Method of modification of WHEY protein at acidic PH by reaction with oxidized phenolic compounds, the modified protein composition, and nutritional uses of the modified protein composition

Abstract

The present invention pertains to a method of preparing modified protein compositions under acidic conditions that expose and modify free thiol groups of beta-lactoglobulin through reaction with oxidized phenolic compounds. The resulting modified protein product has been found to have an excellent performance in e.g. protein-rich beverage products and has particularly been found to give rise to a reduced level of unpleasant odours during sterilizing heat-treatments at neutral pH and during consumption of such beverage products.

Description

[0001] METHOD OF MODIFICATION OF WHEY PROTEIN AT ACIDIC PH BY REACTION WITH OXIDIZED PHENOLIC COMPOUNDS, THE MODIFIED PROTEIN COMPOSITION, AND NUTRITIONAL USES OF THE MODIFIED PROTEIN COMPOSITION

[0002] FIELD OF THE INVENTION

[0003] The present invention pertains to a method of preparing modified protein compositions under acidic conditions that expose and modify free thiol groups of beta-lactoglobulin through reaction with oxidized phenolic compounds. The resulting modified protein product has been found to have an excellent performance in e.g. protein-rich beverage products and has particularly been found to give rise to a reduced level of unpleasant odours during sterilizing heat-treatments at neutral pH and during consumption of such beverage products.

[0004] BACKGROUND

[0005] pH-neutral and alkaline whey protein-rich beverages tend to produce an unpleasant odour similar to the odour of rotten eggs during thermal processing. The beverages are typically bottled immediately after production and therefore expose the consumer to the unpleasant odour when the bottle is opened.

[0006] WO 2021 / 198968 Al discloses a method of making a beverage comprising milk protein concentrate (MPC). The MPC is treated to allow for heat homogenisation of a beverage containing the MPC, while reducing occurrence of unpleasant odour (malodour and / or egg-type or sulfur-type small and / or taste). Specifically, the MPC comprises at least one whey protein, wherein 50-100% of the whey protein are denatured and wherein the calcium content has been depleted by about 5-20 percent by weight.

[0007] Li et al ("Cysteine residues are responsible for the sulfurous off-flavor formed in heated whey protein solutions", Food Chemistry: Molecular Sciences, vol. 5, 1 December 2022 (2022-12-01), page 100120) discloses how the protein composition of a whey protein solution affects the release of H2S during heat-treatment of the solution.

[0008] Poojary et al ("Green Tea Extract Decreases Age-Derived Advanced Glycation End Products but Not Lys-Derived AGEs in UHT Milk during 1-Year Storage", Journal of Agricultural and Food Chemistry, vol. 68, no. 48, 17 November 2020D2) studies the impact of adding green tea extracts to skim milk prior to UHT treatment and investigates the formation of advanced glycation products during long term storage. Waqar et al ("Covalent bonding of 4-methylcatechol to beta-lactoglobulin results in the release of cysteine-4-methylcatechol adducts after in vitro digestion", FOOD CHEMISTRY, vol. 397, 1 December 2022 (2022-12-01), page 133775) discloses the preparation and characterization of conjugates of 4-methylbenzoquinone and beta-lactoglobulin. Waqar does not contain any disclosures suggesting a reduced development of unpleasant odours due to chemical modification of beta-lactoglobulin.

[0009] SUMMARY OF THE INVENTION

[0010] The inventors have found that the unpleasant odour, which typically accompanies thermal processing of pH-neutral, beta-lactoglobulin-rich beverages, is associated with hydrogen sulfide formed from reacting free thiol groups (also referred to as sulfhydryl groups or -SH groups) of the cysteine-residues of the whey protein during the thermal processing. The inventors have furthermore discovered that this problem surprisingly can be reduced or even avoided by reacting beta-lactoglobulin-containing protein with oxidized phenolic compounds at acidic pH.

[0011] The inventors have furthermore found that modification of beta-lactoglobulin-containing protein compositions by reaction with oxidized phenolic compounds at acidic pH gives rise to less colour formation, less side-reactions towards other amino acids, and less protein aggregation than if the protein was modified by reaction with the same oxidized phenolic compounds at neutral pH. The reduced level of aggregation is furthermore advantageous as makes the modified protein more suitable for transparent high protein beverages of low viscosity.

[0012] Thus, an aspect of the invention pertains to a method of producing a protein composition comprising modified beta-lactoglobulin (BLG), the method comprising the steps of

[0013] a) providing:

[0014] - a source comprising one or more phenolic compounds that contain at least two hydroxyl groups bound directly to the same aromatic ring (PCA), and

[0015] - a source comprising BLG,

[0016] b) optionally subjecting a portion of the source comprising one or more PCA to a type of oxidation capable of converting a PCA to a quinone thereby providing a source comprising one or more oxidized PCA (PCA-type oxidation), c) combining a portion of the source comprising one or more PCA and / or a portion of the source comprising one or more oxidized PCA with a source comprising BLG and optionally with further ingredients to provide a protein solution, said protein solution having:

[0017] - a pH in the range of 2.0-4.5, and

[0018] - a BLG content of at least 0.2% w / w,

[0019] - a mole ratio between:

[0020] - the original amount of PCA used for preparing the protein solution, and

[0021] - the content of BLG of the protein solution,

[0022] of at least 0.1:1,

[0023] d) incubating the protein solution within a temperature range and for a duration sufficient to reduce the amount of free thiol groups of the protein solution to at most 15 micromol / g protein, more preferably at most 10 micromol / g protein, with the proviso that if the method does not contain step b), step d) also involves application of a type of oxidation capable of converting a PCA to a quinone, and

[0024] preferably, e) removing at least some of the free PCA and free, oxidized PCA from the incubated protein solution obtained from step d).

[0025] The PCA-type oxidation applied during step b) and / or step d) is preferably sufficient to create a mole ratio between:

[0026] - the theoretical amount of quinone provided to the protein solution during the method determined according to Analysis B, and

[0027] - the content of BLG of the protein solution

[0028] of at least 0.1:1.

[0029] Another aspect of the invention pertains to a protein composition comprising modified BLG, said protein composition is obtainable by the method of the present invention.

[0030] A further aspect of the invention pertains to a process for producing a heat-treated, preferably heat-sterilized, beverage, the process comprising the following steps:

[0031] 1) mixing the protein composition of the present invention with one or more further beverage ingredients to obtain a liquid mixture having a pH of 5.5-8.5, and

[0032] 2) filling the liquid mixture into suitable containers,

[0033] the process furthermore comprising at least one heat-treatment step wherein the liquid mixture is heat-treated, and preferably heat-sterilized, prior to filling and / or after filling,

[0034] preferably wherein the liquid mixture comprises the protein composition in an amount sufficient to contribute with at least 0.5% w / w protein. Yet an aspect of the invention pertains to a heat-treated, preferably heat-sterilized, beverage having a pH of 5.5-8.5, obtainable by the process of the present invention.

[0035] Another aspect of the invention pertains to a food ingredient comprising:

[0036] - the solids of the protein composition of the present invention, and

[0037] - one or more further ingredient(s), preferably selected from:

[0038] - a dairy ingredient, preferably a non-oxidized dairy ingredient,

[0039] - a plant-based ingredient,

[0040] - a non-dairy carbohydrate source,

[0041] - a flavouring agent, and / or

[0042] - a sweetener.

[0043] An even further aspect of the invention pertains to the use of a protein composition comprising modified BLG, preferably the protein composition according to the present invention, as a food ingredient, preferably for:

[0044] - improving the odour, and / or

[0045] - reducing the level of unpleasant odour similar to the odour of rotten eggs, and / or

[0046] - reducing the development of H2S during production, and / or

[0047] - reducing the content of H2S in the headspace of the container,

[0048] of heat-sterilized, beverages having a pH in the range of 5.5-8.5, preferably having a whey protein content of at least 3% w / w, and preferably heat-sterilized using indirect heat-treatment.

[0049] BRIEF SUMMARY OF THE FIGURE

[0050] Figure 1 shows SDS-PAGE gels (both non-reduced and reduced conditions) gels of samples WPI-A1 to WPI-A3 and WPI-B1 to WPI-B3 of Example 1. The Lane "M" contained the molecular weight marker. Sample IDs are on the top of each lane. WPI-A1 and WPI-B1 are samples without added 4MBQ, and WPI-A2 to WPI-A3 and WPI-B2 to WPI-B3 are samples incubated with 4MBQ at 80 degrees C for 15 min.

[0051] DETAILED DESCRIPTION

[0052] An aspect of the invention pertains to a method of producing a protein composition comprising modified BLG, the method comprising the steps of

[0053] a) providing: - a source comprising one or more phenolic compounds that contain at least two hydroxyl groups bound directly to the same aromatic ring (PCA), and

[0054] - a source comprising BLG,

[0055] b) optionally subjecting a portion of the source comprising one or more PCA to a type of oxidation capable of converting a PCA to a quinone thereby providing a source comprising one or more oxidized PCA (referred to as PCA-type oxidation),

[0056] c) combining a portion of the source comprising one or more PCA and / or a portion of the source comprising one or more oxidized PCA with a source comprising BLG and optionally with further ingredients to provide a protein solution, said protein solution having:

[0057] - a pH in the range of 2.0-4.5, and

[0058] - a BLG content of at least 0.2% w / w,

[0059] - a mole ratio between:

[0060] - the original amount of PCA used for preparing the protein solution, and

[0061] - the content of BLG of the protein solution,

[0062] of at least 0.1:1,

[0063] d) incubating the protein solution within a temperature range and for a duration sufficient to reduce the amount of free thiol groups of the protein solution to at most 15 micromol / g protein, more preferably at most 10 micromol / g protein, with the proviso that if the method does not contain step b), step d) also involves application of a type of oxidation capable of converting a PCA to a quinone, and

[0064] preferably, e) removing at least some of the free PCA and free, oxidized PCA from the incubated protein solution obtained from step d).

[0065] The PCA-type oxidation applied during step b) and / or step d) is preferably sufficient to create a mole ratio between:

[0066] - the theoretical amount of quinone provided to the protein solution during the method determined according to Analysis B, and

[0067] - the content of BLG of the protein solution

[0068] of at least 0.1:1.

[0069] In the context of the present invention, the term "beta-lactoglobulin" or BLG, pertains to BLG from mammal species, e.g. in native and / or glycosylated forms and includes the naturally occurring genetic variants. The term BLG also encompasses BLG produced by recombinant technology and / or precision fermentation e.g. based on genetically modified microorganisms or mammalian cells. The term "BLG" or "beta-lactoglobulin" as used herein excludes unfolded and aggregated BLG. The content of BLG is measured according to Analysis L of PCT appl.no.

[0070] PCT / EP2022 / 078739.

[0071] In the context of the present invention, the term "phenolic compounds" has its normal meaning and refers to a molecular compound containing an aromatic ring of which at least one of the six carbon atom is directly bound to a hydroxyl group. The five other carbon atoms of aromatic ring may, individually, be bound to various groups including a hydroxyl group or a hydrogen. The phenolic compounds also comprise polyphenols.

[0072] In the context of the present invention, the term "phenolic compound comprising an aromatic ring to which at least two hydroxyl group are directly bound" (referred to as "PCA" or a Type-A phenolic compound) pertains to molecular compounds, such as e.g. catechol, caffeic acid, or epigallocatechinegallate (EGCG), comprising an aromatic ring of which ring at least two carbon atoms are directly bound to a hydroxy group. The four other carbon atoms of the aromatic ring may, individually, be bound to various groups including a hydroxy group or a hydrogen. A PCA can be oxidized to form a quinone group and the at least two hydroxy groups are therefore positioned in a manner that allows for the formation of a quinone group upon appropriate oxidation. Preferably, at least two of the hydroxyl groups bound to the aromatic ring are in ortho position or para position.

[0073] A PCA preferably has the following structural formula:

[0074]

[0075] wherein at least two of Ri, R2, 3, 4, Rs, and Rsare hydroxyl groups.

[0076] Preferably two or three of Ri, R2, R3, R4, Rs, and Rsare hydroxyl groups.

[0077] It is particularly preferable that at least two of the hydroxyl groups of formula (I) are in ortho or para-position. In the context of the present invention, the term "a type of oxidation capable of converting a PCA to a quinone" or "PCA-type oxidation" pertains to an oxidative treatment capable of oxidizing PCA of the source comprising one or more PCA to a quinone. The PCA-type oxidation typically involves electrochemical oxidation and / or use of chemical oxidizing agents.

[0078] In the context of the present invention, the phrase "the PCA-type oxidation applied during step b) and / or step d) is sufficient to create a mole ratio[...]" is used to define the required level of PCA-type oxidation in a manner that can be applied to all embodiments of the method of the invention. In this context "step b) and / or step d)" means that if the method involves PCA-type oxidation during both a step b) and step d) then the combined PCA-type oxidation of both steps should be considered. However, if the method only involves PCA-type oxidation during step b) then only step b) should be considered. Similarly, if the method only involves PCA-type oxidation during step d) then only step d) should be considered.

[0079] As will be evident to the person skilled in the art, a mole ratio between component X and component Y which must be at least nx:nYmeans that the mole ratio must be at least nxdivided by nY. nxand nYrepresent the amounts of components X and Y in mole. For example, if the mole ratio between component X and components Y must be at least 0.1:1 it means that nxdivided by nYmust be at least 0.1. The same logic applies to weight ratios.

[0080] In the context of the present invention, the phrase "theoretical amount of quinone provided to the protein solution during the method" is a measure of the mole content of quinones that is formed if all PCA (incl. PCA that have been converted to quinones) present in the protein solution of the method was oxidized under the same conditions and under the same oxidizing conditions used in the method but without the presence of proteins or other sources of free thiols or amines. The "theoretical amount of quinone provided to the protein solution during the method" is determined according to Analysis B. For example, if the method of the invention is primarily based on oxidation of PCA during step b) (to provide a source comprising one or more oxidized PCA) prior to step c), then the content of quinones of the portion of the source comprising one or more oxidized PCA that is used for step c) is equal to the of the theoretical amount of quinone provided to the protein solution during the method.

[0081] In some preferred embodiments of the present invention the method of producing a protein composition comprising modified BLG furthermore comprises a step f) of drying a liquid feed comprising at least the protein derived from the incubated protein solution of step d).

[0082] In some preferred embodiments of the invention, the protein solution provided in step c) has a mole ratio between:

[0083] - the original amount of PCA used for preparing the protein solution, and - the content of BLG of the protein solution,

[0084] of at least 1:1,

[0085] and the PCA-type oxidation applied during step b) and / or step d) preferably is sufficient to create a mole ratio between:

[0086] - the theoretical amount of quinone provided to the protein solution during the method determined according to Analysis B, and

[0087] - the content of BLG of the protein solution

[0088] of at least 1:1.

[0089] The term "source comprising one or more PCA" refers to the composition(s) used for providing the PCA for the protein solution of step c) and / or the oxidizing aqueous solution of step b). The term "the portion of the source comprising one or more PCA" pertains to the actual portion used for preparing the protein solution. In some preferred embodiments of the invention substantially all of the source comprising one or more PCA is used.

[0090] If more than one PCA-containing compositions are used for the production of the protein solution of step c) and / or the oxidizing aqueous solution of step b), then each of the used PCA-containing ingredients are considered sub-sources which make up the source comprising one or more PCA.

[0091] In some preferred embodiments of the present invention the PCA comprises a flavonoid, preferably a flavanol or a flavanol ester. Preferred flavonoids are e.g. catechin and a catechin derivative such as epicatechin, gallocatechin, epigallocatechin, catechin 3-gallate, epicatechin 3-gal-late, gallo-catechin 3-gallate, and epigallocatechin 3-gallate (EGCG).

[0092] In other preferred embodiments of the present invention the PCA comprises a stilbenoid, preferably in the form of resveratrol.

[0093] In further preferred embodiments of the present invention the PCA comprises a phenolic acid, preferably one or more of caffeic acid, gallic acid, chlorogenic acid, or a mixture thereof.

[0094] It is often preferred that PCA comprises caffeic acid, gallic acid, chlorogenic acid, catechol, 4-methyl catechol, catechin, epicatechin, gallocatechin, epigallocatechin, catechin 3-gallate, epicatechin 3-gallate, gallocatechin 3-gallate, epigallocatechin 3-gallate (EGCG), resveratrol, car-nosic acid, carnosol, naringenin, or a mixture thereof.

[0095] In some preferred embodiments of the present invention one or more PCAs from the following group caffeic acid, gallic acid, chlorogenic acid, catechol, 4-methyl catechol, catechin, epicatechin, gallocatechin, epigallocatechin, catechin 3-gallate, epicatechin 3-gallate, gallocatechin 3- gallate, epigallocatechin 3-gallate (EGCG), resveratrol, carnosic acid, carnosol, naringenin contribute with at least 40 mol% of the PCAs of the source comprising one or more PCA, more preferably at least 50 mol%, even more preferably at least 60 mol%, even more preferably at least 70 mol%, even more preferably at least 80 mol% and most preferably at least 90 mol% of the PCAs of the source comprising one or more PCA.

[0096] In other preferred embodiments of the present invention one or more PCAs from the following group catechin, epicatechin, gallocatechin, epigallocatechin, catechin 3-gallate, epicatechin 3-gallate, gallocatechin 3-gallate, and epigallocatechin 3-gallate (EGCG) contribute with at least 40 mol% of the PCAs of the source comprising one or more PCA, more preferably at least 50 mol%, even more preferably at least 60 mol%, even more preferably at least 70 mol%, even more preferably at least 80 mol% and most preferably at least 90 mol% of the PCAs of the source comprising one or more PCA.

[0097] In further preferred embodiments of the present invention one or more PCAs from the following group caffeic acid, gallic acid, chlorogenic acid, catechol, 4-methyl catechol, catechin, epicatechin, gallocatechin, epigallocatechin, catechin 3-gallate, epicatechin 3-gallate, gallocatechin 3-gallate, epigallocatechin 3-gallate (EGCG), resveratrol, carnosic acid, carnosol, naringenin contribute with at least 40 mol% of the PCAs of the source comprising one or more PCA, more preferably at least 50 mol%, even more preferably at least 60 mol%, even more preferably at least 70 mol%, even more preferably at least 80 mol% and most preferably at least 90 mol% of the PCAs of the source comprising one or more PCA.

[0098] In even further preferred embodiments of the present invention one or more PCAs from the following group caffeic acid, gallic acid, and chlorogenic acid contribute with at least 40 mol% of the PCAs of the source comprising one or more PCA, more preferably at least 50 mol%, even more preferably at least 60 mol%, even more preferably at least 70 mol%, even more preferably at least 80 mol% and most preferably at least 90 mol% of the PCAs of the source comprising one or more PCA.

[0099] In yet further preferred embodiments of the present invention caffeic acid contributes with at least 40 mol% of the PCAs of the source comprising one or more PCA, more preferably at least 50 mol%, even more preferably at least 60 mol%, even more preferably at least 70 mol%, even more preferably at least 80 mol% and most preferably at least 90 mol% of the PCAs of the source comprising one or more PCA.

[0100] In other preferred embodiments of the present invention epigallocatechin 3-gallate (EGCG) contributes with at least 40 mol% of the PCAs of the source comprising one or more PCA, more preferably at least 50 mol%, even more preferably at least 60 mol%, even more preferably at least 70 mol%, even more preferably at least 80 mol% and most preferably at least 90 mol% of the PCAs of the source comprising one or more PCA.

[0101] In some preferred embodiments of the present invention the PCA has a molecular weight of at most 600 g / mol, more preferably most 400 g / mol, even more preferably most 350 g / mol, and more preferably most 310 g / mol.

[0102] In other preferred embodiments of the present invention the PCA has a molecular weight of at most 250 g / mol, more preferably at most 230 g / mol, even more preferably at most 210 g / mol, and more preferably at most 200 g / mol.

[0103] In some preferred embodiments of the present invention the PCA has a molecular weight in the range of 110-600 g / mol, more preferably 110-400 g / mol, even more preferably 110-350 g / mol, and more preferably 110-310 g / mol.

[0104] In some preferred embodiments of the present invention the PCA has a molecular weight in the range of 110-250 g / mol, more preferably 110-230 g / mol, even more preferably 120-210 g / mol, and more preferably 120-200 g / mol.

[0105] In other preferred embodiments of the present invention the PCA has a molecular weight in the range of 120-1000 g / mol, more preferably 250-700g / mol, even more preferably 300-650 g / mol, and more preferably 350-600 g / mol.

[0106] In other preferred embodiments of the present invention the PCA has a molecular weight in the range of 120-600 g / mol, more preferably 140-500 g / mol, even more preferably 150-400 g / mol, and most preferably 160-300 g / mol.

[0107] In some preferred embodiments of the present invention the PCA neither contain nitrogen nor sulfur.

[0108] In some preferred embodiments of the present invention the PCA do not contain a carboxylic acid groups.

[0109] In some preferred embodiments of the present invention the PCA has a water solubility of at least 8 mM at 25 degrees C, more preferably at least 12 mM, and most preferably at least 16 mM. In some preferred embodiments of the invention the source comprising one or more PCA is selected from a polyphenol extract from a herb, a polyphenol extract from a spice, polyphenol extract from a fruit, polyphenol extract from a berry, and mixtures thereof.

[0110] It is often preferred that the source comprising one or more PCA is preferably selected from the group consisting of a polyphenol extract of tea, more preferably a polyphenol extract of green tea; a polyphenol extract of coffee; polyphenol extract of cocoa; a polyphenol extract of grapes; a polyphenol extract of rosemary; a polyphenol extract of lemon balm; a polyphenol extract of black currant; a single PCA-isolate, and mixtures thereof.

[0111] "Polyphenol extracts" are well-known in the art and pertain to extracts targeting the polyphenol fraction of the given polyphenol source. In some preferred embodiments of the invention the polyphenol extract is a water-based extract. In other preferred embodiments of the invention the polyphenol extract is an extract based on water and ethanol.

[0112] In some preferred embodiments of the present invention the source comprising one or more PCA comprises PCA in an amount of at least 25% w / w relative to the total solids of the source comprising one or more PCA, more preferably at least 40% w / w, even more preferably at least 60% w / w, and most preferably at least 80% w / w relative to the total solids of the source comprising one or more PCA.

[0113] In further preferred embodiments of the present invention the source comprising one or more PCA is a single PCA-isolate comprising at single PCA in an amount of at least 25% w / w relative to the total solids of the source comprising one or more PCA, more preferably at least 40% w / w, even more preferably at least 60% w / w, and most preferably at least 80% w / w relative to the total solids of the source comprising one or more PCA.

[0114] It is preferred that the source comprising one or more PCA does not contribute with bitterness or off-taste to the protein composition comprising modified BLG.

[0115] The term "source comprising BLG" refers to the composition(s) used for providing BLG to the protein solution of step c).

[0116] If more than one BLG-containing composition are used for the production of the protein solution of step c) and / or the oxidizing aqueous solution of step b), then each of the used BLG-containing compositions are considered sub-sources which make up the source comprising BLG.

[0117] In some preferred embodiments of the present invention the protein of the source of BLG is isolated from mammal milk, and preferably from the milk of a ruminant such as e.g. cow, sheep, goat, buffalo, camel, llama, mare and / or deer. Protein isolated from bovine (cow) milk is particularly preferred. The BLG and the additional whey protein are therefore preferably bovine BLG and bovine whey protein. Alternatively, but also preferred, is protein prepared by fermentation of microorganisms during the production of recombinant BLG. If the BLG is recombinant BLG it is preferred that it has a sequence that resembles or even is identical to BLG from ruminant sources such as e.g. cow, sheep, goat, buffalo, camel, llama, mare and / or deer.

[0118] Preferably, the source comprising BLG comprises, or even consists of, a whey protein concentrate, a whey protein isolate, a milk serum protein concentration, a milk serum protein isolate, a BLG isolate, or a combination thereof.

[0119] The source of BLG may be in the form of a liquid or a powder, and its sub-sources may contain a combination of one or more powders comprising BLG and / or one or more liquids comprising BLG.

[0120] The term "whey" pertains to the liquid phase that is left after the casein of milk has been precipitated and removed. Casein precipitation may e.g. be accomplished by acidification of milk and / or by use of rennet enzyme. Several types of whey exist, such as "sweet whey", which is the whey product produced by rennet-based precipitation of casein, and "acid whey" or "sour whey", which is the whey product produced by acid-based precipitation of casein. Acid-based precipitation of casein may e.g. be accomplished by the addition of food acids or by means of bacterial cultures.

[0121] The term "milk serum" pertains to the liquid which remains when casein and milk fat globules have been removed from milk, e.g. by microfiltration or large pore ultrafiltration. Milk serum may also be referred to as "ideal whey".

[0122] In the context of the present invention, the term "whey protein" pertains to the protein that is found in whey or milk serum. Whey protein may be a subset of the protein species found in whey or milk serum, and even a single whey protein species or it may be the complete set of protein species found in whey or / and in milk serum.

[0123] In some preferred embodiments of the invention the protein of the source comprising BLG is isolated from bovine whey or milk serum.

[0124] Unfractionated whey protein typically contains alpha-lactalbumin (ALA), beta-lactoglobulin (BLG), bovine serum albumin, immunoglobulins, osteopontin, lactoferrin, and lactoperoxidase. Whey protein derived from rennet treated milk furthermore comprise caseinomacropeptide (CMP) in addition to the other protein species. In the context of the present invention, the term "whey protein concentrate" (WPC) pertains to dry or aqueous compositions which contain a total amount of protein of 20-89% w / w relative to total solids.

[0125] A WPC preferably contains:

[0126] 30-85% w / w protein relative to total solids,

[0127] 15-90% w / w BLG relative to total protein,

[0128] 4-50% w / w ALA relative to total protein, and

[0129] 0-40% w / w CMP relative to protein.

[0130] Most preferably a WPC contains:

[0131] 70-85% w / w protein relative to total solids,

[0132] 30-90% w / w BLG relative to total protein,

[0133] 4-35% w / w ALA relative to total protein, and

[0134] 0-25% w / w CMP relative to protein.

[0135] WPC based on milk serum protein typically contains no CMP or only traces of CMP.

[0136] The term "whey protein isolate" (WPI) pertains to dry or aqueous compositions which contain a total amount of protein of 86-100% w / w relative to total solids.

[0137] A WPI preferably contains:

[0138] 86-99% w / w protein relative to total solids,

[0139] 30-100% w / w BLG relative to total protein,

[0140] 0-35% w / w ALA relative to total protein, and

[0141] 0-25% w / w CMP relative to total protein.

[0142] Most preferably a WPI contains:

[0143] 90-99% w / w protein relative to total solids,

[0144] 50-99% w / w BLG relative to total protein,

[0145] 0-35% w / w ALA relative to total protein, and

[0146] 0-25% w / w CMP relative to total protein.

[0147] WPI based on milk serum protein typically contains no CMP or only traces of CMP.

[0148] It is particularly preferred that the whey protein source is a WPI. In the context of the present invention, the term "BLG isolate" pertains to a composition that contains BLG in an amount of at least 80% w / w relative to total solids, more preferably at least 90% w / w relative to total solids, and most preferably at least 95% w / w relative to total solids.

[0149] BLG isolates may e.g. be prepared by isolation of BLG from milk serum or whey, preferably according to W02018115520 Al, or alternatively prepared recombinantly by fermentation of a genetically modified microorganisms or mammal cells.

[0150] The source comprising BLG preferably has a degree of protein denaturation of at most 30%, more preferably at most 25%, even more preferably at most 20% and most preferably as most 15%.

[0151] An even lower degree of protein denaturation is often preferred, and in some preferred embodiments of the present invention, the source comprising BLG has a degree of protein denaturation of at most 12%, more preferably at most 10%, even more preferably at most 8%, and most preferably at most 5%.

[0152] In some preferred embodiments of the invention the source comprising BLG has a total fat content of at most 10% w / w relative to total solids, more preferably at most 8% w / w, and most preferably at most 6% w / w relative to total solids.

[0153] In other preferred embodiments of the invention the source comprising BLG has a total fat content of at most 5% w / w relative to total solids, more preferably at most 2% w / w, even more preferably at most 0.5% w / w, and most preferably at most 0.1% w / w relative to total solids.

[0154] In further preferred embodiments of the invention the source comprising BLG has a total fat content in the range of 1-20% w / w relative to total solids, more preferably in the range of 2-16% w / w, even more preferably in the range of 3-12% w / w, and most preferably at most 4-10% w / w relative to total solids.

[0155] In some preferred embodiments of the present invention the source comprising BLG has a total carbohydrate content of at most 10% w / w relative to total solids, more preferably at most 8% w / w, even more preferably at most 6% w / w, and most preferably at most 5% w / w relative to total solids.

[0156] In further preferred embodiments of the present invention the source comprising BLG has a total carbohydrate content of at most 2% w / w relative to total solids, more preferably at most 1% w / w, even more preferably at most 0.5% w / w, and most preferably at most 0.2% w / w relative to total solids. Step b) is optional in the sense that in some preferred embodiments of the present invention, the method comprises b) whereas other preferred embodiments of the present invention do not contain step b) and therefore do employ oxidation of a portion of the source comprising one or more PCA prior to step c).

[0157] Thus, in some preferred embodiments of the present invention the method of producing a protein composition comprising modified BLG comprises step b) of subjecting a portion of the source comprising one or more PCA to oxidation capable of converting a PCA to a quinone (also referred to as PCA-type oxidation) thereby providing a source comprising one or more oxidized PCA. It should be understood that the PCA-type oxidation must be able to oxidize the specific type of PCA specified in step a) to quinones.

[0158] Note that in some instances an oxidized PCA is not a PCA but only carries a quinone functionality.

[0159] In some preferred embodiments of the present invention the oxidation of step b) is involves preparing an oxidizing aqueous solution comprising the source comprising one or more PCA, optionally a chemical oxidizing agent and optionally one or more further ingredients such as water.

[0160] In the context of the present invention the term "oxidizing aqueous solution" is used to describe the aqueous solution in which the PCA-type oxidation of step b) takes place.

[0161] The oxidizing aqueous solution has a content of PCA of preferably at least 0.2 mM, more preferably at least 15 mM, and most preferably at least 50 mM.

[0162] In some preferred embodiments of the present invention the oxidizing aqueous solution has a concentration of PCA of 0.2-600 mM, more preferably 15-550 mM, and most preferably 50-500 mM.

[0163] For some PCA, it is advantageous if the oxidizing aqueous solution comprises one or more amphipathic modifiers to increase PCA solubility. The one or more amphipathic modifiers are preferably food-grade and / or pharmaceutical-grade amphipathic modifiers. Preferred examples of amphipathic modifiers are food grade alchohols, such as e.g. ethanol, dimethyl sulfoxide (DMSO), polysorbates, esters of sorbitan and fatty acids, and a mixture thereof.

[0164] Useful examples of polysorbates may e.g. be selected from the group consisting of polysorbate 20, 40, 60 and 80. Useful examples of esters of sorbitan and fatty acids may e.g. be selected from the group consisting of sorbitan monolaurate, sorbitan monostearate, sorbitan tristearate, and sorbitan monooleate.

[0165] In some preferred embodiments of the present invention the oxidizing aqueous solution has a pH in the range of 2-9.5, more preferably 6.5-9.5, even more preferably 7.1-9.0, even more preferably 7.3-8.7, and most preferably 7.4-8.5.

[0166] pH-values mentioned herein refer to the pH normalized to 25 degrees C and are measured according to Analysis A.

[0167] The inventors have found that it may be advantageous to perform step b) using an oxidizing aqueous solution having a pH in the range of 2-6.4, more preferably 2.2-5, even more preferably 2.6-4, and most preferably 2.8-3.5.

[0168] In some preferred embodiments of the present invention the oxidizing aqueous solution contains one or more further ingredients selected from the group consisting of a food acceptable acid, a food acceptable base, water, an amphipathic modifier, an chemical oxidizing agent, an oxidation catalyst, and a combination thereof.

[0169] Water is typically required as a further ingredient if the source comprising one or more PCA is provided in the form of a powder.

[0170] A chemical oxidizing agent is required in the oxidizing aqueous solution if the PCA-type oxidation of step b) should be partially or completely based on chemical oxidizing agent. It is even feasible to use a combination of several chemical oxidizing agents.

[0171] Food acceptable acids and / or food acceptable bases are typically required if the pH should be adjusted.

[0172] Useful examples of oxidation catalysts are one or more enzymes such as polyphenol oxidase, laccase and / or tyrosinase.

[0173] In some preferred embodiments of the present invention the oxidation of step b) is performed by contacting the portion of the source comprising one or more PCA with a chemical oxidizing agent under conditions that convert at least some of the PCA to quinones. This oxidation is performed in the oxidizing aqueous solution.

[0174] It is often preferred that the chemical oxidizing agent comprises, or even consists of, a peroxide, ozone, dioxygen, or a combination thereof. In other preferred embodiments of the present invention the chemical oxidizing agent comprises, or even consists of, hydrogen peroxide, benzoyl peroxide, or a combination thereof.

[0175] In further preferred embodiments of the present invention the chemical oxidizing agent comprises, or even consists of, hydrogen peroxide, benzoyl peroxide, dioxygen, or a combination thereof.

[0176] The chemical oxidizing agents are preferably used in combination with a catalytic amount of iron ions and / or copper ions.

[0177] In some preferred embodiments of the present invention the oxidation of step b) involves preparing an oxidizing aqueous solution comprising the portion of the source comprising one or more PCA, the chemical oxidizing agent and optionally one or more further ingredients such as water.

[0178] The PCA-type oxidation may also involve enzymatically catalysed oxidation, e.g., involving enzymes such as polyphenol oxidase, laccase and / or tyrosinase.

[0179] Dissolved dioxygen provided by the liquid(s) used to prepare the oxidizing aqueous solution used in step b) and / or the protein solution of step c) may contribute with trace oxidation of PCA but this is typically insufficient to achieve the desired level of oxidation required for the invention.

[0180] It is normally preferred that the oxidizing aqueous solution does not contain protein. Additionally it is preferred that the oxidizing aqueous solution does not contain molecules containing free thiol groups. In some preferred embodiments of the invention the oxidizing aqueous solution furthermore does not contain molecules having amine groups.

[0181] In some preferred embodiments of the invention the oxidizing aqueous solution contains less than 5% w / w protein relative to its weight, more preferably at most 1% w / w, even more preferably at most 0.1%; and most preferably no protein at all.

[0182] In some preferred embodiments of the invention the PCA-type oxidation of step b) involves electrochemical oxidation of the portion of the source comprising one or more PCA under conditions that convert at least some of the PCA to quinones. In some preferred embodiments of the invention the oxidation of step b) involves preparing an oxidizing aqueous solution comprising the portion of the source comprising one or more PCA, and optionally one or more ingredients such as water.

[0183] Preferably, the electrochemical oxidation is operated using a potential difference of -0.1 - 1.5 V, more preferably 0.0 - 1.2 V, and most preferably 0.0 to 0.7V.

[0184] The potential difference, also referred to as the voltage, refers the potential difference between the working electrode(s) and an Ag / AgCI (KCI, c=3 M) reference electrode that are used to perform the electrochemical oxidation of the PCA.

[0185] In some preferred embodiments of the invention the oxidizing aqueous solution has a pH in the range of 6-9 and the electrochemical oxidation is operated using a potential difference of -0.1 to 0.9, even more preferably 0-0.8 V, and most preferably 0.2-0.7 V.

[0186] In other preferred embodiments of the invention the oxidizing aqueous solution has a pH in the range of 2-5 and the electrochemical oxidation is performed using a potential difference of 0.1-1.3 V, and more preferably 0.2-0.9 V, and most preferably 0.3-0.7V.

[0187] For determining an appropriate potential difference to be used for electrochemical oxidation, it is advantageous to identify the oxidation potential of the PCA.

[0188] The required oxidation potential of the PCA, i.e. required for PCA-type oxidation, may e.g. be determined by cyclic voltammetry, wherein the working electrode potential is swept from, e.g., -0.2 V to +1.2 V versus an Ag / AgCI (KCI, c=3 M) reference electrode. At a given applied potential, the current increases, as the PCA begins to oxidize at and / or near the working electrode surface. The current continues increasing as the potential is ramped up, until one or more peak currents are achieved, when the oxidizable groups of the PCA near the electrode are depleted.

[0189] The identified oxidation potential(s) at peak current(s) may be used in electrochemical oxidation of the PCA. If more than one peak is observed (such as the case for e.g. EGCG) the minimum applied potential difference giving rise to a peak current and results in formation of adequate amounts of quinones as determined by Analysis C is selected.

[0190] In some preferred embodiments of the invention, the electrochemical oxidation is implemented as bulk electrolysis, and is preferably performed in constant current mode or in constant potential mode. Agitation during bulk electrolysis is advantageous, e.g., in order to reduce diffusive limitations. In some preferred embodiments of the invention the electrochemical oxidation is implemented as bulk electrolysis in constant potential mode and involves maintaining a constant working electrode potential relative to the reference electrode while monitoring the decline in current over time as the PCA is oxidized to the quinone form. Preferably, the PCA is converted quantitatively into its quinone form. The current may reach a value of zero upon complete oxidation of PCA.

[0191] In other preferred embodiments of the invention the electrochemical oxidation is implemented as bulk electrolysis in constant current mode and involves maintaining a constant current between the working electrode and counter electrode while monitoring the total amount of charge that is passed through the electrolysis cell and monitoring the increase in potential between the working electrode and the reference electrode over time as the PCA is oxidized to the quinone form. The end point of the bulk electrolysis can be determined as when a maximum potential is reached, or as the time when a certain amount of charge has been passed through the electrolysis cell, preferably equal to a theoretical charge of about 2 Faraday per mole desired quinone.

[0192] Bulk electrolysis may be implemented using a potentiostat and a two electrode system or, preferably, a potentiostat and a three electrode system comprising one or more working electrodes, one or more counter electrodes, and a reference electrode.

[0193] The working electrode is preferably the anode. Preferred examples of a working electrode are a glassy carbon electrode, a boron doped diamond electrode, and a graphite electrode.

[0194] The counter electrode is preferably the cathode. A counter electrode is also known as auxiliary electrode. A preferred counter electrode is a platinum electrode, more preferably a platinum electrode selected from a coil type platinum electrode and a platinum flag electrode.

[0195] The reference electrode is preferably a true reference electrode or, more preferably, a quasireference electrode, which is also known as pseudo-reference electrode. A preferred example of a quasi-reference electrode is an Ag / AgCI electrode.

[0196] In some preferred embodiments of the invention, the electrochemical oxidation is performed under nitrogen.

[0197] The amount of quinones formed during electrochemical oxidation is determined by Analysis C.

[0198] The PCA-type oxidation of step b) is partially or completely performed by electrochemical oxidation it is preferred that the oxidizing aqueous solution also comprises electrolytes. The oxidizing aqueous solution preferably comprises electrolytes in a concentration of 0.01- 0.5 M; more preferably 0.05-0.4 M and most preferably 0.1-0.2 M. The used electrolytes are preferably food grade, conductive, and capable of contributing to a stable pH. In some preferred embodiments of the invention, the oxidizing aqueous solution comprises one of more electrolytes selected from the groups consisting of H2SO4, phosphoric acid, and acetic acid.

[0199] In some preferred embodiments of the invention, the electrochemical oxidation is performed at a temperature in the range of 2-80 degrees C, more preferably 5-60 degrees C, even more preferably 10-40 degrees C, and most preferably in the range of 15-30 degrees C.

[0200] In other preferred embodiments of the invention, the electrochemical oxidation is performed at a temperature in the range of 30-90 degrees C, more preferably 40-90 degrees C, even more preferably 50-90 degrees C, and most preferably in the range of 60-90 degrees C.

[0201] In further preferred embodiments of the invention, the electrochemical oxidation is performed at a temperature in the range of 5-25 degrees C, more preferably 10-25 degrees C, even more preferably 15-25 degrees C, and most preferably in the range of 18-25 degrees C.

[0202] The duration of step b) depends on the specific composition of oxidizing aqueous solution and the conditions used for the PCA-type oxidation. If electrochemical oxidation is used the current may be used to monitor the progress of the oxidation. When the current approaches zero at the operating potential difference it is a sign of depletion of PCA. The generated quinone content can be measured according to Analysis C.

[0203] In step c) of the method a portion of the source comprising one or more PCA and / or a portion of the source comprising one or more oxidized PCA are combined with a source comprising BLG and optionally with further ingredients to provide a protein solution having a pH in the range of 2.0-4.5.

[0204] In step c), the term "portion of the source comprising one or more oxidized PCA" pertains to the actual portion of the source comprising one or more oxidized PCA provided in step b) that is used for preparing the protein solution. In some preferred embodiments of the invention substantially all of the source comprising one or more oxidized PCA is used.

[0205] In step c), the term "portion of the source comprising one or more PCA" pertains to the actual portion of the source comprising one or more PCA of step a) that is used for preparing the protein solution. In some preferred embodiments of the invention substantially all of the source comprising one or more PCA is used. In some preferred embodiments of the invention the protein solution has a pH in the range of 2.1-4.5, even more preferably 2.3-4.3, and most preferably 2.5-4.1.

[0206] The inventors have found that the pH ranges defined herein in relation to the protein solution favour selective modification of the free thiol group of BLG and reduce the risk of undesired reactions with the amine groups of BLG.

[0207] In some preferred embodiments of the invention the protein solution has a pH in the range of 2.0-4.5 and a temperature of at least 20 degrees C, more preferably at least 40 degrees C, even more preferably at least 60 degrees C and most preferably at least 70 degrees C.

[0208] In further preferred embodiments of the invention the protein solution has a pH in the range of 2.1-4.3 and a temperature of at least 20 degrees C, more preferably at least 30 degrees C, even more preferably at least 50 degrees C and most preferably at least 60 degrees C.

[0209] In particularly preferred embodiments of the invention the protein solution has a pH in the range of 2.3-4.1 and a temperature of at least 20 degrees C, more preferably at least 30 degrees C, even more preferably at least 50 degrees C and most preferably at least 60 degrees C.

[0210] In even more preferred embodiments of the invention the protein solution has a pH in the range of 2.5-3.9 and a temperature of at least 35 degrees C, more preferably at least 40 degrees C, even more preferably at least 45 degrees C and most preferably at least 50 degrees C.

[0211] Preferably, the protein solution has a pH in the range of 2.0-4.5 and a temperature of 30-95 degrees C, more preferably a pH in the range of 2.1-4.3 and a temperature of 45-95 degrees C, even more preferably a pH in the range of 2.3-4.1 and a temperature of 50-90 degrees C, and most preferably a pH in the range of 2.5-3.9 and a temperature of 55-90 degrees C.

[0212] In other preferred embodiments of the present invention the protein solution has a pH in the range of 2.0-4.5 and a temperature of 3-95 degrees C, more preferably a pH in the range of 2.1-4.3 and a temperature of 40-95 degrees C, even more preferably a pH in the range of 2.3-4.1 and a temperature of 45-95 degrees C, and most preferably a pH in the range of 2.5-3.9 and a temperature of 70-95 degrees C.

[0213] In further preferred embodiments of the present invention the protein solution has a pH in the range of 2.0-4.5 and a temperature of 5-95 degrees C, more preferably a pH in the range of 2.1-4.3 and a temperature of 8-90 degrees C, even more preferably a pH in the range of 2.3- 4.1 and a temperature of 10-85 degrees C, and most preferably a pH in the range of 2.5-3.9 and a temperature of 15-80 degrees C.

[0214] In some preferred embodiments of the invention the protein solution has a BLG content of at least 0.5% w / w, more preferably at least 1% w / w, even more preferably at least 3% w / w, and most preferably at least 6% w / w.

[0215] In further preferred embodiments of the invention the protein solution has a BLG content of 0.5-30% w / w, more preferably 1-20% w / w, even more preferably 3-16% w / w, and most preferably 5-12% w / w.

[0216] In some preferred embodiments of the invention the protein solution has a BLG content of at least 30% w / w relative to total protein, more preferably at least 40% w / w relative to total protein, even more preferably at least 45% w / w relative to total protein, and most preferably at least 50% w / w relative to total protein.

[0217] In further preferred embodiments of the invention the protein solution has a BLG content of 30-99% w / w relative to total protein, more preferably 40-95% w / w relative to total protein, even more preferably 45-90% w / w relative to total protein, and most preferably 50-80% w / w relative to total protein.

[0218] In other preferred embodiments of the invention the protein solution has a BLG content of at least 60% w / w relative to total protein, more preferably at least 80% w / w relative to total protein, even more preferably at least 90% w / w relative to total protein, and most preferably at least 95% w / w relative to total protein

[0219] In some preferred embodiments of the invention the protein solution has a BLG content of at least 30% w / w relative to total solids, more preferably at least 40% w / w relative to total solids, even more preferably at least 45% w / w relative to total solids, and most preferably at least 50% w / w relative to total solids.

[0220] In further preferred embodiments of the invention the protein solution has a BLG content of 30-99% w / w relative to total solids, more preferably 40-95% w / w relative to total solids, even more preferably 45-90% w / w relative to total solids, and most preferably 50-80% w / w relative to total solids.

[0221] In alternative but also preferred embodiments of the invention the protein solution has a BLG content of at least 60% w / w relative to total solids, more preferably at least 80% w / w relative to total solids, even more preferably at least 90% w / w relative to total solids, and most preferably at least 95% w / w relative to total solids.

[0222] In some preferred embodiments of the invention the protein solution has a total fat content of at most 10% w / w relative to total solids, more preferably at most 8% w / w,, and most preferably at most 6% w / w relative to total solids.

[0223] In other preferred embodiments of the invention the protein solution has a total fat content of at most 5% w / w relative to total solids, more preferably at most 2% w / w, even more preferably at most 0.5% w / w, and most preferably at most 0.1% w / w relative to total solids.

[0224] In further preferred embodiments of the invention the protein solution has a total fat content in the range of 1-20% w / w relative to total solids, more preferably in the range of 2-16% w / w, even more preferably in the range of 3-12% w / w, and most preferably 4-10% w / w relative to total solids.

[0225] The inventors have found that an advantage of the present invention is that it causes less fat oxidation than comparable methods for thiol modification. The method of the present invention is therefore suitable for gentle BLG modification in fat-rich systems.

[0226] In some preferred embodiments of the invention the protein solution has a total carbohydrate content of at most 10% w / w relative to total solids, more preferably at most 8% w / w, even more preferably at most 6% w / w, and most preferably at most 5% w / w relative to total solids.

[0227] In further preferred embodiments of the invention the protein solution has a total carbohydrate content of at most 2% w / w relative to total solids, more preferably at most 1% w / w, even more preferably at most 0.5% w / w, and most preferably at most 0.2% w / w relative to total solids.

[0228] In some preferred embodiments of the invention the protein solution has, and is therefore prepared to have, a mole ratio between:

[0229] - the original amount of PCA used for preparing the protein solution, and

[0230] - the content of BLG of the protein solution

[0231] of at least 0.1:1, more preferably at least 0.2:1, even more preferably at least 0.3:1, and most preferably at least 0.4:1.

[0232] In other preferred embodiments of the invention the protein solution has, and is therefore prepared to have, a mole ratio between:

[0233] - the original amount of PCA used for preparing the protein solution, and

[0234] - the content of BLG of the protein solution of at least 1.5:1, more preferably at least 2:1, even more preferably at least 5:1, and most preferably at least 10:1.

[0235] In the context of the present invention the term "the original amount of PCA used for preparing the protein solution" pertains to the amount of the specific PCA mentioned in step a) used for preparing the portion of source comprising one or more PCA that is used for preparing the protein solution in step c) and / or the portion of the source comprising one or more oxidized PCA that is used for preparing the protein solution in step c).

[0236] If step c) only uses a portion of the source comprising one or more oxidized PCA obtained from step b) then the "the original amount of PCA used for preparing the protein solution" pertains to the amount of PCA used for preparing that portion of source comprising one or more oxidized PCA.

[0237] If step c) only uses a portion of the source comprising one or more PCA obtained from step a) then the "the original amount of PCA used for preparing the protein solution" pertains to the amount of PCA used for preparing that portion of source comprising one or more PCA.

[0238] If step c) both uses a portion of the source comprising one or more PCA obtained from step a) and a portion of the source comprising one or more oxidized PCA obtained from step b) then the "the original amount of PCA used for preparing the protein solution" pertains to the amount of PCA used for preparing the portion of source comprising one or more PCA and the portion of source comprising one or more oxidized PCA.

[0239] The PCA referred to in steps b), c), d), e) and f) pertain to the PCA defined in step a).

[0240] In some preferred embodiments of the invention the protein solution has, and is prepared to have, a mole ratio between:

[0241] - the original amount of PCA used for preparing the protein solution, and

[0242] - the content of BLG of the protein solution

[0243] of 0.1:1 - 1000:1, more preferably 0.2:1- 500:1, even more preferably 0.3:1- 200:1, and most preferably 0.4:1- 100:1.

[0244] In further preferred embodiments of the invention the protein solution has, and is prepared to have, a mole ratio between:

[0245] - the original amount of PCA used for preparing the protein solution, and

[0246] - the content of BLG of the protein solution of 0.1:1 - 100:1, more preferably 0.1:1 - 50:1, even more preferably 0.1:1 - 20:1, and most preferably 0.1:1 - 10:1.

[0247] In even further preferred embodiments of the invention the protein solution has, and is prepared to have, a mole ratio between:

[0248] - the original amount of PCA used for preparing the protein solution, and

[0249] - the content of BLG of the protein solution

[0250] of 0.1:1 - 6:1, more preferably 0.2:1 - 5:1, even more preferably 0.3:1 - 4:1, and most preferably 0.4:1 - 3:1.

[0251] In some preferred embodiments of the invention the protein solution has, and is prepared to have, a mole ratio between:

[0252] - the original amount of PCA used for preparing the protein solution, and

[0253] - the content of BLG of the protein solution

[0254] of 1:1 - 1000:1, more preferably 2:1 - 500:1, even more preferably 5:1 - 200:1, and most preferably 10:1 - 100:1.

[0255] In further preferred embodiments of the invention the protein solution has, and is prepared to have, a mole ratio between:

[0256] - the original amount of PCA used for preparing the protein solution, and

[0257] - the content of BLG of the protein solution

[0258] of 1:1 - 100:1, more preferably 1:1- 50:1, even more preferably 1:1-20:1, and most preferably 1:1- 10:1.

[0259] In even further preferred embodiments of the invention the protein solution has, and is prepared to have, a mole ratio between:

[0260] - the original amount of PCA used for preparing the protein solution, and

[0261] - the content of BLG of the protein solution

[0262] of 1:1 - 6:1, more preferably 1.1:1 - 5:1, even more preferably 1.3:1 - 4:1, and most preferably 1.5:1 - 3:1.

[0263] The protein solution is prepared by combining, and preferably mixing, appropriate amounts of the sources described herein to obtain a protein solution having the desired characteristics. Preferably step c) is performed under conditions that do not lead to significant protein denaturation.

[0264] It is often preferred that the mixing of at least step c) is performed by inline mixing, particularly when at least steps b), c), and d) are implemented as a continuous process. In some preferred embodiments of the invention step c) is performed under conditions that lead to less than 15% protein denaturation of the BLG of the source comprising BLG, more preferably less 10%, even more preferably less 5%, and most preferably less than 2%.

[0265] The protein solution is preferably an aqueous solution. Preferably, at least 90% w / w of the nonsolid matter, i.e. the matter that does not contribute to the solids content of the protein solution, of the protein solution is water, more preferably at least 95% w / w, and most preferably at least 99% w / w.

[0266] The protein solution is preferably prepared with a temperature that does not cause protein denaturing, preferably in the range of 0-70 degrees C, and more preferably in the range of 5-65 degrees C, even more preferably in the range of 10-60 degrees C, and most preferably in the range of 20-55 degrees C.

[0267] In other preferred embodiments of the present invention the protein solution is prepared to have a temperature in the range of 0-50 degrees C, more preferably in the range of 2-45 degrees C, even more preferably in the range of 5-40 degrees C, and most preferably in the range of 10-35 degrees C. These ranges of temperatures are often preferred if dissolved dioxygen is intended to contribute with a significant portion of the PCA oxidation.

[0268] Preferably, the protein solution is prepared by mixing the portion of the source comprising one or more PCA obtained from step a), the source comprising BLG obtained from step a), and optionally with further ingredients.

[0269] In some preferred embodiments of the present invention the method does not comprise a step of oxidizing a portion of the source comprising one or more PCA prior to step c), i.e. the method does not comprise step b).

[0270] In other preferred embodiments of the present invention the method comprises step b), i.e. oxidizing a portion of the source comprising one or more PCA prior to the preparation of the protein solution, and the protein solution is prepared by mixing a portion of the source comprising one or more oxidized PCA obtained from step b) with the source comprising BLG, and optionally with further ingredients.

[0271] It is often preferred that most if not all PCA-type oxidation is performed during step b). In some preferred embodiments of the present invention, at less than 20% of the PCA-type oxidation takes place during step d), more preferably at most 10%, even more preferably at most 5%, and most preferably at most 2% of the PCA-type oxidation takes place during step d).

[0272] Preferably, further ingredients comprise water, one or more acids, one or more bases, one of more pH buffers, one or more oxidizing agents, or mixtures thereof.

[0273] In step d) of the method, the protein solution is incubated within a temperature range and for a duration sufficient to reduce the amount of free thiol groups of the protein solution to at most 10 micromol / g protein. If the method does not involve step b), step d) also involves a PCA-type oxidation capable of converting a PCA to a quinone. This oxidation may e.g. be achieved by a chemical oxidizing agent added during the production of the protein solution and / or by electrochemical oxidation during step d). In some preferred embodiments of the present invention the method involves both step b) and PCA-type oxidation during step d).

[0274] In the context of the present invention the term "the incubating protein solution" refers to the protein solution during step d).

[0275] In some preferred embodiments of the present invention the incubation reduces, and is therefore performed to reduce, the amount of free thiol of the protein solution to at most 9 micromol / g protein, more preferably at most 8 micromol / g protein, more preferably at most 5 micromol / g protein, even more preferably at most 3 micromol / g protein, and most preferably at most 2 micromol / g protein.

[0276] In the context of the present application, the amount of free thiol groups is quantified according to Analysis E. The terms "free thiol” and "free thiol groups" are used interchangeably.

[0277] In other preferred embodiments of the present invention the incubation reduces, and is therefore performed to reduce, the amount of free thiol of the protein solution to 0.001-10 micromol / g protein, more preferably 0.01- 9 micromol / g protein, even more preferably 0.02-8 micromol / g protein, even more preferably 0.03-5 micromol / g protein, even more preferably 0.04-3 micromol / g protein, and most preferably 0.04-2 micromol / g protein.

[0278] In some preferred embodiments of the present invention the incubating protein solution has a pH in the range of 2.1-4.5, even more preferably 2.3-4.3, and most preferably 2.5-4.1.

[0279] In some preferred embodiments of the present invention during the incubation of step d) the protein solution is subjected to a pressure in the range of 0.1-1000 bar, more preferably 1-500 bar, even more preferably 20-300 bar, and most preferably 40-200 bar. Typically, the incubation of step d) the protein solution is subjected to a pressure in the range of 0.1-100 bar, more preferably 1-50 bar, even more preferably 1-20 bar, and most preferably 1-10 bar.

[0280] In some preferred embodiments of the present invention during the incubation of step d) the protein solution has a temperature in the range of 0-160 degrees C, more preferably 10-155 degrees C, even more preferably 15-150 degrees C, and most preferably 20-145 degrees C.

[0281] The inventors have found it advantageous that the incubating protein solution is allowed to incubate at a temperature which is at least Tm, BLG minus 15 degrees C in order to speed up the reaction between the oxidized PCA and free thiol groups of BLG. Tm, BLG is the temperature of BLG at which at least 50% of the BLG molecules are on unfolded form a given pH of the incubating protein solution. Tm, BLG can be interpolated from the following table of Tmvalues which have been taken from of Table 1 of Kella et al; "Enhanced thermodynamic stability of / -lacto-globulin at low pH"; Biochem. J. (1988) 255, pages 113-118 .

[0282] PH T m, BLG

[0283] (degrees C)

[0284] 2.0 82.5

[0285] 2.5 78.8

[0286] 3.0 77.2

[0287] 6.5 71.9

[0288]

[0289] It is often preferred that the incubating protein solution of step d) is allowed to incubate at a temperature which is at least Tm, BLG minus 10 degrees C, more preferably at least Tm, BLG minus 5 degrees C, and most preferably at least Tm, BLG minus 2 degrees C. Tm, BLG is the temperature of BLG at which at least 50% of the BLG molecules are on unfolded form a given pH of the incubating protein solution. Tm, BLG can be interpolated from the following table of Tmvalues which have been taken from of Table 1 of Kella et al, Biochem. J. (1988) 255, pages 113-118.

[0290] In some preferred embodiments of the invention the incubating protein solution of step d) is allowed to incubate at a temperature which is in the range of Tm, BLG - 15 degrees C to Tm, BLG + 30 degrees C, more preferably in the range of Tm, BLG - 10 degrees C to Tm, BLG + 20 degrees C, and most preferably in the range of Tm, BLG - 5 degrees C to Tm, BLG + 10 degrees C.

[0291] In some preferred embodiments of the invention the incubating protein solution of step d) has a pH in the range of 2.0-4.5 and a temperature of at least 20 degrees C, more preferably at least 40 degrees C, even more preferably at least 60 degrees C and most preferably at least 70 degrees C.

[0292] In further preferred embodiments of the invention the incubating protein solution of step d) has a pH in the range of 2.1-4.3 and is allowed to incubate at a temperature of at least 20 degrees C, more preferably at least 30 degrees C, even more preferably at least 50 degrees C and most preferably at least 60 degrees C.

[0293] In particularly preferred embodiments of the invention the incubating protein solution of step d) has a pH in the range of 2.3-4.1 and is allowed to incubate at a temperature of at least 20 degrees C, more preferably at least 30 degrees C, even more preferably at least 50 degrees C and most preferably at least 60 degrees C.

[0294] In even more preferred embodiments of the invention the incubating protein solution of step d) has a pH in the range of 2.5-3.9 and is allowed to incubate at a temperature of at least 35 degrees C, more preferably at least 40 degrees C, even more preferably at least 45 degrees C and most preferably at least 50 degrees C.

[0295] Preferably, the incubating protein solution of step d) has a pH in the range of 2.0-4.5 and is allowed to incubate at a temperature of 30-95 degrees C, more preferably has a pH in the range of 2.1-4.3 and is allowed to incubate at a temperature of 45-95 degrees C, even more preferably has a pH in the range of 2.3-4.1 and is allowed to incubate at a temperature of 50-90 degrees C, and most preferably has a pH in the range of 2.5-3.9 and is allowed to incubate at a temperature of 55-90 degrees C.

[0296] Being "allowed to incubate at a temperature" within a certain range means that the temperature of the incubating protein solution will be adjusted to a temperature in this range during step d). If the range contains a upper limit the incubating protein solution will not have temperature above this upper limit during step d).

[0297] In other preferred embodiments of the present invention the incubating protein solution of step d) has a pH in the range of 2.0-4.5 and a temperature of 3-95 degrees C, more preferably a pH in the range of 2.1-4.3 and a temperature of 40-95 degrees C, even more preferably a pH in the range of 2.3-4.1 and a temperature of 45-95 degrees C, and most preferably a pH in the range of 2.5-3.9 and a temperature of 70-95 degrees C.

[0298] In further preferred embodiments of the present invention the incubating protein solution of step d) has a pH in the range of 2.0-4.5 and a temperature of 5-95 degrees C, more preferably a pH in the range of 2.1-4.3 and a temperature of 8-90 degrees C, even more preferably a pH in the range of 2.3-4.1 and a temperature of 10-85 degrees C, and most preferably a pH in the range of 2.5-3.9 and a temperature of 15-80 degrees C.

[0299] In some preferred embodiments of the present invention the duration of the incubation is at most 12 hours, more preferably at most 6 hours, even more preferably at most 3 hours, and most preferably at most 1 hour.

[0300] An advantage of the present method is that it can be operated with a relatively short incubation time. Thus, in further preferred embodiments of the present invention the duration of the incubation of step d) is at most 30 minutes, more preferably at most 10 minutes, even more preferably at most 5 minutes, and most preferably at most 2 minutes.

[0301] The inventors have seen indications that even shorter incubations are technically feasible. Thus, in even further preferred embodiments of the present invention the duration of the incubation of step d) is at most 100 seconds, more preferably at most 60 seconds, even more preferably at most 30 seconds, and most preferably at most 20 seconds.

[0302] In some preferred embodiments of the present invention the oxidation of step d) is involves contacting PCA with a chemical oxidizing agent in the protein solution under conditions that convert at least some of the PCA to quinones. Additional a chemical oxidizing agent(s) may be added during the incubation of step d).

[0303] One or more of the chemical oxidizing agent(s) used in step b) and / or step d) should be capable of PCA-type oxidation during step d) and / or step b).

[0304] In further preferred embodiments of the present invention the chemical oxidizing agent comprises, or even consists of, a peroxide, ozone, dioxygen, or a combination thereof.

[0305] In particular preferred embodiments of the present invention the chemical oxidizing agent comprises, or even consists of, hydrogen peroxide, benzoyl peroxide, or a combination thereof.

[0306] In some preferred embodiments of the present invention the oxidation of step d) involves electrochemical oxidation of PCA of the protein solution under conditions that convert at least some of the PCA to quinones.

[0307] The potential difference should be selected sufficiently high to enable oxidization of PCA to quinones and can be determined as described above in relation to step b). Preferably, the electrochemical oxidation of step d) is performed using a potential difference of -0.1-1.5 V, and most preferably -0.05-0.7 V.

[0308] In some preferred embodiments of the invention the protein solution has a pH in the range of 2.1-4.3 and the electrochemical oxidation is performed using a potential difference of -0.1 to 0.9 V, and more preferably -0.05 to 0.8 V, and most preferably 0 to 0.7 V.

[0309] In other preferred embodiments of the invention the protein solution has a pH in the range of 2.3-4.1 and the electrochemical oxidation is performed using a potential difference of -0.1 to 0.8V, and most preferably 0 to 0.7V.

[0310] In further preferred embodiments of the invention which were found useful in Examples 1-3 the protein solution has a pH in the range of 2.5-3.9 and the electrochemical oxidation is performed using a potential difference of 0.2 to 0.8 V, and most preferably 0.4 to 0.7V.

[0311] Features and embodiments relating to the implementation of electrochemical oxidation described in the context of step b) also applies to the electrochemical oxidation of step d).

[0312] In some preferred embodiments of the present invention the temperature of a first stage of the incubation is in the range of 0-60 degrees C, more preferably 10-50 degrees, and the incubating protein solution subsequently is heated to a temperature in the range of 70-160 degrees C, more preferably 100-150 degrees C in a second stage of the incubation.

[0313] The use of high temperatures during step d) is particularly useful if the incubating protein solution is to be used as a packaged, ready-to-drink beverage, and which has to be filled into suitable containers immediately after step d).

[0314] In some preferred embodiments of the present invention, step d) furthermore comprises addition of further PCA to the incubating protein solution.

[0315] In some preferred embodiments the invention the mole ratio between:

[0316] - the total amount of PCA provided to protein solution in steps c) and d), and - the content of BLG provided to the protein solution in steps c) and d)

[0317] is in the range of 0.1:1 - 1000:1, more preferably 0.2:1 - 500:1, even more preferably 5:1 -200:1, and most preferably 10:1 - 100:1.

[0318] In further preferred embodiments the mole ratio between: - the total amount of PCA provided to protein solution in steps c) and d), and - the content of BLG provided to the protein solution in steps c) and d)

[0319] is in the range of 0.1:1 - 100:1, more preferably 0.1:1- 50:1, even more preferably 0.1:1 -20:1, and most preferably 0.1:1 - 10:1.

[0320] In even further preferred embodiments the mole ratio between:

[0321] - the total amount of PCA provided to protein solution in steps c) and d), and - the content of BLG provided to the protein solution in steps c) and d)

[0322] is in the range of 0.1:1 - 6:1, more preferably 0.2:1 - 5:1, even more preferably 0.3:1 - 4:1, and most preferably 0.4:1 - 3:1.

[0323] In some preferred embodiments the mole ratio between:

[0324] - the total amount of PCA provided to protein solution in steps c) and d), and - the content of BLG provided to the protein solution in steps c) and d)

[0325] is in the range of 1:1 - 1000:1, more preferably 2:1 - 500:1, even more preferably 5:1 -200:1, and most preferably 10:1- 100:1.

[0326] In further preferred embodiments the mole ratio between:

[0327] - the total amount of PCA provided to protein solution in steps c) and d), and - the content of BLG provided to the protein solution in steps c) and d)

[0328] is in the range of 1:1 - 100:1, more preferably 1:1- 50:1, even more preferably 1:1 - 20:1, and most preferably 1:1 - 10:1.

[0329] In even further preferred embodiments of the invention the mole ratio between:

[0330] - the total amount of PCA provided to protein solution in steps c) and d), and - the content of BLG provided to the protein solution in steps c) and d)

[0331] is in the range of 1:1 - 6:1, more preferably 1.1:1 - 5:1, even more preferably 1.3:1 - 4:1, and most preferably 1.5:1 - 3:1.

[0332] In some preferred embodiments of the present invention the PCA-type oxidation applied during step b) and / or step d) is sufficient to create a mole ratio between: - the theoretical amount of quinone provided to the protein solution during the method determined according to Analysis B, and

[0333] - the content of BLG of the protein solution

[0334] of at least 0.1:1, more preferably at least 0.2:1.

[0335] In further preferred embodiments of the present invention the oxidation applied during step b) and / or step d) is sufficient to create a mole ratio between:

[0336] - the theoretical amount of quinone provided to the protein solution during the method determined according to Analysis B, and

[0337] - the content of BLG of the protein solution

[0338] of 0.1:1 - 100:1, more preferably 0.1:1 - 50:1, even more preferably 0.2:1 - 15:1, and most preferably 0.2:1 - 5:1.

[0339] In even further preferred embodiments of the present invention the oxidation applied during step b) and / or step d) is sufficient to create a mole ratio between:

[0340] - the theoretical amount of quinone provided to the protein solution during the method determined according to Analysis B, and

[0341] - the content of BLG of the protein solution

[0342] of 0.1:1 -5:1, more preferably 0.1:1- 4:1, even more preferably 0.2: 1-3:1, and most preferably 0.2:1- 2:1.

[0343] In other preferred embodiments of the present invention the PCA-type oxidation applied during step b) and / or step d) is sufficient to create a mole ratio between:

[0344] - the theoretical amount of quinone provided to the protein solution during the method determined according to Analysis B, and

[0345] - the content of BLG of the protein solution

[0346] of at least 1.1:1, more preferably at least 1.3:1, even more preferably at least 1.5:1, and most preferably at least 2:1.

[0347] In further preferred embodiments of the present invention the oxidation applied during step b) and / or step d) is sufficient to create a mole ratio between:

[0348] - the theoretical amount of quinone provided to the protein solution during the method determined according to Analysis B, and

[0349] - the content of BLG of the protein solution

[0350] of 1:1 - 100:1, more preferably 1:1- 50:1, even more preferably 1:1-15:1, and most preferably 1.5:1- 5:1. If the PCA oxidation mainly or exclusively takes place in step b) it is often preferred that the PCA-type oxidation applied during step b) is sufficient to create a mole ratio between:

[0351] - the quinone content of the portion of the source comprising one or more oxidized PCA that is used in the protein solution, and

[0352] - the content of BLG of the protein solution

[0353] of at least 1.1:1, more preferably at least 1.3:1, even more preferably at least 1.5:1, and most preferably at least 2:1.

[0354] This is e.g. the case when PCA-type oxidation performed during of step d) accounts for less than 20% of the PCA-type oxidation.

[0355] In the above-mentioned preferred embodiments of the present invention it is even more preferred that the oxidation applied during step b) is sufficient to create a mole ratio between:

[0356] - the quinone content of the portion of the source comprising one or more oxidized PCA that is used in the protein solution, and

[0357] - the content of BLG of the protein solution

[0358] of 1:1 - 100:1, more preferably 1:1- 50:1, even more preferably 1:1-15:1, and most preferably 1.5:1- 5:1.

[0359] In some preferred embodiments of the present invention the method does not involve oxidation of a portion of the source comprising one or more PCA prior to step c), i.e. no step b), and the PCA-type oxidation is performed by a chemical oxidizing agent.

[0360] Alternatively, but also preferred, the method does not involve oxidation of a portion of the source comprising one or more PCA prior to step c), i.e. no step b), and the oxidation of PCA is performed by electrochemical oxidization.

[0361] In some preferred embodiments of the present invention the method comprises step b) and the portion of the source comprising one or more oxidized PCA obtained from step b) is used for the preparation of the protein solution of step c), and the oxidation of PCA is performed by a chemical oxidizing agent.

[0362] In further preferred embodiments of the present invention the method comprises step b) and the portion of the source comprising one or more oxidized PCA obtained from step b) is used for the preparation of the protein solution of step c), and the oxidation of PCA is performed by electrochemical oxidization. In other preferred embodiments of the present invention the method comprises oxidation of the PCA during step d), and preferably involves oxidation of PCA during step d) by electrochemical oxidization.

[0363] If the PCA oxidation mainly or exclusively takes place in step d) it is often preferred that the PCA-type oxidation applied during step d) is sufficient to create a mole ratio between:

[0364] - the theoretical amount of quinone provided to the protein solution during the method determined according to Analysis B, and

[0365] - the content of BLG of the protein solution

[0366] of at least 0.1:1, more preferably at least 0.2:1, even more preferably at least 0.2:1, and most preferably at least 0.2:1.

[0367] This is e.g. the case when PCA-type oxidation performed during of step d) accounts for at least 50% of the PCA-type oxidation during the method.

[0368] In other preferred embodiments of the invention the PCA-type oxidation applied during step d) is sufficient to create a mole ratio between:

[0369] - the theoretical amount of quinone provided to the protein solution during the method determined according to Analysis B, and

[0370] - the content of BLG of the protein solution

[0371] of 0.1:1 - 100:1, more preferably 0.1:1- 50:1, even more preferably 0.2:1-15:1, and most preferably 0.2:1- 5:1.

[0372] In even further preferred embodiments of the invention the PCA-type oxidation applied during step d) is sufficient to create a mole ratio between:

[0373] - the theoretical amount of quinone provided to the protein solution during the method determined according to Analysis B, and

[0374] - the content of BLG of the protein solution

[0375] of 0.1:1 -5:1, more preferably 0.1:1- 4:1, even more preferably 0.2: 1-3:1, and most preferably 0.2:1- 2:1.

[0376] In some preferred embodiments of the invention the PCA-type oxidation applied during step d) is sufficient to create a mole ratio between:

[0377] - the theoretical amount of quinone provided to the protein solution during the method determined according to Analysis B, and

[0378] - the content of BLG of the protein solution

[0379] of at least 1.1:1, more preferably at least 1.3:1, even more preferably at least 1.5:1, and most preferably at least 2:1. In the above-mentioned preferred embodiments of the present invention it is even more preferred that the PCA-type oxidation applied during step d) is sufficient to create a mole ratio between:

[0380] - the theoretical amount of quinone provided to the protein solution during the method determined according to Analysis B, and

[0381] - the content of BLG of the protein solution

[0382] of 1:1 - 100:1, more preferably 1:1- 50:1, even more preferably 1:1-15:1, and most preferably 1.5:1- 5:1.

[0383] In some preferred embodiments of the invention the method comprises a step e) which removes at least some of the free oxidized PCA from the incubated protein solution obtained from step d). If free non-oxidized PCA is still present this is removed as well.

[0384] Step e) preferably involves subjecting the incubated protein solution of step d) to membrane based concentration, to diafiltration and / or to chromatography.

[0385] Useful examples of chromatography include size exclusion chromatography, gel filtration chromatography, and / or affinity chromatography.

[0386] In some preferred embodiments of the invention step e) involves subjecting the incubated protein solution of step d) to membrane-based concentration and / or to diafiltration. In order to remove free, oxidized PCA a membrane-based concentration and / or a diafiltration step is / are preferably implemented with a filtration membrane that retains the protein of the incubated protein solution and allows for the permeation of free, oxidized PCA. Ultrafiltration membrane having a molecular weight cut-off of at most 10 kDa are useful for both the concentration and for the diafiltration. If step d) has converted most of protein of the protein solution to protein aggregates even larger molecular weight cut-offs or pore sizes may be used during the mem-brane-based filtration and / or diafiltration.

[0387] In relation to diafiltration it is preferred to use low mineral water source such as demineralised water, reverse osmosis permeate of milk of whey, nanofiltration permeate of milk or whey, or a combination thereof.

[0388] The membrane-based concentration and / or diafiltration of step e) is preferably performed to an extent sufficient to reduce the weight percentage of free PCA and oxidized PCA relative to total solids by at least 30% relative to the weight percentage prior to the membrane-based concentration and / or diafiltration, more preferably by at least 50%, even more preferably by at least 80%, and most preferably by at least 90% relative to the weight percentage prior to the mem-brane-based concentration and / or diafiltration.

[0389] In some preferred embodiments of the invention step e) furthermore involves a pH adjustment which increases the pH to a pH in the range of 6.0-8.5, and most preferably 6.5-7.5. The pH-adjustment is preferably performed after the reduction of free PCA and oxidized PCA.

[0390] The pH adjustment performed by addition of one or more food grade alkalizing agents, preferably selected from the group consisting of sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, a di-basic phosphate salt, a tri-basic phosphate salt, a carbonate salt, a bicarbonate salt, or a combination thereof.

[0391] In some preferred embodiments of the present invention, the method comprises step f) of drying a liquid feed comprising at least the protein derived from the incubated protein solution of step d) or the protein solution resulting from step e).

[0392] In other preferred embodiments of the present invention, the method comprises step f) of drying a liquid feed comprising at least the solids derived from the incubated protein solution of step d) or the protein solution resulting from step e).

[0393] In some preferred embodiments of the present invention, the liquid feed for drying comprises or even consists of the protein solution obtained from step d) or a protein concentrate thereof.

[0394] It is particularly preferred that the liquid feed for drying is a protein concentrate of the protein solution obtained from step d).

[0395] In other preferred embodiments of the present invention, the liquid feed for drying comprises or even consists of the protein solution obtained from step e) or a protein concentrate thereof.

[0396] It is particularly preferred that the liquid feed for drying is the protein concentrate of the protein solution obtained from step e).

[0397] In the context of the present invention, a "protein concentrate" of a first liquid is a second liquid in which at least the protein originates from the first liquid but which has a higher protein content relative to total solids than the first liquid. Preferably, substantially all solids of the protein concentrate originate from the first liquid. A "protein concentrate" of a first liquid is preferably prepared by ultrafiltration, nanofiltration, reverse osmosis, and / or evaporation. Protein concentration ultrafiltration and / or nanofiltration may e.g. be implemented with diafiltration to wash out some of the small non-protein solids. A "protein concentrate" contains the same protein species and preferably has the same weight percentage of the whey protein species relative to total protein as the first liquid. The provision of a protein concentrate may also involve one or more pH adjustments.

[0398] The liquid feed for drying preferably has a high solids content and therefore less water has to be removed and less energy is consumed in the drying operation.

[0399] In some preferred embodiments of the invention the liquid feed for drying has a solids content of at least 15% w / w, more preferably at least 20% w / w, even more preferably at least 25% w / w, and most preferably at least 30% w / w.

[0400] Preferred the liquid feed for drying has a solids content in the range of 15-50% w / w, more preferably 20-45% w / w, even more preferably 25-40% w / w, and most preferably 30-45% w / w.

[0401] Preferably, at least 90% w / w of the solids of the liquid feed for drying originate from the incubated protein solution obtained from step d) or the protein solution obtained from step e), more preferably at least 95% w / w, and most preferably at least 99% w / w. Preferably all the solids of the liquid feed for drying originate from the incubated protein solution obtained from step d) or the protein solution obtained from step e).

[0402] Preferably, at least 90% w / w of the protein of liquid feed for drying originate from the incubated protein solution obtained from step d) or the protein solution obtained from step e), more preferably at least 95% w / w, and most preferably at least 99% w / w. Preferably all the protein of the liquid feed for drying originate from the incubated protein solution obtained from step d) or the protein solution obtained from step e).

[0403] In some preferred embodiments of the invention the liquid feed for drying has a protein content of at least 30% w / w relative to total solids, more preferably at least 40% w / w relative to total solids, even more preferably at least 45% w / w relative to total solids, and most preferably at least 50% w / w relative to total solids.

[0404] In further preferred embodiments of the invention the liquid feed for drying has a protein content of 30-99% w / w relative to total solids, more preferably 40-95% w / w relative to total solids, even more preferably 45-90% w / w relative to total solids, and most preferably 50-80% w / w relative to total solids.

[0405] Particularly preferred embodiments of the invention the liquid feed for drying has a protein content of at least 70% w / w relative to total solids, more preferably at least 80% w / w relative to total solids, even more preferably at least 90% w / w relative to total solids, and most preferably at least 95% w / w relative to total solids. In other preferred embodiments of the present invention the liquid feed is prepared by subjecting the incubated protein solution of step d) or the protein solution obtained from step e) to one or more of:

[0406] - a pH adjustment,

[0407] - a concentration step,

[0408] - a diafiltration, and

[0409] - a heat-treatment.

[0410] In some preferred embodiments of the invention step f) furthermore involves a pH adjustment which provides the liquid feed with a pH in the range of 6.0-8.5, and most preferably 6.5-7.5. Preferably, step f) involves spray-drying the liquid feed.

[0411] Useful examples of spray-drying are e.g. described in WO 2018 / 115520 Al.

[0412] The method of the invention can be implemented as a batch process, a semi-batch process, or a continuous process. Continuous processes are particularly preferred.

[0413] In some preferred embodiments of the invention at least steps b), c), d), and preferably also e) are implemented as a continuous process.

[0414] An aspect of the invention pertains to a protein composition comprising modified BLG and having at most 15 micromol free thiol groups per g protein, more preferably at most 10 micromol / g protein, said protein composition is obtainable by the method of the invention and preferably has one or more of:

[0415] - a protein content of at least 30% w / w relative to total solids,

[0416] - a tryptophan content of at least 0.7% w / w relative to total protein, - a methionine content of at least 0.3% w / w relative to total protein, - a kynurenine content of at most 0.2 micrograms / mg protein,

[0417] - preferably, a fat content of at most 3% w / w relative to total solids, - preferably, a content of protein-bound sulfur in the range of 100-600 micromol / g protein,

[0418] - preferably, a content of protein-bound cysteine residues that form disulfide bonds in the range of 150-400 micromol / g protein.

[0419] In some preferred embodiments of the invention the protein composition comprising modified BLG is protein solution obtained from step d), i.e. the incubated protein solution. In some preferred embodiments of the invention the protein composition comprising modified BLG is protein solution obtained from step e), i.e. the incubated protein solution reduced with respect to PCA and / or oxidized PCA.

[0420] In further preferred embodiments of the invention the protein composition comprising modified BLG is the liquid feed prepared during step f).

[0421] In even further preferred embodiments of the invention the protein composition comprising modified BLG is the powder by step f). It is therefore often preferred that the protein composition comprising modified BLG is obtainable steps a), c), d), e) and f) of the present method.

[0422] In other preferred embodiments of the invention, step e) may be omitted, and in such embodiments the protein composition comprising modified BLG may be obtainable by steps a), c), d) and f) of the present method.

[0423] The protein composition typically has a pH in the range of 2-9.5.

[0424] In some preferred embodiments of the invention, the protein composition has a pH in the range of 5.5-9.5, more preferably 6.0-8.5, even more preferably 6.2-8.0, and most preferably 6.5-7.5. These pH-ranges are particularly preferred when the protein composition should be used for the preparation of heat-treated, pH-neutral beverages.

[0425] In other preferred embodiments of the invention, the protein composition has a pH in the range of 2.0-5.4, more preferably 2.5-5.0, even more preferably 2.8-4.5, and most preferably 3.0-4.0.

[0426] In some preferred embodiments of the invention, the protein composition has a total protein content of at least 30% w / w relative to the total solids of the protein composition, more preferably at least 50% w / w, even more preferably at least 75% w / w and most preferably at least 85% w / w relative to the total solids of the protein composition.

[0427] Preferably, the protein composition has a total protein content in the range of 30-99% w / w relative to the total solids of the protein composition, more preferably 50-97% w / w, even more preferably 75-96% w / w, and most preferably at least 85-95% w / w relative to the total solids of the protein composition.

[0428] In some preferred embodiments of the invention the protein composition has a total fat content of at most 10% w / w relative to total solids, more preferably at most 8% w / w, and most preferably at most 6% w / w relative to total solids. In other preferred embodiments of the invention the protein composition has a total fat content of at most 5% w / w relative to total solids, more preferably at most 2% w / w, even more preferably at most 0.5% w / w, and most preferably at most 0.1% w / w relative to total solids.

[0429] In further preferred embodiments of the invention the protein composition has a total fat content in the range of 1-20% w / w relative to total solids, more preferably in the range of 2-16% w / w, even more preferably in the range of 3-12% w / w, and most preferably at most 4-10% w / w relative to total solids.

[0430] Even lower levels of fat are typically preferred, and it is often preferred that the protein composition has a total fat content of at most 1% w / w relative to total solids, more preferably at most 0.5% w / w, even more preferably at most 0.2% w / w, and most preferably at most 0.1% w / w relative to total solids.

[0431] The protein composition may contain carbohydrate in various amounts.

[0432] However, it is often preferred that the protein composition has a carbohydrate content of at most 65% w / w relative to total solids.

[0433] Even lower levels of carbohydrate are typically preferred, and it is often preferred that the protein composition has a carbohydrate content of at most 20% w / w relative to total solids, more preferably at most 8% w / w, even more preferably at most 2% w / w, and most preferably at most 0.2% w / w relative to total solids.

[0434] The protein composition preferably has an ash content of at most 8% w / w relative to total solids, more preferably at most 6% w / w, even more preferably at most 5% and most preferably at most 4.0%.

[0435] In some preferred embodiments of the present invention, the protein composition has an ash content of 0.4 -8% w / w relative to total solids, more preferably 0.5-6% w / w, even more preferably 0.5-5% w / w and most preferably 0.6-4.0% w / w relative to total solids.

[0436] The protein composition preferably has a combined content of magnesium and calcium at most 1% w / w relative to total solids, more preferably at most 0.7% w / w, even more preferably at most 0.5% and most preferably at most 0.2%.

[0437] In some preferred embodiments of the present invention, the protein composition has combined content of magnesium and calcium of 0.01-1% w / w relative to total solids, more preferably 0.001-0.7% w / w, even more preferably 0.01-0.5% w / w and most preferably 0.01-0.2% w / w relative to total solids.

[0438] The inventors have seen indications that protein composition that contain even up to 15 micromol free thiol groups / g protein can provide a reduced level of unpleasant odours, relative to non-modified whey protein, in heat-treated whey protein beverages that contain 3% whey protein.

[0439] In some preferred embodiments of the invention, the protein composition comprises free thiol groups in an amount of at most 15 micromol / g protein, more preferably at most 14 micromol / g protein, even more preferably at most 13 micromol / g protein, and most preferably at most 12 micromol / g protein.

[0440] In some preferred embodiments of the invention, the protein composition comprises free thiol groups in an amount of 0.001-15 micromol / g protein, more preferably 0.01-14 micromol / g protein, even more preferably 0.01-13 micromol / g protein, and most preferably 0.01-12 micromol / g protein.

[0441] However, it is often preferred that the protein composition contains lower levels of free thiol groups, particularly when the protein composition is to be used for heat-treated, high protein beverages, e.g. containing 6% whey protein or higher. Thus, in some preferred embodiments of the invention the protein composition comprises free thiol groups in an amount of at most 10 micromol / g protein, more preferably at most 8 micromol / g protein, more preferably at most 5 micromol / g protein, even more preferably at most 3 micromol / g protein, and most preferably at most 2 micromol / g protein.

[0442] Preferably, the protein composition comprises free thiol groups in an amount of 0.01-10 micromol / g protein, more preferably 0.01-8 micromol / g protein, more preferably 0.01-5 micromol / g protein, even more preferably 0.01-3 micromol / g protein, and most preferably 0.01-2 micromol / g protein.

[0443] Even lower levels of free thiol groups may be desired, and in some preferred embodiments of the invention, the protein composition comprises free thiol groups in an amount of at most 1 micromol / g protein, more preferably at most 0.7 micromol / g protein, even more preferably at most 0.5 micromol / g protein, and most preferably at most 0.2 micromol / g protein.

[0444] In some preferred embodiments of the invention, the protein composition has a tryptophan content of at least 0.7% w / w relative to total protein, more preferably at least 0.8% w / w, even more preferably at least 0.9% w / w, and most preferably at least 1.0% w / w relative to total protein.

[0445] Preferably, the protein composition has a tryptophan content of 0.7-3% w / w relative to total protein, more preferably 0.8-2.6% w / w, even more preferably 0.9-2.4% w / w, and most preferably 1.0-2.2% w / w relative to total protein.

[0446] Alternatively, but also preferred, the protein composition often has a tryptophan content of 0.7-3% w / w relative to total protein, more preferably 0.8-3% w / w, even more preferably 0.9-3% w / w, and most preferably 1.0-3% w / w relative to total protein.

[0447] In some preferred embodiments of the invention, the protein composition has a methionine content of at least 0.3% w / w relative to total protein, more preferably at least 0.4% w / w, even more preferably at least 0.5% w / w, and most preferably at least 0.6% w / w relative to total protein.

[0448] Preferably, the protein composition has a methionine content of 0.3-3.3% w / w relative to total protein, more preferably 0.4-3.2% w / w, even more preferably 0.5-3.2% w / w, and most preferably 0.6-3.2% w / w relative to total protein.

[0449] Increased lower limits of methionine are often preferred, and in some preferred embodiments of the present invention the protein composition has a methionine content of 1.0-3.3% w / w relative to total protein, more preferably 1.3-3.2% w / w, even more preferably 1.6-3.2% w / w, and most preferably 1.8-3.2% w / w relative to total protein.

[0450] Preferably, the protein composition has a kynurenine content of at most 0.2 micrograms / mg protein, more preferably at most 0.05 micrograms / mg protein, even more preferably at most 0.01 micrograms / mg protein, and most preferably at most 0.001 micrograms / mg protein. It is particularly preferred that the protein composition does not contain detectable kynurenine.

[0451] The content of kynurenine is quantified according to Poojary et al.; "Selective and sensitive UHPLC-ESI-Orbitrap MS method to quantify protein oxidation markers"; Taianta, Volume 234, 1 November 2021 (available online July 2021).

[0452] Kynurenine is a useful marker of tryptophan oxidation, it is believed by the inventors to be partially responsible for the development of yellow colour in heat-sterilized protein beverages based on protein that has been subjected to excessive oxidation, and it is furthermore not desired from a health perspective. Preferably, the protein composition has a content of protein-bound sulfur in the range of 100-600 micromol / g protein, more preferably in the range of 200-500 micromol / g protein, and most preferably in the range of 250-500 micromol / g protein.

[0453] Preferably, the protein composition has a content of protein-bound cysteine residues that form disulfide bonds in the range of 150-400 micromol / g protein, more preferably 160-350, and most preferably 170-300 micromol / g protein.

[0454] The inventors have found that it is advantageous that the particle size of the protein of protein composition is no larger than 10000 kDa and preferably smaller to avoid the development of opaqueness in transparent beverage applications and furthermore to avoid increased viscosity during concentration and drying of the protein.

[0455] In some preferred embodiments of the invention, the protein composition has a weight average molecular weight of the protein in the range of 18 kDa and 10000 kDa, more preferably 30-9000 kDa, even more preferably 50-8000 kDa, and most preferably 80-5000 kDa.

[0456] Preferably, at least 60% w / w of the protein of the protein composition has a molecular weight between 18 kDa and 10000 kDa, more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 99% w / w.

[0457] More preferably, at least 60% w / w of the protein of the protein composition has a molecular weight between 50 kDa and 8000 kDa, more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 99% w / w.

[0458] Even more preferably, at least 60% w / w of the protein of the protein composition has a molecular weight between 80 kDa and 5000 kDa, more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 99% w / w.

[0459] In other preferred embodiments of the invention, the protein composition has a weight average molecular weight of the protein in the range of 18 kDa and 200 kDa, more preferably between 30-150 kDa, and most preferably between 30-100 kDa.

[0460] The inventors have found that the smaller the weight average molecular weight of the protein the higher total protein concentration is feasible during concentration, e.g. by ultrafiltration or nanofiltration, prior to spray-drying. Preferably, at least 60% w / w of the protein of the protein composition has a molecular weight between 18 kDa and 200 kDa, more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 99% w / w.

[0461] More preferably, at least 60% w / w of the protein of the protein composition has a molecular weight between 18 kDa and 150 kDa, more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 99% w / w.

[0462] Even more preferably, at least 60% w / w of the protein of the protein composition has a molecular weight between 18 kDa and 100 kDa, more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 99% w / w.

[0463] The inventors have seen indications that it may be beneficial that a significant protein of the protein composition has a molecular weight of at least 30 kDa, which may be due to dimerisa-tion of modified BLG.

[0464] Thus, preferably, at least 60% w / w of the protein of the protein composition has a molecular weight between 30 kDa and 200 kDa, more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 99% w / w.

[0465] More preferably, at least 60% w / w of the protein of the protein composition has a molecular weight between 30 kDa and 150 kDa, more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 99% w / w.

[0466] Even more preferably, at least 60% w / w of the protein of the protein composition has a molecular weight between 30 kDa and 100 kDa, more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 99% w / w.

[0467] In some preferred embodiments of the invention, the protein composition of the invention is obtainable by the method described herein.

[0468] In some preferred embodiments of the invention, the protein composition is in the form of a liquid, and preferably an aqueous liquid. The protein composition in the form of a liquid preferably has a solids content of 0.1-50% w / w, more preferably 1-35% w / w, even more preferably 5-30% w / w, and most preferably 10-30% w / w.

[0469] In some preferred embodiments of the invention, the protein composition is in the form of a solid, and preferably a powder which preferably has been prepared by spray-drying. The protein composition in the form of a powder preferably has a solids content of at least 90%w / w, more preferably at least 93% w / w, even more preferably at least 94% w / w, and most preferably at least 95%w / w.

[0470] The part of the protein composition and the oxidizing whey protein solution that does not contribute to the solids content is preferably water.

[0471] The part of the protein composition that does not contribute to the solids content preferably comprises water in an amount of at least 80% w / w, more preferably at least 90% w / w, even more preferably at least 95% w / w, and more preferably at least 99% w / w.

[0472] In a particularly preferred embodiment of the invention the protein composition has:

[0473] - a protein content of at least 86% w / w relative to total solids, and most preferably at least 90% relative to total solids,

[0474] - a fat content of at most 1% w / w relative to total solids, and most preferably at most 0.2%, - at most 10 micromol free thiol groups / g protein, and most preferably at most 5 micromol free thiol groups / g protein,

[0475] - a tryptophan content of 0.7-3% w / w relative to total protein, and most preferably 1.0-3% w / w relative to total protein,

[0476] - a methionine content of 0.3-3.3% w / w relative to total protein, and most preferably 1.3-3.2% w / w relative to total protein,

[0477] - a kynurenine content of at most 0.2 micrograms / mg protein, and most preferably at most 0.01 micrograms / mg protein.

[0478] In the above-mentioned particularly preferred embodiment of the invention, the oxidized whey protein composition preferably has:

[0479] - a content of protein-bound sulfur in the range of 100-600 micromol / g protein, and

[0480] - a content of protein-bound cysteine residues that form disulfide bonds in the range of 150-400 micromol / g protein.

[0481] Additionally, in the above-mentioned particularly preferred embodiment of the invention, the protein composition preferably has:

[0482] - a content of protein-bound sulfur in the range of 100-600 micromol / g protein, and

[0483] - a content of protein-bound cysteine residues that form disulfide bonds in the range of 150-400 micromol / g protein.

[0484] It is furthermore often preferred that at least 60% w / w of the protein of the protein composition of the above-mentioned particularly preferred embodiment has a molecular weight between 30 kDa and 9000 kDa, more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 99% w / w. The pH of the above-mentioned particularly preferred embodiment of the protein composition is preferably in the range of 6.2-8.0, and most preferably 6.5-7.5.

[0485] In some preferred embodiments of the present invention the protein composition is a sterile protein composition, and preferably a packaged, sterile protein composition. Preferably in the form of a sterile, liquid protein composition or a sterile, powdered, protein composition.

[0486] Another aspect of the invention pertains to a process for producing a heat-treated, preferably heat-sterilized, beverage, the process comprising the following steps:

[0487] 1) mixing the protein composition of the invention with one or more further beverage ingredients to obtain a liquid mixture having a pH of 5.5-8.5, and

[0488] 2) filling the liquid mixture into suitable containers,

[0489] the process furthermore comprising at least one heat-treatment step wherein the liquid mixture is heat-treated, and preferably heat-sterilized, prior to filling and / or after filling.

[0490] In some preferred embodiments of the present invention the beverage mixture comprises the protein composition in an amount sufficient to contribute with at least 0.5% w / w protein.

[0491] Thus, a more specific aspect of the invention pertains to a process of producing a heat-treated, and preferably heat-sterilized, beverage having a pH of 5.5-8.5, more preferably 6.5-7.5, the process comprises:

[0492] 1) combining the protein composition as described herein with one or more further ingredients to obtain a liquid mixture having a pH of 5.5-8.5, more preferably 6.5-7.5, and comprising: - the protein composition in an amount sufficient to contribute with at least 0.5% w / w protein, and

[0493] - water,

[0494] 2) packaging the liquid mixture in a container, preferably a sterile container, and

[0495] wherein the liquid mixture is heat-treated, and preferably heat-sterilised, prior to and / or after packaging.

[0496] The protein composition as described herein is preferably the only protein source of the food product or of the heat-sterilized beverage, and therefore also of the liquid mixture.

[0497] The inventors have found that it is advantageous that the content of free thiol groups of the liquid mixture is kept low prior to the heat treatment to prevent the formation of unpleasant odours similar to the odour of rotten eggs. Thus, in some preferred embodiments of the invention, the liquid mixture contains, prior to the heat-sterilisation, at most 60 micromol free thiol groups / 100 g liquid mixture, more preferably at most 40 micromol free thiol groups / 100 g liquid mixture, even more preferably at most 30 micromol free thiol groups / 100 g liquid mixture, and most preferably at most 30 micromol free thiol groups / 100 g liquid mixture.

[0498] Even lower contents of free thiol groups are often required, and in some preferred embodiments of the invention the liquid mixture contains, prior to the heat-sterilisation, at most 20 micromol free thiol groups / 100 g liquid mixture, more preferably at most 15 micromol free thiol groups / 100 g liquid mixture, even more preferably at most 10 micromol free thiol groups / 100 g liquid mixture, and most preferably at most 5 micromol free thiol groups / 100 g liquid mixture.

[0499] The liquid mixture preferably comprising a total amount of protein in the range of 0.5-15% w / w relative to the weight of the liquid mixture, more preferably 1-10% w / w relative to the weight of the liquid mixture, even more preferably 2-9% w / w relative to the weight of the liquid mixture, and most preferably 3-8% w / w relative to the weight of the liquid mixture.

[0500] Alternatively, but also preferred, the liquid mixture may comprise a total amount of protein in the range of 4-15% w / w relative to the weight of the liquid mixture, more preferably 5-14% w / w relative to the weight of the liquid mixture, even more preferably 6-13% w / w relative to the weight of the liquid mixture, and most preferably 8-12% w / w relative to the weight of the liquid mixture.

[0501] The protein composition of the invention preferably contributes with at least 30% w / w of the total protein of the liquid mixture, more preferably at least 50% w / w of the total protein, even more preferably at least 70% w / w of the total protein, and most preferably at least 80% w / w of the total protein.

[0502] Even higher contributions are often preferred, and in some preferred embodiments of the present invention, the protein composition of the invention contributes with at least 90% w / w of the total protein of the liquid mixture, more preferably at least 95% w / w of the total protein, even more preferably at least 99% w / w of the total protein, and most preferably 100% w / w of the total protein.

[0503] If the protein composition is used in combination with other protein sources. It is preferred to use sources that have a relatively low content of free thiol groups.

[0504] In some preferred embodiments of the present invention, the liquid mixture comprises total protein in an amount of at least 15% w / w relative to total solids, more preferably at least 20% w / w, and most preferably at least 25% w / w, and most preferably at least 30% w / w relative to total solids.

[0505] The total protein may contribute with an even larger portion of the total solids, e.g. when the beverage is intended as a sports protein beverage. Thus, in some preferred embodiments of the present invention, the liquid mixture comprises total protein in an amount of at least 80% w / w relative to total solids, more preferably at least 90% w / w, even more preferably at least 92% w / w, and most preferably at least 94% w / w relative to total solids.

[0506] The liquid mixture typically has a solids content of 0.5-50% w / w, more preferably 1-35% w / w, even more preferably 2-20% w / w, and most preferably 3-10% w / w.

[0507] The part of the liquid mixture that is not made up of solids preferably comprises water. The part of the liquid mixture that is not made up of solids preferably comprises water in an amount of at least 80% w / w, more preferably at least 90% w / w, even more preferably at least 95% w / w, and more preferably at least 99% w / w.

[0508] In some preferred embodiments of the present invention, the liquid mixture has a calorie content of at most 100 kcal / 100 g, more preferably at most 80 kcal / 100 g, even more preferred at most 70 kcal / 100 g, and most preferably at most 60 kcal / 100 g. Preferably, the liquid mixture may have a calorie content of 2-100 kcal / 100 g, more preferably at 4-80 kcal / 100 g, even more preferred 8-70 kcal / 100 g, and most preferably 12-60 kcal / 100 g. These embodiments are preferred for e.g. sports applications where the protein source is a primary energy source.

[0509] In other preferred embodiments of the present invention, the liquid mixture has a calorie content of more than 100 kcal / 100 g, more preferably at least 120 kcal / 100 g, even more preferred at least 140 kcal / 100 g, and most preferably at least 150 kcal / 100 g. Preferably, the liquid mixture may have a calorie content of 101-300 kcal / 100 g, more preferably at 120-280 kcal / 100 g, even more preferred 140-270 kcal / 100 g, and most preferably 150-260 kcal / 100 g. These embodiments are preferred for e.g. clinical nutrition where the protein source is accompanied by substantial amounts of carbohydrate and fat.

[0510] The compositional features and preferences described in the context of the heat-treated beverage described in pages 65-81 of PCT application no. PCT / EP2022 / 078739 equally apply to the liquid mixture.

[0511] The pH of the liquid mixture may span from slightly acidic to slightly alkaline. Near-pH-neutral liquid mixtures are particularly preferred for the production of near-pH neutral beverages. In some preferred embodiments of the present invention, the liquid mixture has a pH in the range of 5.5-8.0, more preferably 6.0-7.5, even more preferred 6.2-7.3, and most preferred 6.3-7.2.

[0512] In other preferred embodiments of the present invention, the liquid mixture has a pH in the range of 6.0-7.5, more preferably 6.2-7.5, and most preferred 6.3-7.5.

[0513] In further preferred embodiments of the present invention, the liquid mixture has a pH in the range of 6.0-8.0, more preferably 6.6-7.7, even more preferred 6.7-7.6, and most preferred 6.8-7.5.

[0514] Generally, any suitable food acid or food base may be used to adjust the pH of the liquid mixture. Those skilled in the art will recognize suitable means for adjusting the pH. Suitable food bases include sodium or potassium carbonate, sodium or potassium hydrogen carbonate, or ammonium hydroxide. Alternatively, KOH or NaOH may be employed to adjust the pH. Suitable food acids include e.g. citric acid, hydrochloric acid, malic acid or tartaric acid or phosphoric acid.

[0515] In some preferred embodiments of the present invention, the liquid mixture has a viscosity of at most 200 cP at 20 degrees C and at a shear rate of 300 s-1, more preferably at most 100 cP at 20 degrees C and at a shear rate of 300 s-1, even more preferred at most 50 cP at 20 degrees C and a shear rate of 300 s-1, and most preferred at most 20 cP at 20 degrees C and a shear rate of 300 s-1.

[0516] The liquid mixture is typically prepared by mixing the appropriate ingredients with the oxidized whey protein composition. If powder ingredients are used, it is often preferred that these are allowed to hydrate prior to the heat-treatment and similarly if may be preferred that the liquid mixture is homogenized prior to the heat-treatment.

[0517] In some preferred embodiments, the protein composition is provided in the form of a powder, and is preferably mixed water or an aqueous liquid and allow to hydrate prior to the heat-treatment.

[0518] In other preferred embodiments, the protein composition is provided in the form of a liquid, e.g. the protein solution obtained from step d) or step e) of the present method. The protein composition obtained from step d) or e) is then:

[0519] - mixed with one or more further ingredients required to produce the beverage,

[0520] - optionally subjected to homogenisation, - subjected to heat-sterilisation by heating it to a temperature in the range of 140-150 de-grees for 1-10 seconds,

[0521] - cooled, and

[0522] - filled into suitable sterile containers, which are subsequently sealed.

[0523] The packaging of step 2) may be any suitable packaging technique, and any suitable container may be used for packaging the liquid mixture.

[0524] However, in a preferred embodiment of the invention, the packaging of step 2) is aseptic packaging, i.e. the liquid mixture is packaged under aseptic conditions. For example, the aseptic packaging may be performed by using an aseptic filling system, and it preferably involves filling the liquid mixture into one or more aseptic container(s).

[0525] Aseptic filling and sealing are particularly preferred if the liquid mixture already is sterile or very low in microorganisms prior to filling.

[0526] Examples of useful containers are bottles, cartons, bricks, and / or bags.

[0527] The heat-treatment of the process preferably subjects the liquid mixture to a temperature of at least 70 degrees C.

[0528] In some preferred embodiments of the inventive process, the liquid mixture of step 1) is subjected to a heat-treatment comprising at least pasteurisation and then packaged in step 2).

[0529] In another embodiment of the inventive process, the packaged liquid mixture of step 2) is subjected to a heat-treatment comprising at least pasteurisation.

[0530] In some preferred embodiments, the heat-treatment involves heating the liquid mixture to a temperature in the range of 70-80 degrees C.

[0531] In some preferred embodiments of the invention, the temperature of the heat-treatment is in the range of 70-80 degrees C, preferably in the range of 70-79 degrees C, more prefer-ably in the range of 71-78 degrees C, even more preferably in the range of 72-77 degrees C, and most preferably in the range of 73-76 degrees C, such as approx. 75 degrees C.

[0532] Preferably, the duration of the heat-treatment, when performed in the temperature range 70-80, for 1 second to 60 minutes. The highest exposure times are best suited for the low-est temperatures of the temperature range and vice versa. In other preferred embodiments, the temperature of the heat-treatment is at 70 degrees C for at least 60 minutes, or preferably at 75 degrees C for at least 45 minutes, or preferably at 80 degrees C for at least 30 minutes, or preferably at 85 degrees C for at least 22 minutes, or preferably at 90 degrees C for at least 10 minutes.

[0533] In particularly preferred embodiments of the invention, the heat-treatment provides 70-78 degrees C for 1 second to 30 minutes, more preferably 71-77 degrees C for 1 minute to 25 minutes, and even more preferred 72-76 degrees C for 2 minutes to 20 minutes.

[0534] In some preferred embodiments of the invention, the process of the heat-treatment in-volves heating to a temperature of 85°C-95 degrees C for 1 to 30 minutes.

[0535] For example, the temperature of the heat-treatment may be at least 81 degrees C, prefer-ably at least 91 degrees C, preferably at least 95 degrees C, more preferred at least 100 degrees C, even more preferred at least 120 degrees C, and most preferred at least 140 degrees C.

[0536] In some particularly preferred embodiments of the invention, the heat-treatment involves heating the liquid mixture to a temperature in the range of 100-160 degrees C for a duration sufficient to sterilize the liquid mixture. This preferably involves heating the liquid mixture to a temperature in the range of 120 to 155 degrees C for a duration sufficient to obtain sterility, typically 0.1 seconds to 10 minutes, and more preferably 140 to 155 degrees C for a duration sufficient to obtain sterility, typically for 0.1-30 seconds. A heat-treatment of a liquid that renders the liquid sterile is also referred to as a heat-sterilisation.

[0537] Another preferred heat-treatment is a sterilizing UHT-type treatment which typically involves a temperature in the range of 135-146 degrees C and for a duration sufficient to obtain sterility, typically a duration in the range of 1-10 seconds.

[0538] Alternatively, but also preferred, the heat-treatment may involve a temperature in the range of 145-180 degrees C and for a duration sufficient to obtain sterility, typically a duration in the range of 0.01-2 seconds, and more preferably a temperature in the range of 150-180 degrees C and a duration in the range of 0.01-0.3 seconds.

[0539] The implementation of the heat-treatment may involve the use of equipment such as a plate or tubular heat exchanger, scraped surface heat exchanger or a retort system. Alternatively, and particularly preferred for heat-treatments above 95 degrees C, direct steam-based heating may be employed, e.g. using direct steam injection, direct steam infusion, or spray-cooking. Additionally, such direct steam-based heating is preferably used in combination with flash cooling. Suitable examples of implementation of spray-cooking are found in WO2009113858A1, which is incorporated herein for all purposes. Suitable examples of implementation of direct steam injection and direct steam infusion are found in WO2009113858A1 and WO 2010 / 085957 A3, which are incorporated herein for all purposes. General aspects of high-temperature treatment are e.g. found in "Thermal technologies in food processing" ISBN 185573558 X, which is incorporated herein by reference for all purposes.

[0540] In some preferred embodiments of the invention, the heat-treatment involves, or even consists of, retort heat-treatment, preferably at a temperature of at least 80 degrees C, and more preferably at a temperature of at least 95 degrees C, even more preferably at least 100 degrees C, and most preferably at least 120 degrees C, and preferably for a duration sufficient to render the treated liquid sterile.

[0541] In other preferred embodiments of the invention, the heat-treatment involves, or even consists of, steam infusion or spray cooking, preferably at a temperature of at least 100 degrees C, and more preferably at a temperature of at least 120 degrees C, even more preferably at least 130 degrees C, and most preferably at least 140 degrees C, and preferably for a duration sufficient to render the treated liquid sterile.

[0542] In some preferred embodiments of the invention, pasteurisation is combined with a physical microbial reduction.

[0543] Useful examples of physical microbial reduction involve one or more of germ filtration, UV radiation, high pressure treatment, pulsed electric field treatment, and ultrasound.

[0544] In some preferred embodiments of the invention, the heat-treatment is a sterilizing heat-treatment and hence results in a sterile liquid mixture and therefore a sterile beverage. Such sterilisation may e.g. be obtained by combining germ filtration and pasteurisation or by performing heat-treatment at at least 100 degrees C and for a duration sufficient to obtain sterilisation.

[0545] It is beneficial that the liquid mixture is subjected to cooling after the heat-treatment. According to a preferred embodiment of the inventive process, following the heat-treatment, the heat-treated liquid mixture is cooled to preferably 0 to 70 degrees C, preferably 0 to 60 degrees C, even more preferably 0 to 30 degrees C, and most preferably 0-20 degrees C.

[0546] If the heat-treatment does not sterilize the liquid mixture, the heat-treated liquid mixture is preferably cooled to 0 to 15 degrees C after the heat-treatment, more preferably to 1 to 10 degrees C, and most preferably 1-5 degrees C.

[0547] The cooling may take place prior to a filling step or after a filling step. The cooling typically involve flash cooling and / or conventional heat-exchangers.

[0548] At least partial cooling by flash cooling is often preferred, particularly after heat-sterilizing heattreatment. Flash cooling typically strips some of the volatile compounds of the cooled liquid. Whey protein beverages having a pH in the range of 5.5-8.5 are particularly prone to the development of unpleasant odours during heat-treatment and these unpleasant odours are partially stripped from the heat-treated liquid and released in the proximity of the flash cooling system. This is a disadvantage as it exposes the personnel operating the heat-treatment system to an annoying smell and may furthermore be associated with health issues.

[0549] The process of the invention can be implemented as a batch process, a semi-batch process, or a continuous process.

[0550] Another specific aspect of the invention pertains to a process of producing a heat-treated, and preferably heat-sterilized beverage, comprising:

[0551] - performing steps a), c), and d), or

[0552] - performing steps a), b) c), and d), or

[0553] - performing steps a), c), d), and e), or

[0554] - performing steps a), b) c), d), and e)

[0555] of the method described herein to obtain the protein composition on liquid form and subsequently packaging the protein composition or a liquid feed prepared from the protein composition which packaging is according to step 2) of the process as described above.

[0556] If the protein composition is to be used directly as a beverage is preferred that step d) involves a heat-sterilizing heat-treatment, i.e. a heat-treatment that renders the treated liquid sterile.

[0557] Such as a heat-treatment typically requires that the liquid to be treated is heated to a temperature in the range of 100-160 degrees C for a duration sufficient to sterilize the liquid. Suitable time / temperature combinations for such as heat-treatments are described herein.

[0558] Yet another aspect of the invention pertains to a heat-treated, preferably heat-sterilized, beverage having a pH of 5.5-8.5 obtainable by the process of the invention.

[0559] Yet another aspect of the invention pertains to a heat-treated, preferably heat-sterilized, beverage having a pH of 5.5-8.5, and more preferably 6.5-7.5, the beverage comprising the protein composition of the invention in an amount sufficient to contribute with at least 0.5% w / w protein, and preferably having a content of H2S 7 days after production of at most 5 micromol / L, more preferably at most 3 micromol / L, even more preferably at most 1.0 micromol / L, and most preferably at most 0.7 micromol / L.

[0560] A further aspect of the invention pertains to a food ingredient comprising:

[0561] - the protein composition of the invention, and

[0562] - one or more further ingredient(s), preferably selected from:

[0563] - a dairy ingredient, preferably a non-oxidized dairy ingredient,

[0564] - a plant-based ingredient,

[0565] - a non-dairy carbohydrate source,

[0566] - a flavouring agent, and / or

[0567] - a sweetener, e.g. in the form of a sweet carbohydrate, a polyol and / or a high intensity sweetener.

[0568] Preferably, the sweetener comprises one or more of a carbohydrate sweetener, a polyol, a high intensity sweetener, and a combination thereof.

[0569] In one embodiment of the invention, the beverage comprises at least one high-intensity sweetener (HIS). At least one HIS is preferably selected from the group consisting of aspartame, cyclamate, sucralose, acesulfame salt, neotame, saccharin, stevia extract, a steviol glycoside such as e.g. rebaudioside A, or a combination thereof.

[0570] In some embodiments of the invention, it is particularly preferred that the sweetener comprises or even consists of one or more high-intensity sweeteners.

[0571] HIS is both found among natural and artificial sweeteners and typically have a sweetening intensity of at least 10 times that of sucrose.

[0572] If used, the total amount of HIS of the beverage is typically in the range of 0.001-2% w / w. Preferably, the total amount of HIS is in the range of 0.005-1% w / w. Most preferably, the total amount of HIS is in the range of 0.01-0.5% w / w.

[0573] The choice of the sweetener may depend on the beverage to be produced, e.g. high-intensity sweeteners (e.g. aspartame, acesulfame-K or sucralose) may be used in beverages where no energy contribution from the sweetener is desired, whereas for beverages having a natural profile natural sweeteners (e.g. steviol glycosides, sorbitol or sucrose) may be used.

[0574] It may furthermore be preferred that the sweetener comprises or even consists of one or more polyol sweetener(s). Non-limiting examples of useful polyol sweeteners are maltitol, mannitol, lactitol, sorbitol, inositol, xylitol, threitol, galactitol or combinations thereof. If used, the total amount of polyol sweetener of the beverage is typically in the range of 1-20% w / w. More preferably the total amount of polyol sweetener of the beverage is in the range of 2-15% w / w. Even more preferably, the total amount of polyol sweetener may be in the range of 4-10% w / w.

[0575] Yet an aspect of the invention pertains to the use of a protein composition comprising modified BLG, preferably the protein composition comprising modified BLG of the invention, as a food ingredient, preferably for:

[0576] - improving the odour, and / or

[0577] - reducing the level of unpleasant odour similar to the odour of rotten eggs, and / or

[0578] - reducing the development of H2S during production, and / or

[0579] - reducing the content of H2S in the headspace of the container,

[0580] of heat-sterilized, beverages having a pH in the range of 5.5-8.5, preferably having a whey protein content of at least 3% w / w, more preferably at least 6% w / w, and preferably wherein the beverage is heat-sterilized using indirect heat-treatment.

[0581] The protein composition comprising modified BLG of the invention, may advantageously be used for reducing the colour intensity of a food product, preferably a heat-sterilized, beverage having a pH in the range of 5.5-8.5.

[0582] Thus, a further aspect of the invention pertains to the use of a protein composition comprising modified BLG, preferably the protein composition comprising modified BLG of the invention, as a food ingredient, preferably for:

[0583] - improving the odour, and / or

[0584] - reducing the level of unpleasant odour similar to the odour of rotten eggs, and / or

[0585] - reducing the development of H2S during production, and / or

[0586] - reducing the colour intensity, and / or

[0587] - reducing the content of H2S in the headspace of the container,

[0588] of heat-sterilized, beverages having a pH in the range of 5.5-8.5, preferably having a whey protein content of at least 3% w / w, more preferably at least 6% w / w, and preferably wherein the beverage is heat-sterilized using indirect heat-treatment.

[0589] Total protein, viscosity, average molecular weight, content of native proteins, content of total fat, content of lactose, and mineral composition are quantified or determined according to the Analyses described in PCT application no. PCT / EP2022 / 078739 which is incorporated by reference herein for all purposes.

[0590] In the following preferred numbered embodiments of the invention are presented: Numbered embodiment 1. A method of producing a protein composition comprising modified beta-lactoglobulin (BLG), the method comprising the steps of:

[0591] a) providing:

[0592] - a source comprising one or more phenolic compounds that contain at least two hydroxyl groups bound directly to the same aromatic ring (PCA), and

[0593] - a source comprising BLG,

[0594] b) optionally, subjecting a portion of the source comprising one or more PCA to a type of oxidation capable of converting a PCA to a quinone (referred to as PCA-type oxidation) thereby providing a source comprising one or more oxidized PCA,

[0595] c) combining a portion of the source comprising one or more PCA and / or a portion of the source comprising one or more oxidized PCA with a source comprising BLG and optionally with further ingredients to provide a protein solution, said protein solution having:

[0596] - a pH in the range of 2.0-4.5,

[0597] - a BLG content of at least 0.2% w / w, and

[0598] - a mole ratio between:

[0599] - the original amount of PCA used for preparing the protein solution, and

[0600] - the content of BLG of the protein solution,

[0601] of at least 0.1:1,

[0602] d) incubating the protein solution within a temperature range and for a duration sufficient to reduce the amount of free thiol groups of the protein solution to at most 15 micromol / g protein, more preferably at most 10 micromol / g protein, with the proviso that if the method does not contain step b), step d) also involves application of a type of oxidation capable of converting a PCA to a quinone, and

[0603] preferably, e) removing at least some of the free PCA and free, oxidized PCA from the incubated protein solution obtained from step d).

[0604] Numbered embodiment 2. The method according to Numbered embodiment 1, wherein the PCA-type oxidation applied during step b) and / or step d) is sufficient to create a mole ratio between:

[0605] - the theoretical amount of quinone provided to the protein solution during the method determined according to Analysis B, and

[0606] - the amount of BLG of the protein solution

[0607] of at least 0.1:1. Numbered embodiment 3. The method according to Numbered embodiment 1 or 2, wherein the PCA-type oxidation comprises, or even consists of, electrochemical oxidation and wherein the PCA-type electrochemical oxidation applied during the method, preferably during in step b) or during step d), uses a total charge of 1-5 Faraday per mole BLG of the protein solution, more preferably 1.4-4 Faraday per mole BLG, even more preferably 1.6-3 Faraday per mole BLG, and most preferably 1.8-2.5 Faraday per mole BLG of the protein solution.

[0608] Numbered embodiment 4. The method according to any one of the preceding Numbered embodiments, wherein the PCA-type oxidation comprises, of even consists of, electrochemical oxidation and wherein the PCA-type electrochemical oxidation applied during step d) uses a total charge of 1-5 Faraday per mole BLG of the protein solution, more preferably 1.4-4 Faraday per mole BLG, even more preferably 1.6-3 Faraday per mole BLG, and most preferably 1.8-2.5 Faraday per mole BLG of the protein solution.

[0609] Numbered embodiment 5. The method according to any one of the preceding Numbered embodiments, wherein the method involves PCA-type oxidation performed by electrochemical oxidation during step d).

[0610] Numbered embodiment 6. The method according to any one of the preceding Numbered embodiments, wherein the method does not involve electrochemical oxidation prior to step d).

[0611] Numbered embodiment 7. The method according to any one of the preceding Numbered embodiments wherein the PCA comprises:

[0612] - a flavonoid, preferably a flavanol or a flavanol ester, e.g. catechin, epicatechin, gallocatechin, epigallocatechin, catechin 3-gallate, epicatechin 3-gallate, gallo-catechin 3-gallate, and epigal-locatechin 3-gallate (EGCG), and / or

[0613] - a phenolic acid, preferably one or more of gallic acid, caffeic acid, chlorogenic acid, and / or - a stilbenoid, preferably resveratrol.

[0614] Numbered embodiment 8. The method according to any one of the preceding Numbered embodiments wherein the PCA comprises caffeic acid, gallic acid, chlorogenic acid, catechol, 4-me-thyl catechol, catechin, epicatechin, gallocatechin, epigallocatechin, catechin 3-gallate, epicatechin 3-gallate, gallocatechin 3-gallate, epigallocatechin 3-gallate (EGCG), resveratrol, carnosic acid, carnosol, naringenin, or a mixture thereof. Numbered embodiment 9. The method according to any one of the preceding Numbered embodiments wherein the PCA has a molecular weight in the range of 120-1000 g / mol, more preferably 250-700g / mol, even more preferably 300-650 g / mol, and most preferably 350-600 g / mol, and / or

[0615] Numbered embodiment 10. The method according to any one of the preceding Numbered embodiments wherein the PCA has a molecular weight in the range of 120-600 g / mol, more preferably 140-500g / mol, even more preferably 150-400 g / mol, and most preferably 160-300 g / mol.

[0616] Numbered embodiment 11. The method according to any one of the preceding Numbered embodiments wherein the PCA has a has a water solubility of at least 8 mM at 25 degrees C, more preferably at least 12 mM, and most preferably at least 16 mM.

[0617] Numbered embodiment 12. The method according to any one of the preceding Numbered embodiments wherein the source comprising one or more PCA is selected from the group consisting of a polyphenol extract from a herb, a polyphenol extract from a spice, polyphenol extract from a fruit, polyphenol extract from a berry, and mixtures thereof.

[0618] Numbered embodiment 13. The method according to any one of the preceding Numbered embodiments wherein the source comprising one or more PCA is selected from the group consisting of a polyphenol extract of tea, more preferably a polyphenol extract of green tea; a polyphenol extract of coffee; polyphenol extract of cocoa; a polyphenol extract of grapes; a polyphenol extract of rosemary; a polyphenol extract of lemon balm; a polyphenol extract of black currant; a single PCA-isolate, and mixtures thereof.

[0619] Numbered embodiment 14. The method according to any one of the preceding Numbered embodiments wherein the source comprising BLG comprises, or even consists of, a whey protein concentrate, a whey protein isolate, a milk serum protein concentration, a milk serum protein isolate, a BLG isolate, or a combination thereof.

[0620] Numbered embodiment 15. The method according to any one of the preceding Numbered embodiments comprising step b) of subjecting a portion of the source comprising one or more PCA to a type of oxidation capable of converting a PCA to a quinone thereby providing a source comprising one or more oxidized PCA.

[0621] Numbered embodiment 16. The method according to any one of the preceding Numbered embodiments wherein the protein solution has a pH in the range of 2.1-4.5, even more preferably 2.3-4.3, and most preferably 2.5-4.1. Numbered embodiment 17. The method according to any one of the preceding Numbered embodiments wherein the protein solution has a BLG content of at least 0.5% w / w, more preferably at least 1% w / w, even more preferably at least 3% w / w, and most preferably at least 6% w / w.

[0622] Numbered embodiment 18. The method according to any one of the preceding Numbered embodiments wherein the protein solution has a BLG content of 0.5-30% w / w, more preferably 1-20% w / w, even more preferably 3-16% w / w, and most preferably 5-12% w / w.

[0623] Numbered embodiment 19. The method according to any one of the preceding Numbered embodiments wherein the protein solution has a BLG content of at least 30% w / w relative to total protein, more preferably at least 40% w / w relative to total protein, even more preferably at least 45% w / w relative to total protein, and most preferably at least 50% w / w relative to total protein.

[0624] Numbered embodiment 20. The method according to any one of the preceding Numbered embodiments wherein the protein solution has a BLG content of 30-99% w / w relative to total protein, more preferably 40-95% w / w relative to total protein, even more preferably 45-90% w / w relative to total protein, and most preferably 50-80% w / w relative to total protein.

[0625] Numbered embodiment 21. The method according to any one of the preceding Numbered embodiments wherein the protein solution of step c) has a mole ratio between:

[0626] - the original amount of PCA used for preparing the protein solution, and

[0627] - the content of BLG of the protein solution

[0628] of at least 1:1.

[0629] Numbered embodiment 22. The method according to any one of Numbered embodiments 1-20 wherein the protein solution of step c) has a mole ratio between:

[0630] - the original amount of PCA used for preparing the protein solution, and

[0631] - the content of BLG of the protein solution

[0632] of 0.1:1 - 100:1, more preferably 0.1:1 - 50:1, even more preferably 0.1:1 -20:1, and most preferably 0.1:1 - 10:1.

[0633] Numbered embodiment 23. The method according to any one of Numbered embodiments 1-20 wherein the protein solution has a mole ratio between:

[0634] - the original amount of PCA used for preparing the protein solution, and - the content of BLG of the protein solution

[0635] of 0.1:1 -5:1, more preferably 0.1:1- 4:1, even more preferably 0.2: 1-3:1, and most preferably 0.3:1- 2:1.

[0636] Numbered embodiment 24. The method according to any one of Numbered embodiments 1-20 wherein the protein solution has a mole ratio between:

[0637] - the original amount of PCA used for preparing the protein solution, and

[0638] - the content of BLG of the protein solution

[0639] of 1:1 - 100:1, more preferably 1:1 - 50:1, even more preferably 1:1 -20:1, and most preferably 1:1 - 10:1.

[0640] Numbered embodiment 25. The method according to any one of the preceding Numbered embodiments wherein the oxidation applied during step b) and / or step d) is sufficient to create a mole ratio between:

[0641] - the theoretical amount of quinone provided to the protein solution during the method determined according to Analysis B, and

[0642] - the content of BLG of the protein solution

[0643] of 0.1:1 - 100:1, more preferably 0.1:1- 50:1, even more preferably 0.2:1-15:1, and most preferably 0.2:1- 5:1.

[0644] Numbered embodiment 26. The method according to any one of the preceding Numbered embodiments wherein the oxidation applied during step b) and / or step d) is sufficient to create a mole ratio between:

[0645] - the theoretical amount of quinone provided to the protein solution during the method determined according to Analysis B, and

[0646] - the content of BLG of the protein solution

[0647] of 0.1:1 -5:1, more preferably 0.1:1- 4:1, even more preferably 0.2: 1-3:1, and most preferably 0.2:1- 2:1.

[0648] Numbered embodiment 27. The method according to any one of the preceding Numbered embodiments wherein the oxidation applied during step b) and / or step d) is sufficient to create a mole ratio between:

[0649] - the theoretical amount of quinone provided to the protein solution during the method determined according to Analysis B, and

[0650] - the content of BLG of the protein solution

[0651] of at least 1:1. Numbered embodiment 28. The method according to any one of the preceding Numbered embodiments wherein the oxidation applied during step b) and / or step d) is sufficient to create a mole ratio between:

[0652] - the theoretical amount of quinone provided to the protein solution during the method determined according to Analysis B, and

[0653] - the content of BLG of the protein solution

[0654] of 1:1 - 100:1, more preferably 1:1- 50:1, even more preferably 1:1-15:1, and most preferably 1.5:1- 5:1.

[0655] Numbered embodiment 29. The method according to any one of the preceding Numbered embodiments:

[0656] - wherein the method comprises step b) and the portion of the source comprising one or more oxidized PCA obtained from step b) is used for the preparation of the protein solution of step c), and

[0657] - wherein the PCA-type oxidation is performed by electrochemical oxidization.

[0658] Numbered embodiment 29.1. The method according to any one of the preceding Numbered embodiments wherein the PCA-type oxidation applied during step b) is sufficient to create a mole ratio between:

[0659] - the quinone content of the portion of the source comprising one or more oxidized PCA that is used in the protein solution, and

[0660] - the amount of BLG of the protein solution

[0661] of at least 0.1:1, more preferably at least 0.2:1, even more preferably at least 0.4:1, and most preferably at least 0.6:1.

[0662] Numbered embodiment 30. The method according to any one of the preceding Numbered embodiments wherein the PCA-type oxidation applied during step b) is sufficient to create a mole ratio between:

[0663] - the quinone content of the portion of the source comprising one or more oxidized PCA that is used in the protein solution, and

[0664] - the amount of BLG of the protein solution

[0665] of at least 1.1:1, more preferably at least 1.3:1, even more preferably at least 1.5:1, and most preferably at least 2:1.

[0666] Numbered embodiment 31. The method according to any one of the preceding Numbered embodiments comprising step e), and wherein step e) performed to an extent sufficient to reduce the weight percentage of free PCA and oxidized PCA relative to total solids of the protein solution by at least 30% relative to the weight percentage prior to step e) , more preferably by at least 50%, even more preferably by at least 80%, and most preferably by at least 90% relative to the weight percentage prior to step e).

[0667] Numbered embodiment 32. The method according to any one of the preceding Numbered embodiments furthermore comprising step f) of drying a liquid feed comprising at least the protein derived from the incubated protein solution of step d) or e), preferably wherein the liquid feed for drying comprises or even consists of the protein solution obtained from step d) or e) or a protein concentrate thereof.

[0668] Numbered embodiment 33. A protein composition comprising modified BLG and having at most 15 micromol free thiol groups per g protein, more preferably at most 10 micromol free thiol groups per g protein, said protein composition is obtainable by the method according to one or more of the preceding Numbered embodiments, the protein composition preferably having one or more of:

[0669] - a protein content of at least 30% w / w relative to total solids,

[0670] - a tryptophan content of at least 0.7% w / w relative to total protein, - a methionine content of at least 0.3% w / w relative to total protein, - a kynurenine content of at most 0.2 micrograms / mg protein,

[0671] - preferably, a fat content of at most 3% w / w relative to total solids, - preferably, a content of protein-bound sulfur in the range of 100-600 mi- cromol / g protein,

[0672] - preferably, a content of protein-bound cysteine residues that form disulfide bonds in the range of 150-400 micromol / g protein.

[0673] Numbered embodiment 34. The modified whey protein composition according to Numbered embodiment 33 having a protein content of at least 30% w / w relative to total solids.

[0674] Numbered embodiment 35. The modified whey protein composition according to Numbered embodiment 33 or 34 having a tryptophan content of at least 0.7% w / w relative to total protein.

[0675] Numbered embodiment 36. The modified whey protein composition according to any one of Numbered embodiments 33-35 having a methionine content of at least 0.3% w / w relative to total protein.

[0676] Numbered embodiment 37. The modified whey protein composition according to any one of Numbered embodiments 33-36 having a kynurenine content of at most 0.2 micrograms / mg protein. Numbered embodiment 38. The modified whey protein composition according to any one of Numbered embodiments 33-37 having a fat content of at most 3% w / w relative to total solids.

[0677] Numbered embodiment 39. The modified whey protein composition according to any one of Numbered embodiments 33-38 having a content of protein-bound sulfur in the range of 100-600 micromol / g protein.

[0678] Numbered embodiment 40. The modified whey protein composition according to any one of Numbered embodiments 33-39 having a content of protein-bound cysteine residues that form disulfide bonds in the range of 150-400 micromol / g protein.

[0679] Numbered embodiment 41. The modified whey protein composition according to any one of Numbered embodiments 33-40 having a weight average molecular weight of the protein in the range of 18 kDa and 10000 kDa, more preferably 30-9000 kDa, even more preferably 50-8000 kDa, and most preferably 80-5000 kDa.

[0680] Numbered embodiment 42. The protein composition according to any one of Numbered embodiments 33-41 having a weight average molecular weight of the protein in the range of 18 kDa and 200 kDa, more preferably between 30-150 kDa, and most preferably between 30-100 kDa.

[0681] Numbered embodiment 43. The protein composition according to any one of Numbered embodiments 33-42, the protein composition has a pH in the range of 5.5-9.5, more preferably 6.0-8.5, even more preferably 6.2-8.0, and most preferably 6.5-7.5.

[0682] Numbered embodiment 44. A process for producing a heat-treated, preferably heat-sterilized, beverage, the process comprising the following steps:

[0683] 1) mixing the protein composition according to one or more of Numbered embodiments 33-43 with one or more further beverage ingredients to obtain a liquid mixture having a pH of 5.5-8.5, and

[0684] 2) filling the liquid mixture into suitable containers,

[0685] the process furthermore comprising at least one heat-treatment step wherein the liquid mixture is heat-treated, and preferably heat-sterilized, prior to filling and / or after filling,

[0686] preferably wherein the liquid mixture comprises the protein composition in an amount sufficient to contribute with at least 0.5% w / w protein.

[0687] Numbered embodiment 45. The process for producing a heat-treated, preferably heat-sterilized, beverage according to Numbered embodiment 44 wherein the liquid mixture contains, prior to the heat-sterilisation, at most 60 micromol free thiol groups / 100 g liquid mixture, more preferably at most 40 micromol free thiol groups / 100 g liquid mixture, even more preferably at most 30 micromol free thiol groups / 100 g liquid mixture, and most preferably at most 25 micromol free thiol groups / 100 g liquid mixture.

[0688] Numbered embodiment 46. The process for producing a heat-treated, preferably heat-sterilized, beverage according to Numbered embodiment 44 or 45 wherein the liquid mixture contains, prior to the heat-sterilisation, at most 20 micromol free thiol groups / 100 g liquid mixture, more preferably at most 15 micromol free thiol groups / 100 g liquid mixture, even more preferably at most 10 micromol free thiol groups / 100 g liquid mixture, and most preferably at most 5 micromol free thiol groups / 100 g liquid mixture.

[0689] Numbered embodiment 47. The process for producing a heat-treated, preferably heat-sterilized, beverage according to any one of Numbered embodiments 44-46 wherein the liquid mixture comprises a total amount of protein in the range of 0.5-15% w / w relative to the weight of the liquid mixture, more preferably 1-10% w / w relative to the weight of the liquid mixture, even more preferably 2-9% w / w relative to the weight of the liquid mixture, and most preferably 3-8% w / w relative to the weight of the liquid mixture.

[0690] Numbered embodiment 48. The process for producing a heat-treated, preferably heat-sterilized, beverage according to any one of Numbered embodiments 44-47 wherein the liquid mixture comprises a total amount of protein in the range of 4-15% w / w relative to the weight of the liquid mixture, more preferably 5-14% w / w relative to the weight of the liquid mixture, even more preferably 6-13% w / w relative to the weight of the liquid mixture, and most preferably 8-12% w / w relative to the weight of the liquid mixture.

[0691] Numbered embodiment 49. The process for producing a heat-treated, preferably heat-sterilized, beverage according to any one of Numbered embodiments 44-48 wherein the protein composition according to one of more of Numbered embodiments 21-30 contributes with at least 30% w / w of the total protein of the liquid mixture, more preferably at least 50% w / w of the total protein, even more preferably at least 70% w / w of the total protein, and most preferably at least 80% w / w of the total protein.

[0692] Numbered embodiment 50. The process for producing a heat-treated, preferably heat-sterilized, beverage according to any one of Numbered embodiments 44-49 wherein the protein composition according to one of more of Numbered embodiments 21-30 contributes with at least 90% w / w of the total protein of the liquid mixture, more preferably at least 95% w / w of the total protein, even more preferably at least 99% w / w of the total protein, and most preferably 100% w / w of the total protein. Numbered embodiment 51. The process for producing a heat-treated, preferably heat-sterilized, beverage according to any one of Numbered embodiments 44-50 wherein the liquid mixture comprises total protein in an amount of at least 15% w / w relative to total solids, more preferably at least 20% w / w, and most preferably at least 25% w / w, and most preferably at least 30% w / w relative to total solids.

[0693] Numbered embodiment 52. The process for producing a heat-treated, preferably heat-sterilized, beverage according to any one of Numbered embodiments 44-51 wherein the liquid mixture comprises total protein in an amount of at least 80% w / w relative to total solids, more preferably at least 90% w / w, even more preferably at least 92% w / w, and most preferably at least 94% w / w relative to total solids.

[0694] Numbered embodiment 53. The process for producing a heat-treated, preferably heat-sterilized, beverage according to any one of Numbered embodiments 44-52 wherein the liquid mixture has a solids content of 0.5-50% w / w, more preferably 1-35% w / w, even more preferably 2-20% w / w, and most preferably 3-10% w / w.

[0695] Numbered embodiment 54. The process for producing a heat-treated, preferably heat-sterilized, beverage according to any one of Numbered embodiments 44-53 wherein the part of the liquid mixture that is not made up of solids comprises water in an amount of at least 80% w / w, more preferably at least 90% w / w, even more preferably at least 95% w / w, and more preferably at least 99% w / w.

[0696] Numbered embodiment 55. The process for producing a heat-treated, preferably heat-sterilized, beverage according to any one of Numbered embodiments 44-54 wherein the liquid mixture has a calorie content of at most 100 kcal / 100 g, more preferably at most 80 kcal / 100 g, even more preferred at most 70 kcal / 100 g, and most preferably at most 60 kcal / 100 g.

[0697] Numbered embodiment 56. The process for producing a heat-treated, preferably heat-sterilized, beverage according to any one of Numbered embodiments 44-55 wherein the liquid mixture has a calorie content of more than 100 kcal / 100 g, more preferably at least 120 kcal / 100 g, even more preferred at least 140 kcal / 100 g, and most preferably at least 150 kcal / 100 g.

[0698] Numbered embodiment 57. A heat-treated, preferably heat-sterilized, beverage having a pH of 5.5-8.5, obtainable by the process according to one or more of Numbered embodiments 44-56.

[0699] The present invention has been described above with reference to specific embodiments. However, other embodiments than the above described are equally possible within the scope of the invention. The different features and steps of various embodiments and aspects of the invention may be combined in other ways than those described herein unless it is stated otherwise.

[0700] EXAMPLES

[0701] Analysis A: pH

[0702] All pH values are measured using a pH glass electrode and are normalized to 25 degrees C. The pH glass electrode (having a temperature compensation) is rinsed carefully and calibrated before use.

[0703] When the sample is in liquid form, then pH is measured directly in the liquid solution and compensated to 25 degrees C.

[0704] When the sample is a powder, then 10 g powder is dissolved 90 g demineralized water at room temperature while stirring vigorously. The pH of the solution is then measured and normalized to 25 degrees C.

[0705] Analysis B: Determination of the theoretical amount of quinone provided during the method

[0706] The "theoretical amount of quinone provided to the protein solution during the method" is a measure of the mole content of quinones that is formed if all PCA (incl. PCA that have been converted to quinones) present in the protein solution of the method was oxidized under the same conditions and under the same oxidizing conditions used in the method but without the presence of proteins or other sources of free thiols or amines.

[0707] For example, if the method of the invention is based on oxidation of the PCA (to provide a source comprising one or more oxidized PCA) prior to mixing with the source comprising BLG, then the content of quinones in the source comprising one or more oxidized PCA is equal to the theoretical quinone content.

[0708] The content or concentration of quinones is determined according to Analysis C.

[0709] Analysis C: Quantification of the content of quinones To determine the concentration of quinone generated in a sample, a L-Glutathione (GSH) assay was used. The principle of this assay is that quinone of the sample will react with the free thiol in GSH and the residual amount of unreacted GSH is then determined with RP-HPLC. The quinone content thus corresponds to the loss of GSH.

[0710] A 6 mM GSH solution was prepared in 5 mM phosphate buffer pH 4.5. GSH and quinone solutions were mixed in the volumetric ratio 1:1 in an Eppendorf tube and allowed to react for 1 hour at 60 degrees C. The mixture then was separated and quantified at 214nm relative to a standard curve of known GSH concentration by the method of Kurz et al (Kurz, F., Hengst, C., & Kulozik, U. (2020); RP-HPLC method for simultaneous quantification of free and total thiol groups in native and heat aggregated whey proteins; MethodsX, 7:101112). The amount of quinone was then determined as the concentration of lost GSH as compared to a sample of GSH mixed 1:1 with quinone-free buffer (same composition as the quinone-containing sample).

[0711] Analysis D: Color change of the reaction

[0712] The visual color changes were recorded by camera and evaluated qualitatively.

[0713] Analysis E: Determination of available thiol groups

[0714] The content of available thiol groups in WPI and PCA added WPI is quantified using a fluorimet-ric thiol assay kit (Molecular Probes, MAK151, Sigma-Aldrich, https : / / www.siqmaal- drich.com / DK / en / product / siqma / makl51), following the protocol from the manufacturer with minor modifications. The content of free thiols (SH) in the samples is typically reported in micromoles per gram of protein with the protein content determined by the total protein method described below.

[0715] Briefly, the thiol detection reagent provided in the kit is dissolved in dimethyl sulfoxide and then further diluted in 500 pL of phosphate buffer (50 mM, pH 8.0). An appropriately diluted sample (50 pL) is mixed with 50 pL of the diluted thiol detection reagent in an opaque multiwell plate and incubated in the dark for 30 min. After incubation, the fluorescent signal is measured using a spectrofluorimeter (SpectraMax i3x spectrophotometer, Molecular Devices, UK) at excitation and emission wavelengths of 490 nm and 520 nm, respectively.

[0716] The respective buffers (phosphoric acid buffer at pH 3.0 or phosphate buffer at pH 7.5) with the thiol detection reagent and samples with only phosphate buffer served as reagent and sample blanks, respectively. To measure all the unreacted thiols in BLG, 8 M urea is added to the samples. Similarly, 8 M urea are also added to the standards. Therefore, prior to mixing with the thiol reagent, the samples were diluted ten times in respective buffers (pH 3.0 or pH 7.5) containing 8 M urea.

[0717] The corrected absorbance is obtained by subtracting the absorbance of the blanks from the sample absorbance. Thiol groups are quantified based on a glutathione calibration standard curve prepared in the respective buffers within the linear range of 0-30 pM. All calibration standards are freshly prepared on the day of analysis and used within 4 h.

[0718] Analysis F: Molecular weight distribution analysis of whey protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

[0719] SDS-PAGE is performed to analyze the molecular weight distribution of the protein sample and evaluate the presence of disulfide-linked aggregates and non-reducible cross-links.

[0720] The analysis is done according to the description from Jansson et al. (2017) with minor modifications. Briefly, 1 pL of the sample is vortexed with 25 pL lithium dodecyl sulfate (LDS) sample buffer (4x, ThermoFisher Scientific, Carlsbad, CA, USA) and 74 pL MilliQ water. For reduced samples, 64 pL of MilliQ water and 10 pL 1 M dithiothreitol are mixed with 1 pL of the sample and 25 pL LDS sample buffer (4X). The loading samples are heated (80 °C, 10 min).

[0721] Samples (10 pL) and molecular weight markers (LC5800, 3 pL, mass range 3.5-260 kDa;

[0722] Thermo Fisher Scientific, Carlsbad, CA, USA) are loaded on 15-wellNuPAGE 12% Bis-Tris gels (Thermo Fisher Scientific, Carlsbad, CA, USA), and the electrophoresis is run in ice-cold NuPAGE MES SDS running buffer (Thermo Fisher Scientific, Carlsbad, CA, USA) at 200 V for 1 h. The gels is stained with Coomassie Brilliant Blue by incubating gels under shaking at 22 degrees C for 16 h.

[0723] Coomassie Brilliant Blue stained gel is scanned using an Epson Perfection V750 Pro scanner (Epson, Long Beach, CA, USA).

[0724] Analysis G: Amino acid analysis by UHPLC

[0725] Amino acids are quantified by the method described by Zainudin et al. (2019).

[0726] 0.5 mg protein is hydrolyzed using deaerated 4 M methanesulfonic acid (with 0.2%, w / v tryptamine) in a Pico Tag hydrolysis vial in vacuo for 17 h 30 min at 110 degrees C. Neutralized dry hydrolysates are mixed with aminocaproic acid (internal standard) and deri-vatized with o-phthalaldehyde / 3-mercaptopropionic acid and fluorenylmethyloxycarbonyl chloride. The derivatized amino acids are analyzed using a UHPLC-FLD system equipped with an Agilent AdvanceBio AAA column (100 x 3.0 mm, 2.7 pm particle size). Quantification (internal standard calibration) of each amino acid is carried out based on an eight-point calibration curve (0-100 pM) constructed using authentic standards.

[0727] Analysis H: Identification of the protein-PCA adduct by LC-MS analysis

[0728] The identification of the protein-PCA adduct is performed by employing LC-MS / MS as described by Liu at al. (2024).

[0729] A 0.5 mg protein sample is hydrolysed using 6 M HCI (with 0.2% w / v tryptamine) under nitrogen environment in a Pyrex microwave reaction vial. The hydrolysis is performed using a Biotage Initiator-!- microwave system (Biotage, Uppsala, Sweden) by heating samples at 165 degrees C for 1 min, followed by 150 degrees C for 10 min.

[0730] The hydrolyzed samples are dried in a centrifugal vacuum concentrator (Savant SPD 131 DDA with a refrigerated vapour trap Savant RVT 5105, Thermo Scientific, MA, USA). The dried samples are re-dissolved and diluted in 0.1% formic acid and filtered through 0.22 pm syringe filters. and the samples are then injected (10 pL) into an Ultimate 3000 UHPLC system (Thermo Scientific, CA, USA) equipped with a Synchronis Aq C-18 column (Thermo Scientific, CA, USA; 100 mm length, 2.1 mm ID, 1.8 pm particle size).

[0731] The full MS scans are acquired from 70 to 1000 m / z at a resolution of 70,000. The MS / MS fragmentation patterns of amino acids with PCA or oxidized PCA are obtained by HCD-based MS / MS mode (resolution: 17,500) with a normalized collision energy of 30.

[0732] Analysis I: Quantification of H2S

[0733] The level of H2S is measured using the colorimetric assay described by Yusuf et al. (2005) with modifications.

[0734] Briefly, a mixture of zinc acetate (1% w / v, 250 pL) is injected into the sample vial by piercing a needle through the silicon seal of the vial cap to trap the generated H2S. This is followed by the sequential addition of trichloroacetic acid (10% w / v, 250 pL),N,N-dimethyl-p-phenylenediamine sulfate (20 mM; 133 pL) in 7.2 M HCI and FeCI3(30 mM; 133 pL) in 1.2 M HCI. The absorbance (670 nm) of aliquots of the resulting solution (300 pL) is determined after 5 min using a 96-well microplate reader (Tecan Systems, USA). The H2S concentration of each sample is calculated against a calibration curve of ZnS standard solution (purchased from Unisense, Aarhus, Denmark(0.45-35.61 pM).

[0735] Example 1: The benefit of acidic, quinone-based whey protein modification vs. neutral pH modification

[0736] The present inventors have discovered that it is feasible and even advantageous to modify whey protein by reaction with oxidized phenolic compounds at acidic pH relative to reaction at neutral pH.

[0737] In this example, the inventors demonstrate that less color development and less protein aggregation was obtained when reacting whey protein with 4-methylbutyl quinone (oxidized 4-methylcatechol) at acidic pH relative to the same reaction at neutral pH.

[0738] Materials and Methods

[0739] Materials:

[0740] 4-Methylcatechol (4MC, > 95%), sodium phosphate dibasic, phosphoric acid, formic acid (FA), fluorometric thiol assay kit, and acetonitrile (UHPLC grade) were purchased from Merck. Ultra-pure water was obtained through a MilliQ purification system (Millipore, Bedford, MA).

[0741] A BLG-rich whey protein isolate powder (99.6% native BLG, 18.4 kDa) was prepared according to WO 2018 / 115520 Al. The characteristics of the powder are described in Table 2a.

[0742] Table 2a: Composition of the WPI powder. The degree of protein denaturation of the WPI pow-der was less than 2% w / w.

[0743] Content Concentration

[0744] % Protein / dry matter 97

[0745] BLG, % of total protein 99.5

[0746] Na,% of total solids (TS) 0.697

[0747] K, % of TS <0.025

[0748] Mg, % of TS <0.003

[0749] Ca, % of TS <0.025

[0750] P, % of TS <0.025

[0751] pH of 10% solution 6.9

[0752] Lactose, % <0.1

[0753] Fat, % <0.15

[0754]

[0755] Water, % Approx. 4 Protocol:

[0756] Preparation of stock protein solutions:

[0757] Two stock WPI solutions, X and Y, having a of pH 3.0 and pH 9.7 were prepared in the following manner.

[0758] Stock WPI solution X (pH 3.0): WPI powder was dissolved by mixing it into a phosphoric acid buffer (20 mM, pH 2.5) and subsequently allowing the mixture to hydrate under gentle stirring for 1 h at 22 degrees C. The inventors observed no remaining powder particles and the solution became transparent. pH of the solution was then adjusted to pH 3.0 with 5 M phosphoric acid. The concentration of WPI powder in this stock solution was 1.5% (w / w).

[0759] Stock WPI solution Y (pH 9.7) : Solution Y was prepared in the same manner as Solution X, except that the pH was adjusted to pH 9.7.

[0760] BLG is the primary source of free thiols of whey protein (BLG contains one free thiol group per molecule). The molar concentration of native BLG in a whey protein composition is therefore a good approximation to the molar content of free thiol groups of whey protein composition. The molar concentration of the stock WPI solutions was 0.825 mM. Thus, the dosage of quinones was calculated relative to the content of native BLG of the whey protein composition.

[0761] 4-Methyl benzoquinone (4MBQ) generation:

[0762] 4MBQ was prepared by electrochemical oxidation of 4-methylcatechol (4MC) as described by Li et al. (2016) with some modifications.

[0763] 4MC (5 mM) was dissolved in a phosphoric acid buffer solution (10 mM, pH 3.0) containing 100 mM of sodium chloride. The solution was de-oxygenated by purging with nitrogen for 10 min. Cyclic voltammograms of the 4MC solution was obtained by using a voltammetry analyzer CV-50W (BAS Co., Ltd.) with a glassy carbon working electrode (3 mm diameter, BAS Co., Ltd.), a platinum coil counter electrode (5 mm in diameter and 5 cm in length), and an Ag / AgCI (KCI, c=3 M) reference electrode (Metrohm, Switzerland). The bulk electrolysis was performed at an initial potential of 570 mV versus Ag / AgCI under nitrogen using the same voltammetric analyzer with a reticulated vitreous carbon tube working electrode. Electrolysis was terminated when the amount of charge passed was equivalent to two electrons per molecule of 4MC. The 4MBQ concentration was determined by spectrophotometry at 395 nm where 4MBQ has an extinction coefficient of 1350 M”1cm”1.

[0764] The 4MBQ reference solution was used within 30 min after preparation. The 4MBQ working solutions of 0.165 mM and 1.65 mM by mixing samples of 4MBQ reference solution with the electrolysis buffer (10 mM of phosphoric buffer solution, pH 3.0, 100 mM of sodium chloride).

[0765] Mixing and incubation:

[0766] 700 pL Stock WPI solution X or Y was mixed with 350 pL of the electrolysis buffer or one of 4MBQ working solutions in a 2 mL Eppendorf. The final concentration of WPI powder was 1% w / w, and the molar ratios between BLG and 4MBQ were 1:0, 1:0.1, and 1:1. The pH of the final mixture samples were pH 3.0 as acidic condition and pH 7.5 as neutral pH condition.

[0767] The samples having a pH of 3.0 and molar ratios between BLG and 4MBQ of 1:0, 1:0.1, or 1:1 were referred to as WPI-A1, WPI-A2, and WPI-A3. The samples having a pH of 7.5 and molar ratios between BLG and 4MBQ of 1:0, 1:0.1, or 1:1 were referred to as WPI-B1, WPI-B2, and WPI-B3.

[0768] The mixed solutions were quickly transferred into a pre-heated thermomixer (Model Matrix Orbital Delta Plus, IKA Thermoshaker, Germany) and incubated at 80 degrees C for 15 min.

[0769] Removing unreacted 4MC / 4MBQ by spin filtration:

[0770] To avoid further reactions, the unreacted 4MBQ and 4MC were removed by spin filter concentrators. 400 pL reaction mixtures were transferred to 10 kDa MWCO spin filter concentrators (Cytiva, 28932225) and centrifuged at 15,000 g, 4 degrees C for 30 min using a microcentrifuge

[0771] type 157 (Ole Dich Instrumentmakers APS, Hvidovre, Denmark).

[0772] The protein retentate in the spin filters were washed three times by phosphoric acid buffer (20 mM, pH 3.0) for samples prepared at pH 3.0 or phosphate buffer (20 mM pH 7.5) for samples prepared at pH 7.5. The samples were then re-dissolved in respective buffers (400 pL) and used for the thiol determination and amino acid analysis. Table 2b: Overview of the samples prepared in Example 1.

[0773] Stoichiometry

[0774] Sample ID pH [BLG]:[4MBQ]

[0775] mokrnol

[0776] WPI-A1 3.0 1:0

[0777] WPI-A2 3.0 1:0.1

[0778] WPI-A3 3.0 1:1

[0779] WPI-B1 7.5 1:0

[0780] WPI-B2 7.5 1:0.1

[0781] WPI-B3 7.5 1:1

[0782]

[0783] Characterization

[0784] Color difference:

[0785] The color differences of the reaction mixtures at pH 3.0 and pH 7.5 were recorded before heat treatment, before removing unreacted 4MBQ and 4MC by spin filtration, and after removing unreacted 4MBQ and 4MC by spin filtration. The visual evaluation is shown in Table 3a.

[0786] Determination of thiol content:

[0787] The free thiol content has been confirmed to be highly related to the H2S formation, with H2S being identified as the main flavor molecule responsible for the unpleasant odor flavor of dairy products after heat treatment. Therefore, in this example, free thiol content was measured as described in Analysis E. The free thiol content and thiol-reduction with PCA are summarized in Table 3a.

[0788] S DS- PAGE:

[0789] SDS-PAGE was performed as described in Analysis F.

[0790] Amino acid analysis:

[0791] Amino acid analysis was carried out as described in Analysis G.

[0792] Identification of the protein-PCA adduct:

[0793] The protein-PCA adducts were identified after hydrolysis according to Analysis H.

[0794] Results The inventors have studied the feasibility of blocking the free thiol groups of WPI by the reaction with oxidized phenolic compounds at acidic pH and neutral pH. The resulting modified WPI products have been compared with regards to color changes, free thiol contents, molecular weight distributions and amino acid contents.

[0795] Color difference and visual inspection of the samples

[0796] After the incubation step, samples WPI-A1 and WPI-B1 were colorless. The incubated samples WPI-A2, WPI-A3, WPI-B2, and WPI-B3 had an apparent yellow color. The inventors found that this color advantageously could be reduced by e.g. a filtration / washing step to remove unreacted 4MC / 4MBQ.

[0797] After removing unreacted 4MC / 4MBQ using spin filters, the samples WPI-A2 and WPI-B2 were almost colorless with only a slight taint of yellow (the color of WPI-B2 was more intense than the color of WPI-A2). WPI-A3 had a light yellowish color and WPI-B3 had a more intense yellowish color. The color intensities are summarized in Table 3a.

[0798] Effect of the reaction between protein and 4MBQ on the content of free thiol groups (-SH):

[0799] The free thiol content in the samples was measured as described in Analysis E. The results are displayed in Table 3a.

[0800] A free thiol content of 46.8-47.1 micromole SH / g protein was measured in sample WPI-A1 and Bl at pH 3.0 and pH 7.5. Surprisingly, WPI-A2 incubated with 1:0.1 (BLG:4MBQ) at pH 3.0 showed a reduction (24%) in free thiol content relative to WPI-A1 (no 4MBQ) within the 15 min-heating period. Even larger reductions in free thiol content (54%) were observed at the stoichiometry of 1:1 (BLG:4MBQ).

[0801] Under neutral pH conditions, increasing the 4MBQ dosage from 1:0.1 to 1:1 (BLG:4MBQ) in sample WPI-B2 to WPI-B3 showed a similar pattern with free thiol reductions of 3% and 90%, respectively, relative to samples without added 4MC. Table 3a: Residual free thiol and visual appearance after incubation and filtration. Percentages given in brackets denote the level of free thiol reduction relative to the WPI sample (1%) at the same pH without added 4MBQ.

[0802] Stoichiometry

[0803] Free thiols content, micromol Color intensity Sample ID PH [BLG]:[4MBQ]

[0804] SH / g protein (color) mol:mol

[0805] 0

[0806] WPI-A1 3.0 1:0 47.1

[0807] (Colorless)

[0808] +

[0809] WPI-A2 3.0 1:0.1 35.8 (24%)

[0810] (Very slightly yellow) + + +

[0811] WPI-A3 3.0 1:1 21.6 (54%)

[0812] (Slightly yellow) 0

[0813] WPI-B1 7.5 1:0 46.8

[0814] (Colorless)

[0815] + +

[0816] WPI-B2 7.5 1:0.1 45.2 (3%)

[0817] (Slightly yellow) ++++++ WPI-B3 7.5 1:1 4.5 (90%)

[0818] (Intensely yellow)

[0819]

[0820] Note: the data listed in the table are for samples after removing the unreacted 4MBQ / 4MC.

[0821] The more intense color seen at pH 7.5 indicated the occurrence of side reactions. The inventors have seen that lysine-quinone adducts tend to have a higher absorbance in the visible spectrum than e.g. cysteine-quinone adducts.

[0822] The molecular weight distribution of proteins in samples WPI-A1 and WPI-B1 without added 4MBQ and WPI-A2 to WPI-A3 and WPI-B2 to WPI-B3 samples incubated with 4MBQ at 80 degrees C for 15 min was analyzed by SDS-PAGE under both non-reducing and reducing conditions as described in Analysis F. The gel is shown in Figure 1.

[0823] Formation of protein cross-links and aggregation in heated protein systems were evaluated by SDS-PAGE in both non-reducing and reducing conditions. The bands observed at around 18 kDa were related to the monomer of BLG. The bands at around 36 kDa, 54 kDa, 72 kDa and higher than 72 kDa were associated to the dimer, trimer, tetramer and polymer of BLG, respectively. Closer inspection suggested that the fraction of proteins predominantly followed the order of monomer > dimer > trimer > tetramer species > higher order aggregates.

[0824] Most of the covalently linked aggregates in trimer, tetramer and polymer of BLG were reducible, implying that disulfide bonds were the major covalent bonding in these heated protein samples. The inventors surprisingly found that, in the non-reduced gel, protein samples modified under acid conditions exhibited fewer oligomers and polymer of BLG than at neutral pH, where the oligomers and polymer of BLG were more pronounced. Moreover, the addition of 4MBQ to WPI samples consistently resulted in protein bands with smaller molecular weights compared to the WPI samples at the same pH without PCA. This observation suggests that the 4MBQ reduces aggregation during incubation by blocking thiol-d isu Ifide exchange reactions.

[0825] Effect of the reaction between protein and oxidized PCA on the amino acid profiles:

[0826] The amino acid analysis was carried out as described in Analysis G to analyse the changes in the content of essential amino acids in samples WPI-A1, WPI-A3, WPI-B1 and WPI-B3.

[0827] The inventors surprisingly observed that the present modification at acidic pH did not significantly affect the essential amino acid profile of the WPI, confirming that the modification based on oxidized phenolic compounds selectively blocks the cysteine (Cys) residues of whey protein under acidic conditions. In contrast, samples modified under neutral conditions showed a slightly lower level of lysine (Lys), suggesting that Lys of the WPI was involved in the reaction (see Table 3b).

[0828] Table 3b: Comparison of the loss of lysine by acidic reaction with 4MBQ (WPI-A3) vs. reaction at pH 7.5 (WPI-B3). WPI-A1 and WPI-B1 are the controls.

[0829] Samples WPI-A1 WPI-A3 WPI-B1 WPI-B3

[0830] Lys content 7.10 ± 0.09 7.21 ± 0.07 7.07 ± 0.05 6.74 ± 0.07 (g / 100 g of total

[0831] amino acids)

[0832] Reduction % -1.5% 4.7%

[0833]

[0834] Identification of the protein- PCA adducts

[0835] After being hydrolysed, the samples (WPI-A1, WPI-A3, WPI-B1, and WPI-B3) were identified by LC-MS / MS as described in Analysis H. Cys-PCA adduct was in the form of Cys-4MC, Lys-PCA adduct was in the form of Lys-4MBQ. The results are shown in Table 3c.

[0836] As control samples at acidic pH and neutral pH, WPI-A1 and WPI-B1 did not show any peaks related to any adducts.

[0837] At acidic pH (in sample WPI-A3), only Cys adduct was observed. At neutral pH, apart from the Cys adduct, the Lys-4MBQ adduct was observed in sample WPI-B3, which indicated that at neutral pH, Lys was involved into the reaction when more oxidized PCA added ([BLG] :[PCA] = 1:1). This was in accordance with the findings of the amino acid analysis.

[0838] These results proved the adduct formation between Cys and PCA under both acidic and neutral pH conditions, which indicated the Cys-modification occurs under both acidic and neutral pH conditions. Moreover, it provided evidence that Cys-modification is more selective at acidic pH than at neutral pH.

[0839] Table 3c: ESI-MS / MS data for amino acid-PCA adducts. Cys-4MC adduct and Lys-4MBQ adduct were identified. The retention time related to the respective peak in the full-MS spectra. The main ions in the MS / MS spectra at the correlated retention time were listed. The ions with underlines were the respective precursors.

[0840] Cys-PCA adduct (Cys-4MC) Lys-PCA adduct (Lys-4MBQ) Retention Main fragment ions, Retention time Main fragment ions, time (min) m / z (min) m / z

[0841] WPI-A1 Na Na Na Na

[0842] 155.0168, 227.0382,

[0843] WPI-A3 17.82 Na Na

[0844] 224.0647

[0845] WPI-B1 Na Na Na Na

[0846] 155.0168, 227.0382, 84.0817, 128.0713, WPI-B3 17.81 16.40

[0847] 224.0647 206.1181, 267.1352

[0848]

[0849] Note: Na means no related peak was observed in the sample.

[0850] Conclusion

[0851] The inventors have found that the reaction between whey protein and oxidized, PCA-type phenolic compounds at pH 3.0 is sufficient to reduce or eliminate the free thiol groups of BLG in a whey protein solution. The inventors have observed that less color development occurred in the reaction mixture at pH 3.0 than at pH 7.5. Additionally, fewer protein crosslinks were formed and less aggregation occurred in the reaction mixture at pH 3.0 than at pH 7.5. The modification at acidic pH did not affect the essential amino acid profiles of the WPI whereas e.g. Lysine was affected when the reaction took place at pH 7.5. This demonstrates that the quinone-based modification at acidic pH has a high selectivity towards cysteine residues, and therefore leads to less modification of other amino acids.

[0852] Reacting BLG and other whey proteins with oxidized PCA-type phenolic compounds under acidic conditions thus constitutes a feasible route for reducing the content of free thiol groups of the protein. The inventors have previously observed that protein with a high content of free thiol groups gives rise to an unpleasant odor when used in whey protein solutions or beverages that are subjected to e.g. UHT treatments. The inventors have found that it is often preferred to reduce the content of free thiol groups of the whey protein to at most 15 micromol / g protein, more preferably at most 10 micromol / g protein, and most preferably at most 5 micromol / g protein. The higher the protein content of a whey protein beverage, the lower content of free thiol groups are required. The present invention therefore provides a new and surprising approach for reducing the development of unpleasant odors during the production and consumption of heat-treated, pH-neutral, whey protein-rich beverages.

[0853] The content of free thiol groups of the whey protein may be reduced to at most 15 micromol / g protein, more preferably at most 10 micromol / g protein, even more preferably at most 5 micromol / g protein, or even substantially depleted, by increasing the duration of the incubation step and / or increasing the dosage of the oxidized PCA-type phenolic compounds, e.g. by sequential addition of oxidized PCA-type phenolic compounds or in situ generation of oxidized PCA-type phenolic compounds in an incubating mixture containing protein and PCA-type phenolic compounds.

[0854] The method of the present invention also works on BLG-containing protein sources prepared by e.g. fermentation and is therefore not limited to modification of protein isolated from milk or whey.

[0855] Example 2: Acidic quinone-based whey protein modification under a 6% protein condition

[0856] In this example, the inventors demonstrated the feasibility of scaling up the concentration of BLG to 6% for making ready-to drink beverage products with surprisingly low level of unpleasant odor after UHT treatment at acidic pH.

[0857] Materials and Methods

[0858] A WPI stock solution containing WPI powder in an amount of 9% (w / w) and having a pH of 3.0 and 4MBQ working solutions of different 4MBQ concentration were prepared as in Example 1.

[0859] The samples were prepared by mixing 700 pL stock WPI solution (9%) with 350 pL of one or the 4MBQ working solution (or the electrolysis buffer) in a 2 mL Eppendorf. The final concentration of WPI powder was 6%, and the molar ratios between BLG and 4MBQ were 1:0, 1:0.1, 1:0.25 and 1:0.3. The mixed solutions were quickly transferred into a pre-heated thermomixer and incubated at 80 degrees C for 15 min. The samples having a pH of 3.0 and molar ratios between BLG and 4MBQ of 1:0, 1:0.1, 1:0.25, and 1:0.3 were referred to as WPI-C1, WPI-C2, WPI-C3, and WPI-C4.

[0860] The unreacted 4MC / 4MBQ was removed by using 10 kDa MWCO spin filter concentrators (Cytiva, 28932360). The protein fractions were washed three times and re-dissolved in pH 3.0 phosphoric acid buffer (20 mM, pH 3.0) as described in Example 1.

[0861] Analyses were carried out for visual inspection according to to Analysis D and free thiol content according to Analysis E.

[0862] The H2S level was measured according to Analysis I after the samples had been subjected to UHT simulation (see below). The HPLC vials (2 mL 32 x 11.6 mm, part no ML33003A) and the aluminum caps (11 mm, ML 33034L) for UHT simulation were from Mikrolab, Denmark.

[0863] Simulation of UHT treatment:

[0864] 1.0 mL of sample were transferred to 2 mL glass HPLC vials and crimp-sealed using aluminium lids fitted with Teflon septum (Mikrolab ML 33032).

[0865] The sealed vials were transferred to a preheated aluminum heating block (catalog no. 460-0008, VWR, Soborg, Denmark) with holes drilled by the manufacturer to match the dimensions of the 2 mL GC vials. The block was preheated to 160 degrees C, and the samples were kept in the block for 160 seconds. The temperature reached 100 degrees C in about 40 seconds, about 120 degrees C in 65 seconds and reached 140 degrees C after 100 seconds of incubation and 150 degrees C after 160 seconds. After incubation in the heating block, the samples were transferred to an ice-water bath to rapidly quench reactions leading to the development of an unpleasant odour.

[0866] Results:

[0867] The inventors have studied the feasibility of blocking the free thiol groups of WPI with a high protein concentration as a protein drink beverage. The results of the free thiol content, H2S formation, and color changes were summarized in Table 4.

[0868] Effect of the reaction between protein and PCA on the content of free thiols (SH) of the protein

[0869] A free thiol content of 50.2 micromol thiol / g protein was measured in sample WPI-C1 at pH 3.0. Surprisingly, WPI-C2, WPI-C3 and WPI-C4 incubated with oxidized PCA at a stoichiometry of 1:0.1, 1:0.25, 1:0.3 (BLG: PCA) at pH 3.0, showed a reduction in free thiol content of 25%, 35% and 36%, respectively, relative to WPI-C1 that were not incubated with oxidized PCA.

[0870] Effect of the reaction between protein and PCA on H2S development

[0871] The control sample (WPI-C1) generated 59.6 pM H2S. Samples WPI-C2, WPI-C3 and WPI-C4 showed significant reductions of H2S generation by 30%, 80% and 86% relative to WPI-C1.

[0872] Table 4: Residual free thiol content, visual appearance after incubation with oxidized PCA (4MC). Percentages given in brackets denote the level of free thiol or H2S reduction relative to the WPI sample (6%) at the same pH without added PCA.

[0873] Stoichiometry Free thiols, miSample Color

[0874] pH [BLG]:[4MBQ] cromol SH / g proH2S, pM

[0875] ID (visual)

[0876] mol:mol tein

[0877] WPI-C1 3.0 1:0 50.2 59.6 Colorless 41.9 (30%) Very slight WPI-C2 3.0 1:0.1 41.4 (25%)

[0878] brownish WPI-C3 3.0 1:0.25 35.7 (35%) 11.7 (80%) Slight brownish WPI-C4 3.0 1:0.3 35.3 (36%) 8.6 (86%) Slight brownish

[0879]

[0880] Conclusion:

[0881] The present example demonstrated the feasibility of reducing the free thiol and H2S level of 6% WPI solution by quinone-based modification of whey protein with 4MC under acidic conditions. The results provide a possible application of the invention into the ready-drink protein products.

[0882] As mentioned in Example 1, the content of free thiol groups of the whey protein may be reduced to less than 15 micromol / g protein, more preferably less than 10 micromol / g protein, or even sustantually depleted, by increasing the duration of the incubation step and / or increasing the dosage of the oxidized PCA-type phenolic compounds, e.g. by sequential addition of oxidized PCA-type phenolic compounds or in situ generation of oxidized PCA-type phenolic compounds in an incubating mixture containing protein and PCA-type phenolic compounds. Example 3: Documenting acidic, quinone-based whey protein modification using oxidized caffeic acid (CA)

[0883] In this example, the inventors documented the feasibility of using e.g. caffeic acid as PCA in the present invention.

[0884] Materials and Methods

[0885] WPI stock solution at pH 3.0 was prepared as Example 1. Working solutions of oxidized caffeic acid were prepared in the same manner as the 4MBQ working solutions of Example 1 starting from a caffeic acid (CA) isolate instead of 4MC isolate. 700 pL stock WPI solution (1.5%) was mixed with 350 pL of a working solution of oxidized caffeic acid in a 2 mL Eppendorf. The final concentration of WPI powder was 1% w / w, and the molar ratios between WPI and used CA were 1:0, 1:0.5, and 1:1. The mixed solutions were quickly transferred into a pre-heated thermomixer and incubated at 80 degrees C for 15 min.

[0886] Analyses were carried out for visual observation according to Analysis D and the content of free thiol according to Analysis E.

[0887] Results:

[0888] The inventors further investigated the ability of caffeic acid, which is present in coffee, to reduce the free thiol content of whey protein preparations. The results of the free thiol content, H2S formation, and color changes were summarized in Table 5.

[0889] Effect of the reaction between protein and PCA on free thiol (SH) content of the protein

[0890] A free thiol content of 47.8 micromol SH / g protein was measured in sample WPI-D1 at pH 3.0. Surprisingly, WPI-D2 incubated with 1:0.5, 1: 1 (BLG: PCA) at pH 3.0 showed a reduction of 16% and 27%, respectively, in free thiol content relative to WPI-C1 (no PCA) within the 15 min-heating period.

[0891] Effect of the reaction between protein and PCA on H2S development

[0892] The reference (WPI-D1) generated 29.5 pM H2S. H2S contents determined in WPI-D2 and WPI-D3 samples were 15.0 pM and 3.2 pM, with reductions of 49% and 89%, respectively, showing a reduction in off-flavor for the caffeic acid-treated samples. Table 5: Residual free thiol, visual appearance after incubation with oxidized PCA. Percentages given in brackets denote the level of free thiol or H2S reduction relative to the WPI sample at the same pH without added PCA.

[0893] Stoichiometry

[0894] Sample Free thiols, micromol Color

[0895] PH [BLG]:[CA]

[0896] ID SH / g protein (visual)

[0897] mol:mol

[0898] WPI-D1 3.0 1:0 47.8 Colorless

[0899] WPI-D2 3.0 1:0.5 40.1 (16%) Colorless

[0900] WPI-D3 3.0 1:1 34.9 (27%) Colorless

[0901]

[0902] Conclusion:

[0903] The inventors have demonstrated that reduction of the content of free thiols, hence, the reduction of unpleasant odor, can be achieved by the addition of caffeic-quinone at acidic pH with a heat treatment at 80 degrees C for 15 min.

[0904] This experiment confirmed the feasibility of acidic quinone-based whey protein modification in reducing the unpleasant odor generated form dairy products during heat treatment. More promisingly, this example extended the application of acidic quinone-based whey protein modification from a model compound, 4MC to caffeic acid, which is abundant in plants. The inventors have found to use of oxidized caffeic acid to be advantageous as it gives rise to less color development in the modified protein than oxidized 4MC.

[0905] As mentioned in Example 1, the content of free thiol groups of the whey protein may be reduced to at most 15 micromol / g protein, more preferably at most 10 micromol / g protein, or even substantially depleted, by increasing the duration of the incubation step and / or increasing the dosage of the oxidized PCA-type phenolic compounds, e.g. by sequential addition of oxidized PCA-type phenolic compounds or in situ generation of oxidized PCA-type phenolic compounds in an incubating mixture containing protein and PCA-type phenolic compounds. References:

[0906] Whitaker, J. R., Voragen, A. G., & Wong, D. W. (2002). Handbook of food enzymology. CRC Press.

[0907] Kurz, F., Hengst, C., & Kulozik, U. (2020). RP-HPLC method for simultaneous quantification of free and total thiol groups in native and heat aggregated whey proteins. MethodsX, 7, 101112.

[0908] Jansson, T., Rauh, V., Danielsen, B. P., Poojary, M. M., Waehrens, S. S., Bredie, W. L., ... & Lund, M. N. (2017). Green tea polyphenols decrease Strecker aldehydes and bind to proteins in lactosehydrolyzed UHT milk. Journal of Agricultural and Food Chemistry, 65(48), 10550-10561.

[0909] Zainudin MAM, Poojary MM, Jongberg S, Lund MN. Light exposure accelerates oxidative protein polymerization in beef stored in high oxygen atmosphere. Food Chem. 299(2019) 125132.

[0910] Yusuf, M., Huat, B. T. K., Hsu, A., Whiteman, M., Bhatia, M., & Moore, P. K. (2005). Streptozoto-cin-induced diabetes in the rat is associated with enhanced tissue hydrogen sulfide biosynthesis. Biochemical and biophysical research communications, 333(4), 1146-1152.

[0911] Liu, J., Engholm-Keller, K., Poojary, M. M., Bevilacqua, M., Andersen, M. L., & Lund, M. N. (2024). Reactivity and mechanism of the reactions of 4-methylbenzoquinone with amino acid residues in p-lactoglobulin: A kinetic and product investigation. Food chemistry, 434, 137473.

[0912] Li, Y., Jongberg, S., Andersen, M. L., Davies, M. J., & Lund, M. N. (2016). Quinone-induced protein modifications: Kinetic preference for reaction of 1, 2-benzoquinones with thiol groups in proteins. Free Radical Biology and Medicine, 97, 148-157

[0913] Li, C., Paulsen, P. A., Akilhoglu, H. G., Nielsen, S. B., Engholm-Keller, K., & Lund, M. N. (2022). Cysteine residues are responsible for the sulfurous off-flavor formed in heated whey protein solutions. Food Chemistry: Molecular Sciences, 5, 100120.

[0914] Jameson, G. N., Zhang, J., Jameson, R. F., & Lined, W. (2004). Kinetic evidence that cysteine reacts with dopaminoquinone via reversible adduct formation to yield 5-cysteinyl-dopamine: an important precursor of neuromelanin. Organic & Biomolecular Chemistry, 2(5), 777-782.

Claims

CLAIMS1. A method of producing a protein composition comprising modified beta-lactoglobulin (BLG), the method comprising the steps of:a) providing:- a source comprising one or more phenolic compounds that contain at least two hydroxyl groups bound directly to the same aromatic ring (PCA), and- a source comprising BLG,b) optionally, subjecting a portion of the source comprising one or more PCA to a type of oxidation capable of converting a PCA to a quinone (referred to as PCA-type oxidation) thereby providing a source comprising one or more oxidized PCA,c) combining a portion of the source comprising one or more PCA and / or a portion of the source comprising one or more oxidized PCA with a source comprising BLG and optionally with further ingredients to provide a protein solution, said protein solution having:- a pH in the range of 2.0-4.5,- a BLG content of at least 0.2% w / w, and- a mole ratio between:- the original amount of PCA used for preparing the protein solution, and- the content of BLG of the protein solution,of at least 0.1:1,d) incubating the protein solution within a temperature range and for a duration sufficient to reduce the amount of free thiol groups of the protein solution to at most 15 micromol / g protein, more preferably at most 10 micromol / g protein, with the proviso that if the method does not contain step b), step d) also involves application of a type of oxidation capable of converting a PCA to a quinone, andpreferably, e) removing at least some of the free PCA and free, oxidized PCA from the incubated protein solution obtained from step d).

2. The method according to claim 1, wherein the PCA-type oxidation applied during step b) and / or step d) is sufficient to create a mole ratio between:- the theoretical amount of quinone provided to the protein solution during the method determined according to Analysis B, and- the amount of BLG of the protein solutionof at least 0.1:1.

3. The method according to claim 1 or 2, wherein the PCA-type oxidation comprises, or even consists of, electrochemical oxidation and wherein the PCA-type electrochemical oxidation applied during the method, preferably during in step b) or during step d), uses a total charge of 1-5 Faraday per mole BLG of the protein solution, more preferably 1.4-4 Faraday per mole BLG, even more preferably 1.6-3 Faraday per mole BLG, and most preferably 1.8-2.5 Faraday per mole BLG of the protein solution.

4. The method according to any one of the preceding claims wherein the PCA comprises:- a flavonoid, preferably a flavanol or a flavanol ester, e.g. catechin, epicatechin, gallocatechin, epigallocatechin, catechin 3-gallate, epicatechin 3-gallate, gallo-catechin 3-gallate, and epigal-locatechin 3-gallate (EGCG),- a phenolic acid, preferably one or more of gallic acid, caffeic acid, chlorogenic acid, and / or - a stilbenoid, preferably resveratrol.

5. The method according to any one of the preceding claims wherein the PCA has a molecular weight in the range of 120-600 g / mol, more preferably 140-500g / mol, even more preferably 150-400 g / mol, and most preferably 160-300 g / mol.

6. The method according to any one of the preceding claims wherein the source comprising BLG comprises, or even consists of, a whey protein concentrate, a whey protein isolate, a milk serum protein concentration, a milk serum protein isolate, a BLG isolate, or a combination thereof.

7. The method according to any one of the preceding claims wherein the protein solution has one or more of, and more preferably at least two or more of:- a pH in the range of 2.1-4.5, even more preferably 2.3-4.3, and most preferably 2.5-4.1 - a BLG content of at least 0.5% w / w, more preferably at least 1% w / w, even more preferably at least 3% w / w, and most preferably at least 6% w / w,- a BLG content of 0.5-30% w / w, more preferably 1-20% w / w, even more preferably 3-16% w / w, and most preferably 5-12% w / w,- a BLG content of at least 30% w / w relative to total protein, more preferably at least 40% w / w relative to total protein, even more preferably at least 45% w / w relative to total protein, and most preferably at least 50% w / w relative to total protein, and- a BLG content of 30-99% w / w relative to total protein, more preferably 40-95% w / w relative to total protein, even more preferably 45-90% w / w relative to total protein, and most preferably 50-80% w / w relative to total protein.

8. The method according to any one of the preceding claims wherein the protein solution of step c) has a mole ratio between:- the original amount of PCA used for preparing the protein solution, and- the content of BLG of the protein solutionof at least 1:1.

9. The method according to any one of the preceding claims wherein the protein solution of step c) has a mole ratio between:- the original amount of PCA used for preparing the protein solution, and- the content of BLG of the protein solutionof 1:1 - 100:1, more preferably 1:1 - 50:1, even more preferably 1:1 -20:1, and most preferably 1:1 - 10:1.

10. The method according to any one of claims 1-9 wherein the oxidation applied during step b) and / or step d) is sufficient to create a mole ratio between:- the theoretical amount of quinone provided to the protein solution during the method determined according to Analysis B, and- the content of BLG of the protein solutionof at least 1:1.

11. The method according to any one of claims 1-9 wherein the oxidation applied during step b) and / or step d) is sufficient to create a mole ratio between:- the theoretical amount of quinone provided to the protein solution during the method determined according to Analysis B, and- the content of BLG of the protein solutionof 1:1 - 100:1, more preferably 1:1- 50:1, even more preferably 1:1-15:1, and most preferably 1.5:1- 5:1.

12. The method according to any one of the preceding claims furthermore comprising step f) of drying a liquid feed comprising at least the protein derived from the incubated protein solution of step d) or e), preferably wherein the liquid feed for drying comprises or even consists of the protein solution obtained from step d) or e) or a protein concentrate thereof.

13. A protein composition comprising modified BLG and having at most 15 micromol free thiol groups per g protein, more preferably at most 10 micromol free thiol groups per g protein, said protein composition is obtainable by the method according to one or more of the preceding claims, the protein composition preferably having one or more of:- a protein content of at least 30% w / w relative to total solids,- a tryptophan content of at least 0.7% w / w relative to total protein, - a methionine content of at least 0.3% w / w relative to total protein, - a kynurenine content of at most 0.2 micrograms / mg protein,- preferably, a fat content of at most 3% w / w relative to total solids, - preferably, a content of protein-bound sulfur in the range of 100-600 mi- cromol / g protein,- preferably, a content of protein-bound cysteine residues that form disulfide bonds in the range of 150-400 micromol / g protein, and- preferably having a weight average molecular weight of the protein in the range of 18 kDa and 200 kDa, more preferably between 30-150 kDa, and most preferably between 30-100 kDa.

14. The protein composition according to claim 13, having a pH in the range of 5.5-9.5, more preferably 6.0-8.5, even more preferably 6.2-8.0, and most preferably 6.5-7.5.

15. A process for producing a heat-treated, preferably heat-sterilized, beverage, the process comprising the following steps:1) mixing the protein composition according to claim 13 or 14 with one or more further beverage ingredients to obtain a liquid mixture having a pH of 5.5-8.5, and2) filling the liquid mixture into suitable containers,the process furthermore comprising at least one heat-treatment step wherein the liquid mixture is heat-treated, and preferably heat-sterilized, prior to filling and / or after filling, preferably wherein the liquid mixture comprises the protein composition in an amount sufficient to contribute with at least 0.5% w / w protein.

16. A heat-treated, preferably heat-sterilized, beverage having a pH of 5.5-8.5, obtainable by the process according to claim 15.

17. Use of a protein composition comprising modified BLG protein according to claim 13 or 14 as a food ingredient, preferably for:- improving the odour, and / or- reducing the level of unpleasant odour similar to the odour of rotten eggs, and / or- reducing the development of H2S during production, and / or- reducing the colour intensity, and / or- reducing the content of H2S in the headspace of the container,of heat-sterilized, beverages having a pH in the range of 5.5-8.5, preferably having a whey protein content of at least 3% w / w, more preferably at least 6% w / w, and preferably wherein the beverage is heat-sterilized using indirect heat-treatment.

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