments on the whey protein raw material:

[0573] - Adjust pH value;

[0574] - Reduce electrical conductivity;

[0575] - Reduce the temperature;

[0576] - Increase protein concentration;

[0577] - adding an agent that reduces water activity; and

[0578] -Change the ionic composition.

[0579] In some preferred embodiments of the present invention, the preparation of the whey protein solution involves adjusting the pH of the whey protein raw material to 5-6.

[0580] All pH values ​​were measured using a pH glass electrode and normalized to 25° C. Typically, they are normalized to 25° C. by a pH meter. Alternatively, the temperature of the sample is adjusted to 25° C.

[0581] For example, the pH of the whey protein solution may be 4.9 to 6.1. For example, the pH of the whey protein solution may be 5.0 to 6.1. Alternatively, the pH of the whey protein solution may be 5.1 to 6.1. Preferably, the pH of the whey protein solution is 5.1 to 6.0.

[0582] In some preferred embodiments of the present invention, the pH of the whey protein solution is 5.0 to 6.0. Preferably, the pH of the whey protein solution is 5.1 to 6.0. More preferably, the pH of the whey protein solution is 5.1 to 5.9. Even more preferably, the pH of the whey protein solution may be 5.2 to 5.9. Most preferably, the pH of the whey protein solution is 5.2 to 5.8.

[0583] Preferably, the pH is adjusted using a food acceptable acid and / or base. Food acceptable acids are particularly preferred, such as carboxylic acids. For example, useful examples of such acids are 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.

[0584] In some preferred embodiments of the present invention, a lactone (e.g., D-glucono-δ-lactone) is used to adjust the pH, which slowly hydrolyzes and simultaneously lowers the pH of the aqueous solution containing it. The target pH after the lactone hydrolysis is completed can be accurately calculated.

[0585] For example, useful examples of food-acceptable bases are hydroxide sources, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, salts of food acids (eg, trisodium citrate), and / or combinations thereof.

[0586] In another preferred embodiment of the present invention, by adding a cation exchange material (in its H + The pH is adjusted by adding the cation exchange material (in its H form). The magnetic bead / large particle cation exchange material is easily removed from the whey protein solution before or even after crystallization. + The use of a 5-hydroxy-1-(4-(2-hydroxy-2-oxo)-1-yl)-2-nitropropene (whey protein) material to adjust the pH is particularly advantageous in the present invention because it lowers the pH without adding negative counterions that significantly affect the conductivity of the whey protein material.

[0587] In some preferred embodiments of the present invention, the preparation of the whey protein solution involves reducing the conductivity of the whey protein raw material.

[0588] Unless otherwise stated, the conductivity values ​​stated herein have been normalized to 25°C.

[0589] It has been found that reducing the conductivity of the whey protein solution makes the BLG crystal yield higher. The minimum achievable conductivity of the whey protein solution depends on the composition of the protein fraction and the lipid fraction (if any). Some protein species, such as casein macropeptide (CMP), contribute more to the conductivity than other protein species. Therefore, preferably, the conductivity of the whey protein raw material is close to the level where the protein and the counter ions of the protein are the main contributors to the conductivity. Generally, the reduction of conductivity involves removing at least some small free ions that are present in the liquid phase and are not tightly bound to the protein.

[0590] It is generally preferred that the conductivity of the whey protein solution is at most 10 mS / cm. In some preferred embodiments of the present invention, the conductivity of the whey protein solution is at most 5 mS / cm. Preferably, the conductivity of the whey protein solution is at most 4 mS / cm.

[0591] Lower conductivity is even more preferred, which can result in a higher BLG crystal yield. Therefore, preferably, the whey protein solution preferably has a conductivity of at most 3 mS / cm. In some preferred embodiments of the present invention, the conductivity of the whey protein solution is at most 1 mS / cm. Preferably, the conductivity of the whey protein solution is at most 0.5 mS / cm.

[0592] Preferably, the electrical conductivity of the whey protein raw material is reduced by dialysis or diafiltration. Diafiltration by ultrafiltration is particularly preferred because it allows washing out salts and small charged molecules while retaining protein. In some preferred embodiments of the present invention, the whey protein raw material is subjected to UF / diafiltration and subsequent concentration using the same UF unit.

[0593] The ratio between the electrical conductivity (expressed in mS / cm) and the total amount of protein in the whey protein solution (expressed in % total protein by weight relative to the total weight of the whey protein solution) may advantageously be kept below a certain threshold in order to promote crystallization of BLG.

[0594] In some preferred embodiments of the present invention, the ratio between the electrical conductivity of the whey protein solution and the total amount of protein is at most 0.3. Preferably, the ratio between the electrical conductivity of the whey protein solution and the total amount of protein is at most 0.25. Preferably, the ratio between the electrical conductivity of the whey protein solution and the total amount of protein is at most 0.20. More preferably, the ratio between the electrical conductivity of the whey protein solution and the total amount of protein is at most 0.18. Even more preferably, the ratio between the electrical conductivity of the whey protein solution and the total amount of protein is at most 0.12. Most preferably, the ratio between the electrical conductivity of the whey protein solution and the total amount of protein is at most 0.10.

[0595] For example, preferably, the ratio between the electrical conductivity of the whey protein solution and the total amount of protein is about 0.07 or even lower.

[0596] In addition, the inventors have found that the whey protein raw material can be advantageously adjusted to provide a whey protein solution having a UF permeate conductivity of at most 10 mS / cm. The UF permeate conductivity is a measure of the conductivity of the small molecule fraction of a liquid. When the term "conductivity" is used as such herein, it refers to the conductivity of the liquid under investigation. When the term "UF permeate conductivity" is used, it refers to the conductivity of the small molecule fraction of a liquid, which is measured according to Example 1.23.

[0597] Preferably, the UF osmotic conductivity of the whey protein solution is at most 7 mS / cm. More preferably, the UF osmotic conductivity of the whey protein solution is at most 5 mS / cm. Even more preferably, the UF osmotic conductivity of the whey protein solution may be at most 3 mS / cm.

[0598] Even lower UF permeate conductivity can be used, and if a high yield of BLG is to be obtained, lower UF permeate conductivity is even particularly preferred. Therefore, preferably, the UF permeate conductivity of the whey protein solution is at most 1.0 mS / cm. More preferably, the UF permeate conductivity of the whey protein solution is at most 0.4 mS / cm. Even more preferably, the UF permeate conductivity of the whey protein solution may be at most 0.1 mS / cm. Most preferably, the UF permeate conductivity of the whey protein solution may be at most 0.04 mS / cm.

[0599] For example, if MilliQ water is used as a diluent during diafiltration (the conductivity of MilliQ water is about 0.06 μS / cm), even lower UF permeate conductivity can be achieved. Thus, the UF permeate conductivity of the whey protein solution may be at most 0.01 mS / cm. Alternatively, the UF permeate conductivity of the whey protein solution may be at most 0.001 mS / cm. Alternatively, the UF permeate conductivity of the whey protein solution may be at most 0.0001 mS / cm.

[0600] In some preferred embodiments of the present invention, the preparation of the whey protein solution involves reducing the temperature of the whey protein raw material.

[0601] For example, preparing the whey protein solution may comprise reducing the temperature of the whey protein material to at least 5° C., preferably at least 10° C., even more preferably at least 15° C. For example, preparing the whey protein solution may comprise reducing the temperature of the whey protein material to at least 20° C.

[0602] For example, the temperature of the whey protein raw material may be reduced to at most 30° C., preferably at most 20° C., even more preferably at most 10° C. The inventors have found that even lower temperatures provide higher supersaturation, therefore, the temperature of the whey protein raw material may be reduced, for example, to at most 5° C., preferably at most 2° C., even more preferably at most 0° C. The temperature may even be below 0° C. However, preferably, the whey protein solution should remain pumpable, for example in the form of an ice slurry.

[0603] In some preferred embodiments of the present invention, the whey protein solution is an ice slurry before starting BLG crystallization.Alternatively or additionally, during the BLG crystallization of step 2), the crystallized whey protein solution may be converted to or maintained as an ice slurry.

[0604] In some particularly preferred embodiments of the present invention, the preparation of the whey protein solution involves increasing the total protein concentration of the whey protein raw material. For example, the whey protein raw material can be subjected to more than one protein concentration step (e.g., ultrafiltration, nanofiltration, reverse osmosis and / or evaporation) to concentrate the whey protein solution.

[0605] Ultrafiltration is particularly preferred because it allows selective concentration of protein, while the concentrations of salts and carbohydrates are hardly affected. As mentioned above, ultrafiltration is preferably used for diafiltration and concentration of whey protein raw materials.

[0606] In some preferred embodiments of the present invention, the concentration of BLG in the whey protein solution is below the level at which spontaneous crystallization of BLG occurs. Therefore, it is generally preferred that the modification of the whey protein raw material is stopped when the whey protein solution is in the metastable region (i.e., the supersaturated region); wherein, when in the supersaturated region, when using seed crystals, BLG crystals may grow, but crystallization will not start spontaneously.

[0607] In some preferred embodiments of the present invention, the preparation of the whey protein solution involves adding one or more water activity reducing agents to the whey protein raw material.

[0608] Useful but non-limiting examples of such water activity reducing agents are polysaccharides and / or polyethylene glycol (PEG).

[0609] In some preferred embodiments of the invention, the preparation of the whey protein solution involves changing the ionic composition of the whey protein raw material, for example by ion exchange, addition of new ionic species, dialysis or diafiltration.

[0610] Typically, the whey protein solution is prepared by combining two or more of the above-mentioned process steps for producing supersaturation.

[0611] In some preferred embodiments of the present invention, the preparation of the whey protein solution involves at least subjecting the whey protein raw material to:

[0612] - concentration, for example using ultrafiltration, nanofiltration or reverse osmosis at a temperature above 10°C; and

[0613] - Subsequently cooled to a temperature below 10°C.

[0614] In other preferred embodiments of the present invention, the preparation of the whey protein solution involves at least subjecting the whey protein raw material to:

[0615] - Concentration at a pH above 6.0; and

[0616] - followed by addition of an acid (e.g. H + The pH can be lowered by using GDL or cation exchange materials in the form of cation exchange materials.

[0617] In other preferred embodiments of the present invention, the preparation of the whey protein solution involves at least subjecting the whey protein raw material to:

[0618] - Reducing the electrical conductivity, for example by diafiltration using a membrane that retains at least the non-aggregated BLG.

[0619] In other preferred embodiments of the present invention, the preparation of the whey protein solution involves performing at least the following steps on the whey protein raw material:

[0620] - Adjust the pH to 5-6;

[0621] - Reducing the conductivity by diafiltration using a membrane that retains at least non-aggregated BLG;

[0622] - concentrating the protein, for example using ultrafiltration, nanofiltration or reverse osmosis at a temperature above 10°C; and

[0623] - Finally, cooling to a temperature below 10°C.

[0624] In addition, the inventors have found that the yield of BLG in the present process can be increased by controlling the molar ratio between the sum of sodium + potassium and the sum of calcium + magnesium. Unexpectedly, higher relative contents of calcium and magnesium appear to increase the yield of non-aggregated BLG, thereby increasing the efficiency of the present process for recovering BLG.

[0625] In some preferred embodiments of the present invention, the molar ratio between Na+K and Ca+Mg of the whey protein solution of step 1) is at most 4. More preferably, the molar ratio between Na+K and Ca+Mg of the whey protein solution of step 1) is at most 2. Even more preferably, the molar ratio between Na+K and Ca+Mg of the whey protein solution of step 1) is at most 1.5, even more preferably at most 1.0. Most preferably, the molar ratio between Na+K and Ca+Mg of the whey protein solution of step 1) is at most 0.5, such as at most 0.2.

[0626] The molar ratio between Na+K and Ca+Mg can be calculated as (m Na +m K ) / (m Ca +m Mg ), where m Na is the molar content of Na, m K is the molar content of K, m Ca is the molar content of element Ca, m Mg is the molar content of element Mg.

[0627] Particularly preferably, the whey protein solution has been supersaturated with BLG by salting out, so that BLG can be crystallized from the whey protein solution by salting out.

[0628] In some embodiments of the invention, in particular if the edible BLG product of the invention should also have a certain degree of protein denaturation, the whey protein solution has a low content of denatured proteins. Preferably, the whey protein solution has a protein denaturation of at most 2%, preferably at most 1.5%, more preferably at most 1.0%, most preferably at most 0.8%.

[0629] Step 2) of the method involves crystallizing at least some of the BLG of the supersaturated whey protein solution.

[0630] Particularly preferably, the crystallization of step 2) is carried out by salting, i.e. in a liquid of low ionic strength and conductivity. This is in contrast to the salting-out mode, in which a large amount of salt is added to the solution to induce crystallization.

[0631] For example, the crystallization of BLG in step 2) may involve one or more of the following:

[0632] - Wait for crystallization to occur;

[0633] - Adding seed crystals;

[0634] - further increase the supersaturation of BLG; and / or

[0635] - Mechanical stimulation.

[0636] In some preferred embodiments of the invention, step 2) involves adding seed crystals to the whey protein solution. The inventors have found that adding seed crystals allows the control of the time and place of BLG crystallization to avoid sudden blockage of processing equipment and unexpected stops during production. For example, it is often necessary to avoid the start of crystallization when concentrating whey protein raw materials.

[0637] It is particularly preferred that during the operation of step 2), the whey protein solution does not come into contact with a UF membrane or a MF membrane unless a ceramic membrane or a high shear system (eg DCF) is used.

[0638] In principle, any seed material that initiates BLG crystallization can be used. However, preferably, hydrated BLG crystals or dried BLG crystals are used for seeding to avoid adding other impurities to the whey protein solution.

[0639] When added to the whey protein solution, the seeds may be in dry form or may form part of a suspension. It is currently preferred to add a suspension containing seeds (e.g. BLG crystals) as it appears to cause crystallization to start more quickly. Preferably, such a suspension comprises seeds having a pH of 5-6 and a conductivity of at most 10 mS / cm.

[0640] It should be noted that the conductivity unit "mS / cm" means milliSiemens / centimeter, and 1.00 mS corresponds to 1000 μS.

[0641] Particularly preferably, the seeding is performed by a suspension of BLG crystals which have not been dried after BLG crystallization. For example, such a suspension can be a portion of BLG crystals and mother liquor obtained from a previous batch of step 2), or a portion of wet BLG crystals obtained from a previous batch of step 3), step 4) or step 5).

[0642] The inventors have observed that the use of wet BLG crystals as seeds during step 2) provides much larger BLG crystals than the use of dry or poorly hydrated BLG crystals, which again makes the separation of BLG from the mother liquor more efficient. In an experiment (where the whey protein raw material, crystallization conditions, mass and particle size of the seed material, cooling curve and separation method were the same), the inventors found that seeding with non-dried BLG crystals increased the particle size of the resulting crystals by 100% (obtained particle size: 100-130 μm) compared to BLG crystals obtained by seeding with rehydrated, dried BLG crystals (obtained particle size: 40-60 μm).

[0643] Alternatively, if the seeds are based on dried BLG crystals, the crystals are preferably resuspended in an aqueous solution (e.g., water) and the dried BLG crystals are rehydrated for at least 30 minutes, preferably at least 1.0 hour, and even more preferably at least 1.5 hours before the resulting BLG crystal suspension is used to initiate crystallization.

[0644] In some embodiments of the invention, at least some of the seeds are located on a solid phase in contact with the whey protein solution.

[0645] Preferably, the seed crystals have a smaller particle size than the desired BLG crystal size. The size of the seed crystals can be changed by removing the largest seed crystals by sieving or other size fractionation separation methods. For example, the particle size can also be reduced by grinding before particle size fractionation.

[0646] In some embodiments of the invention, at least 90% w / w of the seed crystals have a particle size (measured by sieve analysis) of 0.1 to 600 μm. For example, at least 90% w / w of the seed crystals may have a particle size of 1 to 400 μm. Preferably, at least 90% w / w of the seed crystals may have a particle size of 5 to 200 μm. More preferably, at least 90% w / w of the seed crystals may have a particle size of 5 to 100 μm.

[0647] The particle size and dosage of the seed crystals can be adjusted to provide optimal crystallization of BLG.

[0648] In some preferred embodiments of the invention, seeds are added to the whey protein material before achieving BLG supersaturation, but preferably in such a way that at least some seeds are still present when supersaturation is achieved. This can be achieved by adding seeds when the whey protein material is close to supersaturation (e.g. during cooling, concentration and / or pH adjustment), and supersaturation is achieved before the seeds are completely dissolved.

[0649] In some preferred embodiments of the present invention, step 2) involves further increasing the supersaturation of BLG, preferably to a level that immediately (i.e., within 20 minutes at most, preferably within 5 minutes at most) initiates crystallization of BLG. This is also referred to as the nucleation zone, where crystallites spontaneously form and begin the crystallization process.

[0650] For example, supersaturation can be increased by one or more of the following:

[0651] - Further increase the protein concentration of the whey protein solution;

[0652] - further cooling the whey protein solution;

[0653] - Make the pH of whey protein solution closer to the optimal pH for BLG crystallization;

[0654] -Further reduce the conductivity.

[0655] In some preferred embodiments of the present invention, step 2) includes waiting for BLG crystals to form. This may take several hours, typically for a whey protein solution that is only slightly supersaturated with BLG and to which no seeds are added.

[0656] In some preferred embodiments of the present invention, providing the whey protein solution (step 1) and BLG crystallization (step 2) are performed as two separate steps.

[0657] However, in other preferred embodiments of the present invention, step 2) involves additionally adjusting the crystallized whey protein solution to increase the supersaturation of BLG, or at least maintain supersaturation. This additional adjustment can increase the yield of BLG crystals.

[0658] Such additional adjustments may involve one or more of the following:

[0659] - further increasing the protein concentration of the crystallized whey protein solution;

[0660] - cooling the crystallized whey protein solution to a lower temperature;

[0661] - Bringing the crystallized whey protein solution closer to the optimal pH for BLG crystallization; and

[0662] - Further reduce the conductivity of the crystallized whey protein solution.

[0663] In some preferred embodiments of the present invention, the crystallized whey protein solution is kept in a metastable zone during step 2) to avoid spontaneous formation of new crystals.

[0664] In some preferred embodiments of the present invention, at least some of the BLG crystals obtained during step 2) have an orthorhombic space group P 21 21 21.

[0665] Preferably, at least some of the resulting BLG crystals have an orthorhombic space group of P 21 21 21; a unit cell size of The unit cell angle integral angles are α=90°, β=90°, and γ=90°.

[0666] In some preferred embodiments of the present invention, at least some of the resulting BLG crystals have an orthorhombic space group P 2121 21; a unit cell size The unit cell angle integrals are α=90°, β=90°, and γ=90°.

[0667] Even more preferably, at least some of the resulting BLG crystals may have an orthorhombic space group P 21 21 21, a unit cell size The unit cell angle integrals are α=90°, β=90°, and γ=90°.

[0668] Most preferably, at least some of the resulting BLG crystals have an orthorhombic space group of P 21 21 21; a unit cell size of The unit cell angle integrals are α=90°, β=90°, and γ=90°.

[0669] In some particularly preferred embodiments of the present invention, the method comprises step 3): separating at least some BLG crystals from the remaining whey protein solution. This is particularly preferred when purification of BLG is desired.

[0670] For example, step 3) may comprise separating the BLG crystals to a solid content of at least 30% w / w. Preferably, step 3) comprises separating the BLG crystals to a solid content of at least 40% w / w. Even more preferably, step 3) comprises separating the BLG crystals to a solid content of at least 50% w / w.

[0671] The inventors have found that a high solid content is beneficial for the purification of BLG, because the aqueous portion adhering to the separated BLG crystals generally contains impurities that should be avoided. In addition, a high solid content reduces the energy consumption of converting the separated BLG crystals into a dry product (e.g., powder) and increases the yield of BLG obtained from a given capacity drying unit.

[0672] In some preferred embodiments of the present invention, step 3) comprises separating the BLG crystals to a solid content of at least 60% w / w. Preferably, step 3) comprises separating the BLG crystals to a solid content of at least 70% w / w. Even more preferably, step 3) comprises separating the BLG crystals to a solid content of at least 80% w / w.

[0673] In some preferred embodiments of the present invention, the separation in step 3) involves one or more of the following operations:

[0674] - Centrifugation;

[0675] - decantation;

[0676] -filter;

[0677] - sedimentation; and

[0678] - A combination of the above operations.

[0679] These unit operations are well known and easily accomplished by those skilled in the art. For example, separation by filtration includes the use of vacuum filtration, dynamic cross-flow filtration (DCF), filter presses, or filter centrifuges.

[0680] Different pore sizes can be used for filtering based on the desired results. Preferably, the filter allows native whey protein and small aggregates to pass but retains BLG crystals. Preferably, the nominal pore size of the filter is at least 0.1 μm. For example, the nominal pore size of the filter is at least 0.5 μm. Even more preferably, the nominal pore size of the filter is at least 2 μm.

[0681] Filters with larger pore sizes can also be used and are in fact preferred if mainly larger crystals should be separated from a liquid containing BLG crystals. In some embodiments of the invention, the filter has a nominal pore size of at least 5 μm. Preferably, the filter has a nominal pore size of at least 20 μm. Even more preferably, the filter has a nominal pore size of at least 40 μm.

[0682] For example, the pore size of the filter is 0.03-5000 μm (e.g. 0.1-5000 μm). Preferably, the pore size of the filter may be 0.5-1000 μm. Even more preferably, the pore size of the filter may be 5-800 μm, such as 10-500 μm or 50-500 μm.

[0683] In some preferred embodiments of the present invention, the pore size of the filter is 0.03-100 μm. Preferably, the pore size of the filter may be 0.1-50 μm. More preferably, the pore size of the filter may be 4-40 μm. Even more preferably, the pore size of the filter may be 5-30 μm, such as 10-20 μm.

[0684] The advantage of using filters with pore sizes greater than 1 μm is that bacteria and other microorganisms are at least partially removed during separation and optional washing and / or recrystallization. Thus, the process of the invention allows the production of high-purity BLG with a very low bacterial load and avoids thermal damage to the protein.

[0685] Another advantage of using filters with pore sizes greater than 1 μm is that the removal of water and subsequent drying becomes easier and less energy intensive.

[0686] During the preparation of the whey protein solution, the remaining whey protein solution from which the BLG crystals are separated can be recycled into the whey protein raw material.

[0687] In some preferred embodiments of the present invention, step 3) uses a filter centrifuge. In other preferred embodiments of the present invention, step 3) uses a decanter centrifuge. Preliminary results show that the use of a filter centrifuge and / or a decanter centrifuge to separate BLG crystals from the mother liquor provides a more robust operating method than, for example, vacuum filtration.

[0688] It is generally preferred to dry the formed filter cake with a drying gas to reduce the moisture content of the filter cake, preferably so that the filter cake can be stripped from the filter. If the filter cake is converted directly into a dry, edible BLG composition, the use of a drying gas may constitute part of the separation step, or may be part of the final drying step.

[0689] In some preferred embodiments of the present invention, step 3) uses a dynamic cross-flow filtration (DCF) unit.

[0690] Initial testing has shown that BLG crystals can be effectively separated using a DCF unit with a membrane pore size of 0.03-5 μm (preferably 0.3-1.0 μm), and the inventors have observed that the DCF unit can be operated for a duration sufficient to separate the crystals from even bulk whey protein solutions containing BLG crystals.

[0691] In some preferred embodiments of the present invention, a DCF unit equipped with a membrane capable of retaining BLG crystals is used to carry out step 3), the DCF permeate is recycled to form part of the whey protein solution or whey protein raw material, and the DCF retentate can be recovered or returned to the crystallization tank. Preferably, the DCF permeate is treated, for example, by ultrafiltration / diafiltration to supersaturate the BLG and then mixed with the whey protein solution or whey protein raw material.

[0692] Advantageously, these embodiments do not require raising the temperature of the liquid fluid to above 15°C and are therefore less susceptible to microbial contamination than process variants requiring higher temperatures. Another industrial advantage of these embodiments is that the supersaturation is easily controllable and can be maintained at a level where unwanted spontaneous crystallization does not occur. Therefore, during these process embodiments, the temperature of the liquid fluid is preferably at most 15°C, more preferably at most 12°C, even more preferably at most 10°C, and most preferably at most 5°C.

[0693] These embodiments are illustrated in Example 10 of PCT Application No. PCT / EP2017 / 084553 and are shown in Figure 26. These embodiments can be implemented in a batch process or a continuous process.

[0694] In some preferred embodiments of the present invention, the method comprises a step 4) of washing the BLG crystals, for example, washing the separated BLG crystals of step 3). Washing may comprise a single washing or multiple washing steps.

[0695] Preferably, the washing in step 4) comprises contacting the BLG crystals with a washing liquid without completely dissolving the BLG crystals, and then separating the remaining BLG crystals from the washing liquid.

[0696] Preferably, the wash liquid is selected to avoid complete dissolution of the BLG crystals, for example, the wash liquid may comprise or even consist essentially of cold deionized water, cold tap water, or cold reverse osmosis permeate.

[0697] For example, the wash liquor may comprise, or even consist essentially of, cold demineralized water, cold tap water, or cold reverse osmosis permeate.

[0698] The pH of the washing liquid may be 5 to 6, preferably 5.0 to 6.0, even more preferably 5.1 to 6.0, such as 5.1 to 5.9.

[0699] Alternatively, the pH of the wash liquor may be 6.1 to 8, preferably 6.4 to 7.6, even more preferably 6.6 to 7.4, for example 6.8 to 7.2. This is typically the pH when the wash liquor is deionized water, tap water or the permeate of reverse osmosis. It is generally preferred that the wash liquor has a low mineral content and a low buffering capacity.

[0700] The conductivity of the cleaning liquid is at most 0.1 mS / cm, preferably at most 0.02 mS / cm, even more preferably at most 0.005 mS / cm.

[0701] A washing liquid with a lower conductivity may be used. For example, the washing liquid may have a conductivity of at most 1 μS / cm. Alternatively, the washing liquid may have a conductivity of at most 0.1 μS / cm, such as about 0.05 μS / cm.

[0702] Preferably, the washing step is performed at low temperature to limit dissolution of crystallized BLG. The temperature of the washing liquid is preferably at most 30°C, more preferably at most 20°C, even more preferably at most 10°C.

[0703] For example, the washing step may be performed at up to 5° C., more preferably up to 2° C., such as about 0° C. Temperatures below 0° C. may be used as long as the wash liquid does not freeze at that temperature, for example due to the presence of one or more freezing point depressants.

[0704] In some embodiments of the invention, the wash liquor comprises BLG, for example, in an amount of at least 1% w / w (preferably at least 3% w / w, such as 4% w / w).

[0705] Typically, the washing of step 4) dissolves at most 80% w / w (preferably at most 50% w / w, even more preferably at most 20% w / w) of the initial amount of BLG crystals. Preferably, the washing of step 4) dissolves at most 15% w / w (preferably at most 10% w / w, even more preferably at most 5% w / w) of the initial amount of BLG crystals.

[0706] Typically, the weight ratio between the total amount of wash liquid and the initial amount of separated BLG crystals is at least 1, preferably at least 2, and more preferably at least 5. For example, the weight ratio between the total amount of wash liquid and the initial amount of separated BLG crystals is at least 10. Alternatively, the weight ratio between the total amount of wash liquid and the initial amount of separated BLG crystals is at least 20, such as at least 50 or at least 100.

[0707] The term "total amount of washing liquid" refers to the total amount of washing liquid used during the entire period.

[0708] In some preferred embodiments of the present invention, one or more washing steps are performed in the same filter arrangement as the BLG crystal separation or in a similar filter arrangement. The washing liquid of one or more steps is added to the filter cake containing mainly BLG crystals, which is removed by the filter, while the remaining part of the BLG crystals remains in the filter cake.

[0709] In a particularly preferred embodiment of the present invention, the separation of step 3) is performed using a filter that retains BLG crystals. Subsequently, the filter cake is contacted with one or more amounts of washing liquid that passes through the filter cake and the filter. It is generally preferred that each amount of washing liquid is at most 10 times the volume of the filter cake, preferably at most 5 times the volume of the filter cake, more preferably at most 1 times the volume of the filter cake, even more preferably at most 0.5 times the volume of the filter cake, for example at most 0.2 times the volume of the filter cake. The volume of the filter cake includes the solids and fluids (liquids and gases) of the filter cake. Preferably, the filter cake is washed in this way at least 2 times, preferably at least 4 times, even more preferably at least 6 times.

[0710] For example, the spent washing liquid from step 4) can be recycled into the whey protein raw material or the whey protein solution, from which the washed BLG can be separated again.

[0711] Furthermore, the method may comprise step 5), which involves a recrystallization step comprising the following steps:

[0712] - dissolving the separated BLG crystals in a recrystallization liquid;

[0713] - Adjust the recrystallization liquid to make BLG supersaturated;

[0714] - crystallizing BLG in a supersaturated, conditioned recrystallization liquid; and

[0715] - Separation of BLG crystals from the remaining conditioned recrystallization liquid.

[0716] Step 5) may comprise a single recrystallization step or multiple recrystallization steps.

[0717] In some embodiments of the present invention, the BLG crystals of step 3) or step 4) are recrystallized at least 2 times. For example, the BLG crystals may be recrystallized at least 3 times, for example at least 4 times.

[0718] The washing and recrystallization steps can be combined in any order and performed multiple times if desired.

[0719] For example, the separated BLG crystals of step 3) may be subjected to the following method steps:

[0720] - one or more washing steps (step 4); then

[0721] - One or more recrystallization steps (step 5).

[0722] Alternatively, the separated BLG crystals of step 3) may be subjected to the method steps:

[0723] - one or more recrystallization steps (step 5); then

[0724] - One more washing step (step 4).

[0725] It is also possible to combine a plurality of washing steps and recrystallization steps, for example in the following order:

[0726] - one or more washing steps (step 4);

[0727] - one or more recrystallization steps (step 5);

[0728] - one or more washing steps (step 4); and

[0729] - One or more recrystallization steps (step 5).

[0730] Or, for example, in the following sequence:

[0731] - one or more recrystallization steps (step 5);

[0732] - one or more washing steps (step 4);

[0733] - one or more recrystallization steps (step 5); and

[0734] - One more washing step (step 4).

[0735] The present inventors have noted that the crystallization process (including the preparation of whey protein solutions) is prone to microbial growth and have found that it would be beneficial to improve the process to address this problem.

[0736] Particularly preferably, the total time that the BLG molecules are exposed to a temperature above 12°C from providing the whey protein raw material to separating the BLG molecules in step c) is at most 24 hours, preferably 20 hours, more preferably at most 12 hours, even more preferably at most 6 hours, most preferably at most 3 hours.

[0737] It is possible and often preferred to further reduce the duration; thus, in some preferred embodiments of the present invention, the total time that the BLG molecules are at a temperature above 12°C, from providing the whey protein raw material to separating the BLG molecules in step c), is at most 2 hours, preferably 1 hour, more preferably at most 0.5, even more preferably at most 0.3 hours, most preferably at most 0.1 hours.

[0738] In some embodiments of the invention, the method further involves subjecting the separated BLG to an additional BLG enrichment step, for example, based on chromatography or selective filtration. However, in other preferred embodiments of the invention, the method does not include an additional BLG enrichment step after step 2). The term "additional BLG enrichment step" refers to a method step of enriching BLG relative to the total amount of protein, which step is unrelated to BLG crystallization or processing BLG crystals. An example of such an additional BLG enrichment step is ion exchange chromatography. Washing BLG crystals and / or recrystallizing BLG are not considered "additional BLG enrichment steps".

[0739] In some preferred embodiments of the present invention, the method comprises a drying step, wherein the BLG-enriched composition obtained from step 3), 4) or 5) is converted into a dried composition.

[0740] In a particularly preferred embodiment of the present invention, the method for preparing a BLG-enriched composition comprises the following steps:

[0741] 1) Providing a whey protein solution, the whey protein solution comprising BLG and at least one other whey protein, the whey protein solution is supersaturated with BLG, the pH of the whey protein solution is 5-6, and the whey protein solution comprises:

[0742] - 70-100% w / w protein relative to the total solids;

[0743] - 30-90% w / w, preferably 30-70% non-aggregated BLG relative to the total amount of protein;

[0744] - 4-50% w / w, preferably 8-35% non-aggregated ALA relative to the total amount of protein;

[0745] - 0-25% w / w CMP relative to the total amount of protein;

[0746] - at least 10% w / w protein relative to the total weight of the whey protein solution;

[0747] 2) crystallizing BLG in a supersaturated whey protein solution, preferably by adding seed crystals;

[0748] 3) separating BLG crystals from the remaining whey protein solution;

[0749] 4) optionally, washing the separated BLG crystals obtained from step 3); and

[0750] 5) Optionally, recrystallize the BLG crystals obtained from step 3) or 4).

[0751] Preferably, the whey protein solution is a demineralized whey protein solution, preferably the ratio between the electrical conductivity of the whey protein solution and the total amount of protein is at most 0.3 and / or the UF permeate conductivity is at most 7 mS / cm.

[0752] These embodiments are particularly useful for preparing low mineral and low phosphorus BLG isolates.

[0753] In some preferred embodiments, the method is implemented as a batch process. Alternatively and sometimes preferably, the process can be implemented as a semi-batch process. In other preferred embodiments, the process is implemented as a continuous process.

[0754] An advantage of the present method is that it is much faster than comparable methods of BLG crystallization in the prior art. The duration from initial conditioning of the whey protein raw material to completion of the separation of step 3) may be at most 10 hours, preferably at most 4 hours, more preferably at most 2 hours, even more preferably at most 1 hour.

[0755] Generally, it is preferred to prepare the BLG-enriched composition using mild temperatures that do not compromise the nutritional value of the non-aggregated BLG or other whey proteins of the whey protein material.

[0756] In some preferred embodiments of the invention, non-aggregated BLG is not subjected to temperatures above 90° C. during the process. Preferably, BLG is not subjected to temperatures above 80° C. during the process. Even more preferably, BLG is not subjected to temperatures above 75° C. during the process. It should be noted that even though spray drying typically employs temperatures in excess of 150° C., the short exposure time and simultaneous evaporation of water means that the temperature of the spray-dried protein will not exceed 50-70° C.

[0757] Whichever method is used to prepare the BLG-enriched composition, it may comprise the step of drying the BLG-enriched composition. However, it is currently preferred to use the BLG-enriched composition without drying it to avoid the risk of damaging the protein during drying.

[0758] If the BLG-enriched composition isolated from the whey protein raw material does not yet possess the desired properties for use as a liquid BLG isolate, it may be subjected to one or more steps selected from the group consisting of:

[0759] - Demineralization;

[0760] - Added minerals;

[0761] -dilution;

[0762] -concentrate;

[0763] - Physical reduction of microorganisms; and

[0764] -pH adjustment.

[0765] For instance, non-limiting examples of demineralization include dialysis, gel filtration, UF / diafiltration, NF / diafiltration, and ion exchange chromatography.

[0766] Non-limiting examples of added minerals include adding soluble, edible salts, such as salts of Na, K, Ca and / or Mg. For example, such salts can be phosphates, chlorides, or salts of edible acids, such as citrates or lactates. Minerals can be added in solid, suspension, or dissolved form.

[0767] For example, non-limiting examples of dilution include adding a liquid diluent, such as water, demineralized water, or an aqueous solution of a mineral, acid, or base.

[0768] For example, non-limiting examples of concentration include evaporation, reverse osmosis, nanofiltration, ultrafiltration, and combinations thereof.

[0769] If concentration is necessary to increase the concentration of protein relative to the total amount of solids, a concentration step such as ultrafiltration or dialysis is preferably used. If concentration is not necessary to increase the concentration of protein relative to the total amount of solids, evaporation, nanofiltration and / or reverse osmosis may be used, for example.

[0770] For example, non-limiting examples of physical microbial reduction include heat treatment, bacterial filtration, ultraviolet radiation, high pressure treatment, pulsed electric field treatment and ultrasound. These methods are well known to those skilled in the art.

[0771] Typically, bacterial filtration involves microfiltration or macroporous ultrafiltration, requiring a pore size that can retain microorganisms but allow proteins and other target components to pass through. Useful pore sizes are typically at most 1.5 μm, preferably at most 1.0 μm, more preferably at most 0.8 μm, even more preferably at most 0.5 μm, and most preferably at most 0.2 μm. The pore size for bacterial filtration is typically at least 0.1 μm.

[0772] For example, bacterial filtration may involve membranes with a pore size of 0.02-1 μm, preferably 0.03-0.8 μm, more preferably 0.04-0.6 μm, even more preferably 0.05-0.4 μm, most preferably 0.1-0.2 μm.

[0773] In some preferred embodiments of the invention, the BLG isolate is bacterially filtered and then heat treated using a temperature of at most 80° C., preferably at most 75° C. Preferably, the combination of temperature and duration of the heat treatment is selected to provide a sterile beverage formulation.

[0774] In other preferred embodiments of the invention, the liquid BLG isolate is bacterially filtered and then heat treated for a duration of at most 0.2 seconds, preferably at most 0.1 seconds, using a temperature of at least 150° C. Preferably, the combination of temperature and duration of this heat treatment is selected to provide a sterile beverage preparation.

[0775] For example, non-limiting examples of pH adjustment include the addition of alkali and / or acid (preferably food acceptable alkali and / or acid). Particularly preferably, an acid and / or base capable of chelating divalent metal cations is used. Examples of such acids and bases are: citric acid, citrate, EDTA, lactic acid, lactate, phosphoric acid, phosphates, and combinations thereof.

[0776] A number of preferred embodiments of step a) for providing a liquid BLG separate from a BLG-enriched composition are described below.The process steps mentioned above and below are applied to a BLG-containing product stream subsequent to the BLG-enriched composition.

[0777] In some preferred embodiments of the present invention, for example, if the BLG-enriched composition comprising BLG crystals from the above-mentioned salting process is useful, the providing of a liquid BLG isolate in step a) comprises subjecting the BLG-enriched composition to the following steps in the following order:

[0778] - For example, adjusting the pH to i) 2 to 4.9 or ii) 6.1 to 8.5 to dissolve the BLG crystals of the BLG-rich composition;

[0779] - optionally, demineralization or mineralization; and

[0780] - Either:

[0781] - concentration to the desired protein content followed by physical reduction of microorganisms; or

[0782] - Physical reduction of microorganisms followed by concentration to desired protein content.

[0783] In other preferred embodiments of the present invention, for example, if a BLG-enriched composition comprising BLG crystals from the above-mentioned salting process is useful, the providing of a liquid BLG isolate in step a) comprises subjecting the BLG-enriched composition to the following steps in the following order:

[0784] - For example, adjusting the pH to i) 2 to 4.9 or ii) 6.1 to 8.5 to dissolve the BLG crystals of the BLG-rich composition;

[0785] - optionally, demineralization or mineralization; and

[0786] -Concentrated to desired protein content.

[0787] In other preferred embodiments of the present invention, for example, if a BLG-enriched composition comprising BLG crystals from the above-mentioned salting process is useful, the providing of a liquid BLG isolate in step a) comprises subjecting the BLG-enriched composition to the following steps in the following order:

[0788] - adding minerals and preferably soluble salts to dissolve the BLG crystals of the BLG-rich composition, preferably while maintaining a pH of 5.0 to 6.0; and

[0789] - Either:

[0790] - concentration to the desired protein content followed by physical reduction of microorganisms; or

[0791] - Physical reduction of microorganisms followed by concentration to desired protein content.

[0792] When treating acidic, dissolved BLG-rich compositions, heat treatment alone or in combination with one or more of the other physical microbial reduction methods described herein as a physical microbial reduction is particularly advantageous. The inventors have found that the use of mild heat treatment under acidic conditions is particularly beneficial because it allows the BLG to maintain its native folded state, but still helps reduce the microbial load of the treated stream.

[0793] Particularly preferably, the acidic, solubilized BLG-enriched composition is subjected to a bacterial filtration step while having a total protein concentration of at most 27% w / w, preferably at most 22% w / w, even more preferably at most 17% w / w, followed by a heat treatment step of the bacterially filtered BLG-enriched composition or the liquid BLG isolate.

[0794] Even more preferably, the acidic, solubilized BLG-enriched composition is subjected to a bacterial filtration step while having a total protein concentration of 5-27% w / w, preferably 10-22% w / w, even more preferably up to 12-17% w / w, followed by a heat treatment of the bacterially filtered BLG-enriched composition or the liquid BLG isolate.

[0795] Preferably, the liquid BLG isolate is subjected to a heat treatment step, preferably as the last step before spray drying.

[0796] It is generally preferred to avoid or at least limit the unfolding of BLG during the provision of the liquid BLG isolate. If heat treatment is used at a pH of 2-4.9, the temperature is preferably kept at at most 82°C, preferably at most 80°C, more preferably at most 78°C, to limit or even avoid the unfolding of BLG.

[0797] Preferably, the heat treatment is at least pasteurization.

[0798] In some preferred embodiments of the present invention, the heat treatment temperature is 70-80°C, preferably 70-79°C, more preferably 71-78°C, even more preferably 72-77°C, most preferably 73-76°C, for example about 75°C.

[0799] Preferably, when the heat treatment is carried out at a temperature of 70 to 80° C., the duration of the heat treatment is from 1 second to 30 minutes. The highest exposure time is most suitable for the lowest temperature range, and vice versa.

[0800] In a particularly preferred embodiment of the present invention, the heat treatment provides a temperature of 70-78°C for 1 second to 30 minutes, more preferably a temperature of 71-77°C for 1 minute to 25 minutes, even more preferably a temperature of 72-76°C for 2 minutes to 20 minutes.

[0801] In some embodiments, higher temperatures may also be preferred, particularly if it is desired to expand and optionally aggregate the BLG prior to drying. For example, the temperature of the heat treatment 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.

[0802] For example, the heat treatment may be a UHT type treatment, which generally involves a temperature of 135 to 144° C. and a duration of 2 to 10 seconds.

[0803] Alternatively, but also preferably, the heat treatment may include a temperature of 145-180° C. and a duration of 0.01-1 second, more preferably a temperature of 150-180° C. and a duration of 0.01-0.2 seconds.

[0804] In some preferred embodiments of the present invention, providing the liquid BLG isolate in step a) comprises: sterilizing by heat treatment while the pH is 2-4.9, preferably 2.5-4.7, more preferably 2.8-4.3, even more preferably 3.2-4.0.

[0805] In other preferred embodiments of the present invention, providing a liquid BLG isolate from a BLG-enriched composition in step a) comprises physically reducing microorganisms by heat treatment while the pH is 2-4.9, preferably 2.5-4.7, more preferably 2.8-4.3, even more preferably 3.2-4.0.

[0806] In other preferred embodiments of the present invention, the step a) of providing a liquid BLG isolate from a BLG-rich composition comprises: demineralization, while the pH is 6.1 to 8.5, preferably 6.3 to 8.0, more preferably 6.5 to 7.5. The inventors have found that such demineralization is beneficial to improve the thermal stability of the BLG isolate powder prepared by the method.

[0807] In a particularly preferred embodiment of the present invention, step a) of providing a liquid BLG isolate from a BLG-enriched composition comprises:

[0808] - physical reduction of microorganisms by heat treatment at a pH of 2 to 4.9, preferably 2.5 to 4.7, more preferably 2.8 to 4.3, even more preferably 3.2 to 4.0; and

[0809] - Demineralization with a pH of 6.1 to 8.5, preferably 6.3 to 8.0, more preferably 6.5 to 7.5.

[0810] In some preferred embodiments of the present invention, for example, if the BLG-enriched composition comprising BLG crystals from the above-mentioned salting process is useful, the providing of a liquid BLG isolate in step a) comprises subjecting the BLG-enriched composition to the following steps in the following order:

[0811] - adjusting the pH value to 2-4.9 to dissolve the BLG crystals of the BLG-rich composition;

[0812] - Optionally, concentrated to a desired protein content while the pH is 2 to 4.9, preferably 2.5 to 4.0, more preferably 3.0 to 3.9;

[0813] - Physical reduction of microorganisms by heat treatment while the pH is between 2 and 4.9, preferably between 2.5 and 4.7, more preferably between 2.8 and 4.3, even more preferably between 3.2 and 4.0.

[0814] If the pH of the BLG-enriched composition is already 2-4.9, then preferably, providing a liquid BLG isolate in step a) comprises subjecting the BLG-enriched composition to the following steps in the following order:

[0815] - demineralization with a pH of 2 to 4.9, preferably 2.5 to 4.7, more preferably 2.8 to 4.3, even more preferably 3.2 to 4.0;

[0816] - Optionally, concentrated to a desired protein content while having a pH of 2-4.9, preferably 2.5-4.7, more preferably 2.8-4.3, even more preferably 3.2-4.0.

[0817] - Physical reduction of microorganisms by heat treatment while the pH is between 2 and 4.9, preferably between 2.5 and 4.7, more preferably between 2.8 and 4.3, even more preferably between 3.2 and 4.0.

[0818] In other preferred embodiments of the present invention, it is particularly suitable for providing a liquid BLG isolate having a pH of 5.0 to 8.5, preferably 6.1 to 8.5, and more preferably 6.5 to 8.0, and the step a) of providing a liquid BLG isolate comprises subjecting the BLG-enriched composition to the following steps in the following order:

[0819] - For example, adjusting the pH to 2 to 4.9 to dissolve the BLG crystals of the BLG-rich composition;

[0820] -optionally, concentrated to a desired protein content;

[0821] - Physical reduction of microorganisms by heat treatment at a pH of 2 to 4.9, preferably 2.5 to 4.7, more preferably 2.8 to 4.3, even more preferably 3.2 to 4.0;

[0822] - The pH is adjusted to 5.0 to 8.5, preferably 6.1 to 8.5, more preferably 6.5 to 8.0.

[0823] In other preferred embodiments of the present invention, it is particularly suitable for providing a liquid BLG isolate having a pH of 6.1 to 8.5, preferably 6.3 to 8.0, and more preferably 6.5 to 7.5, and the step a) of providing a liquid BLG isolate comprises subjecting the BLG-enriched composition to the following steps in the following order:

[0824] - For example, adjusting the pH to 2 to 4.9 to dissolve the BLG crystals of the BLG-rich composition;

[0825] - Optionally, concentrated to a desired protein content while having a pH of 2 to 4.9, preferably 2.5 to 4.7, more preferably 2.8 to 4.3, even more preferably 3.2 to 4.0;

[0826] - Physical reduction of microorganisms by heat treatment at a pH of 2 to 4.9, preferably 2.5 to 4.7, more preferably 2.8 to 4.3, even more preferably 3.2 to 4.0;

[0827] - adjusting the pH to 6.1 to 8.5, preferably 6.3 to 8.0, more preferably 6.5 to 7.5; and

[0828] - Demineralization, pH 6.1 to 8.5, preferably 6.3 to 8.0, more preferably 6.5 to 7.5.

[0829] In other preferred embodiments of the present invention, it is particularly suitable for providing a liquid BLG isolate having a pH of 6.1 to 8.5, preferably 6.1 to 8.5, more preferably 6.5 to 8.0, and the step a) of providing a liquid BLG isolate comprises subjecting the BLG-enriched composition to the following steps in the following order:

[0830] - adjusting the pH to at least 6.1 to dissolve the BLG crystals of the BLG-rich composition;

[0831] - demineralization with a pH of 6.1 to 8.5, preferably 6.5 to 8.0, more preferably 6.5 to 7.5;

[0832] - Optionally, concentrated to a desired protein content while the pH is 6.1 to 8.5, preferably 6.5 to 8.0, more preferably 6.5 to 7.5;

[0833] - Optionally, physical reduction of microorganisms is performed by heat treatment while the pH is between 6.1 and 8.5, preferably between 6.5 and 8.0, more preferably between 6.5 and 7.5.

[0834] Typically, the temperature during conversion of the BLG-rich composition to the liquid BLG isolate is 0-82°C, possibly involving even higher temperatures for thermal treatment. If the pH of the BLG stream during treatment is above 4.9, the temperature range is preferably 0-65°C, more preferably 0-15°C or 50-65°C, to reduce microbial growth.

[0835] If the pH of the liquid BLG isolate is at most 4.9, even more preferably at most 4.1, the temperature is preferably 0-82°C, and advantageously 0-15°C or 50-80°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 (if necessary), at a temperature of 50-80°C, more preferably 60-80°C, even more preferably 65-78°C, because high temperatures increase the efficiency of the concentration step and at the same time help to reduce microorganisms. This embodiment makes it possible to produce a liquid BLG isolate with a high BLG content and a very low microbial content, while maintaining the naturalness of the BLG. The resulting spray-dried powder has a high bulk density, a high level of naturalness and very beneficial microbial properties.

[0836] In some preferred embodiments of the present invention, the method of converting the BLG-enriched composition into a liquid BLG isolate is carried out at a temperature of 0-15°C, preferably 1-10°C.

[0837] In other preferred embodiments of the invention, at least some of the process (preferably the entire process) of converting the BLG-enriched composition into a liquid BLG isolate is carried out at a temperature of 50-82° C., preferably 55-80° C., more preferably 60-78° C. This is particularly preferred when the pH of the protein stream during the treatment is at most 4.9, preferably at most 4.3, more preferably at most 3.7, preferably 3.0-4.3.

[0838] In some preferred embodiments of the present invention, at least some of the processes (preferably the entire process) of converting the BLG-rich composition into a liquid BLG isolate are carried out at a temperature of 78 to 82° C., and the pH is 3.0 to 3.7. In other preferred embodiments of the present invention, at least some of the processes (preferably the entire process) of converting the BLG-rich composition into a liquid BLG isolate are carried out at a temperature of 60 to 78° C., and the pH is 3.7 to 4.3.

[0839] Preferably, the BLG enriched composition has substantially the same protein composition as the liquid BLG isolate, and typically, no additional protein fractions need to be added when converting the BLG enriched composition to the liquid BLG isolate.

[0840] In some preferred embodiments of the present invention, the BLG content of the composition enriched with BLG is at least 85% w / w, preferably at least 88% w / w, more preferably at least 90% w / w, even more preferably at least 92%, most preferably at least 95% w / w, relative to the total amount of protein. Sometimes, it is particularly preferred that the BLG content of the composition enriched with BLG is at least 97% w / w, more preferably at least 99% w / w, such as preferably about 100% w / w, relative to the total amount of protein.

[0841] In some preferred embodiments of the invention, the total amount of protein in the BLG-enriched composition is at least 5% w / w, preferably at least 10% w / w, more preferably at least 15% w / w, even more preferably at least 20% w / w, most preferably at least 30% w / w.

[0842] In some preferred embodiments of the present invention, the total amount of protein in the BLG-enriched composition is 5-45% w / w, preferably 10-40% w / w, more preferably 15-38% w / w, even more preferably 20-35% w / w.

[0843] In some preferred embodiments of the present invention, the sum of α-lactalbumin (ALA) and casein macropeptide (CMP) accounts for at least 40% w / w, preferably at least 60% w / w, even more preferably at least 70% w / w, most preferably at least 90% w / w of the non-BLG proteins of the BLG-enriched composition.

[0844] In other preferred embodiments of the present invention, each major non-BLG whey protein in the BLG-enriched composition, expressed as a weight percentage relative to the total amount of protein, is at most 25%, preferably at most 20%, more preferably at most 15%, even more preferably at most 10%, most preferably at most 6% by weight relative to the total amount of protein in a standard whey protein concentrate from sweet whey.

[0845] Even lower concentrations of major non-BLG whey proteins may be desired. Thus, in other preferred embodiments of the invention, each major non-BLG whey protein in the BLG-enriched composition, expressed as a weight percentage relative to the total amount of protein, is at most 4%, preferably at most 3%, more preferably at most 2%, even more preferably at most 1% by weight relative to the total amount of protein in a standard whey protein concentrate from sweet whey.

[0846] The inventors have found that low levels of lactoferrin and / or lactoperoxidase are particularly advantageous for obtaining a whey protein product that is neutral in colour.

[0847] Therefore, in some preferred embodiments of the present invention, the lactoferrin in the BLG-enriched composition, expressed as a percentage by weight relative to the total amount of protein, is at most 25%, preferably at most 20%, more preferably at most 15%, even more preferably at most 10%, most preferably at most 6%, relative to the total amount of protein of a standard whey protein concentrate from sweet whey. Even lower concentrations of lactoferrin may be desired. Therefore, in other preferred embodiments of the present invention, the lactoferrin, expressed as a percentage by weight relative to the total amount of protein, is at most 4%, preferably at most 3%, more preferably at most 2%, even more preferably at most 1%, relative to the total amount of protein of a standard whey protein concentrate from sweet whey.

[0848] Similarly, in some preferred embodiments of the invention, the lactoperoxidase in the BLG-enriched composition is present in a weight percentage relative to the total amount of protein of at most 25%, preferably at most 20%, more preferably at most 15%, even more preferably at most 10%, most preferably at most 6%, relative to the total amount of protein of a standard whey protein concentrate from sweet whey. Even lower concentrations of lactoperoxidase may be required. Therefore, in other preferred embodiments of the invention, the lactoperoxidase is present in a weight percentage relative to the total amount of protein of at most 4%, preferably at most 3%, more preferably at most 2%, even more preferably at most 1%, relative to the total amount of protein of a standard whey protein concentrate from sweet whey.

[0849] In some preferred embodiments of the present invention, the total solid content of the BLG-enriched composition is 5-50% w / w, preferably 10-45% w / w, more preferably 15-40% w / w, even more preferably 20-35% w / w.

[0850] In some preferred embodiments of the present invention, the water content of the BLG-enriched composition is 50-95% w / w, preferably 55-90% w / w, more preferably 60-85% w / w, even more preferably 65-80% w / w.

[0851] In some preferred embodiments of the present invention, the carbohydrate content of the BLG-enriched composition is at most 60% w / w, preferably at most 50% w / w, more preferably at most 20% w / w, even preferably at most 10% w / w, even more preferably at most 1% w / w, most preferably at most 0.1%.

[0852] In some preferred embodiments of the present invention, the lipid content of the BLG-enriched composition is at most 10% w / w, preferably at most 5% w / w, more preferably at most 2% w / w, even more preferably at most 0.1% w / w.

[0853] In some preferred embodiments of the invention, the BLG-enriched composition is used directly as a liquid BLG isolate, for example, if it already has the desired pH and chemical composition.

[0854] Although some embodiments of the present invention do not require the physical reduction of microorganisms of step b), and thus only require steps a) and c); other preferred embodiments require the physical reduction of microorganisms of step b), and thus comprise all three steps a), b) and c).

[0855] Therefore, in some preferred embodiments of the present invention, the liquid BLG isolate is subjected to physical microbial reduction.

[0856] Useful examples of physical microbial reduction include one or more of heating, bacterial filtration, ultraviolet radiation, high pressure treatment, pulsed electric field treatment, and ultrasound.

[0857] In some preferred embodiments of the invention, physical reduction of microorganisms comprises or even consists of heat treatment.

[0858] Preferably, the heat treatment comprises at least pasteurization.

[0859] In a specific embodiment, the heat treatment comprises heating the liquid BLG isolate to a temperature of 70-82°C.

[0860] In some preferred embodiments of the present invention, the heat treatment temperature is 70-80°C, preferably 70-79°C, more preferably 71-78°C, even more preferably 72-77°C, most preferably 73-76°C, such as about 75°C.

[0861] Preferably, the duration of the heat treatment is from 1 second to 30 minutes when carried out at a temperature of 70 to 80° C. The longest exposure time is most suitable for the lowest temperature, and vice versa.

[0862] In a particularly preferred embodiment of the invention, the heat treatment provides a temperature of 70-78°C for 1 second to 30 minutes, more preferably 71-77°C for 1 minute to 25 minutes, even more preferably 72-76°C for 2 minutes to 20 minutes.

[0863] In some embodiments, higher temperatures may also be preferred, particularly if it is desired to allow BLG to expand and optionally aggregate prior to drying. For example, the temperature of the heat treatment 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.

[0864] For example, the heat treatment may involve a temperature of 90 to 130°C and a duration of 5 seconds to 30 minutes. For example, the heat treatment may involve heating to a temperature of 90 to 95°C for 1 to 30 minutes, such as heating to about 120°C for about 20 seconds. Alternatively, the heat treatment may involve heating to a temperature of 115 to 125°C for 5 to 30 seconds, such as heating to about 120°C for about 20 seconds.

[0865] Alternatively, the heat treatment may be, for example, a UHT type treatment, which generally involves a temperature of 135 to 144° C. and a duration of 2 to 10 seconds.

[0866] Alternatively, but also preferably, the heat treatment may involve a temperature of 145-180°C and a duration of 0.01-2 seconds, more preferably a temperature of 150-180°C and a duration of 0.01-0.3 seconds.

[0867] Thermal treatment may involve the use of conventional equipment, such as plate or tube heat exchangers, scraped surface heat exchangers, or retorting systems. Alternatively, for thermal treatments above 95°C, direct steam-based heating may be particularly preferably employed, for example using direct steam injection, direct steam infusion, or spray cooking. In addition, such direct steam-based heating is preferably used in combination with rapid cooling. Suitable examples of spray cooking may be found in WO2009113858A1, the contents of which are incorporated herein by reference for all purposes. Suitable embodiments of direct steam injection and direct steam infusion may be found in WO2009113858A1 and WO 2010 / 085957A3, the contents of which are incorporated herein by reference for all purposes. For example, general aspects of high temperature treatment may be found in Thermal technologies in food processing ISBN 185573558X, the contents of which are incorporated herein by reference for all purposes.

[0868] In some preferred embodiments of the present invention, the physical reduction of microorganisms in step b) is sterilization to obtain a sterile liquid BLG isolate. For example, such sterilization can be achieved by combining bacterial filtration and pasteurization.

[0869] In some preferred embodiments of the present invention, the liquid BLG isolate, preferably at a pH of 2 to 4.9, is subjected to bacterial filtration and then heat treated using a temperature of up to 80° C., preferably up to 75° C. Preferably, the combination of temperature and duration of this heat treatment is selected to provide a liquid BLG isolate.

[0870] In other preferred embodiments of the invention, the liquid BLG isolate is bacterially filtered and then heat treated using a temperature of at least 150°C for at most 0.2 seconds (preferably at most 0.1 seconds). Preferably, the combination of temperature and duration of this heat treatment is selected to provide a liquid BLG isolate.

[0871] Preferably, step c) of the process involves spray drying or freeze drying. Spray drying is particularly preferred.

[0872] The inventors have found that it is particularly advantageous to avoid exposing the liquid BLG isolate to heat treatment regimes that unfold or denature a significant amount of the BLG. Therefore, if the liquid BLG isolate is preheated prior to spraying, it is preferred that the heat load be carefully controlled.

[0873] In some embodiments of the present invention, when reaching the outlet of the spray device (e.g., a nozzle or an atomizer), the temperature of the liquid BLG isolate is at most 70° C., preferably at most 60° C., more preferably at most 50° C. In some preferred embodiments of the present invention, when reaching the outlet of the spray device, the temperature of the liquid BLG isolate is at most 40° C., preferably at most 30° C., more preferably at most 20° C., even more preferably at most 10° C., most preferably at most 5° C.

[0874] For example, the spray device of a spray dryer is a nozzle or an atomizer, which converts the solution or suspension to be dried into droplets, which enter the drying chamber of the spray dryer.

[0875] In some embodiments of the present invention, it is particularly preferred that the temperature of the liquid BLG separator is 0-60°C, preferably 2-40°C, more preferably 4-35°C, and most preferably 5-10°C when reaching the outlet of the spray device.

[0876] The inlet temperature of the gas of the spray dryer is preferably 140-220° C., more preferably 160-200° C., even more preferably 170-190° C., for example, preferably about 180° C. The outlet temperature of the gas of the spray dryer is preferably 50-95° C., more preferably 70-90° C., even more preferably 80-88° C., for example, preferably about 85° C. According to experience, it is considered that the solid to be spray-dried is heated to a temperature 10-15° C. lower than the gas outlet temperature.

[0877] In some preferred embodiments of the present invention, the outlet temperature of the spray dryer is preferably 50-85°C, more preferably 60-80°C, even more preferably 65-75°C, for example preferably about 70°C.

[0878] An advantage of the present process is that the solids content of the liquid BLG isolate to be dried can be very high prior to the drying step, so that less water needs to be removed and less energy is consumed in the drying operation.

[0879] The inventors have found that the lower the degree of expansion of the BLG, the higher the concentration of BLG that can be processed before spray drying.

[0880] In some preferred embodiments of the present invention, the solid content of the liquid BLG isolate is at least 10% w / w. Preferably, the solid content of the liquid BLG isolate is at least 20% w / w. More preferably, the solid content of the liquid BLG isolate is at least 25% w / w. Even more preferably, the solid content of the liquid BLG isolate is at least 30% w / w. Most preferably, the solid content of the liquid BLG isolate is at least 35% w / w.

[0881] In other preferred embodiments of the present invention, the solid content of the liquid BLG isolate is 10-60% w / w. Preferably, the solid content of the liquid BLG isolate is 15-50% w / w. More preferably, the solid content of the liquid BLG isolate is 20-45% w / w. Even more preferably, the solid content of the liquid BLG isolate is a solid content of 25-40% w / w, such as about 35% w / w.

[0882] In some preferred embodiments of the present invention, the liquid BLG isolate comprises a total amount of protein of at least 10% w / w. Preferably, the liquid BLG isolate comprises a total amount of protein of at least 20% w / w. More preferably, the liquid BLG isolate comprises a total amount of protein of at least 25% w / w. Even more preferably, the liquid BLG isolate comprises a total amount of protein of at least 30% w / w. Most preferably, the liquid BLG isolate comprises a total amount of protein of at least 35% w / w.

[0883] In other preferred embodiments of the present invention, the liquid BLG isolate comprises a total amount of protein of 10-50% w / w. Preferably, the liquid BLG isolate comprises a total amount of protein of 15-45% w / w. More preferably, the liquid BLG isolate comprises a total amount of protein of 20-40% w / w. Even more preferably, the liquid BLG isolate comprises a total amount of protein of 25-38% w / w, such as about 35% w / w.

[0884] The inventors have discovered that the protein content reduces the energy consumption for converting the liquid BLG isolate into a powder and increases the BLG yield obtained from a drying unit of a given capacity.

[0885] Preferably, the process of the present invention is operated at a mild temperature that does not compromise the nutritional value of the non-aggregated BLG or other whey proteins of the whey protein solution.

[0886] In some preferred embodiments of the invention, during the process, non-aggregated BLG is not subjected to temperatures above 90° C. Preferably, during the process, non-aggregated BLG is not subjected to temperatures above 80° C. Even more preferably, during the process, non-aggregated BLG is not subjected to temperatures above 75° C. It should be noted that even though spray drying typically employs temperatures in excess of 150° C., the short exposure time and simultaneous evaporation of water means that the temperature of the spray-dried protein will not exceed 40-70° C.

[0887] The inventors have found that prolonged heating during the drying step reduces the amount of BLG in undenatured form. In some preferred embodiments of the invention, the heat exposure during the drying step is kept low enough so that the degree of denaturation of BLG is at most 5%, preferably at most 4%, more preferably at most 2%, even more preferably at most 0.5%, even more preferably at most 0.1%. Most preferably, the drying step does not result in detectable denaturation of BLG at all.

[0888] Furthermore, the drying step may comprise fluidized bed drying, for example integrated in the spray drying apparatus, or carried out as a separate unit after spray drying.

[0889] The combination of spray drying and fluidized bed drying makes it possible to reduce the amount of water removed when the droplets to be dried move in the spray drying chamber, while the residual water is removed from the wet powder by fluidized bed drying. Compared with drying by spray drying alone, this scheme requires less energy for drying, and also makes it possible to modify the powder by, for example, instantization and / or agglomeration. Preferably, instantization is performed by applying lecithin or another useful wetting agent to the surface of the powder. When instantization is performed, the amount of instantization agent (e.g., lecithin) dissolved in edible oil is typically 0.5-2% w / w, preferably 1.0-1.5% w / w, relative to the total weight of the final powder.

[0890] Preferably, the method further comprises the step of packaging the BLG isolate powder.The packaged BLG isolate powder product comprises a container (preferably a sealed container) filled with the BLG isolate powder described herein.

[0891] In some embodiments of the invention, the BLG isolate powder is hermetically sealed in a container, optionally packaged with an inert gas.

[0892] A variety of different containers can be used to store BLG isolate powder. For example, preferred containers are bags, barrels, sachets, boxes, cans and capsules.

[0893] A particularly preferred embodiment of the present invention relates to a method for producing a dry BLG isolate powder having a BLG content of at least 85% w / w, preferably at least 90% w / w, even more preferably at least 94% w / w relative to the total amount of protein, the method comprising the following steps:

[0894] a) providing a liquid BLG isolate having the following characteristics:

[0895] i) pH 2 to 4.9;

[0896] ii) pH 6.1 to 8.5; or

[0897] iii) pH 5.0 to 6.0;

[0898] The liquid BLG isolate has a BLG content of at least 85% w / w, preferably at least 90% w / w, even more preferably at least 94% w / w relative to the total amount of protein;

[0899] b) optionally, subjecting the liquid BLG isolate to physical microbial reduction;

[0900] c) drying the liquid BLG isolate, preferably spray drying;

[0901] Wherein, providing the liquid BLG isolate in step a) comprises:

[0902] - A BLG-enriched composition is prepared by a method comprising the following steps:

[0903] 1) providing a whey protein solution, wherein the whey protein solution comprises non-aggregated BLG and at least one other whey protein, wherein the whey protein solution is supersaturated with BLG and has a pH of 5 to 6;

[0904] 2) Crystallization of BLG in a supersaturated whey protein solution;

[0905] 3) separating BLG crystals from the remaining whey protein solution;

[0906] 4) optionally, washing the BLG crystals, such as the separated BLG crystals obtained from step 3) or 5); and

[0907] 5) optionally, recrystallizing BLG crystals, such as the BLG crystals obtained from step 3) or 4); and

[0908] - treating the BLG-enriched composition to at least dissolve the BLG crystals, thereby obtaining a liquid BLG isolate.

[0909] In the above embodiments, the BLG-rich composition comprises separated BLG crystals from step 3), which are subsequently dissolved by appropriate pH adjustment or alternatively by increasing conductivity and / or raising temperature.

[0910] A particularly preferred embodiment of the present invention relates to a method for producing a dry BLG isolate powder having a BLG content of at least 85% w / w, preferably at least 90% w / w, even more preferably at least 94% w / w relative to the total amount of protein, the method comprising the following steps:

[0911] a) providing a liquid BLG isolate having a pH of 2.5 to 4.9, preferably 2.5 to 4.0, even more preferably 3.0 to 3.9;

[0912] The liquid BLG isolate has a BLG content of at least 85% w / w, preferably at least 90% w / w, even more preferably at least 94% w / w relative to the total amount of protein;

[0913] b) optionally, subjecting the liquid BLG isolate to physical microbial reduction;

[0914] c) drying the liquid BLG isolate, preferably spray drying;

[0915] Wherein, providing the liquid BLG isolate in step a) comprises:

[0916] - A BLG-enriched composition is prepared by a method comprising the following steps:

[0917] 1) providing a whey protein solution, wherein the whey protein solution comprises non-aggregated BLG and at least one other whey protein, wherein the whey protein solution is supersaturated with BLG and has a pH of 5 to 6;

[0918] 2) Crystallization of BLG in a supersaturated whey protein solution;

[0919] 3) separating BLG crystals from the remaining whey protein solution;

[0920] 4) optionally, washing the BLG crystals, such as the separated BLG crystals obtained from step 3) or 5); and

[0921] 5) optionally, recrystallizing BLG crystals, such as the BLG crystals obtained from step 3) or 4); and

[0922] - At least the pH of the BLG-rich composition is adjusted to 2.5 to 4.9, preferably 2.5 to 4.0, even more preferably 3.0 to 3.9.

[0923] Another particularly preferred embodiment of the present invention relates to a method for producing a dry BLG isolate powder having a BLG content of at least 85% w / w, preferably at least 90% w / w, even more preferably at least 94% w / w relative to the total amount of protein, the method comprising the following steps:

[0924] a) providing a liquid BLG isolate having a pH of 6.1 to 8.5, preferably 6.3 to 8.0, more preferably 6.5 to 7.5;

[0925] The liquid BLG isolate has a BLG content of at least 85% w / w, preferably at least 90% w / w, even more preferably at least 94% w / w relative to the total amount of protein;

[0926] b) optionally, subjecting the liquid BLG isolate to physical microbial reduction;

[0927] c) drying the liquid BLG isolate, preferably spray drying;

[0928] Wherein, providing the liquid BLG isolate in step a) comprises:

[0929] - A BLG-enriched composition is prepared by a method comprising the following steps:

[0930] 1) providing a whey protein solution, wherein the whey protein solution comprises non-aggregated BLG and at least one other whey protein, the BLG of the whey protein solution is supersaturated, and the pH of the whey protein solution is 5-6;

[0931] 2) Crystallization of BLG in a supersaturated whey protein solution;

[0932] 3) separating BLG crystals from the remaining whey protein solution;

[0933] 4) optionally washing the BLG crystals, such as the isolated BLG crystals obtained from step 3) or 5); and

[0934] 5) optionally recrystallizing BLG crystals, such as the BLG crystals obtained from step 3) or 4); and

[0935] - At least the pH of the BLG-rich composition is adjusted to 6.1 to 8.5, preferably 6.3 to 8.0, more preferably 6.5 to 7.5.

[0936] An alternative but also preferred embodiment of the present invention relates to a method for producing a dry BLG isolate powder having a BLG content of at least 85% w / w, preferably at least 90% w / w, even more preferably at least 94% w / w relative to the total amount of protein, the method comprising the steps of:

[0937] a) providing a liquid BLG isolate having a pH of 5.0 to 8.5, preferably 6.1 to 8.5, more preferably 6.3 to 8.0, even more preferably 6.5 to 7.5;

[0938] The liquid BLG isolate has a BLG content of at least 85% w / w, preferably at least 90% w / w, even more preferably at least 94% w / w relative to the total amount of protein;

[0939] b) optionally, subjecting the liquid BLG isolate to physical microbial reduction;

[0940] c) drying the liquid BLG isolate, preferably spray drying;

[0941] Wherein, providing the liquid BLG isolate in step a) comprises the following steps in the following order:

[0942] - A BLG-enriched composition is prepared by a method comprising the following steps:

[0943] 1) providing a whey protein solution, wherein the whey protein solution comprises non-aggregated BLG and at least one other whey protein, wherein the whey protein solution is supersaturated with BLG and has a pH of 5 to 6;

[0944] 2) Crystallization of BLG in a supersaturated whey protein solution;

[0945] 3) separating BLG crystals from the remaining whey protein solution;

[0946] 4) optionally, washing the BLG crystals, such as the separated BLG crystals obtained from step 3) or 5); and

[0947] 5) optionally, recrystallizing BLG crystals, such as the BLG crystals obtained from step 3) or 4); and

[0948] - adjusting the pH of the BLG-rich composition to 2.5 to 4.9, preferably 2.5 to 4.0, even more preferably 3.0 to 3.9;

[0949] - physical reduction of microorganisms, which involves at least pasteurization, using a temperature of preferably 70-82°C, even more preferably 70-80°C, and a pH of 2.5-4.9, preferably 2.5-4.0, even more preferably 3.0-3.9;

[0950] - adjusting the pH to 5.0 to 8.5, preferably 6.1 to 8.5, more preferably 6.3 to 8.0, even more preferably 6.5 to 7.5;

[0951] - Optionally, demineralization.

[0952] In some preferred embodiments, the method of the present invention is implemented as a batch process. Alternatively, sometimes preferably, the method can be implemented as a semi-batch process. In other preferred embodiments, the method is implemented as a continuous process.

[0953] Yet another aspect of the present invention relates to the liquid BLG isolate described herein. The liquid BLG isolate is particularly useful for obtaining a spray-dried BLG isolate powder and can therefore be used as a liquid ingredient in the production of liquid or non-liquid foods. Alternatively but also preferably, the liquid BLG isolate can be used as a beverage as is.

[0954] In some preferred embodiments of the present invention, the BLG isolate powder of the present invention can be obtained by the method described herein.

[0955] 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.

[0956] One aspect of the present invention relates to the use of a BLG isolate powder or a liquid BLG isolate as described herein as an ingredient for the production of a food product. For example, the food product may be a beverage or an instant beverage powder.

[0957] Preferably, the use of BLG isolate powder or liquid BLG isolate powder provides one or more of the following effects:

[0958] - Reduce the level of dry mouthfeel;

[0959] - Improve the clarity of the resulting liquid containing BLG isolate powder or liquid BLG isolate;

[0960] - Reduce viscosity;

[0961] - Can increase the protein concentration of heat-treated beverages and foods;

[0962] - has a lower color contribution (the inventors have observed that the BLG isolate of the invention gives eg a protein drink less color than the corresponding WPI solution).

[0963] In some preferred embodiments of the present invention, BLG isolate powder is used as an ingredient for preparing a beverage having a protein content of at least 10-36% w / w, more preferably at least 15-35% w / w, even more preferably 20-34% w / w, and most preferably 25-33% w / w; wherein the BLG isolate powder accounts for at least 90% w / w of the total protein in the beverage, more preferably at least 95% w / w, and most preferably all of the total protein in the beverage.

[0964] Furthermore, the BLG powder of the present invention is particularly suitable for high protein beverages, or shake powders for preparing high protein beverages, because the BLG powder of the present invention contributes less to viscosity than conventional WPI, thus providing a more drinkable beverage.

[0965] For example, the food may be a beverage or instant beverage powder having a pH of 2 to 4.7 and having one or more of the following:

[0966] - Reduced level of dry mouthfeel;

[0967] - improved transparency; and / or

[0968] - Increased protein content, the protein content of the heat treated beverage is preferably at least 3-45% w / w, more preferably 11-40% w / w, even more preferably 15-38% w / w, most preferably 20-36% w / w.

[0969] It should be noted that embodiments and features described in the context of one aspect of the present invention are also applicable to the other aspects of the present invention.

[0970] All patent and non-patent references cited in this application are incorporated herein by reference in their entirety.

[0971] The present invention will now be described in further detail by way of the following non-limiting examples.

[0972] Example

[0973] Example 1: Analytical method

[0974] Example 1.1: Determination of protein nativeness by intrinsic tryptophan fluorescence

[0975] Tryptophan (Trp) fluorescence spectroscopy is a known tool for monitoring protein folding and unfolding. Typically, Trp residues buried in native proteins show the highest fluorescence emission at about 330 nm compared to when the Trp residues are present in more solvent-exposed positions (e.g., unfolded proteins). In unfolded proteins, the wavelength of Trp fluorescence emission typically shifts to higher wavelengths, typically measured at about 350 nm. Here, we exploit this shift to study heat-induced unfolding by calculating the ratio between the fluorescence emission at 330 nm and 350 nm to investigate the effect of heating temperature.

[0976] The analysis consists of the following steps:

[0977] Dilute the beverage composition to 0.6 mg / mL with MQ water;

[0978] Transfer 300 μL of sample to a white 96-well plate, avoiding bubbles, or transfer 3 mL of sample to a 10 mm quartz cuvette;

[0979] Tryptophan fluorescence emission intensity was recorded from the top from 310 to 400 nm by exciting at 295 nm using a 5 nm slit;

[0980] Measure samples at 22°C using a Cary Eclipse fluorescence spectrophotometer equipped with a microplate reader accessory (G9810A) or a single cuvette holder.

[0981] The emission intensity ratio, R = I330 / I350, was calculated by dividing the fluorescence emission intensity measured at 330 nm by the emission intensity measured at 350 nm and was used as a measure of protein nativeness:

[0982] oR of at least 1.11 describes a predominantly native BLG conformation; and

[0983] An oR less than 1.11 indicates that at least partial unfolding and aggregation are involved.

[0984] Example 1.2: Thermal stability at pH 3.9

[0985] Thermal stability at pH 3.9:

[0986] Thermal stability at pH 3.9 is a measure of the ability of a protein composition to remain transparent at pH 3.9 after prolonged pasteurization.

[0987] Thermal stability at pH 3.9 is measured by mixing the powder or liquid sample to be tested with water (or, if a dilute liquid, concentrating it by low temperature evaporation) to form an aqueous solution having a pH of 3.9 and containing 6.0% w / w protein, and adjusting the pH to 3.9 with a minimum amount of 0.1 M NaOH or 0.1 M HCl as necessary.

[0988] The pH adjusted mixture was allowed to stand for 30 minutes, and then 25 mL of the mixture was transferred to a 30 mL thin-walled glass test tube. It was heated to 75.0°C by immersion in a water bath at 75.0°C for 300 seconds. After heating, the glass test tube was immediately transferred to an ice bath, cooled to 1-5°C, and then the turbidity of the heat-treated sample was measured according to Example 1.7.

[0989] Example 1.3: Determination of the degree of protein denaturation of a whey protein composition

[0990] It is known that denatured whey protein has a lower solubility at pH 4.6 than at pH values ​​below or above pH 4.6; therefore, 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 in solution at a pH at which the protein remains stable.

[0991] More specifically, for whey protein, the whey protein composition to be analyzed (e.g. powder or aqueous solution) is converted to:

[0992] - a first aqueous solution comprising 5.0% w / w of total protein and having a pH of 7.0 or 3.0; and

[0993] - A second aqueous solution comprising 5.0% w / w of total protein and having a pH of 4.6.

[0994] pH adjustment was performed using 3% (w / w) NaOH (aq) or 5% (w / w) HCl (aq).

[0995] The total protein content (P pH 7.0或3.0 ).

[0996] The second aqueous solution was stored at room temperature for 2 hours and then centrifuged at 3000 g for 5 minutes. The supernatant sample was recovered and analyzed according to Example 1.5 to obtain the protein concentration (S) in the supernatant. pH4.6 ).

[0997] The protein denaturation degree D of the whey protein composition is calculated as follows:

[0998] D=((P pH 7.0或3.0 -S pH 4.6 ) / P pH 7.0或3.0 )*100%

[0999] Example 1.4: Determination of protein denaturation using reversed phase UPLC analysis (pH 4.6 acid precipitation)

[1000] BLG samples (e.g., unheated reference and heated BLG beverage compositions) 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 allow the denatured protein to precipitate and aggregate at about pH 4.6. The solution was filtered through a 0.22 μm filter to remove aggregates and non-native proteins.

[1001] All samples were diluted to the same extent by adding polished water.

[1002] For each sample, the same volume of sample was loaded onto a UPLC system with a UPLC column (ProteinBEH C4; 1.7 μm; 150×2.1 mm), detected at 214 nm.

[1003] The samples were run under the following conditions:

[1004] Buffer A: Milli-Q water, 0.1% w / w TFA;

[1005] Buffer B: HPLC grade acetonitrile, 0.1% w / w TFA;

[1006] Flow rate: 0.4mL / min;

[1007] 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.

[1008] The concentration of native BLG in the samples was determined using the BLG peak area relative to a protein standard (Sigma L0130) (5-level calibration curve).

[1009] If outside the linear range, dilute the sample further and re-inject.

[1010] Example 1.5: Determination of total protein amount

[1011] The total protein (true protein) of the sample was determined as follows:

[1012] 1) Determine the total nitrogen content of the sample according to ISO 8968-1 / 2 | IDF 020-1 / 2 - Milk - Determination of nitrogen content - Part 1 / 2: Determination of nitrogen content using the Kjeldahl method;

[1013] 2) Determination of non-protein nitrogen in the sample according to ISO 8968-4 | IDF 020-4 - Milk - Determination of nitrogen content - Part 4: Determination of non-protein-nitrogen content; and

[1014] 3) Calculate the total amount of protein: (m 总氮 -m 非蛋白氮 )*6.38.

[1015] Example 1.6: Determination of non-aggregated BLG, ALA and CMP

[1016] The contents of non-aggregated α-lactalbumin (ALA), β-lactoglobulin (BLG) and casein macropeptide (CMP) were analyzed by HPLC at 0.4 mL / min. 25 μL of the filtered sample was injected into 2 TSKgel3000PWxl (7.8 mm 30 cm, Tosohass, Japan) chromatographic columns, which were connected in series with an attached pre-packed column PWxl (6 mm × 4 cm, Tosohass, Japan) equilibrated with an eluent (composed of 465 g Milli-Q water, 417.3 g acetonitrile and 1 mL trifluoroacetic acid), using a UV detector at 210 nm.

[1017] The peak area of ​​the corresponding standard protein was compared with the peak area of ​​the sample to determine the native α-lactalbumin (C α ), β-lactoglobulin (C β ) and casein macropeptide (C CMP ) content was quantitatively determined.

[1018] The total amount of other proteins (non-BLG proteins) was determined by subtracting the amount of BLG from the total amount of protein (determined according to Example 1.5).

[1019] Example 1.7: Determination of turbidity

[1020] Turbidity is the cloudiness or haziness of a fluid caused by large numbers of particles that are not normally visible to the naked eye, similar to smoke in the air.

[1021] Turbidity is measured in Nephelometric Turbidity Units (NTU).

[1022] Add 20mL of beverage / sample into the NTU glass and place 3000IR turbidity meter. Measure the NTU value after stabilization and repeat twice.

[1023] Example 1.8: Determination of viscosity

[1024] Measure the viscosity of the liquid using a Gilson Viscoman viscometer or equivalent, at 300 s. -1 Unless otherwise stated, samples were equilibrated to 15°C before measurement and then measured at this temperature.

[1025] Unless otherwise stated, viscosities are expressed as 300s -1 The higher the cP value measured, the lower the viscosity.

[1026] Example 1.9: Determination of color

[1027] Color was measured using a colorimeter (Konica Minolta, CR-400). 15 g of sample was added to a small petri dish (55 x 14.2 mm, VWR catalog number 391-0895) to avoid bubbles. The protein content of the samples was standardized to 6.0 w / w % protein or less.

[1028] The colorimeter was calibrated with a white calibration plate (No. 19033177). The light source was set to D65 and the observer was set to 2 degrees. The suspension was covered with a lid and the color was measured (CIELAB color space: a* value, b* value, L* value), and the average of three independent readings at different positions of the petri dish was measured.

[1029] The demineralized water reference has the following values:

[1030] L*: 39.97±0.3;

[1031] a*: 0.00±0.06;

[1032] b*: 0.22±0.09.

[1033] The measured values ​​are converted into Δ / difference values ​​based on the measured values ​​of demineralized water.

[1034] ΔL*=L标准化为6.0w / w％蛋白质的样品 *-L 脱矿物质水 *, measured at room temperature.

[1035] Δa*=a 标准化为6.0w / w％蛋白质的样品 *-a 脱矿物质水 *, measured at room temperature.

[1036] Δb*=b 标准化为6.0w / w％蛋白质的样品 *-b 脱矿物质水 *, measured at room temperature.

[1037] Samples were normalized to 6.0 w / w % protein or lower.

[1038] The L*a*b* color space (also known as the CIELAB space) is a unified color space defined by the International Commission on Illumination (CIE) in 1976 for quantitatively reporting brightness and chromaticity (ISO 11664-4:2008(E) / CIE S 014-4 / E:2007).

[1039] In this space, L* represents lightness (values ​​range from 0 to 100), with the darkest black being L*=0 and the lightest white being L*=100.

[1040] The color channels a* and b* represent the true neutral gray value when a* = 0 and b* = 0. The a* axis represents the green-red component, with green being negative and red being positive. The b* axis represents the blue-yellow component, with blue being negative and yellow being positive.

[1041] Example 1.10: Beverage Stability Test / Insoluble Protein Material

[1042] The whey protein beverage composition is considered stable if less than 15% of the total amount of protein precipitated after centrifugation of the heated sample at 3000 g for 5 minutes:

[1043] Add about 20 g of sample to a centrifuge tube and centrifuge at 3000 g for 5 minutes.

[1044] • The protein before centrifugation and the supernatant after centrifugation were analyzed by Kjeldahl method to quantify the protein recovery, see Example 1.5.

[1045] Protein loss was calculated as follows:

[1046]

[1047] This parameter is sometimes also referred to as the level of insoluble protein material and can be used to analyze both liquid and powder samples. If the sample is a powder, 10 g of the powder is suspended in 90 g of demineralized water and hydrated for 1 hour at 22°C with gentle stirring. About 20 g of the sample (e.g., a liquid sample or a suspended powder sample) is placed in a centrifuge tube and centrifuged at 3000 g for 5 minutes. The protein (P) before centrifugation is calculated according to Example 1.5. 总 ) and the supernatant after centrifugation (P 3000×g ) Kjeldahl analysis was performed to quantify protein recovery.

[1048] The amount of insoluble protein material was calculated as follows:

[1049]

[1050] Example 1.11: Sensory Evaluation

[1051] Descriptive sensory evaluation of the heat treated beverage formulations was performed. The beverage formulations were heated using a plate heat exchanger.

[1052] 1 volume of the sample was mixed with 1 volume of water and compared to unheated whey protein isolate, lactic acid and citric acid were also used to develop a table of attributes prior to the final tasting stage:

[1053] category property fragrance Acid whey (acid dairy product) Basic taste Sour, bitter Flavor Whey, citric acid, lactic acid Taste Dry, astringent

[1054] The subjects' mouths were cleaned between each sample using crackers, white tea, melon, and water.

[1055] A small cup was used to provide 15 mL of the test sample at ambient temperature (20-25°C).

[1056] Each test sample was given three times to 10 people in three different areas in random order.

[1057] Attributes (see table above) were scored on a 15 cm scale; where 0 = low intensity and 15 = high intensity.

[1058] Statistical analysis was performed using the "Panelcheck" software, using a 3-way ANOVA test with multiple replicates. Samples were fixed and panels were set to random.

[1059] The Bonferroni correction showing the lowest significant difference value (groups associated with letters for pairwise comparisons) was used to assess significant differences between samples.

[1060] Example 1.12: Determining transparency from an image

[1061] Photographs of the beverage formulations were obtained by placing the sample into a turbidity NTU measurement vial with a paper attached with the text "lorem ipsum". The vial was photographed using a smartphone, and the inventors evaluated whether the text could be clearly observed through the vial.

[1062] Example 1.13: Determination of ash content

[1063] The ash content of foods is measured according to NMKL 173:2005 “Ash, gravimetric determination in foods”.

[1064] Example 1.14: Determination of conductivity

[1065] The "conductivity" (sometimes referred to as "specific conductance") of an aqueous solution is a measure of the ability of a solution to conduct electricity. For example, conductivity can be determined by measuring the AC resistance of a solution between two electrodes, with the result usually expressed in millisiemens per centimeter (mS / cm). For example, conductivity can be measured according to EPA (U.S. Environmental Protection Agency) Method No. 120.1.

[1066] Unless otherwise stated, the conductivity values ​​mentioned herein have been normalized to 25°C.

[1067] The conductivity was measured using a conductivity meter (WTW Cond 3210 with quadrupole 325 electrodes).

[1068] Before use, the system was calibrated according to the instructions in the manual. To avoid local dilution, wash the electrode thoroughly in the same medium as the one in which the measurement is to be made. Lower the electrode into the medium so that the area to be measured is completely immersed. Then stir the electrode to remove any air trapped on the electrode. Then keep the electrode still until a stable value can be obtained and recorded from the display.

[1069] Example 1.15: Determination of the total solid content of a solution

[1070] The total solids content of a solution can be determined according to NMKL 110 2nd edition, 2005 (Total solids (water) – Gravimetric determination in milk and milk products). NMKL is the "Nordisk Method for ” abbreviation.

[1071] The water content of a solution can be calculated as 100% minus the relative content of the total solids (% w / w).

[1072] Example 1.16: Determination of pH

[1073] All pH values ​​were measured using a pH glass electrode and normalized to 25 °C.

[1074] Carefully rinse and calibrate the pH glass electrode (with temperature compensation) before use. When the sample is in liquid form, measure the pH value directly in the liquid solution at 25°C. When the sample is a powder, dissolve 10 g of the powder in 90 mL of demineralized water at room temperature with vigorous stirring. Then measure the pH value of the solution at 25°C.

[1075] Example 1.17: Determination of loose density and bulk density

[1076] The density of a dry powder is defined as the relationship between the weight and volume of a powder, which is analyzed under defined conditions using a special Stampf volumeter (i.e. a graduated cylinder). Density is usually expressed in g / mL or kg / L.

[1077] In this method, a dry powder sample is filled into a measuring cylinder. After a specified number of tappings, the volume of the product is read and the density is calculated.

[1078] This method can define three types of density:

[1079] Poured density is the mass divided by the volume of the powder after transfer to a specified graduated cylinder.

[1080] Bulk density is the mass divided by the volume of the powder after being struck 100 times under the conditions specified in this standard.

[1081] Bulk density is the mass divided by the volume of the powder after being struck 625 times under the conditions specified in this standard.

[1082] This method uses a special measuring cylinder (250 mL, 0-250 mL scale, weight 190 ± 15 g) (J. Engelsmann A.G. 67059 Ludwigshafen / Rh) and a Stampf volumeter (e.g. J. Engelsmann AG)

[1083] The bulk density and tapped density of the dried product were measured by the following procedure.

[1084] Preprocessing :

[1085] The samples to be tested were stored at room temperature.

[1086] The sample is then mixed thoroughly by repeatedly rotating and inverting the container (to avoid breaking up the particles). The container should not be filled more than 2 / 3 full.

[1087] program :

[1088] Weigh 100.0 ± 0.1 g of powder and transfer it into a measuring cylinder. The volume V0 is in milliliters.

[1089] If 100 g of powder does not fit into the cylinder, it should be reduced to 50 g or 25 g.

[1090] Fix the cylinder to the Stampf volumeter and tap it 100 times. Smooth the surface with a spatula and read the volume V in ml. 100 .

[1091] Change the number of taps to 625 (including the 100 taps). After tapping, smooth the surface and read the volume V in milliliters. 625 .

[1092] Calculate density :

[1093] The bulk density and tapped density (expressed in g / mL) are calculated according to the following formula:

[1094] Bulk density = M / V

[1095] Where M represents the mass of the sample (in g), and V represents the volume after 625 taps (in mL).

[1096] Example 1.18: Determination of water content of powder

[1097] The moisture content of foods is measured according to ISO 5537:2004 (Dried milk - Determination of moisture content (Reference method)). NMKL is "Nordisk Method for ” abbreviation.

[1098] Example 1.19: Determination of the amount of calcium, magnesium, sodium, potassium and phosphorus (ICP-MS method)

[1099] The total amounts of calcium, magnesium, sodium, potassium, and phosphorus were determined using the following procedure: microwave digestion was first used to decompose the samples, and then an ICP instrument was used to determine the total amount of minerals.

[1100] instrument:

[1101] The microwave was from Anton Paar and the ICP was an Optima 2000DV from PerkinElmer Inc.

[1102] Material:

[1103] 1M HNO3

[1104] Yttrium in 2% HNO3

[1105] Suitable standard solutions of calcium, magnesium, sodium, potassium and phosphorus in 5% HNO3

[1106] Preprocessing :

[1107] Weigh out a certain amount of powder and transfer it to a microwave digestion tube. Add 5mL of 1M HNO3. Digest the sample in the microwave according to the microwave instructions. Place the digested tube in a fume hood and remove the lid to allow the volatile fumes to evaporate.

[1108] Measurement steps :

[1109] Transfer the pretreated sample to the DigiTUBE using a known amount of Milli-Q water. Add 2% yttrium solution in HNO3 to the digestion tube (approximately 0.25 mL per 50 mL of diluted sample) and dilute to a known volume using Milli-Q water. Analyze the sample on the ICP using the procedure described by the manufacturer.

[1110] Blind samples were prepared by diluting a mixture of 10 mL of 1 M HNO3 and 0.5 mL of a solution of yttrium in 2% HNO3 with Milli-Q water to a final volume of 100 mL.

[1111] Prepare at least 3 standards at the expected sample concentrations.

[1112] Example 1.20: Determination of furosine value

[1113] The method for determining the furosine value is as described in Guerra-Hernandez et al., Maillard Reaction Evaluation by Furosine Determination During Infant Cereal Processing, Journal of Cereal Science 1999, 29: 171-176, and the total protein content is determined according to Example 1.5. The furosine value is expressed in mg creatinine / 100 g protein.

[1114] Example 1.21: Determination of the crystallinity of BLG in liquid

[1115] The following method was used to determine the crystallinity of BLG in liquids at pH 5-6.

[1116] a) Transfer 10 mL of the liquid sample to be studied into a Maxi-Spin filter equipped with a 0.45 μm pore size CA membrane;

[1117] b) Immediately spin the filter at 1500 g for 5 minutes in a centrifuge maintained at 2°C;

[1118] c) 2 mL of cold Milli-Q water (2°C) was added to the retentate side of the spin filter, the filter was immediately spun at 1500 g for 5 minutes while keeping the centrifuge cooled at 2°C, the permeate was collected (Permeate A), the volume was measured, and the concentration of BLG was determined by HPLC using the method described in Example 1.6;

[1119] d) Add 4 mL of 2M NaCl to the retentate side of the filter, stir rapidly, and allow the mixture to stand at 25°C for 15 minutes;

[1120] e) immediately spinning the filter at 1500 g for 5 minutes and collecting the permeate (Permeate B);

[1121] f) Determine the total weight of BLG in permeate A and permeate B using the method described in Example 1.6 and convert the result into the total weight of BLG instead of the weight percentage; the weight of BLG in permeate A is referred to as m 渗透物A , the weight of BLG in permeate B is called m 渗透物B ;

[1122] g) The crystallinity of BLG in liquid was determined as follows:

[1123] Crystallinity = m 渗透物B / (m 渗透物A +m 渗透物B )*100%

[1124] Example 1.22: Determination of the crystallinity of BLG in dry powder

[1125] This method was used to determine the crystallinity of BLG in dry powder.

[1126] a) Mix 5.0 g of powder sample with 20.0 g of cold Milli-Q water (2°C) and let stand at 2°C for 5 minutes;

[1127] b) transferring the liquid sample to be studied to a Maxi-Spin filter with a 0.45 μm CA membrane;

[1128] c) immediately spin the filter at 1500 g for 5 minutes in a centrifuge maintained at 2°C;

[1129] d) Add 2 mL of cold Milli-Q water (2°C) to the retentate side of the spin filter, then immediately spin the filter at 1500 g for 5 minutes, collect the permeate (Permeate A), measure the volume, and determine the BLG concentration by HPLC using the method described in Example 1.6, and convert the result into the total weight of BLG instead of weight percentage. The weight of BLG in Permeate A is referred to as m 渗透物A ;

[1130] f) The crystallinity of BLG in the powder was then calculated using the following formula:

[1131]

[1132] Among them, m 总BLG is the total amount of BLG in the powder sample of step a).

[1133] If the total amount of BLG of a powder sample is unknown, it can be determined as follows: suspend another 5 g powder sample (from the same powder source) in 20.0 g Milli-Q water, adjust the pH to 7.0 by adding aqueous NaOH solution, place the mixture at 25°C for 1 hour under stirring, and finally determine the total amount of BLG of the powder sample using Example 1.6.

[1134] Example 1.23: Determination of UF permeate conductivity

[1135] Transfer 15 mL of sample to an Amicon Ultra-15 centrifugal filter with a cutoff of 3 kDa (3000 NMWL) and centrifuge at 4000 g for 20-30 minutes, or until sufficient UF permeate accumulates at the bottom of the filter unit to measure conductivity. Measure conductivity immediately after centrifugation. Sample handling and centrifugation are performed at the temperature of the sample source.

[1136] Example 1.24: Determination of dried BLG crystals in powder

[1137] The presence of dried BLG crystals in a powder can be determined by the following method:

[1138] The powder sample to be analyzed was resuspended in demineralized water at 4°C in a weight ratio of 2 parts water to 1 part powder, mixed gently, and then rehydrated at 4°C for 1 hour.

[1139] The rehydrated sample is examined microscopically to identify the presence of crystals, preferably using plane polarized light to detect birefringence.

[1140] The crystalline material is isolated and subjected to X-ray crystallographic analysis to verify the presence of the crystalline structure and preferably also that its lattice (space group and unit cell dimensions) corresponds to that of BLG crystals.

[1141] The chemical composition of the isolated crystalline material was analyzed to verify that the solid consisted mainly of non-aggregated BLG.

[1142] Example 1.25: Determination of total lactose content

[1143] The total amount of lactose is measured according to ISO 5765-2:2002 (IDF 79-2:2002) “Dried milk, dried ice-mixes and processed cheese – Determination of lactose content – ​​Part 2: Enzymatic method utilizing the galactose moiety of the lactose”.

[1144] Example 1.26: Determination of total carbohydrate content

[1145] Carbohydrate content was determined by using the Sigma Aldrich Total Carbohydrate Assay Kit (Cat. No. MAK104-1KT), in which carbohydrates are hydrolyzed and converted to furfural and hydroxyfurfural, then converted to a chromogen and monitored spectrophotometrically at 490 nm.

[1146] Example 1.27: Determination of total lipid content

[1147] Lipid content according to ISO 1211:2010 (Determination of fat content – -Gottlieb weight method, Determination of Fat Content– -Gottlieb Gravimetric Method).

[1148] Example 1.28: Determination of Brix

[1149] Brix was measured using a PAL-α digital handheld refractometer (Atago) calibrated with purified water (reverse osmosis filtered water, conductivity up to 0.05 mS / cm).

[1150] Transfer approximately 500 μL of sample to the prism surface of the instrument and begin taking measurements. Read and record the measurement.

[1151] The Brix of whey protein solution is proportional to the total solids (TS), and TS (% w / w) is approximately Brix*0.85.

[1152] Brix is ​​sometimes called degrees Brix or °Brix.

[1153] Example 1.29: Determination of lactoferrin and lactoperoxidase

[1154] Lactoferrin concentration was determined by ELISA immunoassay as outlined by Soyeurt 2012 (Soyeurt et al., Mid-infrared prediction of lactoferrin content in bovine milk: potential indicator of mastitis, Animal 2012, 6: 11: 1830-1838).

[1155] The concentration of lactoperoxidase was determined using a commercially available bovine lactoperoxidase kit.

[1156] Example 1.30: Determination of colony forming units

[1157] The number of colony forming units per gram of sample was determined according to ISO 4833-1:2013(E): Microbiology of food and animal feeding stuffs–horizontal method for the enumeration of microorganisms–Colony-count technique at 30°C.

[1158] Example 1.31: Determination of the total amount of BLG, ALA and CMP

[1159] This procedure is a liquid chromatography (HPLC) method for the quantitative analysis of proteins such as ALA, BLG and CMP as well as other proteins in a composition. Unlike the method of Example 1.6, this method also measures proteins present in aggregates, thus providing a measure of the total amount of protein species in the composition under investigation.

[1160] The separation method was size exclusion chromatography (SEC) using 6M guanidine hydrochloride buffer as the sample solvent and HPLC mobile phase.Mercaptoethanol was used as a reducing agent to reduce disulfide (SS) in proteins or protein aggregates to produce unfolded monomeric structures.

[1161] Sample preparation is easily accomplished by dissolving 10 mg protein equivalent in the mobile phase.

[1162] Two TSK-GEL G3000SWXL (7.7 mm × 30.0 cm) chromatographic columns (GPC chromatographic columns) and a guard column were placed in series to achieve adequate separation of the major proteins in the raw material.

[1163] The eluted analytes were detected and quantified by UV detection (280 nm).

[1164] Equipment / Materials :

[1165] 1. HPLC Pump 515 (Waters) with manual seal wash;

[1166] 2.HPLC pump control module II (Waters);

[1167] 3. Automatic sample injector 717 (Waters);

[1168] 4. Dual absorbance detector 2487 (Waters);

[1169] 5. Computer software capable of generating quantitative reports (Empower 3, Waters);

[1170] 6. Analytical columns: 2 TSK-GEL G3000SWXL (7.8×300mm, P / N: 08541);

[1171] Guard column: TSK-guard column SWxL (6.0×40 mm, P / N: 08543);

[1172] 7. Ultrasonic bath (Branson 5200);

[1173] 8.25 mm syringe filter with 0.2 μm cellulose acetate membrane (514-0060, VWR).

[1174] program :

[1175] Mobile phase :

[1176] A. Stock Buffer

[1177] 1. Weigh 56.6g Na2HPO4, 3.5g NaH2PO4 and 2.9g EDTA into a 1000mL beaker. Dissolve in 800mL water;

[1178] 2. Measure the pH value and, if necessary, adjust to 7.5 ± 0.1 with HCl (to lower the pH) or NaOH (to increase the pH);

[1179] 3. Transfer to a 1000mL volumetric flask and dilute to the mark with water.

[1180] B. 6M guanidine hydrochloride mobile phase

[1181] 1. Weigh 1146 g of guanidine hydrochloride and add it to a 2000 mL beaker, then add 200 mL of stock buffer (A);

[1182] 2. Dilute the solution to about 1600 mL with water while mixing with a magnetic stirring bar (50°C);

[1183] 3. Adjust pH to 7.5 ± 0.1 with NaOH;

[1184] 4. Transfer to a 2000mL volumetric flask and dilute to the mark with water;

[1185] 5. Filter using a solvent filtration device with a 0.22 μm membrane filter.

[1186] Calibration Standards

[1187] Calibration standards for each protein to be quantified were prepared as follows:

[1188] 1. Accurately weigh (to 0.01 mg) about 25 mg of protein reference standard into a 10 mL volumetric flask and dissolve in 10 mL of water. This is the protein stock standard solution (S1).

[1189] 2. Pipette 200 μL S1 into a 20 mL volumetric flask and dilute to volume with mobile phase. This is the low (concentration) working standard solution WS1.

[1190] 3. Pipette 500 μL S1 into a 10 mL volumetric flask and dilute to volume with mobile phase. This is standard solution WS2.

[1191] 4. Pipette 500 μL of S1 into a 5 mL volumetric flask and dilute to volume with mobile phase. This is standard solution WS3.

[1192] 5. Transfer 750 μL of S1 to a 5 mL volumetric flask and dilute to volume with mobile phase. This is standard solution WS4.

[1193] 6. Pipette 1.0 mL of S1 into a 5 mL volumetric flask and dilute to volume with mobile phase. This is the high (concentration) working standard solution WS5.

[1194] 7. Using a graduated disposable pipette, transfer 1.5 mL of WS1 to WS5 into individual vials, add 10 μL of 2-mercaptoethanol to each vial and cap the vial. Vortex the solution for 10 seconds and allow the standards to stand at ambient temperature for approximately 1 hour.

[1195] 8. Filter the standard solution using a 0.22 μm cellulose acetate syringe filter.

[1196] The purity of the protein was measured using the Kjeldahl method (N×6.38), and the area % of the standard solution WS5 was determined using HPLC.

[1197] Protein (mg) = "weight of protein standard" (mg) × P1 × P2;

[1198] P1 = P% (Kjeldahl method);

[1199] P2 = protein area % (HPLC).

[1200] Sample preparation

[1201] 1. Weigh 25 mg of the original protein sample into a 25 mL volumetric flask;

[1202] 2. Add about 20 mL of mobile phase and allow the sample to dissolve for about 30 minutes;

[1203] 3. Add mobile phase to the mark and add 167 μL 2-mercaptoethanol to 25 mL sample solution;

[1204] 4. Ultrasonic treatment for about 30 minutes, and then the sample is placed at ambient temperature for about 1.5 hours;

[1205] 5. The mixed solution was filtered using a 0.22 μL cellulose acetate syringe filter.

[1206] HPLC system / column

[1207] Equilibrate the column

[1208] 1. Connect a GPC guard column and two GPC analytical columns in series. The new column is usually filled with phosphate buffer.

[1209] 2. Within 30 to 60 minutes, gradually flow water through the new column at a rate of 0.1 to 0.5 mL / min. Continue washing for about 1 hour.

[1210] 3. Gradually reduce the flow rate from 0.5 mL / min to 0.1 mL / min and replace the mobile phase in the storage container;

[1211] 4. Gradually increase the pump flow rate from 0.1 mL / min to 0.5 mL / min over 30 to 60 minutes to avoid pressure shock and maintain at 0.5 mL / min;

[1212] 5. Inject 10 samples to saturate the column and wait for the peak to elute. This will help condition the column. Do this without waiting for each injection to complete before the next injection.

[1213] 6. Equilibrate with mobile phase for at least 1 hour.

[1214] Calculation results

[1215] By comparing the peak area of ​​the corresponding standard protein with the peak area of ​​the sample, the quantitative determination of the content of the protein to be quantified (such as α-lactalbumin, β-lactoglobulin and casein macropeptide) is performed. The result is expressed as g specific protein / 100g original sample, or as the weight percentage of the specific protein relative to the weight of the original sample.

[1216] Example 2: Production of spray-dried acidic BLG isolate powder

[1217] Whey protein raw materials

[1218] The lactose-depleted UF retentate of sweet whey from a standard cheese production process was filtered through a 1.2 micron filter and then fat reduced using a Synder FR membrane before being used as feedstock for the BLG crystallization process. The chemical composition of the feedstock is shown in Table 1. We note that all weight percentages of specific proteins (e.g., BLG, ALA) mentioned in this example are related to the weight percentage of non-aggregated protein relative to the total amount of protein.

[1219] adjust

[1220] Use Koch HFK-328 membrane (70m 2 The sweet whey raw material was adjusted to a raw material concentration of 21% total solids (TS) ± 5 on an ultrafiltration device at 20°C, with a feed pressure of 1.5 to 3.0 bar, and purified water (water filtered by reverse osmosis to a conductivity of at most 0.05 mS / cm) was used as the diafiltration medium. The pH was then adjusted to about 5.5 by adding HCl. The diafiltration was continued until the conductivity of the retentate dropped below 0.1 mS / cm in 20 minutes. The retentate was then concentrated until the permeate flow rate was less than 1.43 L / h / m 2 A first sample of the concentrated retentate was collected and centrifuged at 3000 g for 5 minutes. The supernatant of the first sample was used to determine the BLG yield.

[1221] crystallization

[1222] The concentrated retentate was transferred to a 300 L crystallizer where it was seeded with pure BLG crystal material made from rehydrated spray-dried BLG crystals. The seeded whey protein solution was then cooled from 20°C to about 6°C over about 10 hours to allow BLG crystals to form and grow.

[1223] After cooling, a sample of the whey protein solution containing the crystals (second sample) was taken out and the BLG crystals were separated by centrifugation at 3000 g for 5 minutes. The supernatant and crystal precipitate from the second sample were subjected to HPLC analysis as described below. The yield of crystallization was calculated as described below and was determined to be 57%.

[1224] Table 1: Chemical composition of raw materials

[1225]

[1226] Determination of BLG yield using HPLC :

[1227] The supernatants of the first and second samples were diluted to the same extent by adding purified water, and then the diluted supernatants were filtered through a 0.22 μm filter. For each filtered and diluted supernatant, the same volume of the supernatant was injected into a 100 μL / min flow cytometer with a Phenomenex 100 μL flow cytometer. 5μm C4 The LC column was 250×4.6 mm in a HPLC system and the detection was performed at 214 nm.

[1228] The samples were run using the following conditions:

[1229] Buffer A: MilliQ water, 0.1% w / w TFA

[1230] Buffer B: HPLC grade acetonitrile, 0.085% w / w TFA

[1231] Flow rate: 1mL / min

[1232] Column temperature: 40°C

[1233] Gradient: 0-30 min 82-55% A and 18-45% B; 30-32 min 55-10% A and 45-90% B; 32.5-37.5 min 10% A and 90% B; 38-48 min 10-82% A and 90-18% B.

[1234] Data processing:

[1235] Since the supernatants were all processed in the same way, the areas of the BLG peaks could be directly compared to calculate the relative yields. Since the crystals contained only BLG and all samples were processed in the same way, the α-lactalbumin (ALA) concentration in all samples, as well as the ALA area, should be the same, so the ALA area before and after crystallization was used as a correction factor (cf) when calculating the relative yields.

[1236]

[1237] The relative yield was calculated by the following formula:

[1238]

[1239] Acid dissolution of BLC crystals

[1240] A 64-spacer decanter was used to separate the remaining material in the crystallization tank at 350 g, 2750 RPM, and 150 RPM differential speeds, with a feed flow rate of 75 L / h. Before separation, the raw material was mixed with purified water at a ratio of 1:2. The BLG crystals / solid phase in the decanter were then mixed with purified water to make it a more dilute slurry, and then phosphoric acid was added to reduce the pH to about 3.0 to quickly dissolve the crystals.

[1241] After dissolving the BLG crystals, the pure BLG protein liquid was concentrated to 15 degrees Brix on the same ultrafiltration (UF) unit used to prepare the feedstock for crystallization and the pH was adjusted to a final pH of about 3.8. The liquid BLG isolate was then heated to 75°C for 5 minutes and then cooled to 10°C. It was found that the heat treatment reduced the microbial load from 137.000 CFU / g before heat treatment to <1000 CFU / g after heat treatment. The heat treatment did not cause any protein denaturation and the intrinsic tryptophan fluorescence emission ratio (330nm / 350nm) was measured to be 1.20, confirming that the BLG molecule is natural.

[1242] BLG was dried on a pilot plant spray dryer with an inlet temperature of 180°C and an outlet temperature of 75°C. The water content of the resulting powder sampled at the outlet was about 4% w / w; the chemical composition of the powder is shown in Table 2. A sample of the dried powder was dissolved and the degree of protein denaturation was determined to be 1.5%; the intrinsic tryptophan fluorescence emission ratio (I330 / I350) was determined to be 1.20.

[1243] Table 2: Composition of BLG isolate powder (BDL = below detection limit)

[1244]

[1245] The bulk density of the spray-dried powder (beaten 625 times) was estimated to be 0.2-0.3 g / cm 3 .

[1246] in conclusion:

[1247] By using the above methods, we are able to produce a highly pure BLG product that can be heat treated with essentially no protein denaturation or protein unfolding during processing. Heat treatment greatly reduces bacterial levels without damaging the protein product.

[1248] The inventors have found that by increasing the protein content before spray drying, even higher bulk densities can be obtained. In addition, the inventors have observed that even lower denaturation degrees can be obtained if the inlet temperature and / or outlet temperature used for spray drying is reduced.

[1249] Example 3: Production of Spray-Dried pH-Neutral BLG Isolate Powder

[1250] When the same protocol and experimental setup as in Example 2 were used, the lactose-reduced whey protein isolate shown in Table 3 was adjusted and used as the crystallization raw material. The crystallization yield was calculated to be 68%.

[1251] We note that all weight percentages of specific proteins (eg, BLG and ALA) mentioned in this example are related to the weight percentage of non-aggregated protein relative to the total amount of protein.

[1252] Table 3: Composition of raw materials

[1253]

[1254] A 64-spaced decanter was used to separate the remaining material in the crystallization tank at 350 g, 2750 RPM, and 150 RPM differential speeds, with a raw material flow rate of 75 L / h. Before separation, the raw material was mixed with purified water at a ratio of 1:2. The BLG crystals / solid phase in the decanter were then mixed with purified water to make it a more dilute slurry, and then 0.1 M potassium hydroxide was added to adjust the pH to about 7 to quickly dissolve the crystals.

[1255] After dissolving the crystals, the pure BLG protein liquid was concentrated to 15 Brix on the same UF unit 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 using a spray dryer in the pilot plant with an inlet temperature of 180°C and an outlet temperature of 75°C. The water content of the resulting powder sampled at the outlet was about 4% w / w. The composition of the powder is shown in Table 4. After drying, some of the powder was dissolved in demineralized water and the degree of protein denaturation was determined to be 9.0%; the intrinsic tryptophan fluorescence emission ratio (330nm / 350nm) was 1.16.

[1256] Table 4: Chemical composition of BLG isolate powder

[1257]

[1258] The bulk density of the spray-dried powder (beaten 625 times) was estimated to be 0.2-0.3 g / cm 3 .

[1259] in conclusion:

[1260] By using the above method, we are able to produce a pH neutral, high purity BLG product with little or no protein denaturation during processing. The inventors have found that by increasing the protein content before spray drying, even higher bulk densities can be obtained. In addition, the inventors have observed that if the inlet temperature and / or outlet temperature of the spray drying is reduced, even lower denaturation can be obtained. The degree of denaturation can also be further reduced by reducing the mineral content before spray drying.

[1261] Example 4: Wettability of spray-dried BLG isolate powder

[1262] The wettability of the acidic or pH neutral spray dried BLG isolate powders prepared according to Examples 2 and 3 was compared with the wettability of conventional spray dried whey protein isolate (WPI). The wettability was measured as the time taken before the entire powder sample was wetted. 0.5 g of powder was weighed out and placed on the surface of 100 g of demineralized water (10° C.) in a cylindrical container with a diameter of 5 cm. The time from when the powder was placed on the surface of the water to when the powder dissolved or passed through the surface of the water was measured. The results are as follows.

[1263]

[1264] Conclusion: Unexpectedly, the uncoated BLG isolate powders (Sample 2, Sample 3) were much more wettable than the standard WPI (Sample 1). This suggests that the BLG isolate powders of the present invention are useful ingredients for instant beverage powders where fast wettability and solubility are important. In addition, BLG isolate powders have greater utility in the production of protein beverages as they are wetted and dissolved faster than conventional WPIs and are therefore easier to disperse and dissolve during the production process. Ultimately, this could reduce the time required to produce high protein beverages and increase the hourly production capacity of beverage production plants.

[1265] Example 5: Acidic BLG Isolate Has Reduced Levels of Dry Mouthfeel

[1266] The pH of two protein beverages A and B (which contained a sufficient amount of the acidic spray-dried BLG isolate powder prepared in Example 2 to provide 6.0% of the total protein) was adjusted to pH 3.7 and subjected to heat treatment: A: 75°C for 15 seconds or B: 120°C for 20 seconds. 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 of beverage A was still in the native conformation, while obvious unfolding and denaturation occurred in beverage B.

[1267] 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, whereas the turbidity of the comparable standard WPI was above 200 NTU. Thus, the acidic BLG isolate of the present invention is clearly very suitable for the production of clear acidic high protein beverages.

[1268] Less than one week after production, both beverages were sensory tested by a trained sensory panel and found that the dry mouthfeel (characteristic of heat-treated acidic whey protein beverages) of Beverage B (dry mouthfeel score: approximately 10.5 points on a scale of 0 to 12) was more than 100% higher than that of Beverage A (dry mouthfeel score: approximately 5.0 points on a scale of 0 to 12).

[1269] These findings suggest that protein denaturation can cause the dry mouthfeel of acidic protein beverages and that the dry mouthfeel can be significantly reduced by limiting or even avoiding protein denaturation.

[1270] Example 6: Production of ultra-low microbial neutral BLG isolates

[1271] 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 a high protein nativeness. This is demonstrated in the present example where the total processing time is extended by inserting a 6 day storage time at 10°C in the final step of the conditioning process to challenge the microbiological quality of the product.

[1272] Whey protein raw materials:

[1273] The lactose-free UF retentate of sweet whey from a standard cheese production process was filtered through a 1.2 micron filter and had been fat-reduced through a Synder FR membrane before being used as a feedstock for the BLG crystallization process. The chemical composition of the feedstock is shown in Table 5. We note that all weight percentages of specific proteins (e.g., BLG, ALA) mentioned in this example are related to the weight percentage of non-aggregated protein relative to the total amount of protein.

[1274] Table 5: Composition of whey protein raw material used in Example 6

[1275]

[1276] adjust:

[1277] The sweet whey raw material was adjusted to a raw material concentration of 21% total solids (TS) ±5% on an ultrafiltration unit at about 10°C using an Alfa Laval GR82PE membrane with a 30 mill interval and a feed pressure of 1.5 to 3.0 bar, using purified water (water filtered by reverse osmosis with a conductivity of up to 0.05 mS / cm) as the diafiltration medium. The pH was adjusted to about 5.9 using dilute hydrochloric acid and then diafiltration was performed using a batch ultrafiltration unit. The diafiltration was continued until the conductivity of the retentate fell below 0.1 mS / cm over a period of 20 minutes.

[1278] The diafiltered whey protein material was then stored at 10°C for 6 days to challenge the microbiological quality of the resulting product.

[1279] After storage, the whey protein material was heated to 20°C and then the pH was adjusted by adding HCl (using dilute hydrochloric acid) so that the pH was about 5.5.

[1280] The whey protein raw material was adjusted to a raw material concentration of 21% total solids (TS) ± 5% using an Alfa Laval GR82PE membrane with a 30 mill interval and a feed pressure of 1.5 to 3.0 bar on an ultrafiltration unit at 20°C, using purified water as the diafiltration medium. Ultrafiltration was performed using a continuous ultrafiltration unit, with diafiltration added so that the conductivity of the final retentate was 1.9 to 2.2 mS / cm and a TS of 22 ± 5. The retentate was collected in an 800 L tank with a ledge and stirring. After the tank was filled, a first sample of the concentrated retentate was taken for HPLC analysis. The HPLC analysis of this example was performed as described in Example 2.

[1281] crystallization:

[1282] The concentrated retentate was transferred to an 800 L crystallizer where it was seeded with pure BLG crystal material made from rehydrated spray-dried BLG crystals. The crystals were added to 1 L of conditioned WPI and rapidly cooled on ice to below 5° C. The seeded whey protein solution was then cooled from 20° C. to about 6° C. over about 4 hours to allow BLG crystals to form and grow.

[1283] After cooling, a sample of the whey protein solution containing the crystals (second sample) was taken and the BLG crystals were separated by centrifugation at 3000 g for 5 minutes. The supernatant and crystal precipitate of the second sample were subjected to HPLC analysis as described below. The yield of crystallization was calculated and expressed as % as described below.

[1284] Decanter separation:

[1285] The crystals produced in the 800L tank were separated from the mother liquor using a decanter (LEMITECH MD80) with a 64 mill interval (mill represents 1 / 1000 inch) at 600 g and a differential speed of 2750 RPM. The feed rate was 150 L / h, which was a mixture of purified water and raw material from the tank, with a ratio of 1 volume of tank raw material to 2 volumes of purified water.

[1286] Dissolving crystals:

[1287] The crystals collected in the decanter were diluted with purified water to a total solid concentration of about 10%, and then the pH was adjusted to pH 3 using dilute hydrochloric acid. The acidified BLG solution was maintained at pH 3 at 6°C for about 16-48 hours.

[1288] Adjust the pH of the acidified BLG solution:

[1289] The pH of the acidified BLG solution was then adjusted to pH 7 using a dilute mixture of potassium hydroxide and sodium hydroxide and further conditioned on an ultrafiltration unit at about 10°C using an Alfa Laval GR82PE type membrane with a 30 mill interval (feed pressure of 1.5-3.0 bar) to a concentration of about 16% TS.

[1290] Microfiltration:

[1291] 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. The microfiltration was performed at about 10°C and a feed pressure of 3.5. The MF permeate was collected and can now be dried.

[1292] The MF permeate samples were analyzed for chemical composition (see results in Table 6) and microorganisms (Example 1.30) and surprisingly found to be below 10 CFU / g (basically meaning that no colonies were identified and the test sample appeared to be close to sterile). Sterility can be achieved, for example, by using tighter microfiltration membranes and ensuring sterile conditions on the permeate side of the membrane.

[1293] A comparable process without acidification and microfiltration would easily result in a BLG solution containing more than 1.000.000 CFU / g.

[1294] Table 6: Composition of the microfiltration permeate of concentrated BLG solution

[1295]

[1296] in conclusion:

[1297] The present invention can provide a sterile or near-sterile, pH-neutral BLG product with a high protein content even under heavy microbial loads.

[1298] Example 7: Comparison of reverse osmosis concentration and powder bulk density of standard neutral WPI with acidic and neutral BLG

[1299] The present inventors have found that it is possible to prepare spray-dried preparations of purified native BLG which provide powders having higher bulk densities than comparable whey protein isolates spray dried under the same conditions.

[1300] The inventors have also found that liquid high protein preparations of purified native BLG have a lower viscosity than comparable whey protein isolates. This discovery allows BLG isolates to be concentrated to a higher total protein concentration (and lower water content) prior to drying, thus requiring less water to be removed per kg of dried protein than comparable WPI powders, thus requiring less energy. Reduced viscosity also allows for membrane filtration (e.g., microfiltration) with reduced energy consumption, since the energy required for membrane filtration decreases as viscosity decreases.

[1301] The following experiments confirmed the above findings.

[1302] Original materials for reverse osmosis and drying:

[1303] Neutral BLG:

[1304] The MF retentate produced in Example 6 was concentrated using an Alfa Laval RO98pHt membrane with a feed pressure of up to 52 bar, keeping the temperature below 15°C during conditioning. Concentration was continued until the Brix was 32.2. During concentration, samples were taken to measure viscosity and Brix as described in Examples 1.8B and 1.28. The Brix of each sample was converted to protein (w / w%) using the following formula:

[1305]

[1306] 0.85 is an empirically obtained conversion ratio that gives a good relationship between Brix and total solids. Figure 6 shown.

[1307] The total solids of the whey protein solution is about Brix*0.85.

[1308] Acid BLG:

[1309] An acidic BLG solution was prepared as in Example 6 (with the difference that, instead of ultrafiltration at pH 5.92, ultrafiltration was carried out at pH 3 before continuous ultrafiltration at pH 5.5). The composition of the feed can be seen in Table 7. The acidic BLG permeate was subjected to a MF step as described in Example 6 and then concentrated using an Alfa Laval RO98pHt reverse osmosis (RO) membrane with a feed pressure of up to 52 bar. During conditioning, the temperature was kept below 15°C. Concentration was continued until a Brix of 40.0 was obtained. During concentration, samples were taken to measure viscosity and Brix as described in Examples 1.8B and 1.28. The Brix values ​​were converted to protein concentration and the results are shown in Table 7. Figure 6 Before drying, BLG was diluted with purified water to a Brix of 35.5.

[1310] Table 7: Composition of acidic RO raw materials

[1311]

[1312] WPI reference:

[1313] To compare the acidic BLG and pH neutral BLG products with the conventional pH neutral WPI product, a standard liquid concentrate WPI based on sweet whey was extracted from Arla Foods Danmark Protein production and dried on the same pilot plant spray dryer using the same conditions as the acidic BLG and pH neutral BLG samples. The composition of the liquid concentrate WPI is shown in Table 8.

[1314] The viscosity of concentrated WPI was first measured and then the concentrated WPI was gradually diluted with purified water to show the correlation between WPI Brix and viscosity for comparison with the BLG measurements.

[1315] Viscosity curve:

[1316] The temperature of the samples taken during RO and the temperature of the diluted WPI reference were adjusted to 15° C. before viscosity measurements (in triplicate) were performed as described in Example 1.8B. The Brix was measured as described in Example 1.28.

[1317] The concentrated liquid WPI was dried to a Brix of 36.0.

[1318] Table 8: Composition of WPI reference

[1319]

[1320] Spray Drying:

[1321] After concentration by reverse osmosis (RO), all samples were dried on a pilot plant spray dryer with an inlet temperature of 180°C and an outlet temperature of 85°C without any preheating. The temperature of all samples was kept below 12°C until spray drying. The bulk density of the spray-dried powders was then measured as described in Example 1.17: no stomp, 100 stomp, and 625 stomp. Unexpectedly, the bulk density of both the acidic BLG powder and the pH neutral BLG powder was significantly and consistently higher than the WPI reference. On average, the bulk density of the pH neutral BLG was 18.4% higher than the WPI reference powder, while the bulk density of the acidic BLG was 22.2% higher than the WPI reference powder. The results of the bulk density measurements are shown in Table 9 and are shown in Figure 5 .

[1322] Table 9: Bulk density of powders obtained from acidic BLG solution, pH neutral BLG solution and reference WPI solution, having the same total amount of protein and having been spray dried under the same conditions

[1323]

[1324] in conclusion:

[1325] Both the acidic BLG isolate and the pH neutral BLG isolate have lower viscosity and are easier to concentrate with RO than the WPI reference. This discovery allows the BLG isolate to be concentrated to a higher total protein concentration (and lower water content) before drying; therefore, less water needs to be removed than with a comparable WPI powder, resulting in lower energy consumption per kg of dried protein. Reduced viscosity also allows for membrane filtration (e.g., microfiltration) with reduced energy consumption, since the energy required for membrane filtration decreases as viscosity decreases.

[1326] Furthermore, the BLG powder of the present invention is particularly suitable for use in high protein beverages or milkshake powders for preparing high protein beverages because the BLG powder of the present invention contributes less to viscosity than conventional WPI, thereby providing a more drinkable beverage.

[1327] Unexpectedly, the bulk density of the spray-dried BLG powder was higher compared to the WPI reference, even when the BLG was dried at a lower total solids content (as indicated by the Brix value). A higher powder bulk density means a smaller shipping volume per unit weight when shipping, and the denser particles also generate less dust when handled.

Claims

1. A BLG isolate powder having a pH of 2.5 to 4.9, wherein the BLG isolate powder comprises: - at least 30% w / w total protein; - at least 85% w / w beta-lactoglobulin BLG relative to the total amount of protein; - up to 10% w / w water; The BLG isolate powder has: - an intrinsic tryptophan fluorescence emission ratio I330 / I350 of at least 1.11; and / or - The degree of protein denaturation is up to 10%.

2. The BLG isolate powder according to claim 1, wherein The BLG isolate powder was prepared by spray drying.

3. The BLG isolate powder according to claim 1, wherein The BLG isolate powder has one or more of the following: - Bulk density of at least 0.2 g / cm 3 ; - a thermal stability of up to 200 NTU at pH 3.9; and - Up to 15000 colony forming units / g.

4. The BLG isolate powder according to claim 1, wherein The total amount of protein in the BLG isolate powder is at least 70% w / w.

5. The BLG isolate powder according to claim 1, wherein The total amount of protein in the BLG isolate powder is at least 90% w / w.

6. The BLG isolate powder according to claim 1, wherein The BLG isolate powder has a BLG content of at least 90% w / w relative to the total amount of protein.

7. The BLG isolate powder according to claim 1, wherein The BLG isolate powder has a BLG content of at least 95% w / w relative to the total amount of protein.

8. The BLG isolate powder according to claim 1, wherein The lipid content of the BLG isolate powder is at most 10% w / w.

9. The BLG isolate powder according to claim 1, wherein The lipid content of the BLG isolate powder is at most 0.1% w / w.

10. The BLG isolate powder according to claim 1, wherein The bulk density of the BLG isolate powder is at least 0.20 g / cm 3 .

11. The BLG isolate powder according to claim 1, wherein The bulk density of the BLG isolate powder is at least 0.30 g / cm 3 .

12. The BLG isolate powder according to claim 1, wherein The BLG isolate powder has an intrinsic tryptophan fluorescence emission ratio I330 / I350 of at least 1.

12.

13. The BLG isolate powder according to claim 1, wherein The protein denaturation degree of the BLG isolate powder is at most 8%.

14. The BLG isolate powder according to claim 1, wherein: - the total amount of protein in the BLG isolate powder is at least 70% w / w; - the BLG isolate powder has a BLG content of at least 90% w / w relative to the total amount of protein; - the protein denaturation degree of the BLG isolate powder is at most 10%; and - the lipid content of the BLG isolate powder is at most 0.1% w / w.

15. The BLG isolate powder according to claim 14, wherein The BLG isolate powder contained at most 15,000 colony forming units / g.

16. A liquid BLG isolate having a pH of 2 to 4.9, the liquid BLG isolate comprising: - at least 10% w / w total protein; - at least 85% w / w beta-lactoglobulin BLG relative to the total amount of protein; The liquid BLG isolate has: - an intrinsic tryptophan fluorescence emission ratio I330 / I350 of at least 1.11; and / or - The degree of protein denaturation is up to 10%.

17. The liquid BLG isolate according to claim 16, wherein The liquid BLG isolate has one or more of the following: - a thermal stability of up to 200 NTU at pH 3.9; and - Up to 15000 colony forming units / g.

18. The liquid BLG isolate according to claim 17, wherein The liquid BLG isolate has at most 1000 colony forming units / g.

19. A method for producing the BLG isolate powder according to any one of claims 1 to 15, wherein the BLG isolate powder is a dry BLG isolate powder having a BLG content of at least 85% w / w relative to the total amount of protein, the method comprising the following steps: a) providing a liquid BLG isolate having a pH of 2 to 4.9, the liquid BLG isolate having a BLG content of at least 85% w / w relative to the total amount of protein, the liquid BLG isolate having: - an intrinsic tryptophan fluorescence emission ratio I330 / I350 of at least 1.11; and / or - protein denaturation is up to 10%; c) Drying the liquid BLG isolate by spray drying.

20. A method for producing the BLG isolate powder of any one of claims 1 to 15, wherein the BLG isolate powder is a dry BLG isolate powder having a BLG content of at least 85% w / w relative to the total amount of protein, the method comprising the steps of: a) providing a liquid BLG isolate having a pH of 2 to 4.9, the liquid BLG isolate having a BLG content of at least 85% w / w relative to the total amount of protein, the liquid BLG isolate having: - an intrinsic tryptophan fluorescence emission ratio I330 / I350 of at least 1.11; and / or - protein denaturation is up to 10%; b) physical reduction of microorganisms on liquid BLG isolate; c) Drying the liquid BLG isolate by spray drying.

21. The method according to claim 19 or 20, wherein: The total solids content of the liquid BLG isolate is 5 to 50% w / w.

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

12.

23. The method according to claim 19 or 20, wherein: The protein of the liquid BLG isolate has a degree of protein denaturation of at most 6% w / w.

24. The method according to claim 19 or 20, wherein: Providing a liquid BLG isolate involves isolating BLG from a whey protein source to obtain a BLG-enriched composition.

25. The method according to claim 24, wherein: The obtained BLG-enriched composition is subjected to one or more steps selected from the group consisting of: - Demineralization; - Added minerals; -dilution; - Physical reduction of microorganisms; - Concentration; and -pH adjustment.

26. The method of claim 20, wherein: The liquid BLG isolate is subjected to physical microbial reduction.

27. The method according to claim 26, wherein: The physical reduction of microorganisms includes heat treatment.

28. The method according to claim 19 or 20, wherein: The liquid BLG isolate comprises at least 10% w / w total protein.

29. Use of the BLG isolate powder of any one of claims 1 to 15 or the liquid BLG isolate of any one of claims 16 to 18 as an ingredient for producing a food having a pH of 2 to 4.

7.

30. The use according to claim 29, wherein The food has one or more of the following: - Reduced dry mouthfeel; - improved transparency; and / or -Increased protein content.

31. The use according to claim 29, wherein The food is a beverage or instant beverage powder.

32. The use according to claim 29, wherein The food product is a heat-treated beverage having a protein content of 3 to 45% w / w.

33. The use according to claim 29, wherein The food product is a heat-treated beverage having a protein content of 11 to 40% w / w.

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