Processes for preparing beta-lactoglobulin isolates and related methods and uses

Abstract

The present invention relates to novel beta-lactoglobulin (BLG) isolates, to a method for producing such isolates and to the use of the powder in, for example, beverage applications.

Description

[0001] This application is a divisional application of the invention patent application with the application date of June 26, 2019, the application number of 201980052749.1, and the title of "Process for Preparing β-Lactoglobulin Isolate and Related Methods and Uses". Technical Field

[0002] The present invention relates to novel β-lactoglobulin (BLG) isolates and methods for producing such isolates, as well as the use of the powder in, for example, beverage applications. Background Art

[0003] BLG is known as the gel-forming component of whey proteins and is prone to unwanted aggregation and gel formation even at pasteurization temperatures. Compared with traditional whey protein isolates, BLG isolates are considered to be more heat-sensitive; traditional whey protein isolates contain more heat-resistant proteins in addition to BLG: α-lactalbumin (ALA) and caseinomacropeptide (CMP). Previously, due to the high cost of BLG isolates and problems in handling freeze-dried BLG isolates, the industrial food uses of BLG isolates have been limited.

[0004] The separation of β-lactoglobulin (BLG) from milk serum or whey is the subject of many published documents, usually involving multiple separation steps and conventional chromatographic techniques to obtain a purified β-lactoglobulin product.

[0005] 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) described the purification of BLG in 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. It is claimed that the resulting BLG composition contains 0.985 g of β-lactoglobulin / 1 g of protein. The BLG composition was dried by freeze-drying.

[0006] 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.

[0007] 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.

[0008] 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. This method is based on acid whey and involves several steps: precipitating unwanted protein salts, adjusting the pH value, and dialysis to remove other unwanted proteins. Finally, when a high - purity BLG solution is obtained, BLG is crystallized. This method lasts for more than 12 days and requires the addition of toluene. Therefore, the procedure disclosed by Palmer is not compatible with food production safety, and moreover, the product it provides is clearly inedible. Palmer reported that BLG crystals can be dried with ethanol and ether.

[0009] EP 0604684 A1 discloses a method for recovering whey protein concentrates rich in α - lactalbumin and / or β - lactoglobulin from whey protein products. The method includes: a) incubating a solution containing the whey protein product with a calcium - binding ion - exchange resin in acid form, causing the destabilization of α - lactalbumin; b) after separating the resin, adjusting the pH of the treated protein product solution to 4.3 - 4.8; c) incubating the protein product solution at a temperature of 10 - 50 °C to promote the flocculation of α - lactalbumin; d) fractionating the proteins 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 raising the pH of the fraction rich in α - lactalbumin to dissolve the α - lactalbumin fraction; and f) optionally, sufficiently raising the pH of the fraction rich in β - lactoglobulin to neutralize the β - lactoglobulin fraction.

[0010] WO2010 / 037736A1 discloses the separation of whey proteins and the preparation of whey products and whey isolates, in particular the separation of β - lactoglobulin products and the separation of whey protein isolates rich in α - lactalbumin from whey obtained from animals. The whey protein isolate rich in α - lactalbumin provided by the present invention has not only a low β - lactoglobulin content but also a high α - lactalbumin and immunoglobulin G content.

[0011] WO2011 / 112695A1 discloses a nutritional composition containing whey protein micelles and leucine. The nutritional composition provides a sufficient amount of leucine to improve protein synthesis in humans, while also maintaining a fluid matrix with low viscosity and acceptable sensory properties. SUMMARY OF THE INVENTION

[0012] The inventors have found that a highly functional BLG isolate powder can be obtained by drying (preferably spray-drying) a liquid BLG isolate having a high BLG content, the pH of the liquid BLG isolate having a high BLG content being: i) 2 to 4.9; or ii) 6.1 to 8.5; or iii) 5.0 to 6.0.

[0013] Accordingly, one aspect of the present invention relates to a BLG isolate powder preferably prepared by spray-drying, the pH of the BLG isolate powder being i) 2.5 to 4.9, ii) 6.1 to 8.5 or iii) 5.0 to 6.0, the BLG isolate powder comprising:

[0014] - at least 30% w / w of the total protein content;

[0015] - at least 85% w / w of β-lactoglobulin (BLG) relative to the total protein content;

[0016] - at most 10% w / w of water;

[0017] The BLG isolate powder has one or more of the following:

[0018] - a bulk density of at least 0.2 g / cm 3 ;

[0019] - an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.11;

[0020] - a degree of protein denaturation of at most 10%;

[0021] - a thermal stability at pH 3.9 of at most 200 NTU; and

[0022] - at most 15,000 colony forming units / g.

[0023] Another aspect of the present invention relates to a liquid BLG isolate, the pH of the liquid BLG isolate being i) 2 to 4.9, ii) 6.1 to 8.5 or iii) 5.0 to 6.0, the liquid BLG isolate comprising:

[0024] - at least 10% w / w of the total protein content;

[0025] - at least 85% w / w of β-lactoglobulin (BLG) relative to the total protein content;

[0026] The BLG isolate powder has one or more of the following:

[0027] - An intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.11;

[0028] - A protein denaturation degree of at most 10%;

[0029] - A thermal stability at pH 3.9 of at most 200 NTU; and

[0030] - At most 1000 colony forming units / g.

[0031] 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 protein amount, the method comprising the steps of:

[0032] a) Providing a liquid BLG isolate having the following characteristics:

[0033] i) A pH of 2 to 4.9;

[0034] ii) A pH of 6.1 to 8.5; or

[0035] iii) A pH of 5.0 to 6.0;

[0036] The liquid BLG isolate has a BLG content of at least 85% w / w relative to the total protein amount;

[0037] b) Optionally, performing a physical microbial reduction on the liquid BLG isolate;

[0038] c) Drying the liquid BLG isolate, preferably by spray drying.

[0039] Another aspect of the present invention relates to the use of the BLG isolate powder or liquid BLG isolate as described herein as an ingredient for producing a food product (such as a beverage or an instant beverage powder), the food product having a pH of 2 to 4.7 and also having one or more of the following:

[0040] - A reduced dry mouthfeel;

[0041] - Improved transparency; and / or

[0042] - An increased protein content, and the protein content of the heat-treated beverage is preferably at least 3 to 45% w / w, more preferably 11 to 40% w / w, even more preferably 15 to 38% w / w, and most preferably 20 to 36% w / w. Description of the Drawings

[0043] Figure 1 Schematic illustration of the method for producing BLG isolate powder of the present invention is provided.

[0044] Figure 2 Schematic illustration of the method for producing BLG isolate powder starting from whey protein raw materials of the present invention is provided, and the terms used in this patent specification are explained.

[0045] Figure 3 Is a micrograph of BLG crystals.

[0046] Figure 4 Micrographs showing intact BLG crystals and fragmented BLG crystals are shown.

[0047] Figure 5 Indicates that the bulk density of spray-dried BLG isolate is higher compared to equivalent WPI dried under the same conditions.

[0048] Figure 6 Indicates that the viscosity of high-protein liquid BLG isolate is lower compared to equivalent WPI solutions. Detailed Description

[0049] Definition

[0050] In the context of the present invention, the term "β-lactoglobulin" or "BLG" relates to β-lactoglobulin from mammalian species, such as present in native, unfolded, and / or glycosylated forms, including naturally occurring genetic variants. The term also includes aggregated BLG, precipitated BLG, and crystalline 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 typically aggregates with other denatured BLG molecules and / or other denatured whey proteins through hydrophobic interactions and / or covalent bonds.

[0051] BLG is the most abundant protein in bovine and caprine whey, and several genetic variants exist. The major variants in milk are labeled A and B. BLG is a lipocalin that can bind a variety of hydrophobic molecules, demonstrating its role in their transport. It has also been shown that BLG can bind iron via a siderophore and may play a role in combating pathogens. The homologue of BLG is absent in human breast milk.

[0052] Bovine BLG is a relatively small protein with approximately 162 amino acid residues and a molecular weight of approximately 18.3 kDa to 18.4 kDa. As measured using nuclear magnetic resonance spectroscopy, under physiological conditions, it is mainly a dimer, but dissociates into monomers at pH values below approximately 3, retaining its native state. However, BLG also exists as tetramers, octamers, and other multimers under various natural conditions.

[0053] 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, for example, in native, unfolded, and / or glycosylated forms, including naturally occurring genetic variants. However, the term does not include aggregated BLG, precipitated BLG, or crystalline BLG. The amount or concentration of non-aggregated BLG is measured according to Example 1.6.

[0054] By calculating (m 总BLG -m 非聚集BLG ) / m 总BLG *100%, the percentage of non-aggregated BLG relative to total BLG is determined. m 总BLG is the concentration or amount of BLG measured according to Example 1.31, and m 非聚集BLG is the concentration or amount of non-aggregated BLG measured according to Example 1.6.

[0055] In the context of the present invention, the term "crystal" relates to a solid material whose components (such as atoms, molecules, or ions) are arranged in a highly ordered microstructure, forming a lattice that extends in all directions.

[0056] In the context of the present invention, the term "BLG crystal" relates to a protein crystal that mainly contains non-aggregated BLG (preferably native BLG) arranged in a highly ordered microstructure, forming a lattice that extends in all directions. For example, a BLG crystal can be single-crystalline or polycrystalline, and can be, for example, a complete crystal, crystal fragment, or a combination thereof. For example, crystal fragments are formed when a complete crystal is mechanically sheared during processing. Crystal fragments also have the highly ordered microstructure of a crystal, but may lack the uniform surface and / or uniform edges or corners of a complete crystal. For example, see Figure 3 As an example of many complete BLG crystals, Figure 4 is an example of a BLG crystal fragment. In both cases, a BLG crystal or crystal fragment can be visually identified using an optical microscope as a well-defined, compact, and quasi-ordered structure. Generally, a BLG crystal or crystal fragment is at least partially transparent. In addition, protein crystals are known to be birefringent, and this optical property can be used to identify unknown particles with a crystal structure. On the other hand, non-crystalline BLG aggregates usually appear as open or porous masses with ill-defined boundaries, opacity, and irregular sizes.

[0057] 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 a seed crystal.

[0058] "mother liquor"

[0059] In the context of the present invention, the term "mother liquor" relates to the whey protein solution remaining after BLG has been crystallized and at least part of the BLG crystals have been removed. The mother liquor may still contain some BLG crystals, but typically only small BLG crystals that have escaped separation.

[0060] In the context of the present invention, the term "edible composition" relates to a composition that can be safely used for human consumption and as a food ingredient and does not contain problematic toxic components (such as toluene or other harmful organic solvents).

[0061] In the context of the present invention, the term "ALA" or "α-lactalbumin" relates to α-lactalbumin from mammalian species, for example in native and / or glycosylated form, including naturally occurring genetic variants. The term also includes aggregated ALA and precipitated BLG. When referring to the amount of ALA, it means the total amount of ALA (including, for example, 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 is typically aggregated with other denatured ALA molecules and / or other denatured whey proteins through hydrophobic interactions and / or covalent bonds.

[0062] α-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 enable LS to produce lactose by transferring a galactose moiety to glucose. One of the main structural differences of β-lactoglobulin is that ALA does not have any free thiol groups that can serve as a starting point for covalent aggregation reactions.

[0063] In the context of the present invention, the term "non-aggregated ALA" also relates to ALA from mammalian species, for example in native, unfolded and / or glycosylated form, including naturally occurring genetic variants. However, the term does not include aggregated ALA or precipitated ALA. The amount or concentration of non-aggregated BLG is determined according to Example 1.6.

[0064] 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.

[0065] In the context of the present invention, the term "caseinomacropeptide" or "CMP" refers to a hydrophilic peptide, residues 106 - 169, derived from the hydrolysis of "κ-CN" or "κ-casein" of mammalian species by an aspartic protease (such as chymosin), for example in natural and / or glycosylated forms, including naturally occurring genetic variants.

[0066] In the context of the present invention, the term "BLG isolate" refers to a composition in which the BLG content is at least 85% w / w relative to the total amount of protein. Preferably, the total amount of protein in the BLG isolate is at least 30% w / w, preferably at least 80% w / w relative to the total amount of solids.

[0067] In the context of the present invention, the term "BLG isolate powder" refers to a BLG isolate in powder form, which is preferably a free-flowing powder.

[0068] In the context of the present invention, the term "BLG isolate liquid" refers to a BLG isolate in liquid form, which is preferably an aqueous solution.

[0069] The term "whey" refers to the liquid phase remaining after the casein in milk has been precipitated and removed. For example, casein is precipitated by acidifying milk and / or using chymosin. There are several types of whey, such as "sweet whey" and "acid whey", where "sweet whey" is a whey product based on the precipitation of casein by chymosin, and "acidic whey" or "acid whey" is a whey product based on the precipitation of casein by acid. For example, casein is precipitated by adding an edible acid or by bacterial culture.

[0070] The term "milk whey" refers to the liquid remaining when casein and milk fat globules are removed from milk, for example, by microfiltration or macroporous ultrafiltration. Milk whey can also be referred to as "ideal whey".

[0071] The term "whey protein" or "serum protein" refers to the proteins present in whey.

[0072] In the context of the present invention, the term "whey protein" refers to the proteins found in whey or milk whey. Whey protein can be a subset of the protein species found in whey or milk whey, even a single whey protein species, or it can be the complete set of protein species found in whey or / and milk whey.

[0073] In the context of the present invention, the main non-BLG proteins of a 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 weight percentages of the main non-BLG whey proteins of a standard whey protein concentrate from sweet whey are:

[0074] 18% w / w of ALA, relative to the total amount of protein;

[0075] 18% w / w of CMP, relative to the total amount of protein;

[0076] 4% w / w of BSA, relative to the total amount of protein;

[0077] 5% w / w of casein, relative to the total amount of protein;

[0078] 6% w / w of immunoglobulins, relative to the total amount of protein;

[0079] 0.5% w / w of osteopontin, relative to the total amount of protein;

[0080] 0.1% w / w of lactoferrin, relative to the total amount of protein; and

[0081] 0.1% w / w of lactoperoxidase, relative to the total amount of protein.

[0082] The term "casein" refers to the caseins found in milk, including the two native micellar caseins found in raw milk, individual casein species and caseinates.

[0083] 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 field of crystallization (see, for example, Gérard Coquerela, Crystallization of molecular systems from solution: phase diagrams, supersaturation and other basic concepts, Chemical Society Reviews 2014, pages 2286-2300, issue 7), and supersaturation can be determined by a variety of different measurement techniques (such as by spectroscopy or particle size analysis). In the context of the present invention, the supersaturation of BLG is determined by the following procedure.

[0084] Procedure for detecting whether BLG is supersaturated in a liquid under specific conditions:

[0085] 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 keep the sample and its subsequent fractions under the original physical and chemical conditions of the liquid;

[0086] b) Immediately centrifuge the sample at 3000 g for 3.0 minutes, with an acceleration of at most 30 seconds and a deceleration of at most 30 seconds;

[0087] c) Immediately after centrifugation, transfer as much supernatant as possible to a second centrifuge tube (of the same type as in step a) (do not disturb the precipitate if it has formed);

[0088] d) Take a 0.05 mL subsample of the supernatant (subsample A);

[0089] e) Add 10 mg of BLG crystals with a particle size of at most 200 μm (non-aggregated BLG with a purity of at least 98% relative to the total solid content) to the second centrifuge tube and stir the mixture;

[0090] f) Let the second centrifuge tube stand at the original temperature for 60 minutes;

[0091] g) Immediately after step f), centrifuge the second centrifuge tube at 500 g for 10 minutes, and then take another 0.05 mL subsample of the supernatant (subsample B);

[0092] h) Recover the centrifugation precipitate (if any) from step g), resuspend it in MilliQ water, and immediately check for the presence of microscopically observable crystals in the suspension;

[0093] i) Determine the concentration of non-aggregated BLG in subsamples A and B using the method outlined in Example 1.6 – the results are expressed as % w / w of BLG relative to the total weight of the subsample. The concentration of non-aggregated BLG in subsample A is designated as C BLG，A , and the concentration of non-aggregated BLG in subsample B is designated as C BLG，B ;

[0094] j) If C BLG，B is lower than C BLG，A and crystals are observed in step i), then the liquid (under specific conditions) from which the sample in step a) was taken is supersaturated.

[0095] In the context of the present invention, the terms "liquid" and "solution" include: compositions free of particulate matter, as well as compositions comprising a combination of a liquid with solid and / or semi-solid particles (such as protein crystals or other protein particles). Thus, a "liquid" or "solution" can be a suspension or even a slurry. However, "liquid" and "solution" are preferably pumpable.

[0096] 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 from 20 to 89% w / w of protein, based on the total solids.

[0097] Preferably, the WPC or SPC comprises:

[0098] from 20 to 89% w / w of protein, based on the total solids;

[0099] from 15 to 70% w / w of BLG, based on the total protein;

[0100] from 8 to 50% w / w of ALA, based on the total protein; and

[0101] from 0 to 40% w / w of CMP, based on the protein.

[0102] Alternatively, but also preferably, the WPC or SPC may comprise:

[0103] from 20 to 89% w / w of protein, based on the total solids;

[0104] from 15 to 90% w / w of BLG, based on the total protein;

[0105] from 4 to 50% w / w of ALA, based on the total protein; and

[0106] from 0 to 40% w / w of CMP, based on the protein.

[0107] Preferably, the WPC or SPC comprises:

[0108] from 20 to 89% w / w of protein, based on the total solids;

[0109] from 15 to 80% w / w of BLG, based on the total protein;

[0110] from 4 to 50% w / w of ALA, based on the total protein; and

[0111] from 0 to 40% w / w of CMP, based on the protein.

[0112] More preferably, the WPC or SPC comprises:

[0113] from 70 to 89% w / w of protein, based on the total solids;

[0114] from 30 to 90% w / w of BLG, based on the total protein;

[0115] from 4 to 35% w / w of ALA, based on the total protein; and

[0116] 0 to 25% w / w CMP, relative to the protein.

[0117] Typically, SPC does not contain CMP or contains only trace amounts of CMP.

[0118] The terms "whey protein isolate" (WPI) and "serum protein isolate" (SPI) refer to a dry composition or an aqueous composition that contains 90 to 100% w / w protein in total relative to the total solids.

[0119] Preferably, WPI or SPI contains:

[0120] 90 to 100% w / w protein, relative to the total solids;

[0121] 15 to 70% w / w BLG, relative to the total protein;

[0122] 8 to 50% w / w ALA, relative to the total protein; and

[0123] 0 to 40% w / w CMP, relative to the total protein.

[0124] Alternatively, but also preferably, WPI or SPI may contain:

[0125] 90 to 100% w / w protein, relative to the total solids;

[0126] 30 to 95% w / w BLG, relative to the total protein;

[0127] 4 to 35% w / w ALA, relative to the total protein; and

[0128] 0 to 25% w / w CMP, relative to the total protein.

[0129] Preferably, WPI or SPI contains:

[0130] 90 to 100% w / w protein, relative to the total solids;

[0131] 30 to 90% w / w BLG, relative to the total protein;

[0132] 4 to 35% w / w ALA, relative to the total protein; and

[0133] 0 to 25% w / w CMP, relative to the total protein.

[0134] Typically, SPI does not contain CMP or contains only trace amounts of CMP.

[0135] In the context of the present invention, the term "other proteins" refers to proteins other than BLG. Generally, the other proteins present in a whey protein solution comprise one or more non-BLG proteins found in whey or milk serum. Non-limiting examples of such proteins are α-lactalbumin, bovine serum albumin, immunoglobulins, caseinomacropeptide (CMP), osteopontin, lactoferrin, and milk fat globule membrane proteins.

[0136] The terms "consisting essentially of" and "consisting essentially of" mean that the claimed claim or feature covers the specified materials or steps and those materials or steps that do not materially affect the basic and novel features of the claimed invention.

[0137] 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 "n 1 、n 2 、...、n i-1 and / or n i ” means "n 1 ” or "n 2 ” or... or "n i-1 ” or "n i ” or any combination of the components n 1 、n 2 、...、n i-1 and n i ”.

[0138] In the context of the present invention, the term "dry" means that the composition or product under study contains at most 10% w / w, preferably at most 6% w / w, more preferably even less water.

[0139] In the context of the present invention, the term "physically reducing microorganisms" relates to physical interaction with the composition, resulting in a reduction in the total amount of viable microorganisms in the composition. This term does not include the addition of chemicals that kill microorganisms. In addition, this term does not include the thermal exposure of the atomized droplets of the liquid during spray drying, but includes possible preheating before spray drying.

[0140] In the context of the present invention, the pH of the powder refers to the pH of 10 g of powder mixed into 90 g of demineralised water, measured according to Example 1.16.

[0141] In the context of the present invention, unless specifically referred to another reference (e.g., total solids or total protein), the weight percentage (% w / w) of a component of a certain composition, product or material refers to the weight percentage of that component relative to the specific composition, product or material.

[0142] In the context of the present invention, the method step "concentration" and the verb "concentrate" refer to the concentration of proteins, including protein concentration based on the total solids and protein concentration based on the total weight. For example, this means that concentration does not necessarily require an absolute increase in the protein concentration w / w of the composition, as long as the protein content increases relative to the total solids.

[0143] In the context of the present invention, the term "weight ratio" between component X and component Y refers to the value obtained by calculating m X / m Y where m X is the amount (weight) of component X and m Y is the amount (weight) of component Y.

[0144] In the context of the present invention, the term "at least pasteurized" relates to a heat treatment having a microbicidal effect by applying a heat treatment equal to or higher than 70 °C for 10 seconds. The reference for determining the bactericidal effect is Escherichia coli O157:H7.

[0145] In the context of the present invention, the term "whey protein raw material" relates to the whey protein source of a liquid BLG isolate. Compared with the liquid BLG isolate, the content of BLG in the whey protein raw material is lower relative to the total amount of proteins. Usually, the whey protein raw material is WPC, WPI, SPC or SPI.

[0146] In the context of the present invention, the term "BLG-rich composition" relates to a BLG-rich composition obtained by separating BLG from a whey protein raw material. Usually, the BLG-rich composition contains the same whey proteins as the whey protein raw material; however, compared with the whey protein raw material, the BLG is present in the composition at a significantly higher concentration (relative to the total amount of proteins). For example, a BLG-rich composition can be prepared from a whey protein raw material by chromatography, protein crystallization and / or membrane-based protein fractionation. The BLG-rich composition contains at least 85% w / w, preferably at least 90% w / w of BLG relative to the total amount of proteins. In some cases, the BLG-rich composition can be directly used as a liquid BLG isolate. However, usually additional treatment is required to convert the BLG-rich composition into a liquid BLG isolate.

[0147] In the context of the present invention, the term "whey protein solution" is used to describe a special aqueous whey protein composition in which the BLG is supersaturated in a salting-in mode and can be used for the preparation of BLG crystals.

[0148] In the context of the present invention, the term "sterile" means that the studied sterile composition or product does not contain any living microorganisms and thus there is no growth of microorganisms during storage at room temperature. A sterilized composition is sterile.

[0149] When a liquid (such as a beverage preparation) is sterilized and aseptically packaged in a sterile container, the liquid generally has a shelf life of at least six months at room temperature. The sterilization process can kill spores and microorganisms that may cause the liquid to deteriorate.

[0150] In the context of the present invention, the term "protein fraction" relates to the proteins of the composition under study, such as the proteins in a powder or a beverage preparation.

[0151] In the context of the present invention, the term "dry mouthfeel" relates to the sensation in the mouth that feels like dryness in the mouth and teeth and results in minimal saliva production.

[0152] Therefore, the dry mouthfeel is not a taste per se, but a physical mouthfeel and time-related sensation in the mouth.

[0153] In the context of the present invention, unless otherwise specified, the term "mineral" as used herein refers to any of macrominerals, trace minerals or microminerals, other minerals, and combinations thereof. Major minerals include: calcium, phosphorus, potassium, sulfur, sodium, chlorine, magnesium. Trace minerals or microminerals include: iron, cobalt, copper, zinc, molybdenum, iodine, selenium, manganese. Other minerals include: chromium, fluorine, boron, lithium, and strontium.

[0154] In the context of the present invention, unless otherwise specified, the terms "lipid", "fat", and "oil" are used interchangeably and refer to lipid materials derived from plants or animals or obtained by processing plants or animals. These terms also include synthetic lipid materials, provided that such synthetic materials are suitable for human consumption.

[0155] In the context of the present invention, the term "transparent" includes a beverage preparation having a visually clear appearance and allowing light to pass through and different images to appear through it. The turbidity of a transparent beverage is at most 200 NTU.

[0156] In the context of the present invention, the term "opaque" includes a beverage preparation having a visually unclear appearance and a turbidity greater than 200 NTU.

[0157] 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:

[0158] - at least 30% w / w of the total amount of protein;

[0159] - at least 85% w / w of β-lactoglobulin (BLG) relative to the total amount of protein; and

[0160] - Up to 10% w / w of water.

[0161] Preferably, the BLG isolate powder has one or more of the following:

[0162] - A bulk density of at least 0.2 g / cm 3 ;

[0163] - An intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.11;

[0164] - A degree of protein denaturation of at most 10%;

[0165] - A thermal stability at pH 3.9 of at most 200 NTU; and

[0166] - Up to 1000 colony forming units / g.

[0167] Preferably, the BLG isolate powder is an edible composition.

[0168] In some preferred embodiments of the present invention, the pH of the BLG isolate powder is 2 to 4.9. Such a powder is particularly useful for acidic foods (especially acidic beverages).

[0169] In other preferred embodiments of the present invention, the pH of the BLG isolate powder is 6.1 to 8.5.

[0170] In some preferred embodiments of the present invention, the total protein content of 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.

[0171] Even higher protein contents may be required; in some preferred embodiments of the present 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.

[0172] The total protein content is measured according to Example 1.5.

[0173] In some preferred embodiments of the present invention, relative to the total protein content, the BLG isolate powder contains 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 of BLG.

[0174] In some preferred embodiments of the present invention, the BLG isolate powder contains 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) of BLG, relative to the total amount of protein.

[0175] In some preferred embodiments of the present invention, the sum of α-lactalbumin (ALA) and caseinomacropeptide (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 powder.

[0176] 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, has a weight percentage 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.

[0177] An even lower concentration of the major non-BLG whey proteins may be desirable. Thus, 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, has a weight percentage 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.

[0178] In some preferred embodiments of the present invention, ALA accounts for 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 of the BLG isolate powder.

[0179] An even lower content of ALA may be preferred; thus, in some preferred embodiments of the present invention, ALA accounts for 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 proteins of the BLG isolate powder.

[0180] The inventors have observed that reducing lactoferrin and / or lactoperoxidase is particularly advantageous for obtaining a color-neutral whey protein product.

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

[0182] Similarly, in some preferred embodiments of the present invention, lactoperoxidase is present as a weight percentage relative to the total amount of protein, and relative to the total amount of protein in a standard whey protein concentrate from sweet whey, the weight percentage of lactoperoxidase is at most 25%, preferably at most 20%, more preferably at most 15%, even more preferably at most 10%, and most preferably at most 6%. Even lower concentrations of lactoperoxidase may be required. Thus, in other preferred embodiments of the present invention, lactoperoxidase is present as a weight percentage relative to the total amount of protein, and relative to the total amount of protein in a standard whey protein concentrate from sweet whey, the weight percentage of lactoperoxidase is at most 4%, preferably at most 3%, more preferably at most 2%, and even more preferably at most 1%.

[0183] Lactoferrin and lactoperoxidase were quantified according to Example 1.29.

[0184] 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, and most preferably at most 2% w / w.

[0185] 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, and most preferably at most 0.1%. For example, the BLG isolate powder may contain carbohydrates such as lactose, oligosaccharides, and / or hydrolysis products of lactose (i.e., glucose and galactose), sucrose, and / or maltodextrin.

[0186] 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, and even more preferably at most 0.1% w / w.

[0187] The lipid content was measured according to ISO 1211:2010 (Determination of Fat Content – - Gottlieb gravimetric method, Determination of Fat Content – - Gottlieb Gravimetric Method).

[0188] The inventors have found that it may be advantageous to control the mineral content to achieve certain desired properties of the BLG isolate powder.

[0189] 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 of protein. Preferably, the total amount of Na, K, Mg, and Ca in the BLG isolate powder is at most 6 mmol / g of protein, more preferably at most 4 mmol / g of protein, and even more preferably at most 2 mmol / g of protein.

[0190] 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 of protein. Preferably, the total amount of Na, K, Mg, and Ca in the BLG isolate powder is at most 0.6 mmol / g of protein, more preferably at most 0.4 mmol / g of protein, even more preferably at most 0.2 mmol / g of protein, and most preferably at most 0.1 mmol / g of 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 5 mmol / g of protein. Preferably, the total amount of Mg and Ca in the BLG isolate powder is at most 3 mmol / g of protein, more preferably at most 1.0 mmol / g of protein, and even more preferably at most 0.5 mmol / g of protein.

[0192] 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 of protein. Preferably, the total amount of Mg and Ca in the BLG isolate powder is at most 0.2 mmol / g of protein, more preferably at most 0.1 mmol / g of protein, even more preferably at most 0.03 mmol / g of protein, and most preferably 0.01 mmol / g of protein.

[0193] The inventors have found that a low-phosphorus / low-potassium variant of the 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 similarly low amounts of phosphorus and potassium.

[0194] Thus, 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.

[0195] In some preferred embodiments of the present invention, the BLG isolate powder contains at most 600 mg potassium / 100 g protein. More preferably, the BLG isolate powder contains at most 500 mg potassium / 100 g protein. More preferably, the BLG isolate powder contains at most 400 mg potassium / 100 g protein. More preferably, the BLG isolate powder contains at most 300 mg potassium / 100 g protein. Even more preferably, the BLG isolate powder contains at most 200 mg potassium / 100 g protein. Even more preferably, the BLG isolate powder contains at most 100 mg potassium / 100 g protein. Even more preferably, the BLG isolate powder contains at most 50 mg potassium / 100 g protein. Even more preferably, the BLG isolate powder contains at most 10 mg potassium / 100 g protein.

[0196] The phosphorus content is related to the total amount of elemental phosphorus in the composition under study, measured according to Example 1.19. Similarly, the potassium content is related to the total amount of elemental potassium in the composition under study, measured according to Example 1.19.

[0197] In some preferred embodiments of the present invention, the BLG isolate powder contains 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 contains at most 100 mg phosphorus / 100 g protein and at most 340 mg potassium / 100 g protein.

[0198] The low-phosphorus and / or low-potassium compositions of the present invention can be used as food ingredients for producing foods for patient groups with reduced kidney function.

[0199] The present inventors have found that for certain applications (such as acidic foods, especially acidic beverages), it is particularly advantageous for the pH of the acidic BLG isolate powder to be at most 4.9, even more preferably at most 4.3. This is especially true for high-protein, clear acidic beverages.

[0200] In the context of the present invention, the turbidity of the clear liquid measured according to Example 1.7 is at most 200 NTU.

[0201] Thus, in some preferred embodiments of the present invention, the pH of the BLG isolate powder is from 2 to 4.9. Preferably, the pH of the BLG isolate powder is from 2.5 to 4.7, more preferably from 2.8 to 4.3, even more preferably from 3.2 to 4.0, and most preferably from 3.4 to 3.9. Alternatively, but also preferably, the pH of the BLG isolate powder can be from 3.6 to 4.3.

[0202] The present inventors have found that for certain applications (such as pH-neutral foods, especially pH-neutral beverages), a pH-neutral BLG isolate powder is particularly advantageous. This is especially true for high-protein, clear or opaque pH-neutral beverages.

[0203] Thus, in some preferred embodiments of the present invention, the pH of the BLG isolate powder is from 6.1 to 8.5. Preferably, the pH of the powder is from 6.1 to 8.5, more preferably from 6.2 to 8.0, even more preferably from 6.3 to 7.7, and most preferably from 6.5 to 7.5.

[0204] In other preferred embodiments of the present invention, the pH of the BLG isolate powder is from 5.0 to 6.0. Preferably, the pH of the powder is from 5.1 to 5.9, more preferably from 5.2 to 5.8, even more preferably from 5.3 to 5.7, and most preferably from 5.4 to 5.6.

[0205] Advantageously, the bulk density of the BLG isolate powder of the present invention can 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 .

[0206] Low-density powders (such as freeze-dried BLG isolates) are fluffy and prone to getting into the air in the production area during use. This is a problem because it increases the risk of cross-contamination of the freeze-dried powder with other foods, and a dusty environment is known to cause hygiene problems. In extreme cases, a dusty environment can also increase the risk of dust explosion.

[0207] The high-density variants of the present invention are easier to handle and less likely to escape into the surrounding air.

[0208] Another advantage of the high-density variants of the present invention is that they occupy less space during transportation, thereby increasing the weight of the BLG isolate powder that can be transported per unit volume.

[0209] Another advantage of the high-density variants of the present invention is that when used in a powder mixture with other powdered food ingredients, they are less prone to segregation. Examples of powdered food ingredients include powdered sugar (bulk density of about 0.56 g / cm 3 ), granulated sugar (bulk density of about 0.71 g / cm 3 ), and citric acid powder (bulk density of about 0.77 g / cm 3 ).

[0210] For example, the bulk density of the BLG isolate powder of the present invention can be 0.2 - 1.0 g / cm 3 , preferably 0.30 - 0.9 g / cm 3 , more preferably 0.40 - 0.8 g / cm 3 , even more preferably 0.45 - 0.75 g / cm 3 , even more preferably 0.50 - 0.75 g / cm 3 , most preferably 0.6 - 0.75 g / cm 3 .

[0211] In some preferred embodiments of the present invention, the bulk density of the BLG isolate powder is 0.45 - 1.2 g / cm 3 , preferably 0.46 - 1.0 g / cm 3 , more preferably 0.47 - 0.8 g / cm 3 , even more preferably 0.48 - 0.75 g / cm 3 , even more preferably 0.48 - 0.6 g / cm 3 , most preferably 0.50 - 0.6 g / cm 3 .

[0212] The bulk density of the powder is measured according to Example 1.17.

[0213] The inventors have found that it is advantageous to maintain the native conformation of BLG when BLG is used in acidic beverages, and the inventors have observed that an increase in the unfolding of BLG leads to an increase in dry mouthfeel.

[0214] The intrinsic tryptophan fluorescence emission ratio (I330 / I350) is a measure of the degree of unfolding of BLG; the inventors have found that at a high intrinsic tryptophan fluorescence emission ratio (associated with low or non-unfolded BLG), a lower dry mouthfeel is observed. The intrinsic tryptophan fluorescence emission ratio (I330 / I350) is measured according to Example 1.1.

[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 1.11.

[0216] 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, and most preferably at least 1.19.

[0217] 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. Thus, 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.

[0218] 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.

[0219] For example, the protein fraction can be separated from the BLG isolate powder by dissolving the BLG isolate powder in demineralized water and dialyzing or ultrafiltration-based diafiltering the solution using a protein-retaining filter. If the BLG isolate powder contains an interfering level of lipids, the lipids can be removed, for example, by microfiltration. The microfiltration and ultrafiltration / diafiltration steps can be combined to remove lipids and small molecules from the protein fraction.

[0220] Generally preferably, a large amount of 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, approximately 100% of the BLG in the BLG isolate powder is non-aggregated BLG.

[0221] In some preferred embodiments of the present invention, the degree of protein denaturation 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%.

[0222] However, for example, if an opaque beverage is desired, it may also be preferred for the BLG isolate powder to have a significant level of protein denaturation. Thus, in other preferred embodiments of the present invention, the degree of 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%, and most preferably at least 90%.

[0223] If the BLG isolate powder has a significant level of protein denaturation, it is generally preferred to maintain a low level of insoluble protein material (i.e., precipitated protein material), which will precipitate in the beverage during storage. The content of the insoluble material is measured according to Example 1.10.

[0224] In some preferred embodiments of the present invention, the BLG isolate powder contains at most 20% w / w of insoluble protein material, preferably at most 10% w / w of insoluble protein material, more preferably at most 5% w / w of insoluble protein material, even more preferably at most 3% w / w of insoluble protein material, and most preferably at most 1% w / w of insoluble protein material. Even more preferably, the BLG isolate powder does not contain any insoluble protein material at all.

[0225] The inventors have found that the thermal stability of the BLG isolate powder at pH 3.9 is a good indication of its usefulness for clear high-protein beverages. The thermal stability at pH 3.9 is measured according to Example 1.2.

[0226] 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, and 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, and even more preferably at most 2 NTU.

[0227] The microbial content of the BLG isolate powder is preferably kept to a minimum. However, it is challenging to obtain both a high degree of protein nativeness and a low microbial content simultaneously, since the sterilization process tends to cause unfolding and denaturation of the protein. The present invention enables a very low microbial content to be obtained while maintaining a high level of BLG nativeness.

[0228] In some embodiments of the present invention, the liquid BLG isolate contains up to 500,000 CFU / g, preferably up to 100,000 CFU / g, more preferably up to 50,000 CFU / g, and even more preferably up to 25,000 CFU / g.

[0229] Even lower levels of microorganisms are also preferred. Thus, in some preferred embodiments of the present invention, the BLG isolate powder contains up to 15,000 colony forming units (CFU) / g. Preferably, the BLG isolate powder contains up to 10,000 CFU / g. More preferably, the BLG isolate powder contains up to 5,000 CFU / g. Even more preferably, the BLG isolate powder contains up to 1,000 CFU / g. Even more preferably, the BLG isolate powder contains up to 300 CFU / g. Most preferably, the BLG isolate powder contains up to 100 CFU / g, such as up to 10 CFU / g. In particularly preferred embodiments, the powder is sterile. For example, a sterile BLG isolate powder can be prepared by combining several physical methods for reducing microorganisms (such as microfiltration and heat treatment at acidic pH) during the production of the BLG isolate powder. Preferably, the drying is carried out in a sterile drying system (such as a sterile spray dryer).

[0230] 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 contains:

[0231] - at least 30% w / w, preferably at least 80% w / w, and even more preferably at least 90% w / w of the total protein;

[0232] - at least 85% w / w, preferably at least 90% w / w of β-lactoglobulin (BLG) relative to the total protein;

[0233] - up to 6% w / w of water;

[0234] - up to 2% w / w, preferably up to 0.5% w / w of lipids;

[0235] The BLG isolate powder has:

[0236] - an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.11;

[0237] - a degree of protein denaturation of at most 10%; and

[0238] - a thermal stability at pH 3.9 of at most 200 NTU.

[0239] 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:

[0240] - at least 30% w / w, preferably at least 80% w / w, even more preferably at least 90% w / w of the total protein content;

[0241] - at least 85% w / w, preferably at least 90% w / w, more preferably at least 94% w / w of β-lactoglobulin (BLG) relative to the total protein content;

[0242] - at most 6% w / w of water;

[0243] - at most 2% w / w, preferably at most 0.5% w / w of lipids;

[0244] The BLG isolate powder has:

[0245] - an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.11;

[0246] - a degree of protein denaturation of at most 10%, preferably at most 5%; and

[0247] - a thermal stability at pH 3.9 of at most 70 NTU, preferably at most 50 NTU, even more preferably at most 40 NTU.

[0248] 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:

[0249] - at least 30% w / w of the total protein content;

[0250] - at least 85% w / w, preferably at least 90% w / w of β-lactoglobulin (BLG) relative to the total protein content;

[0251] - at most 6% w / w of water;

[0252] The BLG isolate powder has:

[0253] - a bulk density of at least 0.2 g / cm 3 ;

[0254] - an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.11;

[0255] - a degree of protein denaturation of at most 10%; and

[0256] - a thermal stability at pH 3.9 of at most 200 NTU.

[0257] In other preferred embodiments of the present invention, the pH of the BLG isolate powder is 2 to 4.9, and the BLG isolate powder comprises:

[0258] - at least 80% w / w, preferably at least 90% w / w, even more preferably at least 94% w / w of the total protein content;

[0259] - at least 85% w / w, preferably at least 90% w / w, more preferably at least 94% w / w of β-lactoglobulin (BLG) relative to the total protein content;

[0260] - at most 6% w / w of water;

[0261] - at most 2% w / w, preferably at most 0.5% w / w of lipids;

[0262] The BLG isolate powder has:

[0263] - a bulk density of at least 0.2 g / cm 3 , preferably at least 0.3 g / cm 3 , more preferably at least 0.4 g / cm 3 ;

[0264] - an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.11;

[0265] - a degree of protein denaturation of at most 10%, preferably at most 5%, more preferably at most 2%; and

[0266] - a thermal stability at pH 3.9 of at most 50 NTU, preferably at most 30 NTU, even more preferably at most 10 NTU.

[0267] In other preferred embodiments 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:

[0268] - at least 80% w / w, preferably at least 90% w / w, even more preferably at least 94% w / w of the total protein content;

[0269] - at least 90% w / w, preferably at least 92% w / w, even more preferably at least 94% w / w of β-lactoglobulin (BLG) relative to the total protein content;

[0270] - at most 6% w / w of water;

[0271] - at most 2% w / w, preferably at most 0.5% w / w of lipids;

[0272] The BLG isolate powder has:

[0273] - At least 0.2 g / cm 3 、Preferably at least 0.3 g / cm 3 、More preferably at least 0.4 g / cm 3 of bulk density;

[0274] - The intrinsic tryptophan fluorescence emission ratio (I330 / I350) is at least 1.11, preferably at least 1.13;

[0275] - The degree of protein denaturation is at most 10%, preferably at most 5%, more preferably at most 2%; and

[0276] - The thermal stability at pH 3.9 is at most 50 NTU, preferably at most 30 NTU, even more preferably at most 10 NTU.

[0277] In other preferred embodiments of the present invention, the pH of the BLG isolate powder is 6.1 to 8.5, and the BLG isolate powder comprises:

[0278] - At least 80% w / w, preferably at least 90% w / w, even more preferably at least 94% w / w of the total protein;

[0279] - Relative to the total protein, at least 85% w / w, preferably at least 90% w / w, more preferably at least 94% w / w of β-lactoglobulin (BLG);

[0280] - At most 6% w / w of water;

[0281] - At most 2% w / w, preferably at most 0.5% w / w of lipids;

[0282] The BLG isolate powder has:

[0283] - At least 0.2 g / cm 3 、Preferably at least 0.3 g / cm 3 、More preferably at least 0.4 g / cm 3 of bulk density;

[0284] - The degree of protein denaturation is at most 10%, preferably at most 5%, more preferably at most 2%; and

[0285] - The thermal stability at pH 3.9 is at most 50 NTU, preferably at most 30 NTU, even more preferably at most 10 NTU.

[0286] In other preferred embodiments of the present invention, the pH of the BLG isolate powder is 6.1 to 8.5, and the BLG isolate powder comprises:

[0287] - At least 80% w / w, preferably at least 90% w / w, even more preferably at least 94% w / w of the total protein;

[0288] - With respect to the total protein, at least 85% w / w, preferably at least 90% w / w, more preferably at least 94% w / w of β-lactoglobulin (BLG);

[0289] - At most 6% w / w of water;

[0290] - At most 2% w / w, preferably at most 0.5% w / w of lipids;

[0291] The BLG isolate powder has:

[0292] - A bulk density of at least 0.2 g / cm 3 , preferably at least 0.3 g / cm 3 , more preferably at least 0.4 g / cm 3 ;

[0293] - A degree of protein denaturation of at most 10%, preferably at most 5%, more preferably at most 2%; and

[0294] - A thermal stability at pH 3.9 of at most 50 NTU, preferably at most 30 NTU, even more preferably at most 10 NTU.

[0295] In other preferred embodiments of the present invention, the pH of the BLG isolate powder is 5.0 to 6.0, and the BLG isolate powder comprises:

[0296] - At least 80% w / w, preferably at least 90% w / w, even more preferably at least 94% w / w of the total protein;

[0297] - With respect to the total protein, at least 85% w / w, preferably at least 90% w / w, more preferably at least 94% w / w of β-lactoglobulin (BLG);

[0298] - At most 6% w / w of water;

[0299] - At most 2% w / w, preferably at most 0.5% w / w of lipids;

[0300] The BLG isolate powder has:

[0301] - A bulk density of at least 0.2 g / cm 3 , preferably at least 0.3 g / cm 3 , more preferably at least 0.4 g / cm 3 ;

[0302] - A degree of protein denaturation of at most 10%, preferably at most 5%, more preferably at most 2%;

[0303] - The thermal stability at pH 3.9 is at most 50 NTU, preferably at most 30 NTU, even more preferably at most 10 NTU; and

[0304] - Preferably, the BLG crystallinity is less than 10%.

[0305] In some preferred embodiments of the present invention, the pH of the BLG isolate powder is i) 3.0 to 4.3, ii) 6.5 to 7.5 or iii) 5.0 to 6.0, and the BLG isolate powder comprises:

[0306] - At least 90% w / w, preferably at least 92% w / w, even more preferably at least 94% w / w of the total protein;

[0307] - At least 92% w / w, preferably at least 94% w / w of β-lactoglobulin (BLG) relative to the total protein;

[0308] - At most 6% w / w of water;

[0309] - At most 0.5% w / w, preferably at most 0.1% w / w of lipids;

[0310] The BLG isolate powder has:

[0311] - An intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.15;

[0312] - A degree of protein denaturation of at most 5%;

[0313] - A thermal stability at pH 3.9 of at most 40 NTU; and

[0314] - At most 15000 colony forming units / g, preferably at most 1000 colony forming units / g, more preferably at most 100 colony forming units / g, and most preferably the BLG isolate powder is sterile.

[0315] In some preferred embodiments of the present invention, the pH of the BLG isolate powder is i) 3.0 to 4.3, ii) 6.5 to 7.5 or iii) 5.0 to 6.0, and the BLG isolate powder comprises:

[0316] - At least 90% w / w, preferably at least 92% w / w, even more preferably at least 94% w / w of the total protein;

[0317] - At least 92% w / w, preferably at least 94% w / w of β-lactoglobulin (BLG) relative to the total protein;

[0318] - At most 6% w / w of water;

[0319] - Lipids at up to 0.5% w / w, preferably up to 0.1% w / w;

[0320] The BLG isolate powder has:

[0321] - A degree of protein denaturation of up to 5%;

[0322] - A thermal stability at pH 3.9 of up to 40 NTU; and

[0323] - Up to 15,000 colony forming units / g, preferably up to 1,000 colony forming units / g, more preferably up to 100 colony forming units / g, and most preferably the BLG isolate powder is sterile.

[0324] In some preferred embodiments of the present invention, the pH of the BLG isolate powder is i) 3.0 to 4.3, ii) 6.5 to 7.5 or iii) 5.0 to 6.0, and the BLG isolate powder comprises:

[0325] - A total protein content of at least 90% w / w, preferably at least 92% w / w, even more preferably at least 94% w / w;

[0326] - β-lactoglobulin (BLG) of at least 92% w / w, preferably at least 94% w / w, relative to the total protein content;

[0327] - Water at up to 6% w / w;

[0328] - Lipids at up to 0.5% w / w, preferably up to 0.1% w / w;

[0329] The BLG isolate powder has:

[0330] - An intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.15;

[0331] - A thermal stability at pH 3.9 of up to 40 NTU; and

[0332] - Up to 15,000 colony forming units / g, preferably up to 1,000 colony forming units / g, more preferably up to 100 colony forming units / g, and most preferably the BLG isolate powder is sterile.

[0333] In some preferred embodiments of the present invention, the pH of the BLG isolate powder is i) 3.0 to 4.3, ii) 6.5 to 7.5 or iii) 5.0 to 6.0, and the BLG isolate powder comprises:

[0334] - A total protein content of at least 90% w / w, preferably at least 92% w / w, even more preferably at least 94% w / w;

[0335] - At least 92% w / w, preferably at least 94% w / w of β-lactoglobulin (BLG), relative to the total amount of protein;

[0336] - At most 6% w / w of water;

[0337] - At most 0.5% w / w, preferably at most 0.1% w / w of lipids;

[0338] The BLG isolate powder has:

[0339] - An intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.15;

[0340] - A degree of protein denaturation of at most 5%; and

[0341] - At most 15,000 colony forming units / g, preferably at most 1,000 colony forming units / g, more preferably at most 100 colony forming units / g, and most preferably the BLG isolate powder is sterile.

[0342] In some preferred embodiments of the present invention, the pH of the BLG isolate powder is i) 3.0 to 4.3, ii) 6.5 to 7.5 or iii) 5.0 to 6.0, and the BLG isolate powder comprises:

[0343] - At least 90% w / w, preferably at least 92% w / w, even more preferably at least 94% w / w of the total amount of protein;

[0344] - At least 92% w / w, preferably at least 94% w / w of β-lactoglobulin (BLG), relative to the total amount of protein;

[0345] - At most 6% w / w of water;

[0346] - At most 0.5% w / w, preferably at most 0.1% w / w of lipids;

[0347] The BLG isolate powder has:

[0348] - An intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.15;

[0349] - A degree of protein denaturation of at most 5%; and

[0350] - A thermal stability at pH 3.9 of at most 40 NTU.

[0351] In some preferred embodiments of the present invention, the pH of the BLG isolate powder is i) 3.0 to 4.3, ii) 6.5 to 7.5 or iii) 5.0 to 6.0, and the BLG isolate powder comprises:

[0352] - Total protein content of at least 90% w / w, preferably at least 92% w / w, even more preferably at least 94% w / w;

[0353] - β-lactoglobulin (BLG) of at least 92% w / w, preferably at least 94% w / w, relative to the total protein content;

[0354] - Water of at most 6% w / w;

[0355] - Lipids of at most 0.5% w / w, preferably at most 0.1% w / w;

[0356] The BLG isolate powder has:

[0357] - Intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.15;

[0358] - Thermal stability at pH 3.9 of at most 40 NTU; and

[0359] - At most 15,000 colony forming units / g, preferably at most 1,000 colony forming units / g, more preferably at most 100 colony forming units / g, and most preferably the BLG isolate powder is sterile.

[0360] 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 the BLG isolate powder comprises:

[0361] - Total protein content of at least 30% w / w, preferably at least 50% w / w, even more preferably at least 80% w / w;

[0362] - β-lactoglobulin (BLG) of at least 90% w / w, more preferably at least 94% w / w, relative to the total protein content

[0363] - Water of at most 6% w / w;

[0364] - Lipids of at most 0.5% w / w, preferably at most 0.1% w / w;

[0365] The BLG isolate powder has:

[0366] - Intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.15;

[0367] - Degree of protein denaturation of at most 5%, preferably at most 2%; and

[0368] - Thermal stability at pH 3.9 of at most 40 NTU, preferably at most 20 NTU, even more preferably at most 10 NTU.

[0369] 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 it contains:

[0370] - at least 30% w / w of the total protein;

[0371] - at least 85% w / w, preferably at least 90% w / w of β-lactoglobulin (BLG) relative to the total protein;

[0372] - at most 6% w / w of water;

[0373] The BLG isolate powder has:

[0374] - an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.15;

[0375] - a degree of protein denaturation of at most 5%; and

[0376] - a thermal stability at pH 3.9 of at most 40 NTU.

[0377] In other preferred embodiments of the present invention, the pH of the BLG isolate powder is 3.0 to 4.3, and the BLG isolate powder contains:

[0378] - at least 90% w / w, preferably at least 94% w / w of the total protein;

[0379] - at least 94% w / w, preferably at least 96% w / w of β-lactoglobulin (BLG) relative to the total protein;

[0380] - at most 6% w / w of water;

[0381] - at most 0.5% w / w, preferably at most 0.1% w / w of lipids;

[0382] The BLG isolate powder has:

[0383] - a bulk density of 0.45 to 0.8 g / cm 3 and preferably 0.50 to 0.6 g / cm 3 ;

[0384] - an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.15;

[0385] - a degree of protein denaturation of at most 5%, more preferably at most 2%; and

[0386] - a thermal stability at pH 3.9 of at most 30 NTU, preferably at most 10 NTU.

[0387] In other preferred embodiments of the present invention, the pH of the BLG isolate powder is 6.3 to 7.5, and the BLG isolate powder comprises:

[0388] - at least 90% w / w, even more preferably at least 94% w / w of the total protein;

[0389] - at least 94% w / w, even more preferably at least 96% w / w of β-lactoglobulin (BLG) relative to the total protein;

[0390] - at most 6% w / w of water;

[0391] - at most 0.5% w / w, preferably at most 0.1% w / w of lipids;

[0392] The BLG isolate powder has:

[0393] - a bulk density of 0.45 to 0.8 g / cm 3 preferably 0.50 to 0.6 g / cm 3 ;

[0394] - a degree of protein denaturation of at most 10%, preferably at most 5%, more preferably at most 2%; and

[0395] - a thermal stability at pH 3.9 of at most 50 NTU, preferably at most 30 NTU, even more preferably at most 10 NTU.

[0396] In other preferred embodiments of the present invention, the pH of the BLG isolate powder is 5.0 to 6.0, and the BLG isolate powder comprises:

[0397] - at least 90% w / w, even more preferably at least 94% w / w of the total protein;

[0398] - at least 90% w / w, even more preferably at least 94% w / w of β-lactoglobulin (BLG) relative to the total protein;

[0399] - at most 6% w / w of water;

[0400] - at most 0.5% w / w, preferably at most 0.1% w / w of lipids;

[0401] The BLG isolate powder has:

[0402] - a bulk density of 0.45 to 0.8 g / cm 3 preferably 0.50 to 0.6 g / cm 3 ;

[0403] - a degree of protein denaturation of at most 10%, preferably at most 5%, more preferably at most 2%;

[0404] - The thermal stability at pH 3.9 is at most 50 NTU, preferably at most 30 NTU, even more preferably at most 10 NTU; and

[0405] - Preferably, the crystallinity of BLG is less than 10%, more preferably at most 1%.

[0406] Another aspect of the present invention relates to a method for producing a dry BLG isolate powder, the BLG content of which is at least 85% w / w relative to the total amount of protein, the method comprising the steps of:

[0407] a) Providing a liquid BLG isolate having the following characteristics:

[0408] i) The pH is 2 to 4.9;

[0409] ii) The pH is 6.1 to 8.5; or

[0410] iii) The pH is 5.0 to 6.0;

[0411] The BLG content of the liquid BLG isolate is at least 85% w / w relative to the total amount of protein;

[0412] b) Optionally, physically reducing microorganisms in the liquid BLG isolate;

[0413] c) Drying the liquid BLG isolate, preferably by spray drying.

[0414] Preferably, the liquid BLG isolate is an edible composition.

[0415] Preferably, the liquid BLG isolate is prepared from the milk of mammals (preferably ruminants such as cows, sheep, goats, water buffalo, camels, llamas, horses and / or deer). Proteins derived from cow's milk are particularly preferred. Thus, BLG is preferably bovine BLG.

[0416] 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.

[0417] 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, such as about 100.0% w / w relative to the total amount of protein.

[0418] 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, and most preferably at least 30% w / w.

[0419] In some preferred embodiments of the present invention, the total amount of protein in the liquid BLG isolate is 5 - 40% w / w, preferably 10 - 35% w / w, more preferably 15 - 30% w / w, and even more preferably 20 - 25% w / w.

[0420] The inventors have observed that increasing the BLG concentration in the liquid BLG isolate results in a spray-dried powder with a higher bulk density; thus, preferably, the liquid BLG isolate has a relatively high BLG concentration.

[0421] Therefore, in other preferred embodiments of the present invention, the total amount of protein in the liquid BLG isolate is 10 - 40% w / w, preferably 20 - 38% w / w, more preferably 24 - 36% w / w, and even more preferably 28 - 34% w / w.

[0422] In some preferred embodiments of the present invention, the sum of α-lactalbumin (ALA) and caseinomacropeptide (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.

[0423] In some preferred embodiments of the present invention, ALA accounts for at most 80% w / w, preferably at most 60% w / w, even more preferably at most 40% w / w, and most preferably at most 30% w / w of the non-BLG proteins in the liquid BLG isolate.

[0424] An even lower content of ALA may be preferred; thus, in some preferred embodiments of the present invention, ALA accounts for at most 20% w / w, preferably at most 15% w / w, even more preferably 10% w / w, and most preferably at most 5% w / w of the non-BLG proteins in the liquid BLG isolate.

[0425] 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, has a weight percentage of at most 25%, preferably at most 20%, more preferably at most 15%, even more preferably at most 10%, and most preferably at most 6% relative to the total amount of protein in a standard whey protein concentrate from sweet whey.

[0426] Even lower concentrations of the major non-BLG whey proteins are desired. Thus, 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, has a weight percentage 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 in a standard whey protein concentrate from sweet whey.

[0427] The inventors have found that low levels of lactoferrin and / or lactoperoxidase are particularly advantageous for obtaining a color-neutral whey protein product.

[0428] Thus, in some preferred embodiments of the present invention, lactoferrin in the liquid BLG isolate, expressed as a weight percentage relative to the total amount of protein, has a weight percentage 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 in a standard whey protein concentrate from sweet whey. Even lower concentrations of lactoferrin may be desired. Thus, in other preferred embodiments of the present invention, lactoferrin, expressed as a weight percentage relative to the total amount of protein, has a weight percentage 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 in a standard whey protein concentrate from sweet whey.

[0429] Similarly, in some preferred embodiments of the present invention, lactoperoxidase in the liquid BLG isolate, expressed as a weight percentage relative to the total amount of protein, has a weight percentage 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 in a standard whey protein concentrate from sweet whey. Even lower concentrations of lactoperoxidase may be desired. Thus, in other preferred embodiments of the present invention, lactoperoxidase, expressed as a weight percentage relative to the total amount of protein, has a weight percentage 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 in a standard whey protein concentrate from sweet whey.

[0430] In some preferred embodiments of the present invention, the total solids content of the liquid BLG isolate is 5 to 50% w / w, preferably 10 to 40% w / w, more preferably 15 to 35% w / w, even more preferably 20 to 30% w / w.

[0431] Preferably, the fraction that is not counted as part of the total solids content in the liquid BLG isolate consists essentially of water or even consists of water.

[0432] In some preferred embodiments of the present invention, the water content of the liquid BLG isolate is 50 to 95% w / w, preferably 60 to 90% w / w, more preferably 65 to 85% w / w, even more preferably 70 to 80% w / w.

[0433] 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, and most preferably at most 0.1%. For example, the liquid BLG isolate may contain carbohydrates such as lactose, oligosaccharides and / or hydrolysis products of lactose (i.e., glucose and galactose), sucrose and / or maltodextrin.

[0434] 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.

[0435] The inventors have found that it may be advantageous to control the mineral content to achieve certain desired properties of the liquid BLG isolate.

[0436] In some preferred embodiments of the present invention, the total amount of Na, K, Mg and Ca in the liquid BLG isolate is at most 10 mmol / g of protein. Preferably, the total amount of Na, K, Mg and Ca in the liquid BLG isolate is at most 6 mmol / g of protein, more preferably at most 4 mmol / g of protein, even more preferably at most 2 mmol / g of protein.

[0437] In other preferred embodiments of the present invention, the total amount of Na, K, Mg and Ca in the liquid BLG isolate is at most 1.0 mmol / g of protein. Preferably, the total amount of Na, K, Mg and Ca in the liquid BLG isolate is at most 0.6 mmol / g of protein, more preferably at most 0.4 mmol / g of protein, even more preferably at most 0.2 mmol / g of protein, and most preferably at most 0.1 mmol / g of protein.

[0438] In other preferred embodiments of the present invention, the total amount of Mg and Ca in the liquid BLG isolate is at most 5 mmol / g of protein. Preferably, the total amount of Mg and Ca in the liquid BLG isolate is at most 3 mmol / g of protein, more preferably at most 1.0 mmol / g of protein, even more preferably at most 0.5 mmol / g of protein.

[0439] In other preferred embodiments of the present invention, the total amount of Mg and Ca in the liquid BLG isolate is at most 0.3 mmol / g of protein. Preferably, the total amount of Mg and Ca in the liquid BLG isolate is at most 0.2 mmol / g of protein, more preferably at most 0.1 mmol / g of protein, even more preferably at most 0.03 mmol / g of protein, and most preferably at most 0.01 mmol / g of protein.

[0440] The inventors have found that it is possible to prepare a low-phosphorus / low-potassium variant of the BLG isolate powder, which is particularly useful for patients with kidney disease. To produce such a product, the liquid BLG isolate must have similarly low levels of phosphorus and potassium.

[0441] Thus, in some preferred embodiments of the present invention, the total phosphorus content of the liquid BLG isolate is at most 100 mg of phosphorus / 100 g of protein. Preferably, the total phosphorus content of the liquid BLG isolate is at most 80 mg of phosphorus / 100 g of protein. More preferably, the total phosphorus content of the liquid BLG isolate is at most 50 mg of phosphorus / 100 g of protein. Even more preferably, the total phosphorus content of the liquid BLG isolate is at most 20 mg of phosphorus / 100 g of protein. The total phosphorus content of the liquid BLG isolate is at most 5 mg of phosphorus / 100 g of protein.

[0442] In some preferred embodiments of the present invention, the liquid BLG isolate contains at most 600 mg of potassium / 100 g of protein. More preferably, the liquid BLG isolate contains at most 500 mg of potassium / 100 g of protein. More preferably, the liquid BLG isolate contains at most 400 mg of potassium / 100 g of protein. More preferably, the liquid BLG isolate contains at most 300 mg of potassium / 100 g of protein. Even more preferably, the liquid BLG isolate contains at most 200 mg of potassium / 100 g of protein. Even more preferably, the liquid BLG isolate contains at most 100 mg of potassium / 100 g of protein. Even more preferably, the liquid BLG isolate contains at most 50 mg of potassium / 100 g of protein. Even more preferably, the liquid BLG isolate contains at most 10 mg of potassium / 100 g of protein.

[0443] The phosphorus content relates to the total amount of elemental phosphorus in the composition under study, measured according to Example 1.19. Similarly, the potassium content relates to the total amount of elemental potassium in the composition under study, measured according to Example 1.19.

[0444] In some preferred embodiments of the present invention, the liquid BLG isolate contains 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, still more preferably at most 50 mg phosphorus / 100 g protein and at most 400 mg potassium / 100 g protein, or even 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 liquid BLG isolate contains at most 100 mg phosphorus / 100 g protein and at most 340 mg potassium / 100 g protein.

[0445] The low-phosphorus and / or low-potassium composition of the present invention can be used as a food ingredient for producing foods for the patient population with reduced renal function.

[0446] In some preferred embodiments of the present invention, the pH of the liquid BLG isolate is 2 to 4.9, preferably 2.5 to 4.7, more preferably 2.8 to 4.4, even more preferably 3.0 to 4.0, and most preferably 3.4 to 3.9.

[0447] 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.

[0448] In other preferred embodiments of the present invention, the pH of the liquid BLG isolate is 5.0 to 6.0, preferably 5.1 to 5.9, more preferably 5.2 to 5.8, even more preferably 5.3 to 5.7. When the pH of the liquid BLG isolate is 5.0 to 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 the BLG is supersaturated, the liquid BLG isolate can be kept free of crystals as long as it is maintained in the metastable region and the crystallization promoter is not allowed to contact the liquid BLG isolate.

[0449] Preferably, the microbial content of the liquid BLG isolate is low; this is especially likely if the microbial content of the BLG-rich composition is already low.

[0450] In some embodiments of the present invention, the liquid BLG isolate contains at most 500,000 CFU / g, preferably at most 100,000 CFU / g, more preferably at most 50,000 CFU / g, and even more preferably at most 10,000 CFU / g.

[0451] Thus, in some preferred embodiments of the present invention, the liquid BLG isolate contains at most 1000 colony forming units (CFU) / g. Preferably, the liquid BLG isolate contains at most 600 CFU / g. More preferably, the liquid BLG isolate contains at most 300 CFU / g. Even more preferably, the liquid BLG isolate contains at most 100 CFU / g. Even more preferably, the liquid BLG isolate contains at most 50 CFU / g. Most preferably, the liquid BLG isolate contains at most 20 CFU / g, such as at most 10 CFU / g. In a particularly preferred embodiment, the powder is sterile. For example, a sterile liquid BLG isolate can be prepared by combining several physical processes for reducing microorganisms during the production of the BLG isolate powder, such as microfiltration and heat treatment at a low pH (up to pH 4.0).

[0452] Preparing a BLG isolate powder with a low degree of protein unfolding requires that the liquid BLG isolate already has a low degree of protein unfolding, since the unfolding of BLG appears to be an irreversible process.

[0453] If a BLG isolate powder or a liquid BLG isolate with a low degree of BLG unfolding is desired, then preferably the ratio of the intrinsic tryptophan fluorescence emission (I330 / I350) of the liquid BLG isolate is at least 1.11.

[0454] In some preferred embodiments of the present invention, the ratio of the intrinsic tryptophan fluorescence emission (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, and most preferably at least 1.19.

[0455] If the liquid BLG isolate contains a large amount of non-protein substances, then preferably the protein fraction is separated before measuring the ratio of the intrinsic tryptophan fluorescence emission. Thus, in some preferred embodiments of the present invention, the ratio of the intrinsic tryptophan fluorescence emission (I330 / I350) of the protein fraction of the liquid BLG isolate is at least 1.11.

[0456] Preferably, the ratio of the intrinsic tryptophan fluorescence emission (I330 / I350) of the protein fraction of the liquid BLG isolate can 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, and most preferably at least 1.19.

[0457] For example, the protein fraction can be separated from the BLG isolate powder by dialysis or ultrafiltration-based diafiltration using a protein-retaining filter.

[0458] Preparing a BLG isolate powder with a low degree of protein unfolding requires that the liquid BLG isolate already has a low degree of protein denaturation, since the protein denaturation of BLG appears to be an irreversible process. Thus, 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.

[0459] Generally preferably, 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, approximately 100% of the BLG in the liquid BLG isolate is non-aggregated BLG.

[0460] However, it may also be preferably that the protein denaturation level of the liquid BLG isolate is significant, for example if an opaque beverage is desired. Thus, in other preferred embodiments of the present invention, the degree of 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%, and most preferably at least 90%.

[0461] If the protein denaturation level of the liquid BLG isolate is significant, it is generally preferred to keep the level of insoluble protein material low, the insoluble protein material being the precipitated protein material that will settle in the beverage during storage. The level of insoluble material is measured according to Example 1.10.

[0462] In some preferred embodiments of the present invention, the liquid BLG isolate contains at most 20% w / w of insoluble protein material, preferably at most 10% w / w of insoluble protein material, more preferably at most 5% w / w of insoluble protein material, even more preferably at most 3% w / w of insoluble protein material, and most preferably at most 1% w / w of insoluble protein material. Even preferably, the liquid BLG isolate does not contain any insoluble protein material at all.

[0463] As described above, the present inventors have found that the thermal stability of the liquid BLG isolate at pH 3.9 is a good indicator of its usefulness for clear high-protein beverages. The thermal stability at pH 3.9 is measured according to Example 1.2.

[0464] Particularly preferably, the thermal stability of the liquid BLG isolate 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, and most preferably at most 20 NTU. There may be even better thermal stability, and 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.

[0465] 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, and most preferably at most 2 NTU.

[0466] In other preferred embodiments of the present invention, the turbidity of the liquid BLG isolate is more than 200 NTU, preferably at least 400 NTU, more preferably at least 800 NTU, even more preferably at least 1000 NTU, even more preferably at least 2000 NTU, and most preferably at least 5000 NTU. Particularly preferably, such a liquid BLG isolate is used for producing an opaque beverage.

[0467] The inventors have observed that, compared with a comparable liquid WPI, the liquid BLG isolate of the present invention unexpectedly has a lower viscosity. The inventors have found that this makes the liquid BLG isolate particularly suitable for use as a high-protein beverage, as it enables a high protein content to be obtained without resulting in an undesired high viscosity.

[0468] In some preferred embodiments of the present invention, the liquid BLG isolate at 15 °C and 300 s -1 The viscosity at a shear rate is visc(p) ± 50%, more preferably visc(p) ± 40%, even more preferably visc(p) ± 30%, and most preferably visc(p) ± 25%.

[0469] visc(p) is defined as:

[0470] visc(p) = 0.3556e 0.1262*p (for p ≤ 23%) or 0.0254 * e 0.24*p (for p > 23%).

[0471] p is the total protein content of the liquid BLG isolate (expressed as % w / w); thus, for example, if the protein content is 31% w / w, then p is 31.

[0472] This means that, for example, if the liquid BLG isolate (with a protein content of p) at 15 °C and 300 s -1The viscosity at the shear rate is visc(p) ± 25%, and 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:

[0473] Minimum viscosity (in cP): 0.0254 * e 0.24*31 –25% = 43 cP – 25% = 32 cP;

[0474] Maximum viscosity (in cP): 0.0254 * e 0.24*31 +25% = 43 cP + 25% = 54 cP.

[0475] In other preferred embodiments of the present invention, the viscosity of the liquid BLG isolate at 15 °C and 300 s -1 shear rate is visc(p) ± 20%, more preferably visc(p) ± 15%, even more preferably visc(p) ± 10%, and most preferably visc(p) ± 5%.

[0476] Measure the viscosity of the liquid BLG isolate according to Example 1.8 but using a temperature of 15 °C and a shear rate of 300 s -1 of the shear rate.

[0477] In some preferred embodiments of the present invention, the viscosity of the liquid BLG isolate at 15 °C and 300 s -1 shear rate is:

[0478] - at least visc(p) – 20%; and

[0479] - at most visc 最大值 (p) – 20%.

[0480] In other preferred embodiments of the present invention, the viscosity of the liquid BLG isolate at 15 °C and 300 s -1 shear rate is:

[0481] - at least visc(p) – 10%; and

[0482] - at most visc 最大值 (p) – 40%.

[0483] In other preferred embodiments of the present invention, the viscosity of the liquid BLG isolate at 15 °C and 300 s -1 shear rate is:

[0484] - at least visc(p) – 10%; and

[0485] - at most visc最大值 (p) – 50%.

[0486] visc 最大值 (p) is defined as: visc 最大值 (p) ≤ 0.611 * e (0.1494*p) cP.

[0487] In other preferred embodiments of the present invention, the viscosity of the liquid BLG isolate at 15 °C and a shear rate of 300 s -1 is at most visc 最大值 (p) – 10%, more preferably at most visc 最大值 (p) – 20%, even more preferably visc 最大值 (p) – 30%, most preferably visc 最大值 (p) – 50%.

[0488] 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:

[0489] - a total protein content of 20 to 34% w / w, more preferably 24 to 32% w / w, even more preferably 28 to 32% w / w;

[0490] - at least 90% w / w, more preferably at least 94% w / w of β-lactoglobulin (BLG) relative to the total protein content;

[0491] Preferably, the BLG isolate powder has one or more of the following:

[0492] - an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.15;

[0493] - a degree of protein denaturation of at most 2%;

[0494] - a thermal stability at pH 3.9 of at most 20 NTU;

[0495] - sterility; and

[0496] - a viscosity at 15 °C of 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%).

[0497] 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:

[0498] - Total protein content of -20 to 34% w / w, more preferably 24 to 32% w / w, even more preferably 28 to 30% w / w;

[0499] - At least 90% w / w, more preferably at least 94% w / w of β-lactoglobulin (BLG) relative to the total protein content;

[0500] Preferably, the BLG isolate powder has one or more of the following:

[0501] - Intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.15;

[0502] - Degree of protein denaturation of at most 5%;

[0503] - Thermal stability at pH 3.9 of at most 40 NTU;

[0504] - Sterile; and

[0505] - Viscosity at 15 °C of 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%).

[0506] Such acidic high-protein liquid BLG isolates are particularly suitable for the production of high-quality BLG isolate powders and have unexpectedly low viscosities compared to comparable WPI, which results in lower energy consumption during processing (e.g., microfiltration). In addition, compared to the bulk density achievable with traditional WPI of the same protein content, acidic high-protein liquid BLG isolates can produce acidic whey protein powders with much higher bulk densities (see, for example, Example 7).

[0507] Liquid BLG isolates can be provided in a variety of different ways.

[0508] Typically, providing a liquid BLG isolate involves or even consists of: separating BLG from a whey protein raw material by one or more of the following methods to provide a BLG-rich composition:

[0509] - Crystallizing or precipitating BLG by salting in;

[0510] - Crystallizing or precipitating BLG by salting out;

[0511] - Ion exchange chromatography; and

[0512] - Ultrafiltration fractionation of whey proteins.

[0513] A particularly preferred way of providing a BLG-rich composition is by crystallization of BLG, preferably by salting in or salting out.

[0514] Preferably, the whey protein raw material is WPC, WPI, SPC, SPI or a combination thereof.

[0515] The term "whey protein raw material" refers to a composition from which a BLG-rich composition and a subsequent liquid BLG isolate can be obtained. The chemical characteristics and embodiments described for whey protein solutions also apply to whey protein raw materials, except that: generally, the BLG in the whey protein raw material is not supersaturated, and the pH of the raw material is not limited to 5-6.

[0516] In some embodiments of the present invention, the preparation of the BLG-rich composition comprises or even consists of: high-salt BLG crystallization at a pH of 3.6-4.0 according to US2,790,790A1.

[0517] In other embodiments of the present invention, the preparation of the BLG-rich composition comprises or even consists of the methods 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).

[0518] However, in a particularly preferred embodiment of the present invention, as described in PCT application number PCT / EP2017 / 084553 (the content of which is incorporated herein by reference for all purposes), a BLG-rich composition is prepared by crystallization at pH 5-6 under salting-in conditions.

[0519] In some preferred embodiments of the present invention, the BLG-rich composition is an edible BLG composition according to PCT / EP2017 / 084553, which contains at least 90% BLG relative to the total protein and preferably contains BLG crystals.

[0520] Preferably, a BLG-rich composition is prepared by a method comprising the following steps:

[0521] 1) Provide a whey protein solution, the whey protein solution comprising non-aggregated BLG and at least one other whey protein, the whey protein solution being supersaturated with BLG and having a pH of 5 to 6;

[0522] 2) Crystallize non-aggregated BLG in the supersaturated whey protein solution;

[0523] 3) Separate the BLG crystals from the remaining whey protein solution;

[0524] 4) Optionally, wash the BLG crystals, such as the separated BLG crystals obtained from step 3) or 5); and

[0525] 5) Optionally, recrystallize the BLG crystals, such as the BLG crystals obtained from step 3) or 4).

[0526] This method for preparing a BLG-rich composition includes the mandatory steps 1), 2) and 3) and is carried out in that order, and may optionally include steps 4) and / or 5) in any order and number of iterations. However, steps 4) and 5) are generally after step 3). Alternatively or additionally, washing water can be added to the whey protein solution containing crystals before separation.

[0527] In addition, the method may include a step of drying the BLG-rich composition. However, currently it is preferred to use the BLG-rich composition without drying it to avoid the risk of damaging the protein during drying.

[0528] As described above, step 1) of the crystallization process involves providing a whey protein solution, the whey protein solution comprising non-aggregated BLG and at least one other whey protein.

[0529] Preferably, the whey protein solution comprises at least one other non-aggregated whey protein selected from: α-lactalbumin, bovine serum albumin, immunoglobulins, caseinomacropeptide (CMP), osteopontin, lactoferrin, lactoperoxidase, milk fat globule membrane proteins, and combinations thereof.

[0530] In some embodiments of the present invention, relative to the total amount of protein, the whey protein solution contains at most 10% w / w, preferably at most 5% w / w, more preferably at most 1% w / w, and even more preferably at most 0.5% w / w of casein. In some preferred embodiments of the present invention, the whey protein solution does not contain any detectable amount of casein.

[0531] In some preferred embodiments of the present invention, relative to the total amount of protein, the whey protein solution in step 1) contains at least 5% w / w of other whey proteins. Preferably, relative to the total amount of protein, the whey protein solution in step 1) contains at least 10% w / w of other whey proteins. More preferably, relative to the total amount of protein, the whey protein solution in step 1) contains at least 15% w / w of other whey proteins. Even more preferably, relative to the total amount of protein, the whey protein solution in step 1) contains at least 20% w / w of other whey proteins. Most preferably, relative to the total amount of protein, the whey protein solution in step 1) may contain at least 30% w / w of other whey proteins.

[0532] In other preferred embodiments of the present invention, relative to the total amount of protein, the whey protein solution in step 1) contains at least 1% w / w of other whey proteins. Preferably, relative to the total amount of protein, the whey protein solution in step 1) contains at least 2% w / w of other whey proteins. Even more preferably, relative to the total amount of protein, the whey protein solution in step 1) contains at least 3% w / w of other whey proteins. Most preferably, relative to the total amount of protein, the whey protein solution in step 1) may contain at least 4% w / w of other whey proteins.

[0533] In other preferred embodiments of the present invention, relative to the total amount of protein, the whey protein solution in step 1) contains at least 35% w / w of other whey proteins. Preferably, relative to the total amount of protein, the whey protein solution in step 1) may contain at least 40% w / w of other whey proteins. More preferably, for example, relative to the total amount of protein, the whey protein solution in step 1) may contain at least 45% w / w of other whey proteins. Even more preferably, relative to the total amount of protein, the whey protein solution in step 1) may contain at least 50% w / w of other whey proteins.

[0534] In some preferred embodiments of the present invention, relative to the total amount of protein, the whey protein solution in step 1) contains 5 - 90% w / w of other whey proteins. Preferably, relative to the total amount of protein, the whey protein solution in step 1) may contain 10 - 80% w / w of other whey proteins. For example, relative to the total amount of protein, the whey protein solution in step 1) may contain 20 - 70% w / w of other whey proteins. Preferably, relative to the total amount of protein, the whey protein solution in step 1) contains 30 - 70% w / w of other whey proteins.

[0535] As described above, the inventors have found that non-aggregated BLG can be crystallized without using organic solvents. This purification method can also be used to refine a preparation containing whey protein that has undergone some BLG purification, which provides a simple method for further increasing the purity of non-aggregated BLG. Thus, in some preferred embodiments of the present invention, the whey protein solution in step 1) contains 1 to 20% w / w of other whey proteins relative to the total amount of protein. Preferably, the whey protein solution in step 1) can 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 in step 1) can contain 3 to 10% w / w of other whey proteins relative to the total amount of protein.

[0536] In some embodiments of the present invention, the whey protein solution in step 1) contains at least 5% w / w of non-aggregated ALA relative to the total amount of protein. Preferably, the whey protein solution in step 1) contains at least 10% w / w of non-aggregated ALA relative to the total amount of protein. Even more preferably, the whey protein solution in step 1) contains at least 15% w / w of non-aggregated ALA relative to the total amount of protein. Alternatively, the whey protein solution in step 1) can contain at least 20% 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 in step 1) contains at least 25% w / w of non-aggregated ALA relative to the total amount of protein. Preferably, the whey protein solution in step 1) contains at least 30% w / w of non-aggregated ALA relative to the total amount of protein. The whey protein solution in 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 in step 1) can contain at least 40% w / w of non-aggregated ALA relative to the total amount of protein.

[0538] In some preferred embodiments of the present invention, the whey protein solution in step 1) contains 5 to 95% w / w of non-aggregated ALA relative to the total amount of protein. Preferably, the whey protein solution in step 1) contains 5 to 70% w / w of non-aggregated ALA relative to the total amount of protein. Even more preferably, the whey protein solution in step 1) can contain 10 to 60% w / w of non-aggregated ALA relative to the total amount of protein. The whey protein solution in step 1) preferably contains 12 to 50% w / w of non-aggregated ALA relative to the total amount of protein. Even more preferably, the whey protein solution in step 1) can contain 20 to 45% w / w of non-aggregated ALA relative to the total amount of protein.

[0539] In some preferred embodiments of the present invention, the weight ratio of non-aggregated BLG to non-aggregated ALA in the whey protein solution of step 1) is at least 0.01. Preferably, the weight ratio of non-aggregated BLG to non-aggregated ALA in the whey protein solution of step 1) is at least 0.5. Even more preferably, the weight ratio of non-aggregated BLG to non-aggregated ALA in the whey protein solution of step 1) is at least 1, for example at least 2. For example, the weight ratio of non-aggregated BLG to non-aggregated ALA in the whey protein solution of step 1) can be at least 3.

[0540] In some preferred embodiments of the present invention, the weight ratio of non-aggregated BLG to non-aggregated ALA in the whey protein solution of step 1) is from 0.01 to 20. Preferably, the weight ratio of non-aggregated BLG to non-aggregated ALA in the whey protein solution of step 1) is from 0.2 to 10. Even more preferably, the weight ratio of non-aggregated BLG to non-aggregated ALA in the whey protein solution of step 1) is from 0.5 to 4. For example, the weight ratio of non-aggregated BLG to non-aggregated ALA in the whey protein solution of step 1) can be from 1 to 3.

[0541] In some preferred embodiments of the present invention, relative to the total amount of protein, the whey protein solution of step 1) contains at least 1% w / w of non-aggregated BLG. Preferably, relative to the total amount of protein, the whey protein solution of step 1) contains at least 2% w / w of non-aggregated BLG. Even more preferably, relative to the total amount of protein, the whey protein solution of step 1) contains at least 5% w / w of non-aggregated BLG. Preferably, relative to the total amount of protein, the whey protein solution of step 1) may contain at least 10% w / w of non-aggregated BLG.

[0542] In some preferred embodiments of the present invention, relative to the total amount of protein, the whey protein solution of step 1) contains at least 12% w / w of non-aggregated BLG. For example, relative to the total amount of protein, the whey protein solution of step 1) may contain at least 15% w / w of non-aggregated BLG. For example, relative to the total amount of protein, the whey protein solution of step 1) may contain at least 20% w / w of non-aggregated BLG. Alternatively, relative to the total amount of protein, the whey protein solution of step 1) may contain at least 30% w / w of non-aggregated BLG.

[0543] In some particularly preferred embodiments of the present invention, relative to the total amount of protein, the whey protein solution in step 1) contains at most 95% w / w of non-aggregated BLG. Preferably, relative to the total amount of protein, the whey protein solution in step 1) may contain at most 90% w / w of non-aggregated BLG. More preferably, for example, relative to the total amount of protein, the whey protein solution in step 1) may contain at most 85% w / w of non-aggregated BLG. Even more preferably, for example, the whey protein solution in step 1) may contain at most 80% w / w of non-aggregated BLG. Preferably, relative to the total amount of protein, the whey protein solution in step 1) may contain at most 78% w / w of non-aggregated BLG. Preferably, relative to the total amount of protein, the whey protein solution in step 1) may contain at most 75% w / w of non-aggregated BLG.

[0544] In some preferred embodiments of the present invention, relative to the total amount of protein, the whey protein solution in step 1) contains 1-95% w / w of non-aggregated BLG. Preferably, relative to the total amount of protein, the whey protein solution in step 1) may contain 5-90% w / w of non-aggregated BLG. More preferably, relative to the total amount of protein, the whey protein solution in step 1) contains 10-85% w / w of non-aggregated BLG. Even more preferably, relative to the total amount of protein, the whey protein solution in step 1) contains 10-80% w / w of non-aggregated BLG. Most preferably, relative to the total amount of protein, the whey protein solution in step 1) may contain 20-70% w / w of non-aggregated BLG.

[0545] In other preferred embodiments of the present invention, relative to the total amount of protein, the whey protein solution in step 1) contains 10-95% w / w of non-aggregated BLG. Preferably, relative to the total amount of protein, the whey protein solution in step 1) may contain 12-90% w / w of non-aggregated BLG. More preferably, relative to the total amount of protein, the whey protein solution in step 1) contains 15-85% w / w of non-aggregated BLG. Even more preferably, relative to the total amount of protein, the whey protein solution in step 1) contains 15-80% w / w of non-aggregated BLG. Most preferably, relative to the total amount of protein, the whey protein solution in step 1) may contain 30-70% w / w of non-aggregated BLG.

[0546] In some preferred embodiments of the present invention, relative to the weight of the whey protein solution, the whey protein solution in step 1) contains at least 0.4% w / w of non-aggregated BLG. Preferably, the whey protein solution contains at least 1.0% w / w of non-aggregated BLG. More preferably, the whey protein solution contains at least 2.0% w / w of non-aggregated BLG. Even more preferably, the whey protein solution contains at least 4% w / w of non-aggregated BLG.

[0547] Even 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.

[0548] In some preferred embodiments of the present invention, relative to the weight of the whey protein solution, the whey protein solution of step 1) comprises 0.4 to 45% w / w of non-aggregated BLG. Preferably, the whey protein solution comprises 1 to 35% w / w of non-aggregated BLG. More preferably, the whey protein solution comprises 4 to 30% w / w of non-aggregated BLG. Even more preferably, the whey protein solution comprises 10 to 25% w / w of non-aggregated BLG.

[0549] Higher contents of BLG are particularly preferred; thus, in some preferred embodiments of the present invention, relative to the weight of the whey protein solution, the whey protein solution of step 1) comprises 10 to 45% w / w of non-aggregated BLG. Preferably, the whey protein solution preferably comprises 15 to 40% w / w of non-aggregated BLG. More preferably, the whey protein solution comprises 20 to 39% w / w of non-aggregated BLG. Even more preferably, the whey protein solution comprises 25 to 38% w / w of non-aggregated BLG.

[0550] Generally preferably, a large amount of 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, approximately 100% of the BLG in the whey protein solution is non-aggregated BLG.

[0551] Any suitable whey protein source can be used to prepare the whey protein solution. In some preferred embodiments of the present invention, the whey protein solution comprises, or even consists of, whey protein concentrate, whey protein concentrate, whey protein isolate, whey protein isolate or a combination thereof.

[0552] Preferably, the whey protein solution is a demineralised whey protein solution.

[0553] As used herein, the term "demineralised" 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 demineralised 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.

[0554] 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.

[0555] 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.

[0556] For example, the whey protein solution may contain or even consist of demineralized whey protein concentrate. Alternatively, the whey protein solution may contain or even consist of demineralized whey protein concentrate. Alternatively, the whey protein solution may contain demineralized whey protein isolate, or even consist of it. Alternatively, the whey protein solution may contain demineralized whey protein isolate, or even consist of it.

[0557] The BLG-rich composition is preferably prepared from the milk of mammals, preferably from the milk of ruminants (such as cows, sheep, goats, buffalo, camels, llamas, mares, and / or deer). Proteins derived from cow's milk are particularly preferred.

[0558] Preferably, the protein in the whey protein solution is as close as possible to its natural state, and preferably, it has only been subjected to mild heat treatment (if any).

[0559] In some preferred embodiments of the present invention, the furosine value of the whey protein solution is at most 80 mg / 100 g of protein. Preferably, the furosine value of the whey protein solution is at most 40 mg / 100 g of protein. More preferably, the furosine value of the whey protein solution is at most 20 mg / 100 g of protein. Even more preferably, the furosine value of the whey protein solution is at most 10 mg / 100 g of protein. Most preferably, the furosine value of the whey protein solution is at most 5 mg / 100 g of protein. For example, the furosine value is preferably 0 mg / 100 g of protein.

[0560] Generally, in addition to proteins, the whey protein solution also contains other components. The whey protein solution may contain other components commonly found in whey or milk serum, such as minerals, carbohydrates, and / or lipids. Alternatively or additionally, the whey protein solution may contain components that are non-natural to whey or milk serum. However, preferably, such non-natural components should be safe for use in food production and preferably also safe for human consumption.

[0561] The method of the present invention is particularly advantageous for separating BLG from a crude whey protein solution, which contains other solids in addition to BLG.

[0562] For example, the whey protein solution may contain carbohydrates such as lactose, oligosaccharides, and / or hydrolysis products of lactose (i.e., glucose and galactose). For example, the whey protein solution may contain 0 to 40% w / w (such as 1 to 30% w / w or 2 to 20% w / w) of carbohydrates.

[0563] In some preferred embodiments of the present invention, the whey protein solution contains at most 20% w / w of carbohydrates, preferably at most 10% w / w of carbohydrates, more preferably at most 5% w / w of carbohydrates, even more preferably at most 2% w / w of carbohydrates.

[0564] The whey protein solution may also contain lipids, such as lipids in the form of triglycerides and / or other lipid types (such as phospholipids).

[0565] In some embodiments of the present invention, relative to the total amount of solids, the whey protein solution in step 1) contains at most 15% w / w of the total amount of lipids. Preferably, relative to the total amount of solids, the whey protein solution in step 1) contains at most 10% w / w of the total amount of lipids. More preferably, relative to the total amount of solids, the whey protein solution in step 1) contains at most 6% w / w of the total amount of lipids. Even more preferably, relative to the total amount of solids, the whey protein solution in step 1) contains at most 1.0% w / w of the total amount of lipids. Most preferably, relative to the total amount of solids, the whey protein solution in step 1) contains at most 0.5% w / w of the total amount of lipids.

[0566] Generally, relative to the weight of the whey protein solution, the total amount of protein in the whey protein solution is at least 1% w / w. 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.

[0567] In some preferred embodiments of the present invention, the total amount of protein in the whey protein solution is 1 to 50% w / w. Preferably, the total amount of protein in the whey protein solution is 5 to 40% w / w. More preferably, the total amount of protein in the whey protein solution is 10 to 30% w / w. Even more preferably, the total amount of protein in the whey protein solution is 15 to 25% w / w.

[0568] The total amount of protein in the whey protein solution is determined according to Example 1.5.

[0569] Typically, a whey protein solution is prepared by subjecting a whey protein raw material to one or more adjustments that result in a whey protein solution supersaturated with BLG.

[0570] Preferably, the whey protein raw material is WPC, WPI, SPC, SPI, or a combination thereof.

[0571] The term "whey protein raw material" refers to a composition from which a BLG-rich composition and a subsequent liquid BLG isolate are obtained. For example, the whey protein raw material can be converted into a whey protein solution supersaturated with BLG. Typically, the whey protein raw material is an aqueous solution containing BLG and at least one other whey protein, but usually BLG is unsaturated.

[0572] Embodiments regarding the chemical composition of the whey protein solution apply equally 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.

[0573] In some preferred embodiments of the present invention, a supersaturated whey protein solution is prepared by subjecting the whey protein raw material to one or more of the following adjustments:

[0574] - Adjusting the pH value;

[0575] - Lowering the conductivity;

[0576] - Lowering the temperature;

[0577] - Increasing the protein concentration;

[0578] - Adding a reagent that reduces the water activity; and

[0579] - Changing the ionic composition.

[0580] 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 to 6.

[0581] All pH values are measured using a pH glass electrode and normalized to 25°C. Typically, it is normalized to 25°C using a pH meter. Alternatively, the temperature of the sample is adjusted to 25°C.

[0582] For example, the pH of the whey protein solution can be 4.9 to 6.1. For example, the pH of the whey protein solution can be 5.0 to 6.1. Alternatively, the pH of the whey protein solution can be 5.1 to 6.1. Preferably, the pH of the whey protein solution is 5.1 to 6.0.

[0583] 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 can be 5.2 to 5.9. Most preferably, the pH of the whey protein solution is 5.2 to 5.8.

[0584] Preferably, the pH is adjusted using food acceptable acids and / or bases. 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.

[0585] In some preferred embodiments of the present invention, lactones (such as D - glucono - δ - lactone) are used to adjust the pH, which slowly hydrolyzes and simultaneously reduces the pH of the aqueous solution containing it. The target pH after the completion of lactone hydrolysis can be accurately calculated.

[0586] For example, useful examples of food acceptable bases are hydroxide sources such as sodium hydroxide, potassium hydroxide, calcium hydroxide, salts of edible acids (such as trisodium citrate), and / or combinations thereof.

[0587] In other preferred embodiments of the present invention, the pH is adjusted by adding a cation exchange material (in its H + form). Before or even after crystallization, the magnetic bead type / large particle type cation exchange material is easily removed from the whey protein solution. Adjusting the pH by adding a cation exchange material (in its H + form) is particularly advantageous in the present invention because it reduces the pH without adding negative counterions that significantly affect the conductivity of the whey protein raw material.

[0588] 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.

[0589] Unless otherwise specified, the conductivity values described herein have been normalized to 25 °C.

[0590] It has been found that reducing the conductivity of the whey protein solution results in a higher yield of BLG crystals. The lowest 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 caseinomacropeptide (CMP), contribute more to the conductivity than other protein species. Therefore, preferably, the conductivity of the whey protein raw material is brought close to the level at which the protein and its counterions are the main contributors to the conductivity. Generally, reducing the conductivity involves removing at least some of the small free ions present in the liquid phase and not tightly bound to the protein.

[0591] Generally preferably, 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.

[0592] Even lower conductivity is even more preferred, which can result in a higher yield of BLG crystals. Therefore, preferably, the preferred conductivity of the whey protein solution is 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.

[0593] Preferably, the conductivity of the whey protein raw material is reduced by dialysis or diafiltration. Diafiltration by ultrafiltration is particularly preferred because it allows the washing out of salts and small charged molecules while retaining the protein. In some preferred embodiments of the present invention, the same UF unit is used for UF / diafiltration and subsequent concentration of the whey protein raw material.

[0594] Advantageously, the ratio between the conductivity (expressed in mS / cm) and the total amount of protein in the whey protein solution (expressed as the total weight percentage of protein relative to the total weight of the whey protein solution) is kept below a certain threshold to promote the crystallization of BLG.

[0595] In some preferred embodiments of the present invention, the ratio between the conductivity of the whey protein solution and the total amount of protein is at most 0.3. Preferably, the ratio between the conductivity of the whey protein solution and the total amount of protein is at most 0.25. Preferably, the ratio between the conductivity of the whey protein solution and the total amount of protein is at most 0.20. More preferably, the ratio between the conductivity of the whey protein solution and the total amount of protein is at most 0.18. Even more preferably, the ratio between the conductivity of the whey protein solution and the total amount of protein is at most 0.12. Most preferably, the ratio between the conductivity of the whey protein solution and the total amount of protein is at most 0.10.

[0596] For example, preferably, the ratio between the conductivity of the whey protein solution and the total amount of protein is about 0.07 or even lower.

[0597] Furthermore, the inventors have found that the whey protein raw material can be advantageously conditioned 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 the liquid. When the term "conductivity" is used herein in this context, 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 the liquid, which is measured according to Example 1.23.

[0598] Preferably, the UF permeate conductivity of the whey protein solution is at most 7 mS / cm. More preferably, the UF permeate conductivity of the whey protein solution is at most 5 mS / cm. Even more preferably, the UF permeate conductivity of the whey protein solution can be at most 3 mS / cm.

[0599] Even lower UF permeate conductivities can be used, and if a high yield of BLG is to be obtained, even lower UF permeate conductivities are particularly preferred. Thus, preferably, the UF permeate conductivity of the whey protein solution is at most 1.0 mS / cm. More preferably, the UF permeate conductivity of the whey protein solution is at most 0.4 mS / cm. Even more preferably, the UF permeate conductivity of the whey protein solution can be at most 0.1 mS / cm. Most preferably, the UF permeate conductivity of the whey protein solution can be at most 0.04 mS / cm.

[0600] For example, if MilliQ water (conductivity about 0.06 μS / cm) is used as a diluent during diafiltration, even lower UF permeate conductivities can be achieved. Thus, the UF permeate conductivity of the whey protein solution can be at most 0.01 mS / cm. Alternatively, the UF permeate conductivity of the whey protein solution can be at most 0.001 mS / cm. Alternatively, the UF permeate conductivity of the whey protein solution can be at most 0.0001 mS / cm.

[0601] In some preferred embodiments of the present invention, the preparation of the whey protein solution involves lowering the temperature of the whey protein raw material.

[0602] For example, the preparation of the whey protein solution can include lowering the temperature of the whey protein raw material by at least 5 °C, preferably at least 10 °C, even more preferably at least 15 °C. For example, the preparation of the whey protein solution can include lowering the temperature of the whey protein raw material by at least 20 °C.

[0603] For example, the temperature of the whey protein raw material can 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 a higher supersaturation. Thus, the temperature of the whey protein raw material can, for example, be reduced to at most 5 °C, preferably at most 2 °C, even more preferably at most 0 °C. The temperature can even be below 0 °C. However, preferably, the whey protein solution should remain pumpable, for example in the form of a slush.

[0604] In some preferred embodiments of the present invention, the whey protein solution is a slush before the start of BLG crystallization. Alternatively or additionally, during the BLG crystallization in step 2), the crystallized whey protein solution can be converted to or maintained as a slush.

[0605] 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, one or more protein concentration steps (such as ultrafiltration, nanofiltration, reverse osmosis, and / or evaporation) can be performed on the whey protein raw material to obtain a concentrated whey protein solution by concentration.

[0606] Ultrafiltration is particularly preferred because it allows selective concentration of proteins with little effect on the concentration of salts and carbohydrates. As described above, ultrafiltration is preferably used for diafiltration and concentration of the whey protein raw material.

[0607] 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. Thus, generally preferably, when the whey protein solution is in the metastable region (i.e., the supersaturated region), the modification of the whey protein raw material is stopped; wherein, when in the supersaturated region, when using seed crystals, BLG crystals can grow, but crystallization does not start spontaneously.

[0608] 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.

[0609] Useful but non-limiting examples of such water activity reducing agents are polysaccharides and / or polyethylene glycol (PEG).

[0610] In some preferred embodiments of the present invention, the preparation of the whey protein solution involves changing the ionic composition of the whey protein raw material, for example, by ion exchange, adding new ionic species, dialysis, or diafiltration.

[0611] Generally, the whey protein solution is prepared by combining two or more of the above-described method steps for generating supersaturation.

[0612] In some preferred embodiments of the present invention, the preparation of the whey protein solution involves at least performing on the whey protein raw material:

[0613] - Concentration, for example by using ultrafiltration, nanofiltration or reverse osmosis at a temperature above 10 °C; and

[0614] - Subsequently cooling to a temperature below 10 °C.

[0615] 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:

[0616] - Concentration at a pH above 6.0; and

[0617] - Subsequently reducing the pH by adding an acid (such as GDL in the form of H + or a cation exchange material).

[0618] 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:

[0619] - Reducing the conductivity, for example by using a membrane that at least retains non-aggregated BLG for diafiltration to reduce the conductivity.

[0620] In other preferred embodiments of the present invention, the preparation of the whey protein solution involves at least a combination of the following steps for the whey protein raw material:

[0621] - Adjusting the pH to 5 - 6;

[0622] - Using a membrane that at least retains non-aggregated BLG for diafiltration to reduce the conductivity;

[0623] - Concentrating the protein, for example by using ultrafiltration, nanofiltration or reverse osmosis at a temperature above 10 °C to concentrate the protein; and

[0624] - Finally, cooling to a temperature below 10 °C.

[0625] Furthermore, the inventors of the present invention have found that by controlling the molar ratio between the sum of sodium + potassium and the sum of calcium + magnesium, the yield of BLG of the present method can be increased. Unexpectedly, a higher relative content of calcium and magnesium seems to increase the yield of non-aggregated BLG, thus improving the efficiency of recovering BLG by the method of the present invention.

[0626] In some preferred embodiments of the present invention, the molar ratio of Na+K to Ca+Mg in the whey protein solution of step 1) is at most 4. More preferably, the molar ratio of Na+K to Ca+Mg in the whey protein solution of step 1) is at most 2. Even more preferably, the molar ratio of Na+K to Ca+Mg in the whey protein solution of step 1) is at most 1.5, even more preferably at most 1.0. Most preferably, the molar ratio of Na+K to Ca+Mg in the whey protein solution of step 1) is at most 0.5, for example at most 0.2.

[0627] 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 the element Ca, and m Mg is the molar content of the element Mg.

[0628] Particularly preferably, the BLG in the whey protein solution has been supersaturated by salting in, so that the BLG can be crystallized from the whey protein solution by salting in.

[0629] In some embodiments of the present invention, particularly, if the edible BLG product of the present invention is also to have a certain degree of protein denaturation, the whey protein solution has a low content of denatured protein. Preferably, the degree of protein denaturation of the whey protein solution is at most 2%, preferably at most 1.5%, more preferably at most 1.0%, and most preferably at most 0.8%.

[0630] Step 2) of the method involves crystallizing at least some of the BLG in the supersaturated whey protein solution.

[0631] Particularly preferably, the crystallization in step 2) is carried out by salting in, that is, in a liquid with low ionic strength and conductivity. This is contrary to the salting-out mode, in which a large amount of salt is added to the solution to cause crystallization.

[0632] For example, the crystallization of BLG in step 2) may involve more than one of the following:

[0633] - Waiting for crystallization to occur;

[0634] - Adding seed crystals;

[0635] - Further increasing the supersaturation of BLG; and / or

[0636] - Mechanical stimulation.

[0637] In some preferred embodiments of the present invention, step 2) involves adding seed crystals to the whey protein solution. The inventors have found that adding seed crystals enables control of the time and location at which BLG crystallization occurs, so as to avoid sudden blockage of the processing equipment and unexpected stoppage during production. For example, it is usually necessary to avoid the start of crystallization when concentrating the whey protein raw material.

[0638] Particularly preferably, during the operation of step 2), the whey protein solution does not come into contact with a UF membrane or an MF membrane, unless a ceramic membrane or a high-shear system (such as DCF) is used.

[0639] In principle, any seed material that induces the crystallization of BLG can be used. However, preferably, hydrated BLG crystals or dry BLG crystals are used for inoculation to avoid adding other impurities to the whey protein solution.

[0640] When added to the whey protein solution, the seed can be in dry form or can be part of a suspension. Currently preferably, a suspension containing seeds (such as BLG crystals) is added because it seems to make the crystallization start faster. Preferably, such a suspension contains seeds with a pH of 5 to 6 and a conductivity of at most 10 mS / cm.

[0641] It should be noted that the conductivity unit "mS / cm" represents millisiemens per centimeter, and 1.00 mS corresponds to 1000 μS.

[0642] Particularly preferably, the seeds are added through a suspension of BLG crystals that have not been dried after BLG crystallization. For example, such a suspension can be a part of the BLG crystals and the mother liquor obtained from step 2) of a previous batch, or a part of the wet BLG crystals obtained from step 3), step 4), or step 5) of a previous batch.

[0643] The inventors have observed that using wet BLG crystals as seeds during step 2) provides much larger BLG crystals compared to using dry or poorly hydrated BLG crystals, which again makes the separation of BLG from the mother liquor more effective. In an experiment (where the raw materials of whey protein, crystallization conditions, the quality and particle size of the seed material, the cooling curve, and the separation method are the same), the inventors found that compared to the BLG crystals obtained by inoculating with rehydrated, dry BLG crystals (the obtained particle size: 40 - 60 μm), using non-dry BLG crystals for inoculation increased the particle size of the obtained crystals (the obtained particle size: 100 - 130 μm) by 100%.

[0644] Alternatively, if the seeds are based on dry BLG crystals, it is preferred to redisperse the crystals in an aqueous solution (such as water), and rehydrate the dry BLG crystals for at least 30 minutes, preferably at least 1.0 hour, and even more preferably at least 1.5 hours before using the obtained BLG crystal suspension to initiate crystallization.

[0645] In some embodiments of the present invention, at least some of the seeds are located on a solid phase in contact with the whey protein solution.

[0646] Preferably, the seeds have a smaller particle size compared to the desired BLG crystal size. The size of the seeds can be changed by removing the largest seeds through sieving or other size classification separation methods. For example, the particle size can also be reduced by grinding before size classification.

[0647] In some embodiments of the present 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 can have a particle size of 1 to 400 μm. Preferably, at least 90% w / w of the seed crystals can have a particle size of 5 to 200 μm. More preferably, at least 90% w / w of the seed crystals can have a particle size of 5 to 100 μm.

[0648] The particle size and dosage of the seed crystals can be adjusted to provide optimal crystallization of BLG.

[0649] In some preferred embodiments of the present invention, the seed crystals are added to the whey protein raw material before BLG supersaturation is achieved, but preferably in such a way that at least some seed crystals are still present when supersaturation is reached. This can be achieved by adding the seed crystals when the whey protein raw material approaches supersaturation (e.g., during cooling, concentration, and / or pH adjustment), and reaching a supersaturated state before the seed crystals are completely dissolved.

[0650] In some preferred embodiments of the present invention, step 2) involves further increasing the supersaturation of BLG, preferably to an extent that immediately (i.e., within at most 20 minutes, preferably within at most 5 minutes) initiates BLG crystallization. This is also referred to as the nucleation zone, where microcrystals spontaneously form and initiate the crystallization process.

[0651] For example, the supersaturation can be increased by one or more of the following:

[0652] - Further increasing the protein concentration of the whey protein solution;

[0653] - Further cooling the whey protein solution;

[0654] - Making the pH of the whey protein solution closer to the optimal pH for BLG crystallization;

[0655] - Further reducing the conductivity.

[0656] 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 where BLG is only slightly supersaturated and no seed crystals are added.

[0657] In some preferred embodiments of the present invention, providing the whey protein solution (step 1) and BLG crystallization (step 2) are carried out as two separate steps.

[0658] 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.

[0659] Such additional adjustments may involve more than one of the following:

[0660] - Further increasing the protein concentration of the crystallized whey protein solution;

[0661] - Cooling the crystallized whey protein solution to a lower temperature;

[0662] - Bringing the crystallized whey protein solution closer to the optimal pH for BLG crystallization; and

[0663] - Further reducing the conductivity of the crystallized whey protein solution.

[0664] In some preferred embodiments of the present invention, the crystallized whey protein solution is maintained in the metastable region during step 2) to avoid spontaneous formation of new crystals.

[0665] In some preferred embodiments of the present invention, at least some of the resulting BLG crystals during step 2) have an orthorhombic space group P 2 1 2 1 2 1 .

[0666] Preferably, at least some of the resulting BLG crystals have an orthorhombic space group P 2 1 2 1 2 1 ; unit cell dimensions Unit cell integral angle α = 90°, β = 90°, γ = 90°.

[0667] In some preferred embodiments of the present invention, at least some of the resulting BLG crystals have an orthorhombic space group P 2 1 2 1 2 1 ; unit cell dimensions Unit cell integral angle α = 90°, β = 90°, γ = 90°.

[0668] Even more preferably, at least some of the resulting BLG crystals may have an orthorhombic space group P 2 1 2 1 2 1 , unit cell dimensions Unit cell integral angle α = 90°, β = 90°, γ = 90°.

[0669] Most preferably, at least some of the resulting BLG crystals have an orthorhombic space group P 2 1 2 1 2 1 ; unit cell dimensions The unit cell angles are α = 90°, β = 90°, and γ = 90°.

[0670] 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 it is desired to purify BLG.

[0671] For example, step 3) may comprise separating the BLG crystals to a solids content of at least 30% w / w. Preferably, step 3) comprises separating the BLG crystals to a solids content of at least 40% w / w. Even more preferably, step 3) comprises separating the BLG crystals to a solids content of at least 50% w / w.

[0672] The inventors have found that a high solids content is beneficial for the purification of BLG because the aqueous portion adhering to the separated BLG crystals typically contains impurities that should be avoided. In addition, a high solids content reduces the energy consumption for converting the separated BLG crystals into a dry product (such as a powder) and increases the yield of BLG obtained from a given capacity of the drying unit.

[0673] In some preferred embodiments of the present invention, step 3) comprises separating the BLG crystals to a solids content of at least 60% w / w. Preferably, step 3) comprises separating the BLG crystals to a solids content of at least 70% w / w. Even more preferably, step 3) comprises separating the BLG crystals to a solids content of at least 80% w / w.

[0674] In some preferred embodiments of the present invention, the separation in step 3) involves more than one of the following operations:

[0675] - Centrifugation;

[0676] - Decantation;

[0677] - Filtration;

[0678] - Sedimentation; and

[0679] - Combinations of the above operations.

[0680] These unit operations are well known and readily achievable by those skilled in the art. For example, separation by filtration includes the use of vacuum filtration, dynamic cross-flow filtration (DCF), a filter press, or a filtration centrifuge.

[0681] Depending on the desired result, different pore sizes can be used for filtration. Preferably, the filter allows native whey proteins and small aggregates to pass through 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.

[0682] Filters with larger pore sizes can also be used, and in fact are preferred if larger crystals are to be separated mainly from a liquid containing BLG crystals. In some embodiments of the present invention, the nominal pore size of the filter is at least 5 μm. Preferably, the nominal pore size of the filter is at least 20 μm. Even more preferably, the nominal pore size of the filter is at least 40 μm.

[0683] 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 can be 0.5 - 1000 μm. Even more preferably, the pore size of the filter can be 5 - 800 μm, such as 10 - 500 μm or 50 - 500 μm.

[0684] 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 can be 0.1 - 50 μm. More preferably, the pore size of the filter can be 4 - 40 μm. Even more preferably, the pore size of the filter can be 5 - 30 μm, such as 10 - 20 μm.

[0685] The advantage of using a filter with a pore size greater than 1 μm is that during separation and optionally during washing and / or recrystallization, bacteria and other microorganisms are at least partially removed. Thus, the method of the present invention enables the production of high-purity BLG with a very low bacterial load and avoids thermal damage to the protein.

[0686] Another advantage of using a filter with a pore size greater than 1 μm is that water removal and subsequent drying become easier and consume less energy.

[0687] During the preparation of the whey protein solution, the remaining whey protein solution from which the BLG crystals have been separated can be recycled into the whey protein raw material.

[0688] In some preferred embodiments of the present invention, a filtration centrifuge is used in step 3). In other preferred embodiments of the present invention, a decanter centrifuge is used in step 3). Preliminary results indicate that using a filtration 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.

[0689] Typically and preferably, the formed filter cake is dried with a dry gas to reduce the moisture content of the filter cake, preferably such that the filter cake can be peeled off from the filter. If the filter cake is directly converted into a dry edible BLG composition, the use of the dry gas can form part of the separation step or can be part of the final drying step.

[0690] In some preferred embodiments of the present invention, step 3) employs a dynamic cross-flow filtration (DCF) unit.

[0691] Initial tests showed that a DCF unit with a membrane pore size of 0.03 - 5 μm (preferably 0.3 - 1.0 μm) can effectively separate BLG crystals, and the inventors have observed that the DCF unit can operate for a sufficient duration to separate the crystals from even a large batch of whey protein solution containing BLG crystals.

[0692] In some preferred embodiments of the present invention, step 3) is carried out using a DCF unit equipped with a membrane capable of retaining BLG crystals, the DCF permeate is recycled to form part of the whey protein solution or whey protein feedstock, and the DCF retentate can be recovered or returned to the crystallization tank. Preferably, for example, the DCF permeate is treated by ultrafiltration / diafiltration to supersaturate the BLG and then mixed again with the whey protein solution or whey protein feedstock.

[0693] Advantageously, these embodiments do not require raising the temperature of the liquid fluid above 15 °C and are thus less susceptible to microbial contamination compared to method variants that require higher temperatures. Another industrial advantage of these embodiments is that the supersaturation is easily controlled and can be maintained at a level where no unwanted spontaneous crystallization occurs. Therefore, during these method 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.

[0694] These embodiments are illustrated in Example 10 of PCT application No. PCT / EP2017 / 084553 and shown in Figure 26. These embodiments can be carried out in a batch process or a continuous process.

[0695] In some preferred embodiments of the present invention, the method includes step 4) of washing the BLG crystals, for example, washing the separated BLG crystals of step 3). The washing can include a single washing or multiple washing steps.

[0696] Preferably, the washing in step 4) includes 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.

[0697] Preferably, the washing liquid is selected to avoid complete dissolution of the BLG crystals. For example, the washing liquid may comprise or even consist essentially of cold deionized water, cold tap water or cold reverse osmosis permeate.

[0698] For example, the washing liquid may comprise or even consist essentially of cold demineralized water, cold tap water or cold reverse osmosis permeate.

[0699] The pH of the washing liquid may be from 5 to 6, preferably from 5.0 to 6.0, even more preferably from 5.1 to 6.0, such as from 5.1 to 5.9.

[0700] Alternatively, the pH of the washing liquid may be from 6.1 to 8, preferably from 6.4 to 7.6, even more preferably from 6.6 to 7.4, such as from 6.8 to 7.2. This is typically the pH when the washing liquid is deionized water, tap water or reverse osmosis permeate. Generally preferably, the washing liquid has a low mineral content and a low buffering capacity.

[0701] The conductivity of the washing liquid is at most 0.1 mS / cm, preferably at most 0.02 mS / cm, even more preferably at most 0.005 mS / cm.

[0702] Washing liquids with even lower conductivity may be used. For example, the conductivity of the washing liquid may be at most 1 μS / cm. Alternatively, the conductivity of the washing liquid may be at most 0.1 μS / cm, such as about 0.05 μS / cm.

[0703] Preferably, the washing step is carried out at a low temperature to limit the dissolution of the 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.

[0704] For example, the washing step may be carried out at most 5 °C (more preferably at most 2 °C, such as about 0 °C). Temperatures below 0 °C may be used, provided that the washing liquid does not freeze at that temperature, for example due to the presence of more than one freezing point depressant.

[0705] In some embodiments of the present invention, the washing liquid comprises BLG. For example, the amount of BLG is at least 1% w / w (preferably at least 3% w / w, such as 4% w / w).

[0706] Generally, the washing in 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 in 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.

[0707] Typically, the weight ratio between the total amount of the washing liquid and the initial amount of the separated BLG crystals is at least 1, preferably at least 2, more preferably at least 5. For example, the weight ratio between the total amount of the washing liquid and the initial amount of the separated BLG crystals is at least 10. Alternatively, the weight ratio between the total amount of the washing liquid and the initial amount of the separated BLG crystals is at least 20, such as at least 50 or at least 100.

[0708] The term "total amount of the washing liquid" refers to the total amount of the washing liquid used during the entire period.

[0709] In some preferred embodiments of the present invention, more than one washing step is carried out in the same filter arrangement or a similar filter arrangement as the separation of the BLG crystals. The washing liquid for more than one step is added to the filter cake mainly containing the BLG crystals, and the washing liquid is removed through the filter, while the remaining part of the BLG crystals remains in the filter cake.

[0710] In a particularly preferred embodiment of the present invention, the separation in step 3) is carried out using a filter that retains the BLG crystals. Subsequently, the filter cake is contacted with more than one amount of the washing liquid that passes through the filter cake and the filter. Generally preferably, each amount of the washing liquid is at most 10 times the volume of the filter cake, preferably at most 5 times the volume of the filter cake, more preferably at most 1 time the volume of the filter cake, even more preferably at most 0.5 times the volume of the filter cake, such as 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.

[0711] For example, the used washing liquid from step 4) can be recycled into the whey protein raw material or the whey protein solution, from which the washed-out BLG can be separated again.

[0712] In addition, the method may include step 5), and step 5) involves a recrystallization step including the following steps:

[0713] - Dissolving the separated BLG crystals in a recrystallization liquid;

[0714] - Adjusting the recrystallization liquid so that the BLG is supersaturated;

[0715] - Crystallizing the BLG in the supersaturated and adjusted recrystallization liquid; and

[0716] - Separating the BLG crystals from the remaining adjusted recrystallization liquid.

[0717] Step 5) may include a single recrystallization step or multiple recrystallization steps.

[0718] In some embodiments of the present invention, the BLG crystal in step 3) or step 4) is recrystallized at least 2 times. For example, the BLG crystal can be recrystallized at least 3 times, such as at least 4 times.

[0719] The washing and recrystallization steps can be combined in any order and can be carried out multiple times if needed.

[0720] For example, the following method steps can be performed on the separated BLG crystal in step 3):

[0721] - More than one washing step (step 4); then

[0722] - More than one recrystallization step (step 5).

[0723] Alternatively, the following method steps can be performed on the separated BLG crystal in step 3):

[0724] - More than one recrystallization step (step 5); then

[0725] - More than one washing step (step 4).

[0726] Multiple washing steps and recrystallization steps can also be combined, for example, in the following order:

[0727] - More than one washing step (step 4);

[0728] - More than one recrystallization step (step 5);

[0729] - More than one washing step (step 4); and

[0730] - More than one recrystallization step (step 5).

[0731] Or, for example, in the following order:

[0732] - More than one recrystallization step (step 5);

[0733] - More than one washing step (step 4);

[0734] - More than one recrystallization step (step 5); and

[0735] - More than one washing step (step 4).

[0736] The inventors have noticed that the crystallization process (including the preparation of whey protein solution) is prone to growing microorganisms, and it is found beneficial to improve the process to solve this problem.

[0737] Particularly preferably, the total time that the BLG molecules are at a temperature above 12 °C from the provision of the whey protein raw material to the isolation of 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, and most preferably at most 3 hours.

[0738] Possibly and generally preferably, the duration is further reduced; thus, in some preferred embodiments of the present invention, the total time that the BLG molecules are at a temperature above 12 °C from the provision of the whey protein raw material to the isolation of the BLG molecules in step c) is at most 2 hours, preferably 1 hour, more preferably at most 0.5 hour, even more preferably at most 0.3 hour, and most preferably at most 0.1 hour.

[0739] In some embodiments of the present invention, the method further involves subjecting the isolated BLG to an additional BLG enrichment step, for example, based on chromatography or selective filtration. However, in other preferred embodiments of the present invention, the method does not include an additional BLG enrichment step after step 2). The term "additional BLG enrichment step" refers to a method step for enriching BLG relative to the total amount of protein, and this step has nothing to do with BLG crystallization or treating BLG crystals. An example of such an additional BLG enrichment step is ion exchange chromatography. Washing the BLG crystals and / or recrystallizing the BLG are not considered "additional BLG enrichment steps".

[0740] In some preferred embodiments of the present invention, the method includes a drying step, wherein the BLG-rich composition obtained from step 3), 4) or 5) is converted into a dry composition.

[0741] In a particularly preferred embodiment of the present invention, the method for preparing a BLG-rich composition includes the following steps:

[0742] 1) Providing a whey protein solution, the whey protein solution containing BLG and at least one other whey protein, the whey protein solution being supersaturated with BLG, the pH of the whey protein solution being 5 to 6, and the whey protein solution containing:

[0743] - 70 to 100% w / w of protein relative to the total amount of solids;

[0744] - 30 to 90% w / w, preferably 30 to 70% of non-aggregated BLG relative to the total amount of protein;

[0745] - 4 to 50% w / w, preferably 8 to 35% of non-aggregated ALA relative to the total amount of protein;

[0746] - 0 to 25% w / w of CMP relative to the total amount of protein;

[0747] - At least 10% w / w protein, relative to the total weight of the whey protein solution;

[0748] 2) Crystallize BLG in a supersaturated whey protein solution, preferably by seeding;

[0749] 3) Separate the BLG crystals from the remaining whey protein solution;

[0750] 4) Optionally, wash the separated BLG crystals obtained in step 3); and

[0751] 5) Optionally, recrystallize the BLG crystals obtained in step 3) or 4).

[0752] Preferably, the whey protein solution is a demineralized whey protein solution. Preferably, the ratio between the 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.

[0753] These embodiments are particularly useful for preparing a BLG isolate with low minerals and low phosphorus.

[0754] In some preferred embodiments, the method is implemented as a batch method. Alternatively and sometimes preferably, the process can be implemented as a semi-batch method. In other preferred embodiments, the process is implemented as a continuous method.

[0755] One advantage of the present method is that it is much faster than comparable methods of BLG crystallization in the prior art. The duration from initially conditioning the whey protein raw material to the completion of the separation in step 3) can be at most 10 hours, preferably at most 4 hours, more preferably at most 2 hours, and even more preferably at most 1 hour.

[0756] Generally, it is preferred to use mild temperatures to prepare the BLG-rich composition, and the mild temperatures do not damage the nutritional value of the non-aggregated BLG or other whey proteins in the whey protein raw material.

[0757] In some preferred embodiments of the present invention, during the method, the non-aggregated BLG is not subjected to a temperature higher than 90 °C. Preferably, during the method, the BLG is not subjected to a temperature higher than 80 °C. Even more preferably, during the method, the BLG is not subjected to a temperature higher than 75 °C. It should be noted that even though spray drying typically uses temperatures above 150 °C, the short exposure time and the simultaneous evaporation of water mean that the temperature of the spray-dried protein does not exceed 50 - 70 °C.

[0758] Regardless of the method used to prepare the BLG-rich composition, it can include a step of drying the BLG-rich composition. However, currently it is preferred to use the BLG-rich composition without drying it to avoid the risk of damaging the protein during drying.

[0759] If a BLG-rich composition isolated from a whey protein source does not yet have the properties required to be used as a liquid BLG isolate, it can be subjected to one or more steps selected from the group below as part of providing a liquid BLG isolate:

[0760] - Demineralization;

[0761] - Mineral addition;

[0762] - Dilution;

[0763] - Concentration;

[0764] - Physical reduction of microorganisms; and

[0765] - pH adjustment.

[0766] For example, non-limiting examples of demineralization include dialysis, gel filtration, UF / filtration, NF / filtration, and ion exchange chromatography.

[0767] Non-limiting examples of mineral addition include addition of 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 citrate or lactate. Minerals can be added in solid, suspension, or dissolved form.

[0768] For example, non-limiting examples of dilution include addition of a liquid diluent, such as water, demineralized water, or an aqueous solution of minerals, acids, or bases.

[0769] For example, non-limiting examples of concentration include evaporation, reverse osmosis, nanofiltration, ultrafiltration, and combinations thereof.

[0770] If concentration must increase the concentration of protein relative to the total solids, a concentration step, such as ultrafiltration or dialysis, is preferably used. If concentration need not increase the concentration of protein relative to the total solids, evaporation, nanofiltration, and / or reverse osmosis can be employed, for example.

[0771] For example, non-limiting examples of physical reduction of microorganisms 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.

[0772] Typically, bacterial filtration involves microfiltration or macroporous ultrafiltration and requires a pore size capable of retaining microorganisms but allowing proteins and other target components to pass through. Useful pore sizes are generally 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 generally at least 0.1 μm.

[0773] For example, bacterial filtration may involve a membrane with a pore size of 0.02 to 1 μm, preferably 0.03 to 0.8 μm, more preferably 0.04 to 0.6 μm, even more preferably 0.05 to 0.4 μm, and most preferably 0.1 to 0.2 μm.

[0774] In some preferred embodiments of the present invention, the BLG isolate is subjected to bacterial filtration and then heat-treated at a temperature of at most 80 °C (preferably at most 75 °C). Preferably, the combination of the temperature and duration of this heat treatment is selected to provide a sterile beverage formulation.

[0775] In other preferred embodiments of the present invention, the liquid BLG isolate is subjected to bacterial filtration and then heat-treated at a temperature of at least 150 °C for a duration of at most 0.2 seconds (preferably at most 0.1 second). Preferably, the combination of the temperature and the duration of this heat treatment is selected to provide a sterile beverage formulation.

[0776] For example, non-limiting examples of pH adjustment include adding a base and / or an acid (preferably a food-acceptable base and / or acid). Particularly preferably, an acid and / or a 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, phosphate, and combinations thereof.

[0777] Many preferred embodiments of providing a liquid BLG isolate from a BLG-rich composition in step a) are described below. The method steps mentioned in the context are applied to the BLG-containing product stream following the BLG-rich composition.

[0778] In some preferred embodiments of the present invention, for example, if it is useful that the BLG-rich composition contains BLG crystals from the above-mentioned salting-out process, providing the liquid BLG isolate in step a) includes subjecting the BLG-rich composition to the following steps in the following order:

[0779] - For example, adjusting the pH to i) 2 to 4.9 or ii) 6.1 to 8.5 to dissolve the BLG crystals in the BLG-rich composition;

[0780] - Optionally, demineralizing or adding minerals; and

[0781] - One of the following two:

[0782] - Concentrating to the desired protein content and then physically reducing microorganisms; or

[0783] - Physically reducing microorganisms and then concentrating to the desired protein content.

[0784] In other preferred embodiments of the present invention, for example, if it is useful for the BLG-rich composition to contain BLG crystals from the above salting-in process, the providing of the liquid BLG isolate in step a) comprises subjecting the BLG-rich composition to the following steps in the following order:

[0785] - 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;

[0786] - Optionally, demineralizing or adding minerals; and

[0787] - Concentrating to the desired protein content.

[0788] In other preferred embodiments of the present invention, for example, if it is useful for the BLG-rich composition to contain BLG crystals from the above salting-in process, the providing of the liquid BLG isolate in step a) comprises subjecting the BLG-rich composition to the following steps in the following order:

[0789] - Adding minerals and preferably soluble salts to dissolve the BLG crystals of the BLG-rich composition, preferably while maintaining the pH at 5.0 to 6.0; and

[0790] - One of the following two:

[0791] - Concentrating to the desired protein content and then physically reducing microorganisms; or

[0792] - Physically reducing microorganisms and then concentrating to the desired protein content.

[0793] When treating an acidic, dissolved BLG-rich composition, it is particularly advantageous to perform heat treatment alone as a physical method for reducing microorganisms, or to combine heat treatment with one or more other physical methods for reducing microorganisms as described herein. The inventors have found that using mild heat treatment under acidic conditions is particularly beneficial because it allows BLG to maintain its native folded state but still helps to reduce the microbial load of the treated stream.

[0794] Particularly preferably, a bacterial filtration step is performed on the acidic, dissolved BLG-rich composition while its total protein concentration is at most 27% w / w, preferably at most 22% w / w, and even more preferably at most 17% w / w, followed by a heat treatment step on the bacterially filtered BLG-rich composition or liquid BLG isolate.

[0795] Even more preferably, a bacterial filtration step is carried out on the acidic, dissolved BLG-rich composition while the concentration of its total protein is 5 to 27% w / w, preferably 10 to 22% w / w, even more preferably at most 12 to 17% w / w, and then the bacterially filtered BLG-rich composition or the liquid BLG isolate is heat-treated.

[0796] Preferably, a heat treatment step is carried out on the liquid BLG isolate, preferably as the last step before spray drying.

[0797] Generally preferably, during the provision of the liquid BLG isolate, the unfolding of BLG is avoided or at least restricted. If heat treatment is carried out at a pH of 2 to 4.9, the temperature is preferably maintained at at most 82 °C, preferably at most 80 °C, more preferably at most 78 °C, to restrict or even avoid the unfolding of BLG.

[0798] Preferably, the heat treatment is at least pasteurization.

[0799] In some preferred embodiments of the present invention, the temperature of the heat treatment is 70 to 80 °C, preferably 70 to 79 °C, more preferably 71 to 78 °C, even more preferably 72 to 77 °C, most preferably 73 to 76 °C, for example about 75 °C.

[0800] 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.

[0801] In a particularly preferred embodiment of the present invention, the heat treatment provides a temperature of 70 to 78 °C for 1 second to 30 minutes, more preferably a temperature of 71 to 77 °C for 1 minute to 25 minutes, even more preferably a temperature of 72 to 76 °C for 2 minutes to 20 minutes.

[0802] In some embodiments, higher temperatures may also be preferred, especially if it is desired to unfold and optionally aggregate BLG before drying. For example, the temperature of the heat treatment can be at least 81 °C, preferably at least 91 °C, more preferably at least 100 °C, even more preferably at least 120 °C, most preferably at least 140 °C.

[0803] For example, the heat treatment can be a UHT-type treatment, which generally involves a temperature of 135 to 144 °C and a duration of 2 to 10 seconds.

[0804] Or, but also preferably, the heat treatment can include a temperature of 145 to 180 °C and a duration of 0.01 to 1 second, more preferably a temperature of 150 to 180 °C and a duration of 0.01 to 0.2 second.

[0805] In some preferred embodiments of the present invention, providing the liquid BLG isolate in step a) includes: heat sterilization while the pH is 2 to 4.9, preferably 2.5 to 4.7, more preferably 2.8 to 4.3, and even more preferably 3.2 to 4.0.

[0806] In other preferred embodiments of the present invention, providing the liquid BLG isolate from the BLG-rich composition in step a) includes: physically reducing microorganisms by heat treatment while the pH is 2 to 4.9, preferably 2.5 to 4.7, more preferably 2.8 to 4.3, and even more preferably 3.2 to 4.0.

[0807] In other preferred embodiments of the present invention, providing the liquid BLG isolate from the BLG-rich composition in step a) includes: demineralization while the pH is 6.1 to 8.5, preferably 6.3 to 8.0, and more preferably 6.5 to 7.5. The inventors have found that such demineralization is beneficial to improving the thermal stability of the BLG isolate powder prepared by this method.

[0808] In a particularly preferred embodiment of the present invention, providing the liquid BLG isolate from the BLG-rich composition in step a) includes:

[0809] - Physically reducing microorganisms by heat treatment while the pH is 2 to 4.9, preferably 2.5 to 4.7, more preferably 2.8 to 4.3, and even more preferably 3.2 to 4.0; and

[0810] - Demineralization while the pH is 6.1 to 8.5, preferably 6.3 to 8.0, and more preferably 6.5 to 7.5.

[0811] In some preferred embodiments of the present invention, for example, if it is useful that the BLG-rich composition contains BLG crystals from the above salting-out process, providing the liquid BLG isolate in step a) includes performing the following steps on the BLG-rich composition in the following order:

[0812] - Adjusting the pH value to 2 to 4.9 to dissolve the BLG crystals in the BLG-rich composition;

[0813] - Optionally, concentrating to the desired protein content while the pH is 2 to 4.9, preferably 2.5 to 4.0, and more preferably 3.0 to 3.9;

[0814] - Physically reducing microorganisms by heat treatment while the pH is 2 to 4.9, preferably 2.5 to 4.7, more preferably 2.8 to 4.3, and even more preferably 3.2 to 4.0.

[0815] If the pH of the BLG-rich composition is already between 2 and 4.9, then preferably, providing the liquid BLG isolate in step a) comprises subjecting the BLG-rich composition to the following steps in the following order:

[0816] - Demineralizing 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;

[0817] - Optionally, concentrating to the desired protein content 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] - Physically reducing 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.

[0819] In other preferred embodiments of the present invention, which are particularly suitable for providing a liquid BLG isolate having a pH of 5.0 to 8.5, preferably 6.1 to 8.5, more preferably 6.5 to 8.0, providing the liquid BLG isolate in step a) comprises subjecting the BLG-rich composition to the following steps in the following order:

[0820] - For example, adjusting the pH to 2 to 4.9 to dissolve the BLG crystals of the BLG-rich composition;

[0821] - Optionally, concentrating to the desired protein content;

[0822] - Physically reducing 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;

[0823] - Adjusting the pH to 5.0 to 8.5, preferably 6.1 to 8.5, more preferably 6.5 to 8.0.

[0824] In other preferred embodiments of the present invention, which are particularly suitable for 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, providing the liquid BLG isolate in step a) comprises subjecting the BLG-rich composition to the following steps in the following order:

[0825] - For example, adjusting the pH to 2 to 4.9 to dissolve the BLG crystals of the BLG-rich composition;

[0826] - Optionally, concentrating to the desired protein content 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;

[0827] - Physically reducing microorganisms by heat treatment, with a pH of 2 to 4.9, preferably 2.5 to 4.7, more preferably 2.8 to 4.3, and even more preferably 3.2 to 4.0;

[0828] - Adjusting the pH to 6.1 to 8.5, preferably 6.3 to 8.0, more preferably 6.5 to 7.5; and

[0829] - Demineralizing, with a pH of 6.1 to 8.5, preferably 6.3 to 8.0, more preferably 6.5 to 7.5.

[0830] In other preferred embodiments of the present invention, which are particularly suitable for providing a liquid BLG isolate with a pH of 6.1 to 8.5, preferably 6.1 to 8.5, more preferably 6.5 to 8.0, the provision of the liquid BLG isolate in step a) comprises subjecting the BLG-rich composition to the following steps in the following order:

[0831] - Adjusting the pH to at least 6.1 to dissolve the BLG crystals of the BLG-rich composition;

[0832] - Demineralizing, with a pH of 6.1 to 8.5, preferably 6.5 to 8.0, more preferably 6.5 to 7.5;

[0833] - Optionally, concentrating to the desired protein content, with a pH of 6.1 to 8.5, preferably 6.5 to 8.0, more preferably 6.5 to 7.5;

[0834] - Optionally, physically reducing microorganisms by heat treatment, with a pH of 6.1 to 8.5, preferably 6.5 to 8.0, more preferably 6.5 to 7.5.

[0835] Generally, the temperature during the conversion of the BLG-rich composition into a liquid BLG isolate is 0 to 82 °C, and even higher temperatures may be involved for heat treatment. If the pH of the BLG stream during treatment is higher than 4.9, the temperature range is preferably 0 to 65 °C, more preferably 0 to 15 °C or 50 to 65 °C, to reduce microbial growth.

[0836] If the pH of the liquid BLG isolate is at most 4.9, even more preferably at most 4.1, the preferred temperature is from 0 to 82 °C, and advantageously from 0 to 15 °C or from 50 to 80 °C, to reduce microbial growth. The inventors have found that it is particularly advantageous to carry out the process or at least the concentration step (if required) at a temperature of from 50 to 80 °C, more preferably from 60 to 80 °C, even more preferably from 65 to 78 °C, since the high temperature increases the efficiency of the concentration step and at the same time helps to reduce the micro-organisms. This embodiment makes it possible to produce a liquid BLG isolate with a high BLG content and a very low micro-organism 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 characteristics.

[0837] In some preferred embodiments of the invention, the process for converting the BLG-rich composition into a liquid BLG isolate is carried out at a temperature of from 0 to 15 °C, preferably from 1 to 10 °C.

[0838] In other preferred embodiments of the invention, at least some (preferably the whole) of the process for converting the BLG-rich composition into a liquid BLG isolate is carried out at a temperature of from 50 to 82 °C, preferably from 55 to 80 °C, more preferably from 60 to 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 from 3.0 to 4.3.

[0839] In some preferred embodiments of the invention, at least some (preferably the whole) of the process for converting the BLG-rich composition into a liquid BLG isolate is carried out at a temperature of from 78 to 82 °C, with a pH of from 3.0 to 3.7. In other preferred embodiments of the invention, at least some (preferably the whole) of the process for converting the BLG-rich composition into a liquid BLG isolate is carried out at a temperature of from 60 to 78 °C, with a pH of from 3.7 to 4.3.

[0840] Preferably, the BLG-rich composition has substantially the same protein composition as the liquid BLG isolate and generally no additional protein fraction needs to be added when converting the BLG-rich composition into a liquid BLG isolate.

[0841] In some preferred embodiments of the invention, the BLG content of the BLG-rich composition 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, particularly preferably, the BLG content of the BLG-rich composition is at least 97% w / w, more preferably at least 99% w / w, for example preferably about 100% w / w, relative to the total amount of protein.

[0842] In some preferred embodiments of the present invention, the total protein content of the BLG-rich 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, and most preferably at least 30% w / w.

[0843] In some preferred embodiments of the present invention, the total protein content of the BLG-rich composition is 5 - 45% w / w, preferably 10 - 40% w / w, more preferably 15 - 38% w / w, even more preferably 20 - 35% w / w.

[0844] In some preferred embodiments of the present invention, the sum of α-lactalbumin (ALA) and caseinomacropeptide (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 of the BLG-rich composition.

[0845] In other preferred embodiments of the present invention, each major non-BLG whey protein in the BLG-rich composition, expressed as a weight percentage relative to the total protein content, has a weight percentage of at most 25%, preferably at most 20%, more preferably at most 15%, even more preferably at most 10%, and most preferably at most 6% relative to the total protein content of a standard whey protein concentrate from sweet whey.

[0846] It may be desirable to have an even lower concentration of the major non-BLG whey proteins. Thus, in other preferred embodiments of the present invention, each major non-BLG whey protein in the BLG-rich composition, expressed as a weight percentage relative to the total protein content, has a weight percentage of at most 4%, preferably at most 3%, more preferably at most 2%, and even more preferably at most 1% relative to the total protein content of a standard whey protein concentrate from sweet whey.

[0847] The inventors have found that low levels of lactoferrin and / or lactoperoxidase are particularly advantageous for obtaining a color-neutral whey protein product.

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

[0849] Similarly, in some preferred embodiments of the present invention, lactoperoxidase in the BLG-rich composition is present as a weight percentage relative to the total amount of protein. Relative to the total amount of protein in the standard whey protein concentrate from sweet whey, the weight percentage of lactoperoxidase is at most 25%, preferably at most 20%, more preferably at most 15%, even more preferably at most 10%, and most preferably at most 6%. Even lower concentrations of lactoperoxidase may be required. Thus, in other preferred embodiments of the present invention, lactoperoxidase is present as a weight percentage relative to the total amount of protein. Relative to the total amount of protein in the standard whey protein concentrate from sweet whey, the weight percentage of lactoperoxidase is at most 4%, preferably at most 3%, more preferably at most 2%, and even more preferably at most 1%.

[0850] In some preferred embodiments of the present invention, the total solids content of the BLG-rich composition is 5 - 50% w / w, preferably 10 - 45% w / w, more preferably 15 - 40% w / w, and even more preferably 20 - 35% w / w.

[0851] In some preferred embodiments of the present invention, the water content of the BLG-rich composition is 50 - 95% w / w, preferably 55 - 90% w / w, more preferably 60 - 85% w / w, and even more preferably 65 - 80% w / w.

[0852] In some preferred embodiments of the present invention, the carbohydrate content of the BLG-rich 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, and most preferably at most 0.1%.

[0853] In some preferred embodiments of the present invention, the lipid content of the BLG-rich composition is at most 10% w / w, preferably at most 5% w / w, more preferably at most 2% w / w, and even more preferably at most 0.1% w / w.

[0854] In some preferred embodiments of the present invention, the BLG-rich composition is directly used as a liquid BLG isolate, for example, if it already has the desired pH and chemical composition.

[0855] Although some embodiments of the present invention do not require the physical reduction of microorganisms in step b) and thus only require steps a) and c); other preferred embodiments require the physical reduction of microorganisms in step b) and thus include all three steps a), b) and c).

[0856] Thus, in some preferred embodiments of the present invention, the liquid BLG isolate is physically reduced in microorganisms.

[0857] Useful examples of physical reduction of microorganisms include one or more of heating, bacterial filtration, ultraviolet radiation, high pressure treatment, pulsed electric field treatment and ultrasound.

[0858] In some preferred embodiments of the present invention, the physical reduction of microorganisms comprises or even consists of heat treatment.

[0859] Preferably, the heat treatment includes at least pasteurization.

[0860] In a specific embodiment, the heat treatment includes heating the liquid BLG isolate to a temperature of 70 to 82 °C.

[0861] In some preferred embodiments of the present invention, the temperature of the heat treatment is 70 to 80 °C, preferably 70 - 79 °C, more preferably 71 to 78 °C, even more preferably 72 to 77 °C, most preferably 73 to 76 °C, for example about 75 °C.

[0862] Preferably, when carried out at a temperature of 70 to 80 °C, the duration of the heat treatment is from 1 second to 30 minutes. The maximum exposure time is most suitable for the lowest temperature and vice versa.

[0863] In a particularly preferred embodiment of the present invention, the heat treatment provides a temperature of 70 to 78 °C for 1 second to 30 minutes, more preferably a temperature of 71 to 77 °C for 1 minute to 25 minutes, even more preferably a temperature of 72 to 76 °C for 2 minutes to 20 minutes.

[0864] In some embodiments, higher temperatures may also be preferred, especially if it is necessary to unfold and optionally aggregate the BLG before drying. For example, the temperature of the heat treatment can be at least 81 °C, preferably at least 91 °C, more preferably at least 100 °C, even more preferably at least 120 °C, most preferably at least 140 °C.

[0865] 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.

[0866] Optionally, for example, the heat treatment may be a UHT-type treatment, which typically involves a temperature of 135 to 144 °C and a duration of 2 to 10 seconds.

[0867] Or, but also preferably, the heat treatment may involve a temperature of 145 to 180 °C and a duration of 0.01 to 2 seconds, more preferably a temperature of 150 to 180 °C and a duration of 0.01 to 0.3 seconds.

[0868] Carrying out the heat treatment may involve using conventional equipment, such as plate or tubular heat exchangers, scraped surface heat exchangers or dry distillation systems. Alternatively, for heat treatments above 95 °C, particularly preferably, direct steam-based heating may be employed, such as using direct steam injection, direct steam infusion or spray cooking. Furthermore, such direct steam-based heating is preferably used in combination with rapid cooling. Suitable examples of carrying out spray cooking can be found in WO2009113858A1, the content of which is incorporated herein by reference for all purposes. Suitable embodiments of carrying out direct steam injection and direct steam infusion can be found in WO2009113858A1 and WO 2010 / 085957A3, the content of which is incorporated herein by reference for all purposes. For example, general aspects of high-temperature treatment can be found in "Thermal technologies in food processing" ISBN 185573558 X, the content of which is incorporated herein by reference for all purposes.

[0869] 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 obtained by combining bacterial filtration and pasteurization.

[0870] In some preferred embodiments of the present invention, a liquid BLG isolate having a preferred pH of 2 to 4.9 is subjected to bacterial filtration and then heat-treated at a temperature of at most 80 °C (preferably at most 75 °C). Preferably, the combination of the temperature and duration of this heat treatment is selected to provide a liquid BLG isolate.

[0871] In other preferred embodiments of the present invention, the liquid BLG isolate is bacterially filtered and then heat treated at a temperature of at least 150 °C for up to 0.2 seconds (preferably up to 0.1 second). Preferably, the combination of the temperature and duration of this heat treatment is selected to provide the liquid BLG isolate.

[0872] Preferably, step c) of the method involves spray drying or freeze drying. Spray drying is particularly preferred.

[0873] The inventors have found that it is particularly advantageous to avoid exposing the liquid BLG isolate to heat treatment regimes that cause a large amount of BLG to unfold or denature. Therefore, if the liquid BLG isolate is preheated before spraying, the heat load is preferably carefully controlled.

[0874] In some embodiments of the present invention, when reaching the outlet of the spraying device (such as a nozzle or 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 spraying 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, and most preferably at most 5 °C.

[0875] For example, the spraying device of the spray dryer is a nozzle or atomizer, which converts the solution or suspension to be dried into droplets that enter the drying chamber of the spray dryer.

[0876] In some embodiments of the present invention, particularly preferably, when reaching the outlet of the spraying device, the temperature of the liquid BLG isolate is 0 - 60 °C, preferably 2 - 40 °C, more preferably 4 - 35 °C, and most preferably 5 - 10 °C.

[0877] 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. Empirically, it is considered that the solid to be spray dried is heated to a temperature 10 - 15 °C lower than the gas outlet temperature.

[0878] 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.

[0879] The advantage of this method is that, before the drying step, the solid content of the liquid BLG isolate to be dried can be very high, so less water needs to be removed in the drying operation and less energy is consumed.

[0880] The inventors found that the lower the degree of unfolding of BLG, the higher the concentration of BLG that can be processed before spray drying.

[0881] 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.

[0882] 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 25 - 40% w / w, such as about 35% w / w.

[0883] In some preferred embodiments of the present invention, the total amount of protein contained in the liquid BLG isolate is at least 10% w / w. Preferably, the total amount of protein contained in the liquid BLG isolate is at least 20% w / w. More preferably, the total amount of protein contained in the liquid BLG isolate is at least 25% w / w. Even more preferably, the total amount of protein contained in the liquid BLG isolate is at least 30% w / w. Most preferably, the total amount of protein contained in the liquid BLG isolate is at least 35% w / w.

[0884] In other preferred embodiments of the present invention, the total amount of protein contained in the liquid BLG isolate is 10 - 50% w / w. Preferably, the total amount of protein contained in the liquid BLG isolate is 15 - 45% w / w. More preferably, the total amount of protein contained in the liquid BLG isolate is 20 - 40% w / w. Even more preferably, the total amount of protein contained in the liquid BLG isolate is 25 - 38% w / w, such as about 35% w / w.

[0885] The inventors found that reducing the protein content decreases the energy consumption for converting the liquid BLG isolate into powder and increases the BLG yield obtained from a drying unit of a given capacity.

[0886] Preferably, the method 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 in the whey protein solution.

[0887] In some preferred embodiments of the present invention, during the method, non-aggregated BLG is not subjected to a temperature higher than 90 °C. Preferably, during the method, non-aggregated BLG is not subjected to a temperature higher than 80 °C. Even more preferably, during the method, non-aggregated BLG is not subjected to a temperature higher than 75 °C. It should be noted that even though spray drying typically uses temperatures above 150 °C, the short exposure time and the simultaneous evaporation of water mean that the temperature of the spray-dried protein does not exceed 40 - 70 °C.

[0888] The inventors have found that prolonging the heating during the drying step reduces the amount of BLG in the non-denatured form. In some preferred embodiments of the present 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 any detectable denaturation of BLG.

[0889] Furthermore, the drying step may include fluidized bed drying, for example integrated in a spray drying device, or performed as a separate unit after spray drying.

[0890] The combination of spray drying and fluidized bed drying makes it possible to reduce the amount of water removed as the droplets to be dried move through the spray drying chamber, and the residual water is removed from the moist powder by fluidized bed drying. This solution requires less energy for drying compared to drying only by spray drying, and also makes it possible to modify the powder by, for example, instantization and / or agglomeration. Preferably, instantization is carried out by applying lecithin or another useful wetting agent to the surface of the powder. When instantization is carried out, the amount of instantization reagent (such as lecithin) dissolved in edible oil is usually 0.5 - 2% w / w, preferably 1.0 - 1.5% w / w, relative to the total weight of the final powder.

[0891] Preferably, the method further includes the step of packaging the BLG isolate powder. The packaged BLG isolate powder product includes a container (preferably a sealed container) containing the BLG isolate powder described herein.

[0892] In some embodiments of the present invention, the BLG isolate powder is hermetically sealed in a container and optionally packaged with an inert gas.

[0893] Various different containers can be used to store the BLG isolate powder. For example, preferred containers are bags, drums, sachets, boxes, cans, and capsules.

[0894] One particularly preferred embodiment of the present invention relates to a method for producing a dry BLG isolate powder, which 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 protein content. The method comprises the following steps:

[0895] a) Providing a liquid BLG isolate, which has the following characteristics:

[0896] i) The pH is 2 to 4.9;

[0897] ii) The pH is 6.1 to 8.5; or

[0898] iii) The pH is 5.0 to 6.0;

[0899] The BLG content of the liquid BLG isolate is at least 85% w / w, preferably at least 90% w / w, even more preferably at least 94% w / w, relative to the total protein content;

[0900] b) Optionally, physically reducing the microorganisms in the liquid BLG isolate;

[0901] c) Drying the liquid BLG isolate, preferably by spray drying;

[0902] Wherein, providing the liquid BLG isolate in step a) includes:

[0903] - Preparing a BLG-rich composition by a method comprising the following steps:

[0904] 1) Providing a whey protein solution, the whey protein solution containing non-aggregated BLG and at least one other whey protein, the whey protein solution being BLG supersaturated and having a pH of 5 to 6;

[0905] 2) Crystallizing BLG in the supersaturated whey protein solution;

[0906] 3) Separating the BLG crystals from the remaining whey protein solution;

[0907] 4) Optionally, washing the BLG crystals, such as the separated BLG crystals obtained from step 3) or 5); and

[0908] 5) Optionally, recrystallizing the BLG crystals, such as the BLG crystals obtained from step 3) or 4); and

[0909] - Treating the BLG-rich composition to at least dissolve the BLG crystals to obtain a liquid BLG isolate.

[0910] In the above embodiments, the BLG-rich composition comprises isolated BLG crystals from step 3), which are subsequently dissolved by appropriate pH adjustment or alternatively by increasing the conductivity and / or raising the temperature.

[0911] 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:

[0912] 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;

[0913] 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;

[0914] b) optionally, physically reducing the microorganisms in the liquid BLG isolate;

[0915] c) drying the liquid BLG isolate, preferably by spray drying;

[0916] wherein providing the liquid BLG isolate in step a) comprises:

[0917] - preparing a BLG-rich composition by a method comprising the following steps:

[0918] 1) providing a whey protein solution comprising non-aggregated BLG and at least one other whey protein, the whey protein solution being supersaturated with BLG and having a pH of 5 to 6;

[0919] 2) crystallizing BLG in the supersaturated whey protein solution;

[0920] 3) separating BLG crystals from the remaining whey protein solution;

[0921] 4) optionally, washing the BLG crystals, such as the isolated BLG crystals obtained from step 3) or 5); and

[0922] 5) optionally, recrystallizing the BLG crystals, such as the BLG crystals obtained from step 3) or 4); and

[0923] - adjusting the pH of the BLG-rich composition to at least 2.5 to 4.9, preferably 2.5 to 4.0, even more preferably 3.0 to 3.9.

[0924] Another particularly preferred embodiment of the present invention relates to a method for producing a dry BLG isolate powder, which 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 protein content. The method comprises the following steps:

[0925] 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;

[0926] 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 protein content;

[0927] b) optionally, physically reducing microorganisms in the liquid BLG isolate;

[0928] c) drying the liquid BLG isolate, preferably by spray drying;

[0929] wherein providing the liquid BLG isolate in step a) comprises:

[0930] - preparing a BLG-rich composition by a method comprising the following steps:

[0931] 1) providing a whey protein solution comprising non-aggregated BLG and at least one other whey protein, the whey protein solution being supersaturated with BLG and having a pH of 5 to 6;

[0932] 2) crystallizing BLG in the supersaturated whey protein solution;

[0933] 3) separating BLG crystals from the remaining whey protein solution;

[0934] 4) optionally washing the BLG crystals, such as the separated BLG crystals obtained from step 3) or 5); and

[0935] 5) optionally recrystallizing the BLG crystals, such as the BLG crystals obtained from step 3) or 4); and

[0936] - adjusting the pH of the BLG-rich composition to at least 6.1 to 8.5, preferably 6.3 to 8.0, more preferably 6.5 to 7.5.

[0937] An alternative but also preferred embodiment of the present invention relates to a method for producing a dry BLG isolate powder, which 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 protein content. The method comprises the following steps:

[0938] a) Provide 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, and even more preferably 6.5 to 7.5;

[0939] The BLG content of the liquid BLG isolate is at least 85% w / w, preferably at least 90% w / w, and even more preferably at least 94% w / w, relative to the total protein content;

[0940] b) Optionally, physically reduce the microorganisms in the liquid BLG isolate;

[0941] c) Dry the liquid BLG isolate, preferably by spray drying;

[0942] Wherein, providing the liquid BLG isolate in step a) includes the following steps in the following order:

[0943] - Prepare a BLG-rich composition by a method including the following steps:

[0944] 1) Provide a whey protein solution, the whey protein solution containing non-aggregated BLG and at least one other whey protein, the whey protein solution being BLG supersaturated and having a pH of 5 to 6;

[0945] 2) Crystallize BLG in the supersaturated whey protein solution;

[0946] 3) Separate the BLG crystals from the remaining whey protein solution;

[0947] 4) Optionally, wash the BLG crystals, such as the separated BLG crystals obtained from step 3) or 5); and

[0948] 5) Optionally, recrystallize the BLG crystals, such as the BLG crystals obtained from step 3) or 4); and

[0949] - Adjust the pH of the BLG-rich composition to 2.5 to 4.9, preferably 2.5 to 4.0, and even more preferably 3.0 to 3.9;

[0950] - Physically reduce the microorganisms, the physical reduction of microorganisms involving at least pasteurization, using a temperature of preferably 70 to 82 °C, and even more preferably 70 to 80 °C, while the pH is 2.5 to 4.9, preferably 2.5 to 4.0, and even more preferably 3.0 to 3.9;

[0951] - Adjust the pH to 5.0 to 8.5, preferably 6.1 to 8.5, more preferably 6.3 to 8.0, and even more preferably 6.5 to 7.5;

[0952] - Optionally, demineralize.

[0953] In some preferred embodiments, the method of the present invention is implemented as a batch method. Alternatively, and sometimes preferably, the method can be implemented as a semi-batch method. In other preferred embodiments, the method is implemented as a continuous method.

[0954] Another aspect of the present invention relates to the liquid BLG isolate described herein. The liquid BLG isolate is particularly useful for obtaining spray-dried BLG isolate powder and can thus 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 such.

[0955] In some preferred embodiments of the present invention, the BLG isolate powder of the present invention can be obtained by the method described herein.

[0956] In some preferred embodiments of the present invention, the liquid BLG isolate of the present invention can be obtained by the method described herein, with the drying step omitted.

[0957] One aspect of the present invention relates to the use of the BLG isolate powder or liquid BLG isolate described herein as an ingredient for producing foods. For example, the food can be a beverage or an instant beverage powder.

[0958] Preferably, the use of the BLG isolate powder or liquid BLG isolate powder provides one or more of the following effects:

[0959] - Reducing the level of dry mouthfeel;

[0960] - Improving the transparency of the resulting liquid containing the BLG isolate powder or liquid BLG isolate;

[0961] - Reducing viscosity;

[0962] - Can increase the protein concentration of heat-treated beverage foods;

[0963] - Having a lower color contribution (the inventors have observed that the BLG isolate of the present invention gives less color to, for example, protein beverages compared to the corresponding WPI solution).

[0964] In some preferred embodiments of the present invention, the BLG isolate powder is used as an ingredient for preparing a beverage, and the protein content of the beverage is 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, more preferably at least 95% w / w, and most preferably all of the total protein in the beverage.

[0965] In addition, the BLG powder of the present invention is particularly suitable for high-protein beverages, or for preparing shake powder for high-protein beverages, because the BLG powder of the present invention contributes less to viscosity than conventional WPI, thus providing more drinkable beverages.

[0966] For example, the food can be a beverage or an instant beverage powder, having a pH of 2 to 4.7 and having one or more of the following:

[0967] - Reduced level of dry mouthfeel;

[0968] - Improved transparency; and / or

[0969] - Increased protein content, and the protein content of the heat-treated beverage is preferably at least 3 to 45% w / w, more preferably 11 to 40% w / w, even more preferably 15 to 38% w / w, and most preferably 20 to 36% w / w.

[0970] It should be noted that the embodiments and features described in the content of one aspect of the present invention are also applicable to other aspects of the present invention.

[0971] All patents and non-patent references cited in this application are incorporated herein by reference in their entirety.

[0972] The present invention will now be described in further detail by the following non-limiting examples.

[0973] Examples

[0974] Example 1: Analytical method

[0975] Example 1.1: Determination of protein naturality by intrinsic tryptophan fluorescence

[0976] Tryptophan (Trp) fluorescence spectroscopy is a known tool for monitoring protein folding and unfolding. Generally, Trp residues buried in native proteins exhibit the highest fluorescence emission at approximately 330 nm compared to when Trp residues are in more solvent-exposed positions (such as unfolded proteins). In unfolded proteins, the wavelength of Trp fluorescence emission usually shifts to higher wavelengths, typically measured at approximately 350 nm. Here, we studied the heat-induced unfolding by calculating the ratio between the fluorescence emissions at 330 nm and 350 nm to study the effect of heating temperature.

[0977] The analysis includes the following steps:

[0978] · Dilute the beverage composition with MQ water to 0.6 mg / mL;

[0979] · Transfer 300 μL of the sample to a white 96-well plate, avoiding the formation of air bubbles, or transfer 3 mL of the sample to a 10 mm quartz cuvette;

[0980] · The tryptophan fluorescence emission intensity from 310 to 400 nm was recorded from the top by excitation at 295 nm using a 5 nm slit.

[0981] · Samples were measured at 22 °C using a Cary Eclipse fluorescence spectrophotometer equipped with a microplate reader attachment (G9810A) or a single cuvette holder.

[0982] · 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 used as a measure of protein native state:

[0983] ο An R of at least 1.11 describes predominantly native BLG conformation; and

[0984] ο An R less than 1.11 indicates at least partial unfolding and aggregation.

[0985] Example 1.2: Thermal stability at pH 3.9

[0986] Thermal stability at pH 3.9:

[0987] Thermal stability at pH 3.9 is a measure of the ability of the protein composition to remain transparent at pH 3.9 after long-term pasteurization.

[0988] The thermal stability at pH 3.9 was measured as follows: By mixing the powder or liquid sample to be tested with water (or, if it is a dilute liquid, concentrating it by low-temperature evaporation), an aqueous solution with a pH of 3.9 and containing 6.0% w / w protein was formed, and the pH was adjusted to 3.9 with a minimum amount of 0.1 M NaOH or 0.1 M HCl as needed.

[0989] The pH-adjusted mixture was allowed to stand for 30 minutes, 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.

[0990] Example 1.3: Determination of the degree of protein denaturation of the whey protein composition

[0991] It is known that denatured whey protein has lower solubility at pH 4.6 compared to its solubility at pH values below or above pH 4.6; therefore, the degree of denaturation of the whey protein composition was measured by determining the amount of soluble protein at pH 4.6 (relative to the total amount of protein at the pH at which the protein in the solution remains stable).

[0992] More specifically, for whey proteins, the whey protein composition to be analyzed (e.g., powder or aqueous solution) is converted to:

[0993] - A first aqueous solution containing 5.0% w / w total protein and having a pH of 7.0 or 3.0; and

[0994] - A second aqueous solution containing 5.0% w / w total protein and having a pH of 4.6.

[0995] pH adjustment is carried out using 3% (w / w) NaOH (aqueous solution) or 5% (w / w) HCl (aqueous solution).

[0996] The total protein amount (P pH 7.0或3.0 ) of the first aqueous solution is measured according to Example 1.5.

[0997] The second aqueous solution is stored at room temperature for 2 hours and then centrifuged at 3000 g for 5 minutes. The supernatant sample is recovered and analyzed according to Example 1.5 to obtain the protein concentration (S pH4.6 ) in the supernatant.

[0998] The degree of protein denaturation D of the whey protein composition is calculated as follows:

[0999] D = ((P pH 7.0或3.0 - S pH 4.6 ) / P pH 7.0或3.0 ) * 100%

[1000] Example 1.4: Determination of protein denaturation (pH 4.6 acid precipitation) using reversed-phase UPLC analysis

[1001] The BLG sample (e.g., unheated reference and heated BLG beverage composition) is diluted to 2% with MQ water. 5 mL of the protein solution, 10 mL of Milli-Q, 4 mL of 10% acetic acid, and 6 mL of 1.0 M NaOAc are mixed and stirred for 20 minutes to cause the denatured proteins to precipitate and aggregate at approximately pH 4.6. The solution is filtered through a 0.22 μm filter to remove the aggregates and non-native proteins.

[1002] All samples are diluted to the same extent by adding polished water.

[1003] For each sample, the same volume of the sample is loaded onto a UPLC system equipped with a UPLC column (ProteinBEH C4; 1.7 μm; 150 × 2.1 mm), and detected at 214 nm.

[1004] Run the sample under the following conditions:

[1005] Buffer A: Milli-Q water, 0.1% w / w TFA;

[1006] Buffer B: HPLC-grade acetonitrile, 0.1% w / w TFA;

[1007] Flow rate: 0.4 mL / min;

[1008] Gradient: 0 - 6.00 minutes, 24 - 45% B; 6.00 - 6.50 minutes, 45 - 90% B; 6.50 - 7.00 minutes, 90% B; 7.00 - 7.50 minutes, 90 - 24% B; and 7.50 - 10.00 minutes, 24% B.

[1009] Determine the concentration of native BLG in the sample using the BLG peak area relative to a protein standard (Sigma L0130) (calibration curve of 5 levels).

[1010] If outside the linear range, further dilute the sample and re-inject.

[1011] Example 1.5: Determination of total protein

[1012] The total protein content (true protein) of the sample is determined as follows:

[1013] 1) Determine the total nitrogen 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;

[1014] 2) Determine the non-protein nitrogen of the sample according to ISO 8968-4|IDF 020-4 - Milk - Determination of nitrogen content - Part 4: Determination of non-protein-nitrogen content; and

[1015] 3) Calculate the total protein: (m 总氮 - m 非蛋白氮 ) * 6.38.

[1016] Example 1.6: Determination of non-aggregated BLG, ALA, and CMP

[1017] The contents of non-aggregated α-lactalbumin (ALA), β-lactoglobulin (BLG), and caseinomacropeptide (CMP) were analyzed by HPLC at 0.4 mL / min. 25 μL of the filtered sample was injected into two TSKgel 3000PWxl (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 of Milli-Q water, 417.3 g of acetonitrile, and 1 mL of trifluoroacetic acid), and a UV detector at 210 nm was used.

[1018] The contents of native α-lactalbumin (C α ), β-lactoglobulin (C β ), and caseinomacropeptide (C CMP ) were quantitatively determined by comparing the peak areas of the corresponding standard proteins with those of the sample.

[1019] The total amount of other proteins (non-BLG proteins) was determined by subtracting the amount of BLG from the total amount of proteins (measured according to Example 1.5).

[1020] Example 1.7: Determination of turbidity

[1021] Turbidity is the cloudiness or haziness of a fluid caused by large numbers of particles that are generally invisible to the naked eye, similar to smoke in the air.

[1022] Turbidity is measured in nephelometric turbidity units (NTU).

[1023] 20 mL of the beverage / sample was added to an NTU glass and placed in a 3000IR nephelometer. After stabilization, the NTU value was measured and repeated twice.

[1024] Example 1.8: Determination of viscosity

[1025] The viscosity of the liquid was measured using a Gilson Viscoman viscometer or an equivalent viscometer, expressed in a shear rate of 300 s -1 . Unless otherwise specified, the sample was equilibrated to 15 °C before measurement and then measured at this temperature.

[1026] Unless otherwise specified, the viscosity is expressed in centipoise (cP) at a shear rate of 300 s -1 . The higher the measured cP value, the lower the viscosity.

[1027] Example 1.9: Determination of color

[1028] Measure the color using a colorimeter (Konica Minolta, CR-400). Add 15 g of the sample to a small petri dish (55×14.2 mm, VWR catalog number 391-0895), avoiding the formation of air bubbles. Normalize the protein content of the sample to 6.0 w / w% protein or less.

[1029] Calibrate the colorimeter with a white calibration plate (number 19033177). Set the light source to D65 and the field of view (observer) to 2 degrees. Cover the suspension with a lid and measure the color (CIELAB color space: a* value, b* value, L* value), taking the average of three independent readings at different positions of the petri dish.

[1030] The demineralized water reference has the following values:

[1031] L*: 39.97 ± 0.3;

[1032] a*: 0.00 ± 0.06;

[1033] b*: 0.22 ± 0.09.

[1034] Based on the measured values of demineralized water, convert the measured values to Δ / difference.

[1035] 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.

[1036] 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.

[1037] 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.

[1038] Normalize the sample to 6.0 w / w% protein or less.

[1039] The L*a*b* color space (also known as the CIELAB space) is a uniform color space defined by the International Commission on Illumination (CIE) in 1976 for quantitatively reporting lightness and chromaticity (ISO 11664-4:2008(E) / CIE S 014-4 / E:2007).

[1040] In this space, L* represents lightness (values from 0 to 100), with the darkest black being L* = 0 and the brightest white being L* = 100.

[1041] 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 in the negative direction and red in the positive direction. The b* axis represents the blue - yellow component, with blue in the negative direction and yellow in the positive direction.

[1042] Example 1.10: Beverage Stability Test / Insoluble Protein Substances

[1043] The whey protein beverage composition is considered stable if the total amount of protein precipitated after centrifuging the heated sample at 3000 g for 5 minutes is less than 15%:

[1044] · Add approximately 20 g of the sample to a centrifuge tube and centrifuge at 3000 g for 5 minutes.

[1045] · Analyze the protein before centrifugation and the supernatant after centrifugation by the Kjeldahl method to quantify the protein recovery, see Example 1.5.

[1046] Calculate the protein loss as follows:

[1047]

[1048] This parameter is sometimes also referred to as the level of insoluble protein substances and can be used to analyze liquid and powder samples. If the sample is a powder, suspend 10 g of the powder in 90 g of demineralized water and hydrate it with gentle stirring at 22 °C for 1 hour. Place approximately 20 g of the sample (e.g., liquid sample or suspended powder sample) in a centrifuge tube and centrifuge at 3000 g for 5 minutes. According to Example 1.5, use the protein (P 总 ) before centrifugation and the supernatant (P 3000×g ) after centrifugation for Kjeldahl analysis to quantify the protein recovery.

[1049] Calculate the amount of insoluble protein substances as follows:

[1050]

[1051] Example 1.11: Sensory Evaluation

[1052] Conduct a descriptive sensory evaluation of the heat - treated beverage formulation. Heat the beverage formulation using a plate heat exchanger.

[1053] Mix 1 volume of the sample with 1 volume of water, compare it with the unheated whey protein isolate, and also use lactic acid and citric acid to form an attribute table before the final tasting stage:

[1054] Category Attribute Fragrance Acid whey (acid dairy product) Basic taste Acid, bitter Flavor Whey, citric acid, lactic acid Taste Dry, astringent

[1055] Use crackers, white tea, melon, and water to clean the subjects' mouths between each sample.

[1056] Provide 15 mL of the test sample at ambient temperature (20 - 25 °C) in a small cup.

[1057] Each test sample was supplied three times to 10 individuals in three different areas in random order.

[1058] Rate the attributes (see table above) on a 15 - cm scale; where 0 = low intensity and 15 = high intensity.

[1059] Perform statistical analysis using “Panelcheck” software, with multiple repetitions using a three - way ANOVA test. Fix the sample and set the panel as random.

[1060] Use the Bonferroni correction that shows the lowest significance difference value (paired comparison with groups related to letters) to evaluate the significant differences between samples.

[1061] Example 1.12: Determine transparency through images

[1062] Take a photo of the beverage preparation by placing the sample in a turbidity NTU measurement vial with a piece of paper with the words “lorem ipsum” written on it. Take a photo of the vial using a smartphone, and the inventor evaluated whether the words could be clearly observed through the vial.

[1063] Example 1.13: Determination of ash content

[1064] The ash content of the food was measured according to NMKL 173:2005 “Ash in foods - gravimetric determination”.

[1065] Example 1.14: Determination of conductivity

[1066] The “conductivity” of an aqueous solution (sometimes called “specific conductance”) is a measure of the solution's ability to conduct electricity. For example, conductivity can be determined by measuring the alternating current resistance of the solution between two electrodes, and the result is usually expressed in units of millisiemens per centimeter (mS / cm). For example, conductivity can be measured according to Method 120.1 of the EPA (U.S. Environmental Protection Agency).

[1067] Unless otherwise stated, the conductivity values mentioned in this article have been standardized to 25 °C.

[1068] Conductivity was measured using a conductivity meter (WTW Cond 3210 with a four - pole 325 electrode).

[1069] Before use, the system has been calibrated according to the instructions in the manual. To avoid local dilution, thoroughly wash the electrode in the same medium as that for measurement. Lower the electrode into the medium so that the area to be measured is completely submerged. Then agitate the electrode to remove any air remaining on the electrode. Then keep the electrode stationary until a stable value is obtained and recorded from the display.

[1070] Example 1.15: Determination of total solids in solution

[1071] The total solids content of the solution can be determined according to NMKL 110, 2nd Edition, 2005 (Total solids (Water) – Gravimetric determination in milk and milk products). NMKL is the abbreviation of "Nordisk Metodikkomité for ".

[1072] The water content of the solution can be calculated as 100% minus the relative content (%) of the total solids (w / w).

[1073] Example 1.16: Determination of pH

[1074] Measure all pH values using a pH glass electrode and normalize them to 25 °C.

[1075] 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 in powder form, dissolve 10 g of the powder in 90 mL of demineralized water with vigorous stirring at room temperature. Then measure the pH value of the solution at 25 °C.

[1076] Example 1.17: Determination of bulk density and tapped density

[1077] The density of dry powder is defined as the relationship between the weight and volume of the powder, and the powder is analyzed using a special Stampf volumeter (i.e., a graduated cylinder) under specified conditions. Density is usually expressed in g / mL or kg / L.

[1078] In this method, the dry powder sample is packed into the graduated cylinder. After a specified number of tapping times, read the volume of the product and calculate the density.

[1079] This method can define three types of density:

[1080] Poured density, which is the mass divided by the volume of the powder after it has been transferred to the specified graduated cylinder.

[1081] The apparent density, which is the mass divided by the volume of the powder after being tapped 100 times under the conditions specified in this standard.

[1082] The bulk density, which is the mass divided by the volume of the powder after being tapped 625 times under the conditions specified in this standard.

[1083] This method uses a special graduated cylinder (250 mL, graduated from 0 to 250 mL, weighing 190 ± 15 g) (J.Engelsmann A.G. 67059 Ludwigshafen / Rh) and a Stampf volumeter (such as J.Engelsmann A.G.)

[1084] The apparent density and bulk density of the dried product are measured by the following procedure.

[1085] Pretreatment :

[1086] The sample to be measured is stored at room temperature.

[1087] Then, the sample is thoroughly mixed by repeatedly rotating and inverting the container (avoiding particle breakage). The container is filled to no more than 2 / 3.

[1088] Procedure :

[1089] Weigh 100.0 ± 0.1 g of the powder and transfer it to the graduated cylinder. The volume V 0 is in milliliters.

[1090] If 100 g of the powder cannot be filled into the graduated cylinder, it should be reduced to 50 g or 25 g.

[1091] Fix the graduated cylinder to the Stampf volumeter and tap it 100 times. Level the surface with a spatula and read the volume V in milliliters 100 .

[1092] Change the number of light taps to 625 times (including the 100 taps). After tapping, level the surface and read the volume V in milliliters 625 .

[1093] Calculation of density :

[1094] Calculate the apparent density and bulk density (in g / mL) according to the following formula:

[1095] Bulk density = M / V

[1096] where M represents the sample mass (in g) and V represents the volume after 625 taps (in mL).

[1097] Example 1.18: Determination of water content of powder

[1098] The moisture content of the food was measured according to ISO 5537:2004 (Dried milk - Determination of moisture content (Reference method)). NMKL is the abbreviation for "Nordisk Metodikkomité for ".

[1099] Example 1.19: Determination of the amounts of calcium, magnesium, sodium, potassium, and phosphorus (ICP-MS method)

[1100] The total amounts of calcium, magnesium, sodium, potassium, and phosphorus were determined using the following procedure: First, microwave digestion was used to decompose the sample, and then an ICP device was used to determine the total amount of minerals.

[1101] Instrument:

[1102] The microwave was from Anton Paar, and the ICP was the Optima 2000DV from PerkinElmer Inc.

[1103] Materials:

[1104] 1M HNO 3

[1105] Yttrium in 2% HNO 3

[1106] Appropriate standard solutions of calcium, magnesium, sodium, potassium, and phosphorus in 5% HNO3

[1107] Pretreatment :

[1108] Weigh out a certain amount of the powder and then transfer it to a microwave digestion tube. Add 5 mL of 1M HNO 3 . Digest the sample in the microwave according to the microwave instructions. Place the digested tube in a fume hood, remove the lid, and allow the volatile fumes to evaporate.

[1109] Measurement steps :

[1110] Transfer the pretreated sample to a DigiTUBE using a known amount of Milli-Q water. Add the yttrium solution in 2% HNO 3 (about 0.25 mL for every 50 mL of diluted sample), and dilute to a known volume with Milli-Q water. Analyze the sample on the ICP using the steps described by the manufacturer.

[1111] By diluting 10 mL of 1M HNO with Milli-Q water 3 ​and diluted to a final volume of 100 mL with a mixture of 0.5 mL of a solution of yttrium in 2% HNO 3 to prepare a blind sample.

[1112] Prepare at least 3 standard samples with concentrations at the expected sample concentrations.

[1113] Example 1.20: Determination of furosine value

[1114] 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. The total protein was determined according to Example 1.5. The furosine value is expressed in mg creatinine / 100 g protein.

[1115] Example 1.21: Determination of crystallinity of BLG in liquid

[1116] The following method is used to determine the crystallinity of BLG in a liquid with a pH of 5–6.

[1117] a) Transfer 10 mL of the liquid sample under study to a Maxi-Spin filter equipped with a 0.45 μm pore size CA membrane;

[1118] b) Immediately spin the filter at 1500 g for 5 minutes, keeping the centrifuge at 2 °C;

[1119] c) Add 2 mL of cold Milli-Q water (2 °C) to the retentate side of the spin filter. Immediately spin the filter at 1500 g for 5 minutes while keeping the centrifuge cooled to 2 °C. Collect the permeate (permeate A), measure the volume, and determine the concentration of BLG by HPLC using the method described in Example 1.6;

[1120] d) Add 4 mL of 2 M NaCl to the retentate side of the filter and stir rapidly. Let the mixture stand at 25 °C for 15 minutes;

[1121] e) Immediately spin the filter at 1500 g for 5 minutes and collect the permeate (permeate B);

[1122] f) Determine the total weight of BLG in permeates A and B using the method described in Example 1.6. Convert the results to the total weight of BLG and substitute the weight percentage; the weight of BLG in permeate A is designated as m 渗透物A , and the weight of BLG in permeate B is designated as m 渗透物B ;

[1123] g) The crystallinity of BLG in the liquid is determined as follows:

[1124] Crystallinity = m 渗透物B / (m 渗透物A + m 渗透物B ) * 100%

[1125] Example 1.22: Determination of crystallinity of BLG in dry powder

[1126] This method is used to determine the crystallinity of BLG in the dry powder.

[1127] a) Mix 5.0 g of the powder sample with 20.0 g of cold Milli-Q water (2 °C) and let stand at 2 °C for 5 minutes;

[1128] b) Transfer the liquid sample under study to a Maxi-Spin filter with a 0.45 μm CA membrane;

[1129] c) Immediately spin the filter at 1500 g for 5 minutes, keeping the centrifuge at 2 °C;

[1130] d) Add 2 mL of cold Milli-Q water (2 °C) to the retentate side of the spun filter, then immediately spin the filter at 1500 g for 5 minutes, collect the permeate (permeate A), measure the volume, determine the BLG concentration by HPLC using the method described in Example 1.6, convert the result to the total weight of BLG, and substitute the weight percentage. The weight of BLG in permeate A is designated as m 渗透物A ;

[1131] f) Then calculate the crystallinity of BLG in the powder using the following formula:

[1132]

[1133] where m 总BLG is the total amount of BLG in the powder sample of step a).

[1134] If the total amount of BLG in the 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 of Milli-Q water, adjust the pH to 7.0 by adding an aqueous NaOH solution, let the mixture stand at 25 °C for 1 hour with stirring, and finally determine the total amount of BLG in the powder sample using Example 1.6.

[1135] Example 1.23: Determination of UF permeate conductivity

[1136] Transfer 15 mL of the sample to an Amicon Ultra-15 centrifugal filter with a 3 kDa (3000 NMWL) cutoff and centrifuge at 4000 g for 20 - 30 minutes, or until sufficient UF permeate accumulates at the bottom of the filter unit to measure the conductivity. Measure the conductivity immediately after centrifugation. The sample handling and centrifugation are carried out at the temperature of the sample source.

[1137] Example 1.24: Determination of dry BLG crystals in powder

[1138] The presence of dry BLG crystals in the powder can be determined by the following method:

[1139] Resuspend the powder sample to be analyzed in demineralized water at 4 °C at a weight ratio of 2 parts water to 1 part powder, mix gently, and then rehydrate at 4 °C for 1 hour.

[1140] Examine the rehydrated sample by microscopy to identify the presence of crystals, preferably detecting birefringence using plane-polarized light.

[1141] Separate the crystalline material, perform X-ray crystallography analysis to verify the presence of the crystal structure, and preferably also verify that its crystal lattice (space group and unit cell dimensions) corresponds to that of BLG crystals.

[1142] Analyze the chemical composition of the separated crystalline material to verify that its solid is mainly composed of non-aggregated BLG.

[1143] Example 1.25: Determination of total lactose

[1144] 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".

[1145] Example 1.26: Determination of total carbohydrates

[1146] Determine the carbohydrate content by using the Sigma Aldrich Total Carbohydrate Assay Kit (Catalog No. MAK104-1KT), where the carbohydrates are hydrolyzed and converted to furfural and hydroxymethylfurfural, and then converted to a chromogen, and spectrophotometric monitoring is carried out at 490 nm.

[1147] Example 1.27: Determination of total lipids

[1148] The lipid content was measured according to ISO 1211:2010 (Determination of Fat Content – - Gottlieb Gravimetric Method, Determination of Fat Content – - Gottlieb Gravimetric Method).

[1149] Example 1.28: Determination of Brix

[1150] The Brix was measured using a PAL-α digital handheld refractometer (Atago) calibrated with purified water (water filtered by reverse osmosis, with a conductivity of at most 0.05 mS / cm).

[1151] Approximately 500 μL of the sample was transferred to the prism surface of the instrument, and then the measurement was started. The measured value was read and recorded.

[1152] The Brix of the whey protein solution is proportional to the total solids (TS), and TS (% w / w) is approximately Brix * 0.85.

[1153] The Brix is sometimes also referred to as degrees Brix or °Brix.

[1154] Example 1.29: Determination of lactoferrin and lactoperoxidase

[1155] The concentration of lactoferrin was determined by the ELISA immunoassay 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).

[1156] The concentration of lactoperoxidase was determined using a commercially available bovine milk peroxidase kit.

[1157] Example 1.30: Determination of colony forming unit number

[1158] 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.

[1159] Example 1.31: Determination of total amounts of BLG, ALA, and CMP

[1160] This procedure is a liquid chromatography (HPLC) method for the quantitative analysis of proteins (such as ALA, BLG, and CMP) and other proteins in a composition. Different from the method of Example 1.6, this method also measures the proteins present in aggregates, thus providing a measure of the total amount of protein species in the composition under study.

[1161] The separation mode is size exclusion chromatography (SEC). The method uses 6M guanidine hydrochloride buffer as the sample solvent and HPLC mobile phase. Mercaptoethanol is used as a reducing agent to reduce the disulfides (S-S) in proteins or protein aggregates to produce unfolded monomeric structures.

[1162] Sample preparation can be easily achieved by dissolving 10 mg protein equivalent in the mobile phase.

[1163] Two TSK-GEL G3000SWXL (7.7 mm × 30.0 cm) chromatographic columns (GPC columns) and a guard column were placed in series to achieve sufficient separation of the main proteins in the raw material.

[1164] The eluted analytes were detected and quantified by ultraviolet detection (280 nm).

[1165] Equipment / Materials :

[1166] 1. HPLC Pump 515 (Waters) with manual seal wash;

[1167] 2. HPLC Pump Control Module II (Waters);

[1168] 3. Autosampler 717 (Waters);

[1169] 4. Dual Absorbance Detector 2487 (Waters);

[1170] 5. Computer software capable of generating quantitative reports (Empower 3, Waters);

[1171] 6. Analytical column: 2 x TSK-GEL G3000SWXL (7.8×300mm, P / N: 08541);

[1172] Guard column: TSK-Guard column SWxL (6.0×40mm, P / N: 08543);

[1173] 7. Ultrasonic bath (Branson 5200);

[1174] 8. 25mm syringe filter with 0.2μm cellulose acetate membrane (514-0060, VWR).

[1175] Procedure :

[1176] Mobile phase :

[1177] A. Stock buffer

[1178] 1. Weigh 56.6g of Na 2 HPO 4 、3.5g of NaH 2 PO 4 and 2.9g of EDTA, and add them to a 1000mL beaker. Dissolve in 800mL of water;

[1179] 2. Measure the pH value. If necessary, adjust it to 7.5±0.1 with HCl (to lower the pH) or NaOH (to increase the pH);

[1180] 3. Transfer to a 1000mL volumetric flask and dilute to the mark with water.

[1181] 6M guanidine hydrochloride mobile phase

[1182] 1. Weigh 1146g of guanidine hydrochloride, add it to a 2000mL beaker, and then add 200mL of stock buffer (A);

[1183] 2. Dilute the solution with water to approximately 1600mL while mixing with a magnetic stir bar (50℃);

[1184] 3. Adjust the pH to 7.5±0.1 with NaOH;

[1185] 4. Transfer to a 2000mL volumetric flask and dilute to the mark with water;

[1186] 5. Filter using a solvent filtration device with a 0.22μm membrane filter.

[1187] Calibration standard

[1188] The calibration standard for each protein to be quantified is prepared as follows:

[1189] 1. Accurately weigh (weigh to 0.01 mg) approximately 25 mg of the protein reference standard into a 10 mL volumetric flask, dissolve it in 10 mL of water, which is the protein stock standard solution (S1) of the protein;

[1190] 2. Pipette 200 μL of S1 into a 20 mL volumetric flask and dilute it to the mark with the mobile phase, which is the low (concentration) working standard solution WS1;

[1191] 3. Pipette 500 μL of S1 into a 10 mL volumetric flask and dilute it to the mark with the mobile phase, which is the standard solution WS2;

[1192] 4. Pipette 500 μL of S1 into a 5 mL volumetric flask and dilute it to the mark with the mobile phase, which is the standard solution WS3;

[1193] 5. Pipette 750 μL of S1 into a 5 mL volumetric flask and dilute it to the mark with the mobile phase, which is the standard solution WS4;

[1194] 6. Pipette 1.0 mL of S1 into a 5 mL volumetric flask and dilute it to the mark with the mobile phase, which is the high (concentration) working standard solution WS5;

[1195] 7. Using a graduated disposable pipette, transfer 1.5 mL of WS1 to WS5 into separate vials, add 10 μL of 2-mercaptoethanol to each vial, cap the vials, vortex the solution for 10 seconds, and let the standard stand at ambient temperature for approximately 1 hour.

[1196] 8. Filter the standard solution using a 0.22 μm cellulose acetate syringe filter.

[1197] Measure the purity of the protein using the Kjeldahl method (N × 6.38), and determine the area % of the standard solution WS5 using HPLC.

[1198] Protein (mg) = "Protein standard weight" (mg) × P1 × P2;

[1199] P1 = P% (Kjeldahl method);

[1200] P2 = Protein area % (HPLC).

[1201] Sample preparation

[1202] 1. Weigh 25 mg equivalent of the original sample of the protein into a 25 mL volumetric flask;

[1203] 2. Add about 20 mL of mobile phase and dissolve the sample for about 30 minutes;

[1204] 3. Add mobile phase to the mark and add 167 μL of 2-mercaptoethanol to 25 mL of the sample solution;

[1205] 4. Sonicate for about 30 minutes and then let the sample stand at ambient temperature for about 1.5 hours;

[1206] 5. Mix the solution and filter using a 0.22 μL cellulose acetate syringe filter.

[1207] HPLC system / Column

[1208] Equilibrate the column

[1209] 1. Connect the GPC guard column and two GPC analytical columns in series. The new chromatographic columns are usually filled with phosphate buffer solution;

[1210] 2. Gradually pass water through the new chromatographic columns at a rate of 0.1 to 0.5 mL / min within 30 to 60 minutes and continue washing for about 1 hour;

[1211] 3. Gradually reduce the flow rate from 0.5 mL / min to 0.1 mL / min and replace it with the mobile phase in the storage container;

[1212] 4. Gradually increase the pump flow rate from 0.1 mL / min to 0.5 mL / min within 30 to 60 minutes to avoid pressure shock and maintain at 0.5 mL / min;

[1213] 5. Inject 10 samples to saturate the chromatographic column and wait for the peaks to elute. This will help to condition the chromatographic column. It is not necessary to wait for each injection to complete before the next injection;

[1214] 6. Equilibrate with the mobile phase for at least 1 hour.

[1215] Calculation of results

[1216] The quantitative determination of the content of the protein to be quantified (such as α-lactalbumin, β-lactoglobulin, and caseinomacropeptide) is carried out by comparing the peak area of the corresponding standard protein with the peak area of the sample. The results are expressed as g of specific protein / 100 g of the original sample, or as the weight percentage of the specific protein relative to the weight of the original sample.

[1217] Example 2: Production of spray-dried acidic BLG isolate powder

[1218] Whey protein raw material

[1219] The lactose-depleted UF retentate of sweet whey from a standard cheese production process is filtered through a 1.2 micron filter, then de-fatted using a Synder FR membrane, and then used as a raw material for the BLG crystallization process. The chemical composition of the raw material 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 proteins relative to the total amount of proteins.

[1220] Adjustment

[1221] Using a Koch HFK-328 type membrane (70m 2 membrane) with a 46mill spacing, the feed pressure is 1.5 - 3.0 bar. The sweet whey raw material is adjusted on a ultrafiltration device at 20 °C to a raw material concentration of 21% total solids (TS) ± 5, and refined water (filtered by reverse osmosis to a conductivity of at most 0.05 mS / cm) is used as the diafiltration medium. Then the pH is adjusted to approximately 5.5 by adding HCl. Diafiltration is continued until the conductivity of the retentate drops below 0.1 mS / cm within 20 minutes. Then the retentate is concentrated until the permeate flow rate is below 1.43 L / h / m 2 . A first sample of the concentrated retentate is collected and centrifuged at 3000g for 5 minutes. The supernatant of the first sample is used to determine the BLG yield.

[1222] Crystallization

[1223] The concentrated retentate is transferred to a 300L crystallization tank, where the concentrated retentate is inoculated with pure BLG crystal material made from rehydrated spray-dried BLG crystals. Subsequently, the inoculated whey protein solution is cooled from 20 °C to approximately 6 °C in about 10 hours to allow BLG crystal formation and growth.

[1224] After cooling, a sample (second sample) of the whey protein solution containing crystals is taken, and the BLG crystals are separated by centrifuging at 3000g for 5 minutes. HPLC analysis is performed on the supernatant and crystal precipitate from the second sample as described below. The crystallization yield is calculated as described below and determined to be 57%.

[1225] Table 1: Chemical composition of the raw material

[1226]

[1227] Determination of BLG yield using HPLC :

[1228] By adding purified water, the supernatants of the first and second samples were diluted to the same extent, 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 an HPLC system with a Phenomenex 5 μm C4 LC column 250×4.6 mm and detected at 214 nm.

[1229] The samples were run using the following conditions:

[1230] Buffer A: MilliQ water, 0.1% w / w TFA

[1231] Buffer B: HPLC-grade acetonitrile, 0.085% w / w TFA

[1232] Flow rate: 1 mL / min

[1233] Column temperature: 40 °C

[1234] Gradient: 0 - 30 minutes 82 - 55% A and 18 - 45% B; 30 - 32 minutes 55 - 10% A and 45 - 90% B; 32.5 - 37.5 minutes 10% A and 90% B; 38 - 48 minutes 10 - 82% A and 90 - 18% B.

[1235] Data processing:

[1236] Since the supernatants were all treated in the same way, the areas of the BLG peaks could be directly compared to calculate the relative yield. Since the crystals only contain BLG and all samples were treated in the same way, the concentrations and areas of α-lactalbumin (ALA) in all samples should be the same. Therefore, when calculating the relative yield, the ALA areas before and after crystallization were used as the correction factor (cf).

[1237]

[1238] The relative yield was calculated using the following formula:

[1239]

[1240] Dissolve BLC crystals with acid

[1241] A decanter with 64 spacers was used to differentially separate the remaining material in the crystallization tank at 350 g, 2750 RPM, and a differential speed of 150 RPM. The feed flow rate was 75 L / h. Before separation, the raw material was mixed with purified water at a ratio of 1:2. Then, the BLG crystals / solids in the decanter were mixed with purified water to make a thinner slurry, and then phosphoric acid was added to lower the pH to about 3.0 to rapidly dissolve the crystals.

[1242] After dissolving the BLG crystals, the pure BLG protein liquid was concentrated to 15 °Bx on the same ultrafiltration (UF) device used for preparing the raw material for crystallization, and the pH was adjusted to a final pH of about 3.8. Then, the liquid BLG isolate was 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 (330 nm / 350 nm) was measured to be 1.20, confirming that the BLG molecules were native.

[1243] The BLG was dried on a pilot-scale 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 measured to be 1.5%; the intrinsic tryptophan fluorescence emission ratio (I330 / I350) was measured to be 1.20.

[1244] Table 2: Composition of the BLG isolate powder (BDL = below the detection limit)

[1245]

[1246] The bulk density (tapped 625 times) of the spray-dried powder was estimated to be 0.2 - 0.3 g / cm 3 。

[1247] Conclusion:

[1248] By using the above method, we were able to produce a high-purity BLG product that can be heat-treated with substantially no protein denaturation or protein unfolding during the treatment. The heat treatment significantly reduced the bacterial level without damaging the protein product.

[1249] The inventors have found that even higher bulk densities can be obtained by increasing the protein content before spray drying. In addition, the inventors have observed that even lower degrees of denaturation can be obtained if the inlet temperature and / or outlet temperature used for spray drying are reduced.

[1250] Example 3: Production of spray-dried pH-neutral BLG isolate powder

[1251] When using the same protocol and experimental setup as in Example 2, the lactose-reduced whey protein isolate shown in Table 3 was conditioned and used as the crystallization feedstock. The crystallization yield was calculated to be 68%.

[1252] We note that all weight percentages of the specific proteins (e.g., BLG and ALA) mentioned in this example are related to the weight percentage of non-aggregated protein relative to the total amount of protein.

[1253] Table 3: Composition of the feedstock

[1254]

[1255]

[1256] A 64-compartment decanter was used to separate the remaining material in the crystallization tank at 350 g, 2750 RPM, and a differential speed of 150 RPM. The feedstock flow rate was 75 L / h. Before separation, the feedstock was mixed with demineralized water at a ratio of 1:2. Then the BLG crystals / solids in the decanter were mixed with demineralized water to make a thinner slurry, and 0.1 M potassium hydroxide was added to adjust the pH to about 7 to rapidly dissolve the crystals.

[1257] After dissolving the crystals, the pure BLG protein liquid was concentrated to 15 °Bx on the same UF device 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 pilot-scale 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 approximately 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 ratio of intrinsic tryptophan fluorescence emission (330 nm / 350 nm) was 1.16.

[1258] Table 4: Chemical composition of the BLG isolate powder

[1259]

[1260]

[1261] The bulk density (tapped 625 times) of the spray-dried powder was estimated to be 0.2 - 0.3 g / cm 3 .

[1262] Conclusion:

[1263] By using the above method, we are able to produce a high-purity BLG product with a pH-neutrality and with little or no protein denaturation during processing. The inventors have found that an even higher bulk density can be obtained by increasing the protein content before spray drying. In addition, the inventors have observed that an even lower degree of denaturation can be obtained if the inlet temperature and / or the outlet temperature of the spray drying is reduced. The degree of denaturation can also be further reduced by reducing the mineral content before spray drying.

[1264] Example 4: Wettability of spray-dried BLG isolate powder

[1265] The wettability of the acidic or pH-neutral spray-dried BLG isolate powder prepared according to Example 2 and Example 3 was compared with the wettability of a conventional spray-dried whey protein isolate (WPI). 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 placing the powder on the water surface until the powder dissolved or passed through the water surface was measured. The results are as follows.

[1266]

[1267] Conclusion: Unexpectedly, the uncoated BLG isolate powder (Sample 2, Sample 3) has much better wettability than the standard WPI (Sample 1). This shows that the BLG isolate powder of the present invention is a useful ingredient for instant beverage powders (where rapid wettability and solubility are important). In addition, compared with conventional WPI, the BLG isolate powder has higher practicality in the production of protein beverages because they are wetted and dissolved faster, and thus are more easily dispersed and dissolved during the production process. Ultimately, this can reduce the time required to produce high-protein beverages and increase the hourly production capacity of beverage production plants.

[1268] Example 5: Acidic BLG isolate has a reduced level of dry mouthfeel

[1269] The pH values 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) were adjusted to pH 3.7 and they were heat-treated: 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 ratio of intrinsic fluorescence emission (I330 / I350), it was confirmed that the protein in beverage A was still in its native conformation, while significant unfolding and denaturation occurred in beverage B.

[1270] The turbidity of beverages A and B was measured according to Example 1.7. The turbidity of both beverages was less than 40 NTU, while the turbidity of the equivalent standard WPI was higher than 200 NTU. Thus, the acidic BLG isolate of the present invention is clearly very suitable for the production of clear acidic high-protein beverages.

[1271] Less than one week after production, a trained sensory test panel performed sensory tests on both beverages. It was found that the dry mouthfeel of beverage B (dry mouthfeel score: approximately 10.5 on a scale of 0 to 12) (characteristic of heat-treated acidic whey protein beverages) was more than 100% higher than that of beverage A (dry mouthfeel score: approximately 5.0 on a scale of 0 to 12).

[1272] This finding indicates that protein denaturation causes the dry mouthfeel of acidic protein beverages, and the dry mouthfeel can be significantly reduced by limiting or even avoiding protein denaturation.

[1273] Example 6: Production of a neutral BLG isolate with an ultra-low microbial content

[1274] The inventors have found that the present invention enables the production of a pH-neutral BLG powder with a very low bacterial content and a high degree of protein naturality. This is demonstrated in this example, in which the total processing time was extended by inserting a 6-day storage period at 10 °C in the final step of the adjustment process to challenge the microbial quality of the product.

[1275] Whey protein raw material:

[1276] The lactose-removed UF retentate of sweet whey from a standard cheese production process was filtered through a 1.2 µm filter and had its fat reduced through a Synder FR membrane before being used as a raw material for the BLG crystallization process. The chemical composition of the raw material is shown in Table 5. We note that all weight percentages of the specific proteins (e.g., BLG, ALA) mentioned in this example relate to the weight percentage of non-aggregated protein relative to the total protein content.

[1277] Table 5: Composition of the whey protein raw material used in Example 6

[1278]

[1279]

[1280] Adjustment:

[1281] Using Alfa Laval GR82PE membranes with a 30 mill spacing, at a feed pressure of 1.5 - 3.0 bar, the sweet whey raw material is conditioned on an ultrafiltration unit at approximately 10 °C to a raw material concentration of 21% total solids (TS) ± 5%. Refined water (water filtered by reverse osmosis with a conductivity of at most 0.05 mS / cm) is used as the diafiltration medium. The pH is adjusted to approximately 5.9 using dilute hydrochloric acid, and then diafiltration is carried out using a batch ultrafiltration unit. Diafiltration continues until the conductivity of the retentate drops below 0.1 mS / cm within 20 minutes.

[1282] Then, the diafiltered whey protein raw material is stored at 10 °C for 6 days to challenge the microbiological quality of the resulting product.

[1283] After storage, the whey protein raw material is heated to 20 °C, and then the pH is adjusted by adding HCl (using dilute hydrochloric acid) to a pH of approximately 5.5.

[1284] Using Alfa Laval GR82PE membranes with a 30 mill spacing, at a feed pressure of 1.5 - 3.0 bar, on an ultrafiltration unit at 20 °C, the whey protein raw material is conditioned to a raw material concentration of 21% total solids (TS) ± 5%. Refined water is used as the diafiltration medium. Ultrafiltration is carried out using a continuous ultrafiltration unit, and diafiltration is added to bring the conductivity of the final retentate to 1.9 - 2.2 mS / cm and the TS to 22 ± 5. The retentate is collected in an 800 L tank with ledges and agitation. After the tank is full, a first sample of the concentrated retentate is taken for HPLC analysis. The HPLC analysis of this example is carried out as described in Example 2.

[1285] Crystallization:

[1286] The concentrated retentate is transferred to an 800 L crystallization tank where the concentrated retentate is inoculated with pure BLG crystal material made from rehydrated spray-dried BLG crystals. The crystals are added to 1 L of conditioned WPI and rapidly cooled on ice to below 5 °C. Subsequently, the inoculated whey protein solution is cooled from 20 °C to approximately 6 °C in about 4 hours to allow BLG crystal formation and growth.

[1287] After cooling, a sample (second sample) of the whey protein solution containing crystals is taken, and the BLG crystals are separated by centrifuging at 3000 g for 5 minutes. The supernatant and crystal precipitate of the second sample are subjected to HPLC analysis as described below. The yield of crystallization is calculated and expressed as a percentage as described below.

[1288] Decanter separation:

[1289] Using a decanter (LEMITECH MD80) with a 64 - mill (mill means 1 / 1000 inch) interval, separate the crystals and mother liquor generated in an 800 - L tank at a differential speed of 600 g and 2750 RPM. The feed flow rate is 150 L / h, which is a mixture of purified water and raw materials from the tank, and the ratio is 1 volume of tank raw materials to 2 volumes of purified water.

[1290] Dissolve the crystals:

[1291] Dilute the crystals collected in the decanter with purified water to a total solid concentration of about 10%, and then adjust the pH to pH 3 using dilute hydrochloric acid. At 6 °C, keep the acidified BLG solution at pH 3 for about 16 - 48 hours.

[1292] Adjust the pH of the acidified BLG solution:

[1293] Then adjust the pH of the acidified BLG solution to pH 7 using a dilute mixture of potassium hydroxide and sodium hydroxide, and further adjust it on an ultrafiltration device 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.

[1294] Microfiltration:

[1295] Use a Membralox EP - 1940 - GL - UTP membrane with a nominal pore size of 0.8 microns to microfilter a portion of the concentrated BLG solution. The microfiltration is carried out at about 10 °C and a feed pressure of 3.5. Collect the MF permeate, which can now be dried.

[1296] Analyze the chemical composition (see the results in Table 6) and microorganisms (Example 1.30) of the MF permeate sample. Unexpectedly, it is found to be less than 10 CFU / g (basically meaning no colonies are identified, and the test sample seems to be close to sterile). For example, using a tighter microfiltration membrane and ensuring sterile conditions on the permeate side of the membrane can achieve sterility.

[1297] A comparable process without acidification and microfiltration is likely to result in the BLG solution containing more than 1,000,000 CFU / g.

[1298] Table 6: Composition of the microfiltration permeate of the concentrated BLG solution

[1299]

[1300] Conclusion:

[1301] Even under a heavy microbial load, the present invention can provide a sterile or nearly sterile pH - neutral BLG product with high protein naturalness.

[1302] Example 7: Comparison of Reverse Osmosis Concentration and Powder Bulk Density of Standard Neutral WPI with Acidic and Neutral BLG

[1303] The present inventors have found that spray-dried preparations of purified native BLG can be prepared which provide powders having a higher bulk density than equivalent whey protein isolates spray-dried under the same conditions.

[1304] The inventors have also found that the viscosity of liquid high-protein preparations of purified native BLG is lower than that of equivalent whey protein isolates. This finding enables the BLG isolate to be concentrated to a higher total protein concentration (and lower water content) prior to drying, and thus, compared to equivalent WPI powders, less water needs to be removed per kilogram of dry protein, and thus energy consumption is lower. The reduced viscosity also enables membrane filtration (e.g., microfiltration) with reduced energy consumption, since the energy required for membrane filtration decreases with decreasing viscosity.

[1305] The following experiments confirm the above findings.

[1306] Raw materials for reverse osmosis and drying:

[1307] Neutral BLG:

[1308] The MF retentate produced in Example 6 was concentrated using an Alfa Laval RO98pHt membrane with a feed pressure of up to 52 bar, and the temperature was maintained below 15 °C during conditioning. Concentration was continued until the Brix was 32.2. During concentration, viscosity and Brix were measured by sampling as described in Examples 1.8B and 1.28. The Brix of each sample was converted to protein (w / w%) using the following formula:

[1309]

[1310] 0.85 is the conversion factor obtained empirically which gives a good relationship between Brix and total solids. The results are as Figure 6 shown.

[1311] The total solids of the whey protein solution is approximately Brix * 0.85.

[1312] Acidic BLG:

[1313] As in Example 6, an acidic BLG solution was prepared (difference: before continuous ultrafiltration at pH 5.5, instead of ultrafiltration at pH 5.92, ultrafiltration was carried out at pH 3). The composition of the raw materials can be seen in Table 7. As described in Example 6, the acidic BLG permeate was subjected to the MF step and then concentrated using an Alfa Laval RO98pHt reverse osmosis (RO) membrane, with a feed pressure of at most 52 bar. During the adjustment period, the temperature was maintained below 15 °C. Concentration was continued until a Brix of 40.0 was obtained. During concentration, viscosity and Brix were measured by sampling as described in Examples 1.8B and 1.28. The Brix values were converted to protein concentrations, and the results are as Figure 6 shown. Before drying, the BLG was diluted to a Brix of 35.5 with purified water.

[1314] Table 7: Composition of acidic RO raw materials

[1315]

[1316] WPI reference:

[1317] To compare the acidic BLG product and the pH-neutral BLG product with a traditional pH-neutral WPI product, a standard liquid concentrated WPI based on sweet whey was extracted from Arla Foods Danmark Protein production and dried on the same pilot-scale spray dryer, using the same conditions as for the acidic BLG sample and the pH-neutral BLG sample. The composition of the liquid concentrated WPI is shown in Table 8.

[1318] First, the viscosity of the concentrated WPI was measured, and then the concentrated WPI was gradually diluted with purified water to show the correlation between the WPI Brix and viscosity for comparison with the BLG measurements.

[1319] Viscosity curve:

[1320] As described in Example 1.8B, before viscosity measurement (in triplicate), the temperature of the samples collected during RO and the temperature of the diluted WPI reference were adjusted to 15 °C. The Brix was measured as described in Example 1.28.

[1321] The concentrated liquid WPI was dried to a Brix of 36.0.

[1322] Table 8: Composition of WPI reference

[1323]

[1324]

[1325] Spray drying:

[1326] After concentration by reverse osmosis (RO), all samples were dried on a pilot plant spray dryer at an inlet temperature of 180 °C and an outlet temperature of 85 °C without any preheating. The temperature of 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: without stomping, stomping 100 times, and stomping 625 times. Unexpectedly, the bulk density of both the acidic BLG powder and the pH-neutral BLG powder was significantly and consistently higher than that of the WPI reference. On average, the bulk density of pH-neutral BLG was 18.4% higher than that of the WPI reference powder, while the bulk density of acidic BLG was 22.2% higher than that of the WPI reference powder. The results of the bulk density measurements are shown in Table 9 and are shown in Figure 5 。

[1327] Table 9: Bulk density of powders obtained from acidic BLG solution, pH-neutral BLG solution, and reference WPI solution, the powders having the same total protein content and having been spray dried under the same conditions

[1328]

[1329] Conclusion:

[1330] Compared to the WPI reference, both the acidic BLG isolate and the pH-neutral BLG isolate have lower viscosities and are more easily concentrated by RO. This finding allows the BLG isolate to be concentrated to a higher total protein concentration (and lower water content) before drying; thus, less water needs to be removed compared to comparable WPI powders, and thus the energy consumption per kilogram of dried protein is lower. The reduced viscosity also allows for membrane filtration (e.g., microfiltration) with reduced energy consumption, as the energy required for membrane filtration decreases with decreasing viscosity.

[1331] In addition, the BLG powder of the present invention is particularly suitable for 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, and thus provides more drinkable beverages.

[1332] Unexpectedly, compared to the WPI reference, the spray-dried BLG powder has a higher bulk density even when the BLG is dried at a lower total solids content (as indicated by the Brix value). A higher powder bulk density means a smaller transport volume per unit weight during transportation, and denser particles also generate less dust during handling.

Claims

1. A BLG isolate powder, preferably prepared by spray drying, having a pH of 6.1 to 8.5, and comprising: - at least 30% w / w of the total protein content; - at least 85% w / w of β-lactoglobulin (BLG) relative to the total protein content; - at most 10% w / w of water; The BLG isolate powder has one or more of the following: - The bulk density is at least 0.2 g / cm 3 ; - an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.11; - a protein denaturation degree of at most 10%; - a thermal stability at pH 3.9 of at most 200 NTU; and - at most 15,000 colony forming units / g.

2. The BLG isolate powder according to claim 1, wherein the total protein content of the BLG isolate powder is at least 70% w / w.

3. The BLG isolate powder according to claim 1 or 2, wherein the BLG content of the BLG isolate powder is at least 88% w / w relative to the total protein content.

4. The BLG isolate powder according to claim 1, wherein the lipid content of the powder is at most 0.1% w / w.

5. 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 .

6. The BLG isolate powder according to claim 1, wherein the intrinsic tryptophan fluorescence emission ratio (I330 / I350) of the BLG isolate powder is at least 1.

11.

7. The BLG isolate powder according to claim 1, wherein the protein denaturation degree of the BLG isolate powder is at most 10%.

8. A liquid BLG isolate having a pH of 6.1 to 8.5 and comprising: - at least 10% w / w of the total protein content; - at least 85% w / w of β-lactoglobulin (BLG) relative to the total protein content; The BLG isolate has one or more of the following: - an intrinsic tryptophan fluorescence emission ratio (I330 / I350) of at least 1.11; - a protein denaturation degree of at most 10%; - a thermal stability at pH 3.9 of at most 200 NTU; and - at most 15,000 colony forming units / g, preferably at most 1,000 colony forming units / g.

9. A method for producing the dried BLG isolate powder according to claim 1, having a BLG content of at least 85% w / w relative to the total protein content, the method comprises the following steps: a) providing a liquid BLG isolate having the following characteristics: pH of 6.1 to 8.5; a BLG content of at least 85% w / w relative to the total protein content of the liquid BLG isolate; b) optionally, physically reducing microorganisms in the liquid BLG isolate; c) drying the liquid BLG isolate by spray drying.

10. The method according to claim 9, wherein the total solids content of the liquid BLG isolate is 5 to 50% w / w.

11. The method according to claim 9 or 10, wherein, the intrinsic tryptophan fluorescence emission ratio (I330 / I350) of the protein fraction of the liquid BLG isolate is at least 1.

11.

12. The method according to claim 9 or 10, wherein, the degree of protein denaturation of the protein in the liquid BLG isolate is at most 10% w / w.

13. The method according to claim 9, wherein, providing the liquid BLG isolate involves: separating BLG from a whey protein raw material, and optionally, subjecting the resulting BLG-rich composition to one or more steps selected from the group consisting of: - demineralization; - addition of minerals; - dilution; - physical reduction of microorganisms; - concentration; and - pH adjustment.

14. The method according to claim 9, wherein, physical reduction of microorganisms is carried out on the liquid BLG isolate, preferably, the physical reduction of microorganisms comprises or even consists of heat treatment.

15. Use of the BLG isolate powder according to any one of claims 1 to 7 or the liquid BLG isolate according to claim 8 as an ingredient for producing food.

16. A BLG isolate powder, preferably prepared by spray drying, having a pH of 5.0 to 6.0, the BLG isolate powder comprising: - at least 30% w / w of the total protein content; - at least 85% w / w of β-lactoglobulin (BLG) relative to the total protein content; - at most 10% w / w of water; and - at most 15,000 colony forming units / g.

17. A BLG isolate powder, preferably prepared by spray drying, having a pH of 5.0 to 6.0, the BLG isolate powder comprising: - at least 30% w / w of the total protein content; - at least 85% w / w of β-lactoglobulin (BLG) relative to the total protein content; - at most 10% w / w of water; and - at most 0.1% w / w of lipids.

18. A BLG isolate powder, preferably prepared by spray drying, having a pH of 5.0 to 6.0, the BLG isolate powder comprising: - at least 30% w / w of the total protein content; - at least 85% w / w of β-lactoglobulin (BLG) relative to the total protein content; - at most 10% w / w of water; the BLG isolate powder has a degree of protein denaturation of at most 10%.

19. A liquid BLG isolate, having a pH of 5.0 to 6.0, the liquid BLG isolate comprising: - at least 10% w / w of the total protein content; - at least 85% w / w of β-lactoglobulin (BLG) relative to the total protein content; and - at most 15,000 colony forming units / g.

20. A method for producing the dried BLG isolate powder according to any one of claims 16 to 18, having a BLG content of at least 85% w / w relative to the total protein content, the method comprises the following steps: a) Provide a liquid BLG isolate having the following characteristics: pH is 5.0 to 6.0; The BLG content of the liquid BLG isolate is at least 85% w / w relative to the total protein; b) Optionally, physically reduce the microorganisms in the liquid BLG isolate; c) Dry the liquid BLG isolate by spray drying.

21. Use of the BLG isolate powder according to any one of claims 16 to 18 or the liquid BLG isolate according to claim 19 as an ingredient for producing food.

22. A BLG isolate powder, preferably prepared by spray drying, having a pH of 2.5 to 4.9, the BLG isolate powder comprising: - at least 30% w / w of the total protein; - at least 85% w / w of β-lactoglobulin (BLG) relative to the total protein; - at most 10% w / w of water; and - at most 15,000 colony forming units / g.

23. A BLG isolate powder, preferably prepared by spray drying, having a pH of 2.5 to 4.9, the BLG isolate powder comprising: - at least 30% w / w of the total protein; - at least 85% w / w of β-lactoglobulin (BLG) relative to the total protein; - at most 10% w / w of water; and - A bulk density of at least 0.4 g / cm 3 is provided.

24. A BLG isolate powder, preferably prepared by spray drying, having a pH of 2.5 to 4.9, the BLG isolate powder comprising: - at least 30% w / w of the total protein; - at least 85% w / w of β-lactoglobulin (BLG) relative to the total protein; - at most 10% w / w of water; - at most 0.1% w / w of lipids.

25. A liquid BLG isolate having a pH of 2 to 4.9, the liquid BLG isolate comprising: - at least 10% w / w of the total protein; - at least 85% w / w of β-lactoglobulin (BLG) relative to the total protein; and - at most 15,000 colony forming units / g; The BLG isolate powder has one or more of the following: - The intrinsic tryptophan fluorescence emission ratio (I330 / I350) is at least 1.11; - The degree of protein denaturation is at most 10%; and - The lipid content is at most 0.1% w / w.

26. A method for producing the dried BLG isolate powder according to any one of claims 22 to 24, the BLG content of the dried BLG isolate powder being at least 85% w / w relative to the total protein, the method comprises the following steps: a) Provide a liquid BLG isolate having the following characteristics: pH is 2 to 4.9; The BLG content of the liquid BLG isolate is at least 85% w / w relative to the total protein; b) Optionally, physically reduce the microorganisms in the liquid BLG isolate; c) Dry the liquid BLG isolate by spray drying.

27. Use of the BLG isolate powder according to any one of claims 22 to 24 or the liquid BLG isolate according to claim 25 as an ingredient for producing a food product (such as a beverage or an instant beverage powder), the food product having a pH of 2 to 4.7 and further having one or more of the following: - Reduced dry mouthfeel; - Improved transparency; and / or - Increased protein content, the protein content of the heat-treated beverage preferably being at least 3 to 45% w / w, more preferably 11 to 40% w / w, even more preferably 15 to 38% w / w, and most preferably 20 to 36% w / w.

Images