Method for producing modified protein composition by reaction with oxidized phenolic compound, modified protein composition and nutritional use of modified protein composition

Abstract

The present invention relates to a method of preparing a modified protein composition by reaction with an oxidized phenolic compound under conditions of exposure and modification of the free thiol groups of beta-lactoglobulin. The resulting modified protein products have been found to have excellent performance in, for example, protein-rich beverage products, and have particularly been found to produce reduced levels of unpleasant odors during neutral pH sterilization heat treatments as well as during consumption of such beverage products.

Description

Invention Field

[0001] This invention relates to a method for preparing modified protein compositions by reacting with oxidized phenolic compounds under conditions of exposure and modification of the free thiol groups of β-lactoglobulin. The resulting modified protein products have been found to have excellent performance in, for example, protein-rich beverage products, and have been found to produce reduced levels of unpleasant odors during neutral pH sterilization heat treatment and during consumption of such beverage products. background

[0002] pH-neutral, whey protein-rich beverages tend to develop an unpleasant odor, similar to rotten eggs, during heat processing. These beverages are typically bottled immediately after production, thus exposing consumers to this unpleasant odor when the bottles are opened.

[0003] WO 2021 / 198968 A1 discloses a method for preparing a beverage containing milk protein concentrate (MPC). The MPC is treated to allow for heat homogenization of the beverage containing it, while reducing the presence of unpleasant odors (malodorous and / or egg-like or sulfurous odors and / or tastes). Specifically, the MPC contains at least one whey protein, wherein 50%-100% of the whey protein is denatured, and wherein approximately 5-20% by weight of calcium has been consumed.

[0004] Li et al. (“Cysteine ​​residues are responsible for the sulfurous off-flavor formed in heated whey protein solutions”, Food Chemistry: Molecular Sciences, Vol. 5, December 1, 2022 (2022-12-01), pp. 100-120) disclosed how the protein composition of whey protein solutions affects the release of H2S during heat treatment of the solution.

[0005] Poojary et al. (“Green Tea Extract Decreases Age-Derived Advanced Glycation Endproducts but Not Lys-Derived AGEs in UHT Milk during 1-Year Storage”, Journal of Agricultural and Food Chemistry, Vol. 68, No. 48, November 17, 2020, D2) investigated the effects of adding green tea extract to skim milk before UHT treatment and studied the formation of advanced glycation products during long-term storage.

[0006] 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, December 1, 2022 (2022-12-01), p. 133775) disclosed the preparation and characterization of conjugates of 4-methylbenzoquinone and β-lactoglobulin. Waqar did not include any disclosure indicating a reduction in unpleasant odor production due to chemical modification of β-lactoglobulin. Invention Overview

[0007] The inventors have noted that the unpleasant odor that typically accompanies pH-neutral, whey protein-rich beverages is associated with hydrogen sulfide formed from free thiol groups of cysteine ​​residues in whey protein during heat processing.

[0008] The inventors have also discovered that this problem can be surprisingly reduced or even avoided by reacting proteins containing β-lactoglobulin with oxidized phenolic compounds.

[0009] Therefore, aspects of the present invention relate to a method for producing a protein composition comprising modified β-lactoglobulin (BLG), the method comprising the following steps: a) Provide: - A source comprising one or more phenolic compounds (PCA), said one or more phenolic compounds comprising at least two hydroxyl groups directly bonded to the same aromatic ring, and - Sources containing BLG (BoG) b) Optionally, a portion of the source containing one or more PCA undergoes a type of oxidation (PCA-type oxidation) capable of converting PCA into quinone, thereby providing a source of PCA containing one or more oxidations. c) Combining a portion of the source containing one or more PCA and / or a portion of the source containing one or more oxidized PCA with a source containing BLG and optional additional components to provide a protein solution having: - pH in the range of 6.5-9.5, and - At least 0.2% w / w BLG content, - A molar ratio of at least 0.1:1 among the following: - The original amount of PCA used to prepare the protein solution, and - The BLG content of the protein solution, d) Incubate the protein solution at a temperature sufficient to reduce the amount of free thiol groups in the protein solution to at most 10 μmol / g protein for a duration sufficient to reduce the amount of free thiol groups in the protein solution to at most 10 μmol / g protein, provided that if the method does not include step b), then step d) also involves applying a type of oxidation capable of converting PCA to quinone. Preferably, the PCA-type oxidation applied during step b) and / or step d) is sufficient to produce a molar ratio of at least 0.1:1 among the following: - Based on the theoretical amount of quinone supplied to the protein solution during the method determined by Analysis 1, and - The BLG content of the protein solution.

[0010] Another aspect of the invention relates to a protein composition comprising modified BLG, said protein composition being obtainable by the method of the invention.

[0011] Another aspect of the invention relates to a process for producing a heat-treated, preferably heat-sterilized, beverage, the process comprising the following steps: 1) The protein composition of the present invention is mixed with one or more other beverage ingredients to obtain a liquid mixture having a pH of 5.5-8.5, and 2) Fill the liquid mixture into a suitable container. The process further includes at least one heat treatment step, wherein the liquid mixture is heat-treated, preferably heat-sterilized, before and / or after filling. Preferably, the liquid mixture comprises a protein composition sufficient to contribute at least 0.5% w / w of protein.

[0012] Another aspect of the invention relates to a heat-treated, preferably heat-sterilized beverage having a pH of 5.5-8.5, which can be obtained by the process of the invention.

[0013] Another aspect of the present invention relates to a food ingredient comprising: - The solid form of the protein composition of the present invention, and - One or more additional ingredients, preferably selected from: - Dairy ingredients, preferably unoxidized dairy ingredients. - Plant-based ingredients, - Non-dairy carbohydrate sources, - Flavorings, and / or - Sweeteners.

[0014] Another aspect of the invention relates to a protein composition containing modified BLG in a heat-sterilized beverage, and preferably to the use of the protein composition according to the invention as a food ingredient, preferably for: - Improves odor, and / or - Reduces unpleasant odor levels similar to rotten eggs, and / or - Reduce H2S generation during production, and / or - Reduce the H2S content in the headspace of the container. The heat-sterilized beverage has a pH in the range of 5.5-8.5, preferably has a whey protein content of at least 3% w / w, and is preferably heat-sterilized using indirect heat treatment. Detailed Explanation

[0015] An aspect of the present invention relates to a method for producing a protein composition comprising modified BLG, the method comprising the following steps: a) Provide: - A source comprising one or more phenolic compounds (PCA), said one or more phenolic compounds comprising at least two hydroxyl groups directly bonded to the same aromatic ring, and - Sources containing BLG (BoG) b) Optionally, a portion of the source containing one or more PCA undergoes a type of oxidation capable of converting PCA to quinone (referred to as PCA-type oxidation), thereby providing a source of PCA containing one or more oxidations. c) Combining a portion of the source containing one or more PCA and / or a portion of the source containing one or more oxidized PCA with a source containing BLG and optional additional components to provide a protein solution having: - pH in the range of 6.5-9.5, and - At least 0.2% w / w BLG content, - A molar ratio of at least 0.1:1 among the following: - The original amount of PCA used to prepare the protein solution, and - The BLG content of the protein solution, d) Incubate the protein solution at a temperature sufficient to reduce the amount of free thiol groups in the protein solution to at most 10 μmol / g protein for a duration sufficient to reduce the amount of free thiol groups in the protein solution to at most 10 μmol / g protein, provided that if the method does not include step b), then step d) also involves applying a type of oxidation capable of converting PCA to quinone. Preferably, the PCA-type oxidation applied during step b) and / or step d) is sufficient to produce a molar ratio of at least 0.1:1 among the following: - Based on the theoretical amount of quinone supplied to the protein solution during the method determined by Analysis 1, and - The BLG content of the protein solution.

[0016] In the context of this invention, the term "β-lactoglobulin" or BLG refers to BLG derived from mammalian species, such as BLG in its natural and / or glycosylated form, and includes naturally occurring genetic variants. The term BLG also covers BLG produced through recombinant technologies and / or precise fermentation, such as BLG produced through recombinant technologies and / or precise fermentation based on genetically modified microorganisms or mammalian cells. The term "BLG" or "β-lactoglobulin" as used herein does not include denatured and aggregated BLG. The content of BLG was determined according to the analytical method described in PCT application number PCT / EP2022 / 078739.

[0017] In the context of this invention, the term "phenolic compound" has its general meaning and refers to a molecular compound containing an aromatic ring, at least one of the six carbon atoms of said aromatic ring being directly bonded to a hydroxyl group. The other five carbon atoms of the aromatic ring may be individually bonded to a variety of groups, including hydroxyl groups or hydrogen. Phenolic compounds also include polyphenols.

[0018] In the context of this invention, the term "phenolic compound comprising an aromatic ring with at least two hydroxyl groups directly bonded thereto" (referred to as "PCA") refers to a molecular compound comprising an aromatic ring, such as catechol or epigallocatechin gallate (EGCG), wherein at least two carbon atoms of the aromatic ring are directly bonded to hydroxyl groups. The other four carbon atoms of the aromatic ring may individually bond to a variety of groups, including hydroxyl groups or hydrogen.

[0019] PCA preferably has the following structural formula:

[0020] At least two of R1, R2, R3, R4, R5, and R6 are hydroxyl groups.

[0021] Preferably, two or three of R1, R2, R3, R4, R5 and R6 are hydroxyl groups.

[0022] In the context of this invention, the term "a type of oxidation capable of converting PCA to quinone" or "PCA-type oxidation" refers to an oxidation process capable of oxidizing PCA in a source containing one or more types of PCA to quinone. PCA-type oxidation typically involves electrochemical oxidation and / or the use of chemical oxidants.

[0023] In the context of this invention, the phrase “the PCA-type oxidation applied during step b) and / or step d) is sufficient to produce a molar ratio [...]” is used to define the desired level of PCA-type oxidation in a manner applicable to all embodiments of the method of this invention. In this context, “step b) and / or step d)” means that if the method involves PCA-type oxidation during both steps b) and d), then the combined PCA-type oxidation of both steps should be considered. However, if the method involves PCA-type oxidation only during step b), then only step b) should be considered. Similarly, if the method involves PCA-type oxidation only during step d), then only step d) should be considered.

[0024] As is obvious to those skilled in the art, there must be at least n X :n Y The molar ratio between component X and component Y means that the molar ratio must be at least n. X Divide by n Y n X and n Y This represents the molar amounts of component X and component Y. For example, if the molar ratio between component X and component Y must be at least 0.1:1, then it means n X Divide by n Y It must be at least 0.1. The same logic applies to weight ratio.

[0025] In the context of this invention, the phrase "theoretical amount of quinone supplied to the protein solution during the method" is a measure of the molar amount of quinone compounds formed when all PCA (including PCA that has been converted to quinone compounds) present in the protein solution of the method is oxidized under the same conditions and the same oxidation conditions used in the method, but in the absence of a source of protein or other free thiols or free amines. The "theoretical amount of quinone supplied to the protein solution during the method" is determined according to Analysis 1. For example, if the method of the present invention is primarily based on the oxidation of PCA during step b) prior to step c) (to provide a source containing one or more oxidized PCA), then the amount of quinone compounds in a portion of the source containing one or more oxidized PCA used in step c) is equal to the theoretical amount of quinone supplied to the protein solution during the method.

[0026] In some preferred embodiments of the invention, the method for producing a protein composition comprising modified BLG further includes step e): drying a liquid feed, said liquid feed comprising at least a protein derived from the incubated protein solution of step d).

[0027] In some preferred embodiments of the invention, the protein solution provided in step c) has a molar ratio of at least 1:1 among the following: - The original amount of PCA used to prepare the protein solution, and - The BLG content of the protein solution, Furthermore, the PCA-type oxidation applied during step b) and / or step d) is preferably sufficient to produce a molar ratio of at least 1:1 among the following: - Based on the theoretical amount of quinone supplied to the protein solution during the method determined by Analysis 1, and - The BLG content of the protein solution.

[0028] The term "source containing one or more PCA" refers to a composition used to provide PCA for the protein solution in step c) and / or the oxidizing aqueous solution in step b). The term "a portion of a source containing one or more PCA" refers to the actual portion used to prepare the protein solution. In some preferred embodiments of the invention, substantially all sources containing one or more PCA are used.

[0029] If more than one PCA-containing composition is used to produce the protein solution of step c) and / or the oxidizing aqueous solution of step b), then each of the PCA-containing components used is considered a sub-source constituting a source containing one or more PCA.

[0030] In some preferred embodiments of the invention, the PCA comprises flavonoids, preferably flavanols or flavanol esters. Preferred flavonoids are, for example, catechins and catechin derivatives, such as epicatechin, gallatechin, epigallocatechin, catechin 3-gallate, epicatechin 3-gallate, gallatechin 3-gallate, and epigallocatechin 3-gallate (EGCG).

[0031] In other preferred embodiments of the invention, PCA comprises a stilbene compound, preferably in the form of resveratrol.

[0032] Preferably, the PCA comprises caffeic acid, gallic acid, chlorogenic acid, catechin, 4-methylcatechin, catechin, epicatechin, gallocatechin, epigallocatechin, catechin 3-gallate, epicatechin 3-gallate, gallocatechin 3-gallate, epigallocatechin 3-gallate (EGCG), resveratrol, sarsaparilla acid, sarsaparillaol, naringenin, or mixtures thereof.

[0033] In some preferred embodiments of the invention, one or more PCA sources from the group consisting of caffeic acid, gallic acid, chlorogenic acid, catechin, 4-methylcatechin, catechin, epicatechin, gallocatechin, epigallocatechin, catechin 3-gallate, epicatechin 3-gallate, gallocatechin 3-gallate, epigallocatechin 3-gallate (EGCG), resveratrol, sarsaparilla acid, sarsaparillaol, and naringenin contribute at least 40 mol% of the PCA containing one or more PCA sources, 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 PCA containing one or more PCA sources.

[0034] In other preferred embodiments of the invention, one or more PCA sources from the group consisting of catechin, epicatechin, gallocatechin, epigallocatechin, catechin 3-gallate, epicatechin 3-gallate, gallocatechin 3-gallate, and epigallocatechin 3-gallate (EGCG) contribute at least 40 mol% of the PCA containing one or more PCA sources, 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 PCA containing one or more PCA sources.

[0035] In another preferred embodiment of the invention, one or more PCA sources from the group consisting of caffeic acid, gallic acid, chlorogenic acid, catechin, 4-methylcatechin, catechin, epicatechin, gallocatechin, epigallocatechin, catechin 3-gallate, epicatechin 3-gallate, gallocatechin 3-gallate, epigallocatechin 3-gallate (EGCG), resveratrol, sarsaparilla acid, sarsaparillaol, and naringenin contribute at least 40 mol% of the PCA containing one or more PCA sources, 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 PCA containing one or more PCA sources.

[0036] In even more preferred embodiments of the invention, one or more PCA sources from the group consisting of caffeic acid, gallic acid, and chlorogenic acid contribute at least 40 mol% of the PCA containing one or more PCA sources, 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 PCA containing one or more PCA sources.

[0037] In other preferred embodiments of the invention, epigallocatechin 3-gallate (EGCG) contributes at least 40 mol% of the PCA containing one or more PCA sources, 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 PCA containing one or more PCA sources.

[0038] In some preferred embodiments of the invention, PCA has a molecular weight of up to 600 g / mol, more preferably up to 400 g / mol, even more preferably up to 350 g / mol and more preferably up to 310 g / mol.

[0039] In other preferred embodiments of the invention, the PCA has a molecular weight of up to 250 g / mol, more preferably up to 230 g / mol, even more preferably up to 210 g / mol and more preferably up to 200 g / mol.

[0040] In some preferred embodiments of the invention, PCA has a molecular weight in the range of 110 g / mol to 600 g / mol, more preferably 110 g / mol to 400 g / mol, even more preferably 110 g / mol to 350 g / mol, and even more preferably 110 g / mol to 310 g / mol.

[0041] In some preferred embodiments of the invention, PCA has a molecular weight in the range of 110 g / mol-250 g / mol, more preferably 110 g / mol-230 g / mol, even more preferably 120 g / mol-210 g / mol, and even more preferably 120 g / mol-200 g / mol.

[0042] In other preferred embodiments of the invention, PCA has a molecular weight in the range of 120 g / mol-1000 g / mol, more preferably 250 g / mol-700 g / mol, even more preferably 300 g / mol-650 g / mol, and even more preferably 350 g / mol-600 g / mol.

[0043] In some preferred embodiments of the invention, the PCA contains neither nitrogen nor sulfur.

[0044] In some preferred embodiments of the present invention, PCA does not contain a carboxylic acid group.

[0045] In some preferred embodiments of the invention, PCA has a water solubility of at least 8 mM, more preferably at least 12 mM, and most preferably at least 16 mM at 25°C.

[0046] In some preferred embodiments of the invention, the source of one or more PCA is selected from polyphenol extracts from herbs, polyphenol extracts from spices, polyphenol extracts from fruits, polyphenol extracts from berries, and mixtures thereof.

[0047] Generally preferred sources containing one or more PCA are preferably selected from the group consisting of: polyphenol extracts of tea, more preferably polyphenol extracts of green tea; polyphenol extracts of coffee; polyphenol extracts of cocoa; polyphenol extracts of grapes; polyphenol extracts of rosemary; polyphenol extracts of lemon balm; polyphenol extracts of blackcurrant; single PCA isolates and mixtures thereof.

[0048] "Polyphenol extract" is well known in the art and relates to an extract of the polyphenol fraction from a 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 a water- and ethanol-based extract.

[0049] In some preferred embodiments of the invention, the source containing one or more PCA contains at least 25% w / w of PCA relative to the total solids of the source containing one or more PCA, more preferably at least 40% w / w of the total solids of the source containing one or more PCA, even more preferably at least 60% w / w of the total solids of the source containing one or more PCA, and most preferably at least 80% w / w of the total solids of the source containing one or more PCA.

[0050] In another preferred embodiment of the invention, the source containing one or more PCA is a single PCA isolate containing at least 25% w / w, more preferably at least 40% w / w, even more preferably at least 60% w / w, and most preferably at least 80% w / w of the total solids of the source containing one or more PCA, relative to the total solids of the source containing one or more PCA.

[0051] Preferably, the source containing one or more PCA does not contribute bitterness or off-flavor to the protein composition containing modified BLG.

[0052] The term "source containing BLG" refers to the composition used to provide BLG to the protein solution in step c).

[0053] If more than one BLG-containing composition is used to produce the protein solution of step c) and / or the oxidizing aqueous solution of step b), then each of the BLG-containing compositions used is considered a sub-source constituting a BLG-containing source.

[0054] In some preferred embodiments of the invention, the source protein of the BLG is isolated from mammalian milk, and preferably from the milk of ruminant animals such as, for example, cows, sheep, goats, buffalo, camels, llamas, mares, and / or deer. Particularly preferred are proteins isolated from bovine (dairy cow) milk. Therefore, the BLG and other whey proteins are preferably bovine BLG and bovine whey protein. Optionally, but still preferably, the protein is prepared by microbial fermentation during the production of the recombinant BLG. If the BLG is recombinant, it is preferred that the BLG has a sequence similar to or even identical to that of BLG from ruminant animal sources such as, for example, cows, sheep, goats, buffalo, camels, llamas, mares, and / or deer.

[0055] Preferably, the source containing BLG includes or even consists of the following: whey protein concentrate, whey protein isolate, milk serum protein concentrate, milk serum protein isolate, BLG isolate, or a combination thereof.

[0056] The source of BLG can be in liquid or powder form, and its sub-sources can contain a combination of one or more BLG-containing powders and / or one or more BLG-containing liquids.

[0057] The term "whey" refers to the liquid phase remaining after casein in milk has been precipitated and removed. Casein precipitation can be achieved, for example, by acidification of the milk and / or by using rennet enzyme. Several types of whey exist, such as "sweet whey," a whey product produced by rennet-based precipitation of casein; and "acid whey" or "sour whey," a whey product produced by acid-based precipitation of casein. Acid-based precipitation of casein can be accomplished, for example, by adding food acid or by bacterial culture.

[0058] The term "milk serum" refers to the liquid remaining after casein and milk fat globules have been removed from milk, for example, through microfiltration or macroporous ultrafiltration. Whey can also be referred to as "ideal whey."

[0059] In the context of this invention, the term "whey protein" refers to proteins present in whey or milk serum. A whey protein can be a subset of proteinaceous substances present in whey or milk serum, and even a single whey protein substance, or it can be a whole suite of proteinaceous substances present in whey and / or milk serum.

[0060] In some preferred embodiments of the invention, the protein containing BLG is isolated from bovine whey or milk serum.

[0061] Ungraded whey proteins typically contain alpha-lactalbumin (ALA), beta-lactoglobulin (BLG), bovine serum albumin, immunoglobulins, osteopontin, lactoferrin, and lactoperoxidase. In addition to other proteins, whey proteins derived from rennet-treated milk also include casein macropeptide (CMP).

[0062] In the context of this invention, the term "whey protein concentrate (WPC)" refers to a dry or aqueous composition comprising a total amount of protein of 20% w / w to 89% w / w relative to total solids.

[0063] WPC preferably includes: Protein accounts for 30% w / w to 85% w / w of the total solids. BLG accounts for 15% w / w-90% w / w of total protein. ALA relative to 4% w / w-50% w / w of total protein, and CMP relative to protein: 0% w / w-40% w / w.

[0064] Most preferably, the WPC comprises: Compared to 70% w / w-85% w / w of protein in total solids, BLG accounts for 30% w / w-90% w / w of total protein. ALA relative to total protein is 4% w / w-35% w / w, and CMP relative to protein: 0% w / w-25% w / w.

[0065] WPCs based on lactose serum albumin typically contain no CMP or only trace amounts of CMP.

[0066] The term "whey protein isolate (WPI)" refers to a dry or aqueous composition comprising a total amount of protein of 86% w / w to 100% w / w relative to total solids.

[0067] WPI preferably includes: Compared to the total solids of 86% w / w-99% w / w protein, BLG, relative to 30% w / w-100% w / w of total protein, ALA relative to total protein of 0% w / w-35% w / w, and CMP relative to total protein: 0% w / w-25% w / w.

[0068] Preferably, the WPI includes: Relative to 90% w / w-99% w / w of total solids protein, BLG accounts for 50% w / w-99% w / w of total protein. ALA relative to total protein of 0% w / w-35% w / w, and CMP relative to total protein: 0% w / w-25% w / w.

[0069] WPIs based on lactose serum albumin typically contain no CMP or only trace amounts of CMP.

[0070] Of particular preference is whey protein source WPI.

[0071] In the context of this invention, the term "BLG isolate" refers to a composition comprising a BLG amount of at least 80% w / w, more preferably at least 90% w / w, and most preferably at least 95% w / w, relative to total solids.

[0072] BLG isolates can be prepared, for example, by isolating BLG from milk serum or whey, preferably according to WO2018115520 A1, or alternatively, by recombinantly preparing them through fermentation of genetically modified microorganisms or mammalian cells.

[0073] The source containing BLG preferably has a protein denaturation degree of up to 30%, more preferably up to 25%, even more preferably up to 20%, and most preferably up to 15%.

[0074] Even lower levels of protein denaturation are generally preferred, and in some preferred embodiments of the invention, the source containing BLG has a protein denaturation level of up to 12%, more preferably up to 10%, even more preferably up to 8%, and most preferably up to 5%.

[0075] In some preferred embodiments of the invention, the source containing BLG has a total fat content of up to 10% w / w relative to total solids, more preferably up to 8% w / w relative to total solids, and most preferably up to 6% w / w relative to total solids.

[0076] In other preferred embodiments of the invention, the source containing BLG has a total fat content of up to 5% w / w relative to total solids, more preferably up to 2% w / w relative to total solids, even more preferably up to 0.5% w / w relative to total solids, and most preferably up to 0.1% w / w relative to total solids.

[0077] In another preferred embodiment of the invention, the source containing BLG has a total fat content in the range of 1% w / w to 20% w / w relative to total solids, more preferably in the range of 2% w / w to 16% w / w relative to total solids, even more preferably in the range of 3% w / w to 12% w / w relative to total solids, and most preferably in the range of up to 4% w / w to 10% w / w relative to total solids.

[0078] In some preferred embodiments of the invention, the source containing BLG has a total carbohydrate content of up to 10% w / w relative to total solids, more preferably up to 8% w / w relative to total solids, even more preferably up to 6% w / w relative to total solids, and most preferably up to 5% w / w relative to total solids.

[0079] In another preferred embodiment of the invention, the source containing BLG has a total carbohydrate content of up to 2% w / w relative to total solids, more preferably up to 1% w / w relative to total solids, even more preferably up to 0.5% w / w relative to total solids, and most preferably up to 0.2% w / w relative to total solids.

[0080] Step b) is optional, meaning that in some preferred embodiments of the invention, the method includes b), while other preferred embodiments of the invention do not include step b), and thus oxidation of a source containing one or more PCA is indeed used prior to step c).

[0081] Therefore, in some preferred embodiments of the invention, a method for producing a protein composition comprising modified BLG includes step b), subjecting a portion of a source containing one or more PCA types to an oxidation capable of converting PCA to quinones (also referred to as PCA-type oxidation), thereby providing a source of PCA containing one or more oxidations. It should be understood that PCA-type oxidation must be capable of oxidizing the specific type of PCA specified in step a) to quinone compounds.

[0082] Note that in some cases, oxidized PCA is not PCA, but only contains quinone functional groups.

[0083] In some preferred embodiments of the invention, the oxidation in step b) involves the preparation of an oxidizing aqueous solution containing a source of one or more PCA, optionally a chemical oxidant, and optionally one or more other components such as water.

[0084] In the context of this invention, the term "oxidizing aqueous solution" is used to describe an aqueous solution in which the PCA-type oxidation of step b) occurs.

[0085] The oxidizing aqueous solution has a PCA content of preferably at least 0.2 mM, more preferably at least 15 mM, and most preferably at least 50 mM.

[0086] In some preferred embodiments of the invention, the oxidizing aqueous solution has a PCA concentration of 0.2 mM-600 mM, more preferably 15 mM-550 mM, and most preferably 50 mM-500 mM.

[0087] For some PCAs, it is advantageous if the oxidizing aqueous solution contains one or more amphiphilic modifiers to increase the solubility of the PCA. These one or more amphiphilic modifiers are preferably food-grade and / or pharmaceutical-grade amphiphilic modifiers. Preferred examples of amphiphilic modifiers are food-grade alcohols, such as, for example, ethanol, dimethyl sulfoxide (DMSO), polysorbate, esters of sorbitol and fatty acids, and mixtures thereof.

[0088] Useful examples of polysorbate esters may be selected, for example, from the group consisting of: polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80. Useful examples of esters of sorbitol and fatty acids may be selected, for example, from the group consisting of: sorbitol monolaurate, sorbitol monostearate, sorbitol tristearate, and sorbitol monooleate.

[0089] In some preferred embodiments of the 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.

[0090] The pH value mentioned in this article refers to the pH value standardized to 25°C.

[0091] The inventors have found that it can be advantageous to perform step b) using an oxidizing aqueous solution having a pH in the range of 2-6.4, more preferably 3.0-6.0, even more preferably 3.5-5.5, and most preferably 4.0-5.0.

[0092] In some preferred embodiments of the invention, the oxidizing aqueous solution contains one or more additional ingredients selected from the group consisting of: food-acceptable acids, food-acceptable bases, water, amphiphilic modifiers, chemical oxidants, oxidation catalysts, and combinations thereof.

[0093] If a source containing one or more PCA is provided in powder form, water is usually required as an additional component.

[0094] If the PCA-type oxidation in step b) is to be based partly or entirely on a chemical oxidant, then a chemical oxidant is required in an oxidizing aqueous solution. Even the use of a combination of several chemical oxidants is feasible.

[0095] If pH needs to be adjusted, then food-acceptable acids and / or food-acceptable bases are usually required.

[0096] Useful examples of oxidation catalysts are one or more enzymes, such as polyphenol oxidase, laccase, and / or tyrosinase.

[0097] In some preferred embodiments of the invention, the oxidation in step b) is carried out by contacting a portion of a source containing one or more PCA compounds with a chemical oxidizing agent under conditions that convert at least some of the PCA into quinone compounds. This oxidation is performed in an oxidizing aqueous solution.

[0098] Typically, it is preferred that the chemical oxidant contains or even consists of the following: peroxide, ozone, hydrogen peroxide, or a combination thereof.

[0099] In other preferred embodiments of the invention, the chemical oxidant includes or is composed of hydrogen peroxide, benzoyl peroxide, or a combination thereof.

[0100] In another preferred embodiment of the invention, the chemical oxidant includes or is composed of hydrogen peroxide, benzoyl peroxide, hydrogen peroxide, or combinations thereof.

[0101] In some preferred embodiments of the invention, the oxidation in step b) involves preparing an oxidizing aqueous solution containing a portion of a source containing one or more PCA, a chemical oxidant, and optionally one or more other components such as water.

[0102] PCA-type oxidation can also involve enzymatically catalyzed oxidation, such as that involving enzymes like polyphenol oxidase, laccase, and / or tyrosinase.

[0103] The dissolved hydrogen peroxide provided by the liquid used to prepare the oxidizing aqueous solution used in step b) and / or the protein solution in step c) can contribute to the trace oxidation of PCA, but this is generally insufficient to achieve the desired oxidation level required by the present invention.

[0104] Generally, it is preferred that the oxidizing aqueous solution does not contain proteins. 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 also does not contain molecules having amine groups.

[0105] In some preferred embodiments of the invention, the oxidizing aqueous solution contains less than 5% w / w, more preferably at most 1% w / w, and even more preferably at most 0.1% protein relative to its weight; and most preferably contains no protein at all.

[0106] In some preferred embodiments of the invention, step b) of the PCA-type oxidation involves electrochemical oxidation of a portion of a source containing one or more PCA compounds under conditions that convert at least some of the PCA into quinone compounds.

[0107] In some preferred embodiments of the invention, the oxidation in step b) involves preparing an oxidizing aqueous solution containing a portion of a source containing one or more PCA and optionally one or more components such as water.

[0108] Preferably, electrochemical oxidation is operated using a potential difference of -0.1 V to 1.5 V, more preferably 0.0 V to 1.2 V, and most preferably 0.0 V to 0.7 V.

[0109] Potential difference, also known as voltage, refers to the potential difference between the working electrode and the counter electrode used for electrochemical oxidation in PCA.

[0110] 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 V to 0.9 V, even more preferably 0 V to 0.8 V, and most preferably 0.2 V to 0.7 V.

[0111] In other preferred embodiments of the invention, the oxidizing aqueous solution has a pH in the range of 3-5, and electrochemical oxidation is performed using a potential difference of 0.1 V-1.3 V, more preferably 0.2 V-0.9 V, and most preferably 0.3 V-0.7 V.

[0112] To determine the appropriate potential difference for electrochemical oxidation, it is advantageous to determine the oxidation potential of PCA.

[0113] The required PCA oxidation potential can be determined, for example, by cyclic voltammetry, where the electrode potential is scanned from, for example, -0.2 V to +1.2 V. At a given application potential, the current increases as the PCA begins to oxidize at and / or near the working electrode surface. The current continues to increase with the potential ramp until one or more peak currents are reached when the oxidizable groups of the PCA near the electrode are depleted.

[0114] The oxidation potential determined at the peak current can be used for the electrochemical oxidation of PCA. If more than one peak is observed (as in the case of, for example, EGCG), the minimum applicable potential difference that produces the peak current and results in the formation of a sufficient amount of quinone compound, as determined by Analysis 2, is selected.

[0115] In some preferred embodiments of the invention, electrochemical oxidation is carried out as bulk electrolysis and involves maintaining the current at a constant potential while monitoring the decrease in current over time as PCA is oxidized to its quinone form. Preferably, PCA is quantitatively converted to its quinone form. When PCA is completely oxidized, the current can reach a value of zero.

[0116] Stirring during bulk electrolysis is advantageous, for example, to reduce diffusion limitations.

[0117] Bulk electrolysis can be carried out using a potentiostat and a two-electrode system, or preferably using a potentiostat and a three-electrode system comprising one or more working electrodes, one or more counter electrodes, and a reference electrode.

[0118] Preferably, the working electrode is an anode. Preferred examples of working electrodes are glassy carbon electrodes, boron-doped diamond electrodes, and graphite electrodes.

[0119] Preferably, the counter electrode is a cathode. The counter electrode is also referred to as an auxiliary electrode. The preferred counter electrode is a platinum electrode, more preferably a platinum electrode selected from coil-type platinum electrodes and platinum flagelectrodes.

[0120] Preferably, the reference electrode is a true reference electrode, or more preferably a quasi-reference electrode, which is also referred to as a pseudo-reference electrode. A preferred example of a quasi-reference electrode is an Ag / AgCl electrode.

[0121] In some preferred embodiments of the invention, electrochemical oxidation is carried out under nitrogen atmosphere.

[0122] The amount of quinone compounds formed during electrochemical oxidation can be determined by analysis 2. If analysis 2 is unavailable, alternatively, the content of quinone compounds can be determined using the GSH assay described in Example 2.

[0123] The PCA-type oxidation in step b) is carried out partially or completely by electrochemical oxidation. Preferably, the oxidizing aqueous solution also contains an electrolyte. Preferably, the oxidizing aqueous solution contains an electrolyte with a concentration of 0.01 M-0.5 M, more preferably 0.05 M-0.4 M, and most preferably 0.1 M-0.2 M. Preferably, the electrolyte used is food-grade, conductive, and contributes to a stable pH. In some preferred embodiments of the invention, the oxidizing aqueous solution contains one or more electrolytes selected from the group consisting of H₂SO₄, phosphoric acid, and acetic acid.

[0124] In some preferred embodiments of the invention, electrochemical oxidation is carried out at temperatures ranging from 2°C to 80°C, more preferably from 5°C to 60°C, even more preferably from 10°C to 40°C, and most preferably from 15°C to 30°C.

[0125] In other preferred embodiments of the invention, the electrochemical oxidation is carried out at a temperature in the range of 30°C-90°C, more preferably 40°C-90°C, even more preferably 50°C-90°C, and most preferably in the range of 60°C-90°C.

[0126] In another preferred embodiment of the invention, the electrochemical oxidation is carried out at a temperature in the range of 5°C-25°C, more preferably 10°C-25°C, even more preferably 15°C-25°C, and most preferably in the range of 18°C-25°C.

[0127] The duration of step b) depends on the specific composition of the oxidizing aqueous solution and the conditions used for PCA-type oxidation. If electrochemical oxidation is used, the oxidation process can be monitored using an electric current. When the current approaches zero at the operating potential difference, this is an indication of PCA depletion. The amount of quinone produced can be measured according to Analysis 2.

[0128] In step c) of the method, a portion of a source containing one or more PCA and / or a portion of a source containing one or more oxidized PCA is combined with a source containing BLG and optional other components to provide a protein solution having a pH in the range of 6.5-9.5.

[0129] In step c), the term "a portion of a source containing one or more oxidized PCA" refers to the actual portion of the source containing one or more oxidized PCA provided in step b) and used to prepare the protein solution. In some preferred embodiments of the invention, substantially all sources containing one or more oxidized PCA are used.

[0130] In step c), the term "part of a source containing one or more PCA" refers to the actual portion of the source containing one or more PCA used in step a) to prepare the protein solution. In some preferred embodiments of the invention, substantially all sources containing one or more PCA are used.

[0131] In some preferred embodiments of the invention, the protein solution has a pH in the range of 6.7-9.5, even more preferably 7.1-9.0, even more preferably 7.3-8.7, and most preferably 7.4-8.5.

[0132] The inventors have discovered that the pH range defined herein for protein solutions facilitates the selective modification of the free thiol groups of BLG and reduces the risk of undesirable reactions with the amine groups of BLG.

[0133] In some preferred embodiments of the invention, the protein solution has a pH in the range of 6.5-9.5 and a temperature of at least 20°C, more preferably at least 40°C, even more preferably at least 60°C, and most preferably at least 70°C.

[0134] In another preferred embodiment of the invention, the protein solution has a pH in the range of 7.1-9.0 and a temperature of at least 20°C, more preferably at least 30°C, even more preferably at least 50°C, and most preferably at least 60°C.

[0135] In a particularly preferred embodiment of the invention, the protein solution has a pH in the range of 7.3-8.7 and a temperature of at least 20°C, more preferably at least 30°C, even more preferably at least 50°C, and most preferably at least 60°C.

[0136] In a more preferred embodiment of the invention, the protein solution has a pH in the range of 7.4-8.5 and a temperature of at least 35°C, more preferably at least 40°C, even more preferably at least 45°C, and most preferably at least 50°C.

[0137] Preferably, the protein solution has a pH in the range of 6.5-9.5 and a temperature of 20°C-70°C, more preferably a pH in the range of 7.1-9.0 and a temperature of 20°C-60°C, even more preferably a pH in the range of 7.3-8.7 and a temperature of 20°C-60°C, and most preferably a pH in the range of 7.4-8.5 and a temperature of 35°C-50°C.

[0138] In other preferred embodiments of the invention, the protein solution has a pH in the range of 6.5-9.5 and a temperature of 3°C-90°C, more preferably a pH in the range of 7.1-9.0 and a temperature of 40°C-80°C, even more preferably a pH in the range of 7.3-8.7 and a temperature of 45°C-75°C, and most preferably a pH in the range of 7.4-8.5 and a temperature of 50°C-70°C.

[0139] In another preferred embodiment of the invention, the protein solution has a pH in the range of 6.5-9.5 and a temperature of 5°C-90°C, more preferably a pH in the range of 7.1-9.0 and a temperature of 8°C-80°C, even more preferably a pH in the range of 7.3-8.7 and a temperature of 10°C-75°C, and most preferably a pH in the range of 7.4-8.5 and a temperature of 15°C-70°C.

[0140] 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.

[0141] In another preferred embodiment of the invention, the protein solution has a BLG content of 0.5% w / w-30% w / w, more preferably 1% w / w-20% w / w, even more preferably 3% w / w-16% w / w, and most preferably 5% w / w-12% w / w.

[0142] In some preferred embodiments of the invention, the protein solution has a BLG content of at least 30% w / w relative to the total protein, more preferably at least 40% w / w relative to the total protein, even more preferably at least 45% w / w relative to the total protein, and most preferably at least 50% w / w relative to the total protein.

[0143] In another preferred embodiment of the invention, the protein solution has a BLG content of 30% w / w-99% w / w, more preferably 40% w / w-95% w / w, even more preferably 45% w / w-90% w / w, and most preferably 50% w / w-80% w / w, relative to the total protein.

[0144] In other preferred embodiments of the invention, the protein solution has a BLG content of at least 60% w / w relative to the total protein, more preferably at least 80% w / w relative to the total protein, even more preferably at least 90% w / w relative to the total protein, and most preferably at least 95% w / w relative to the total protein.

[0145] 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.

[0146] In another preferred embodiment of the invention, the protein solution has a BLG content of 30% w / w-99% w / w, more preferably 40% w / w-95% w / w, even more preferably 45% w / w-90% w / w, and most preferably 50% w / w-80% w / w, relative to the total solids.

[0147] In an alternative but also preferred embodiment 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.

[0148] In some preferred embodiments of the invention, the protein solution has a total fat content of up to 10% w / w relative to total solids, more preferably up to 8% w / w relative to total solids, and most preferably up to 6% w / w relative to total solids.

[0149] In other preferred embodiments of the invention, the protein solution has a total fat content of up to 5% w / w relative to total solids, more preferably up to 2% w / w relative to total solids, even more preferably up to 0.5% w / w relative to total solids, and most preferably up to 0.1% w / w relative to total solids.

[0150] In another preferred embodiment of the invention, the protein solution has a total fat content in the range of 1% w / w to 20% w / w relative to total solids, more preferably in the range of 2% w / w to 16% w / w relative to total solids, even more preferably in the range of 3% w / w to 12% w / w relative to total solids, and most preferably in the range of 4% w / w to 10% w / w relative to total solids.

[0151] The inventors have discovered that an advantage of this invention is that it induces less fatty acid oxidation compared to comparable methods used for thiol modification. Therefore, the method of this invention is suitable for mild BLG modification in fatty acid-rich systems.

[0152] In some preferred embodiments of the invention, the protein solution has a total carbohydrate content of up to 10% w / w relative to total solids, more preferably up to 8% w / w relative to total solids, even more preferably up to 6% w / w relative to total solids, and most preferably up to 5% w / w relative to total solids.

[0153] In another preferred embodiment of the invention, the protein solution has a total carbohydrate content of up to 2% w / w relative to total solids, more preferably up to 1% w / w relative to total solids, even more preferably up to 0.5% w / w relative to total solids, and most preferably up to 0.2% w / w relative to total solids.

[0154] In some preferred embodiments of the invention, the protein solution has a molar ratio 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, and is therefore prepared to have a molar ratio 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: - The original amount of PCA used to prepare the protein solution, and - The BLG content of the protein solution.

[0155] In other preferred embodiments of the invention, the protein solution has a molar ratio 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, and is therefore prepared to have a molar ratio 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: - The original amount of PCA used to prepare the protein solution, and - The BLG content of the protein solution.

[0156] In the context of this invention, the term "raw amount of PCA for preparing a protein solution" refers to the amount of a specific PCA mentioned in step a) for preparing the following: a portion of a source containing one or more PCA for preparing a protein solution in step c), and / or a portion of a source containing one or more oxidized PCA for preparing a protein solution in step c).

[0157] If step c) uses only a portion of the source containing one or more oxidized PCA obtained from step b), then “original amount of PCA for preparing protein solution” refers to the amount of PCA used to prepare that portion of the source containing one or more oxidized PCA.

[0158] If step c) uses only a portion of the source containing one or more PCA obtained from step a), then “original amount of PCA for preparing protein solution” refers to the amount of PCA used to prepare that portion of the source containing one or more PCA.

[0159] If step c) uses both a portion of a source containing one or more types of PCA obtained from step a) and a portion of a source containing one or more types of oxidized PCA obtained from step b), then “the original amount of PCA for preparing the protein solution” refers to the amount of PCA used to prepare that portion of the source containing one or more types of PCA and that portion of the source containing one or more types of oxidized PCA.

[0160] The PCA mentioned in steps b), c), d), and e) refers to the PCA defined in step a).

[0161] In some preferred embodiments of the invention, the protein solution has a molar ratio 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, and is prepared to have a molar ratio 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. - The original amount of PCA used to prepare the protein solution, and - The BLG content of the protein solution.

[0162] In another preferred embodiment of the invention, the protein solution has a molar ratio 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, and is therefore prepared to have a molar ratio 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: - The original amount of PCA used to prepare the protein solution, and - The BLG content of the protein solution.

[0163] In even more preferred embodiments of the invention, the protein solution has a molar ratio 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, and is prepared to have a molar ratio 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: - The original amount of PCA used to prepare the protein solution, and - The BLG content of the protein solution.

[0164] In some preferred embodiments of the invention, the protein solution has a molar ratio 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, and is prepared to have a molar ratio 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: - The original amount of PCA used to prepare the protein solution, and - The BLG content of the protein solution.

[0165] In another preferred embodiment of the invention, the protein solution has a molar ratio 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, and is therefore prepared to have a molar ratio 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: - The original amount of PCA used to prepare the protein solution, and - The BLG content of the protein solution.

[0166] In even more preferred embodiments of the invention, the protein solution has a molar ratio 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, and is prepared to have a molar ratio 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: - The original amount of PCA used to prepare the protein solution, and - The BLG content of the protein solution.

[0167] Protein solutions are prepared by combining and preferably mixing appropriate amounts of the sources described herein to obtain protein solutions with desired properties. Preferably, step c) is performed under conditions that do not cause significant protein denaturation.

[0168] It is generally preferred that at least step c) is mixed by inline mixing, especially when at least steps b), c) and d) are carried out as a continuous process.

[0169] In some preferred embodiments of the invention, step c) is performed under conditions that result in protein denaturation of less than 15%, more preferably less than 10%, even more preferably less than 5%, and most preferably less than 2% of the BLG from the source containing BLG.

[0170] Preferably, the protein solution is an aqueous solution. Preferably, at least 90% w / w, more preferably at least 95% w / w, and most preferably at least 99% w / w of the non-solid substance of the protein solution, i.e., the substance that does not contribute to the solid content of the protein solution, is water.

[0171] Preferably, the protein solution is prepared at a temperature that does not cause protein denaturation, said temperature preferably in the range of 0°C-70°C, more preferably in the range of 5°C-65°C, even more preferably in the range of 10°C-60°C, and most preferably in the range of 20°C-55°C.

[0172] In other preferred embodiments of the invention, the protein solution is prepared to have a temperature in the range of 0°C-50°C, more preferably in the range of 2°C-45°C, even more preferably in the range of 5°C-40°C, and most preferably in the range of 10°C-35°C. These temperature ranges are generally preferred if the dissolved hydrogen peroxide is expected to contribute significantly to the oxidation of PCA.

[0173] Preferably, the protein solution is prepared by mixing a portion of a source containing one or more PCA obtained from step a), a source containing BLG obtained from step a), and optional additional components.

[0174] In some preferred embodiments of the invention, the method does not include the step of oxidizing a portion of a source containing one or more PCA prior to step c), i.e., the method does not include step b).

[0175] In other preferred embodiments of the invention, the method includes step b), which involves oxidizing a portion of a source containing one or more PCA prior to preparing a protein solution, and the protein solution is prepared by mixing a portion of the source containing one or more oxidized PCA obtained from step b) with a source containing BLG and optional additional components.

[0176] It is generally preferred that most (if not all) of the PCA-type oxidation is carried out during step b).

[0177] In some preferred embodiments of the invention, less than 20% of PCA-type oxidation occurs during step d), more preferably up to 10%, even more preferably up to 5%, and most preferably up to 2% of PCA-type oxidation occurs during step d).

[0178] Preferably, the additional components include water, one or more acids, one or more bases, one or more pH buffers, one or more oxidants, or mixtures thereof.

[0179] In step d) of the method, the protein solution is incubated at a temperature sufficient to reduce the amount of free thiol groups in the protein solution to at most 10 μmol / g protein for a duration sufficient to reduce the amount of free thiol groups in the protein solution to at most 10 μmol / g protein. If the method does not involve step b), step d) also involves PCA-type oxidation capable of converting PCA to quinone. This oxidation can be achieved, for example, by a chemical oxidant added during the production of the protein solution and / or by electrochemical oxidation during step d). In some preferred embodiments of the invention, the method includes both step b) and PCA-type oxidation during step d).

[0180] In the context of this invention, the term "incubating protein solution" refers to the protein solution during step d).

[0181] In some preferred embodiments of the invention, incubation reduces the amount of free thiols in the protein solution to at most 9 μmol / g protein, more preferably at most 8 μmol / g protein, more preferably at most 5 μmol / g protein, even more preferably at most 3 μmol / g protein, and most preferably at most 2 μmol / g protein, and thus incubation is carried out to reduce the amount of free thiols in the protein solution to at most 9 μmol / g protein, more preferably at most 8 μmol / g protein, more preferably at most 5 μmol / g protein, even more preferably at most 3 μmol / g protein, and most preferably at most 2 μmol / g protein.

[0182] In other preferred embodiments of the invention, incubation reduces the amount of free thiols in the protein solution to 0.001-10 μmol / g protein, more preferably 0.01-9 μmol / g protein, even more preferably 0.02-8 μmol / g protein, even more preferably 0.03-5 μmol / g protein, even more preferably 0.04-3 μmol / g protein, and most preferably 0.04-2 μmol / g protein, and thus incubation is performed to reduce the amount of free thiols in the protein solution to 0.001-10 μmol / g protein, more preferably 0.01-9 μmol / g protein, even more preferably 0.02-8 μmol / g protein, even more preferably 0.03-5 μmol / g protein, even more preferably 0.04-3 μmol / g protein, and most preferably 0.04-2 μmol / g protein.

[0183] In some preferred embodiments of the invention, the incubation protein solution has a pH in the range of 6.7-9.5, even more preferably 7.1-9.0, even more preferably 7.3-8.7, and most preferably 7.4-8.5.

[0184] In some preferred embodiments of the invention, during the incubation in step d), the protein solution is subjected to a pressure in the range of 1-1000 bar, more preferably 20-500 bar, even more preferably 30-300 bar, and most preferably 40-200 bar.

[0185] In some preferred embodiments of the invention, during the incubation in step d), the protein solution has a temperature in the range of 0°C-160°C, more preferably 10°C-155°C, even more preferably 15°C-150°C, and most preferably 20°C-145°C.

[0186] In other preferred embodiments of the invention, the protein solution being incubated, i.e. the protein solution during step d), has a temperature in the range of 0°C-67°C, more preferably 10°C-65°C, even more preferably 15°C-60°C, and most preferably 20°C-55°C.

[0187] Preferably, the incubation protein solution has a temperature in the range of 5°C-65°C, more preferably 10°C-65°C, even more preferably 30°C-60°C, and most preferably 40°C-55°C.

[0188] In other preferred embodiments, the protein solution is incubated at a temperature in the range of 60°C-98°C, more preferably 65°C-95°C, and most preferably 70°C-90°C.

[0189] In some embodiments of the present invention, the pH of the incubated protein solution is in the range of 6.5-7.0, and the temperature of the incubated protein solution is in the range of 40°C-65°C, more preferably 45°C-65°C, even more preferably 50°C-65°C, and most preferably 55°C-65°C.

[0190] In other preferred embodiments of the invention, the pH of the incubation protein solution in step d) is in the range of 7.1-9.5, and the temperature of the incubation protein solution is in the range of 5°C-65°C, more preferably 10°C-65°C, even more preferably 30°C-60°C, and most preferably 40°C-55°C.

[0191] In another preferred embodiment of the invention, the pH of the incubation protein solution in step d) is in the range of 8.5-9.5, and the temperature of the incubation protein solution is in the range of 0°C-65°C, more preferably 0°C-50°C, even more preferably 0°C-30°C, and most preferably 5°C-25°C.

[0192] The inventors have found that, particularly preferably, the pH of the incubation protein solution in step d) is in the range of 7.5-8.5, and the temperature of the incubation protein solution is in the range of 5°C-60°C, more preferably 10°C-60°C, even more preferably 15°C-60°C, and most preferably 20°C-60°C. These ranges appear to favor both the selective modification of the free thiols of BLG and the relatively rapid reaction kinetics.

[0193] Furthermore, the inventors have found that it is particularly preferred that the pH of the incubation protein solution in step d) is in the range of 7.7-8.5, and the temperature of the incubation protein solution is in the range of 25°C-55°C, more preferably 30°C-55°C, even more preferably 35°C-50°C, and most preferably 35°C-45°C. These ranges appear to also benefit both the selective modification of the free thiols of BLG and the relatively rapid reaction kinetics.

[0194] In another preferred embodiment of the invention, the pH of the incubation protein solution in step d) is in the range of 7.5-8.5, and the temperature of the incubation protein solution is in the range of 60°C-98°C, more preferably 65°C-95°C, and most preferably 70°C-90°C.

[0195] In a more preferred embodiment of the invention, the pH of the incubation protein solution in step d) is in the range of 7.5-8.5, and the temperature of the incubation protein solution is in the range of 45°C-160°C, more preferably 50°C-155°C, and most preferably 55°C-150°C.

[0196] In some preferred embodiments of the invention, the incubation duration is up to 12 hours, more preferably up to 6 hours, even more preferably up to 3 hours, and most preferably up to 1 hour.

[0197] The advantage of the method of the present invention is that it can be operated with a relatively short incubation time. Therefore, in another preferred embodiment of the invention, the incubation duration 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.

[0198] The inventors have observed that even shorter incubation times are technically feasible. Therefore, in an even more preferred embodiment of the invention, the incubation duration 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.

[0199] In some preferred embodiments of the invention, the oxidation in step d) involves contacting the PCA with a chemical oxidant in a protein solution under conditions that convert at least some of the PCA into quinone compounds. Additional chemical oxidants may be added during the incubation in step d).

[0200] One or more of the chemical oxidants used in step b) and / or step d) should be capable of PCA-type oxidation during step d) and / or step b).

[0201] In another preferred embodiment of the invention, the chemical oxidant includes or is composed of the following: peroxide, ozone, hydrogen peroxide, or combinations thereof.

[0202] In a particularly preferred embodiment of the invention, the chemical oxidant includes or is composed of hydrogen peroxide, benzoyl peroxide, or a combination thereof.

[0203] In some preferred embodiments of the invention, the oxidation in step d) involves the electrochemical oxidation of the PCA in a protein solution under conditions that convert at least some of the PCA into quinone compounds.

[0204] The potential difference should be chosen to be high enough to allow PCA to be oxidized to quinone compounds, and can be determined as described above regarding step b).

[0205] Preferably, the electrochemical oxidation in step d) is performed using a potential difference of -0.1 V to 1.5 V, and most preferably -0.05 V to 0.7 V.

[0206] In some preferred embodiments of the invention, the protein solution has a pH in the range of 7.1-9.0, and electrochemical oxidation is performed using a potential difference of -0.1 V to 0.9 V, more preferably -0.05 V to 0.8 V, and most preferably 0 V to 0.7 V.

[0207] In other preferred embodiments of the invention, the protein solution has a pH in the range of 7.3-8.7, and electrochemical oxidation is performed using a potential difference of -0.1 V to 0.8 V, and most preferably 0 V to 0.7 V.

[0208] In another preferred embodiment of the invention, the protein solution has a pH in the range of 7.4-8.5, and electrochemical oxidation is performed using a potential difference of -0.1 V to 0.8 V, and most preferably -0.05 V to 0.7 V.

[0209] In addition to the acidic pH range suggested in the context of step b), the features and implementation schemes related to the implementation of electrochemical oxidation described in the context of step b) also apply to the electrochemical oxidation in step d).

[0210] In some preferred embodiments of the invention, the temperature of the first stage of incubation is in the range of 0°C-60°C, more preferably 10°C-50°C, and then in the second stage of incubation, the incubated protein solution is heated to a temperature in the range of 70°C-160°C, more preferably 100°C-150°C.

[0211] If the incubated protein solution is to be used as a packaged ready-to-drink beverage and must be filled into a suitable container immediately after step d), then using high temperature during step d) is particularly useful.

[0212] In some preferred embodiments of the invention, step d) further includes adding additional PCA to the incubated protein solution.

[0213] In some preferred embodiments of the invention, the molar ratios between the following items are 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: - The total amount of PCA supplied to the protein solution in steps c) and d), and - The amount of BLG supplied to the protein solution in steps c) and d).

[0214] In another preferred embodiment, the molar ratio between the following items 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: - The total amount of PCA supplied to the protein solution in steps c) and d), and - The amount of BLG supplied to the protein solution in steps c) and d).

[0215] In even more preferred embodiments, the molar ratio between the following items 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: - The total amount of PCA supplied to the protein solution in steps c) and d), and - The amount of BLG supplied to the protein solution in steps c) and d).

[0216] In some preferred embodiments, the molar ratio between the following items 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: - The total amount of PCA supplied to the protein solution in steps c) and d), and - The amount of BLG supplied to the protein solution in steps c) and d).

[0217] In another preferred embodiment, the molar ratio between the following items 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: - The total amount of PCA supplied to the protein solution in steps c) and d), and - The amount of BLG supplied to the protein solution in steps c) and d).

[0218] In even more preferred embodiments of the invention, the molar ratio between the following items 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: - The total amount of PCA supplied to the protein solution in steps c) and d), and - The amount of BLG supplied to the protein solution in steps c) and d).

[0219] In some preferred embodiments of the invention, the PCA-type oxidation applied during step b) and / or step d) is sufficient to produce a molar ratio of at least 0.1:1, more preferably at least 0.2:1, between the following: - Based on the theoretical amount of quinone supplied to the protein solution during the method determined by Analysis 1, and - The BLG content of the protein solution.

[0220] In another preferred embodiment of the invention, the oxidation applied during step b) and / or step d) is sufficient to produce a molar ratio between 0.1:1 and 100:1, more preferably 0.1:1 and 50:1, even more preferably 0.2:1 and 15:1, and most preferably 0.2:1 and 5:1: - Based on the theoretical amount of quinone supplied to the protein solution during the method determined by Analysis 1, and - The BLG content of the protein solution.

[0221] In even more preferred embodiments of the invention, the oxidation applied during step b) and / or step d) is sufficient to produce a molar ratio 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: - Based on the theoretical amount of quinone supplied to the protein solution during the method determined by Analysis 1, and - The BLG content of the protein solution.

[0222] In other preferred embodiments of the invention, the PCA-type oxidation applied during step b) and / or step d) is sufficient to produce a molar ratio 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 among the following: - Based on the theoretical amount of quinone supplied to the protein solution during the method determined by Analysis 1, and - The BLG content of the protein solution.

[0223] In another preferred embodiment of the invention, the oxidation applied during step b) and / or step d) is sufficient to produce a molar ratio between 1:1 and 100:1, more preferably 1:1 and 50:1, even more preferably 1:1 and 15:1, and most preferably 1.5:1 and 5:1: - Based on the theoretical amount of quinone supplied to the protein solution during the method determined by Analysis 1, and - The BLG content of the protein solution.

[0224] If PCA oxidation occurs primarily or only in step b), it is generally preferred that the PCA-type oxidation applied during step b) is sufficient to produce a molar ratio 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: The quinone content of the portion of the source containing one or more oxidized PCA used in the protein solution, and - The BLG content of the protein solution.

[0225] This is, for example, when the PCA-type oxidation performed during step d) accounts for less than 20% of the total PCA-type oxidation.

[0226] In the preferred embodiments of the invention described above, more preferably, the oxidation applied during step b) is sufficient to produce a molar ratio between 1:1 and 100:1, more preferably 1:1 and 50:1, even more preferably 1:1 and 15:1, and most preferably 1.5:1 and 5:1: The quinone content of the portion of the source containing one or more oxidized PCA used in the protein solution, and - The BLG content of the protein solution.

[0227] In some preferred embodiments of the invention, the method does not involve oxidation of a portion of a source containing one or more PCA prior to step c), i.e., there is no step b), and the PCA-type oxidation is carried out by a chemical oxidant.

[0228] Optionally, but still preferred, the method does not involve the oxidation of a portion of a source containing one or more PCA prior to step c), i.e., there is no step b), and the oxidation of PCA is carried out by electrochemical oxidation.

[0229] In some preferred embodiments of the invention, the method includes step b), and a portion of the source of PCA containing one or more oxidations obtained from step b) is used to prepare the protein solution of step c), and the oxidation of PCA is carried out by a chemical oxidant.

[0230] In another preferred embodiment of the invention, the method includes step b), and a portion of the source of PCA containing one or more oxidations obtained from step b) is used to prepare the protein solution of step c), and the oxidation of PCA is carried out by electrochemical oxidation.

[0231] In other preferred embodiments of the invention, the method includes oxidation of PCA during step d), and preferably involves oxidizing PCA by electrochemical oxidation during step d).

[0232] If PCA oxidation occurs primarily or only in step d), it is generally preferred that the PCA-type oxidation applied during step d) is sufficient to produce a molar ratio 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: - Based on the theoretical amount of quinone supplied to the protein solution during the method determined by Analysis 1, and - The BLG content of the protein solution.

[0233] This is, for example, when the PCA oxidation performed during step d) accounts for at least 50% of the PCA oxidation during the process.

[0234] In other preferred embodiments of the invention, the PCA-type oxidation applied during step d) is sufficient to produce a molar ratio between 0.1:1 and 100:1, more preferably 0.1:1 and 50:1, even more preferably 0.2:1 and 15:1, and most preferably 0.2:1 and 5:1: - Based on the theoretical amount of quinone supplied to the protein solution during the method determined by Analysis 1, and - The BLG content of the protein solution.

[0235] In even more preferred embodiments of the invention, the PCA-type oxidation applied during step d) is sufficient to produce a molar ratio 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: - Based on the theoretical amount of quinone supplied to the protein solution during the method determined by Analysis 1, and - The BLG content of the protein solution.

[0236] In some preferred embodiments of the invention, the PCA-type oxidation applied during step d) is sufficient to produce a molar ratio 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 among the following: - Based on the theoretical amount of quinone supplied to the protein solution during the method determined by Analysis 1, and - The BLG content of the protein solution.

[0237] In the preferred embodiments of the invention described above, more preferably, the PCA-type oxidation applied during step d) is sufficient to produce a molar ratio between 1:1 and 100:1, more preferably 1:1 and 50:1, even more preferably 1:1 and 15:1, and most preferably 1.5:1 and 5:1: - Based on the theoretical amount of quinone supplied to the protein solution during the method determined by Analysis 1, and - The BLG content of the protein solution.

[0238] Step d) may also include a step of reducing the levels of unreacted and oxidized PCA in the incubated protein solution. This may involve, for example, subjecting the incubated protein solution to a separation step, such as dialysis, nanofiltration, ultrafiltration, or chromatography. Ultrafiltration is particularly preferred and may be performed in conjunction with dialysis.

[0239] In some preferred embodiments of the present invention, the product obtained by step d) is a protein concentrate of an incubated protein solution.

[0240] In some preferred embodiments of the invention, the method includes step e): drying a liquid feed, the liquid feed containing at least proteins derived from the incubated protein solution of step d).

[0241] In other preferred embodiments of the invention, the method includes step e): drying the liquid feed, which contains at least a solid derived from the incubated protein solution of step d).

[0242] In some preferred embodiments of the invention, the liquid feed for drying comprises a protein solution or a protein concentrate thereof obtained from step d), or even consists of a protein solution or a protein concentrate thereof obtained from step d).

[0243] Particularly preferred is that the liquid feed for drying is a protein concentrate of the protein solution obtained in step d).

[0244] In the context of this invention, the "protein concentrate" of the first liquid is a second liquid wherein at least the protein is derived from the first liquid, but the second liquid has a higher protein content relative to total solids than the first liquid. Preferably, substantially all the solids of the protein concentrate are derived from the first liquid. The "protein concentrate" of the first liquid is preferably prepared by ultrafiltration, nanofiltration, reverse osmosis, and / or evaporation. Protein concentration ultrafiltration and / or nanofiltration can be carried out, for example, in conjunction with percolation, to wash out some small non-protein solids. The "protein concentrate" contains the same protein material as the first liquid and preferably has the same whey protein content relative to total protein weight percentage as the first liquid. The provision of the protein concentrate may also involve one or more pH adjustments.

[0245] In other preferred embodiments of the invention, a liquid feed is prepared by subjecting the incubated protein solution of step d) to one or more of the following: - pH adjustment, - Concentration steps, - Percolation, and - Heat treatment.

[0246] In some preferred embodiments of the invention, the preparation of the liquid feed in step e) involves subjecting the protein solution obtained from step d), i.e., the incubated protein solution, to ultrafiltration / percolation, preferably to a degree sufficient to reduce the weight percentage of free phenolic compounds relative to the total solids by at least 30%, more preferably at least 50%, even more preferably at least 80%, and most preferably at least 90%. Preferably, such ultrafiltration / percolation is performed using an ultrafiltration membrane that retains the protein during operation but allows molecules with a molecular weight less than 2000 Da to permeate.

[0247] In some preferred embodiments of the invention, step e) also involves pH adjustment, which provides a liquid feed having a pH in the range of 6.0-8.5 and most preferably 6.5-7.5.

[0248] Preferably, step e) involves the spray-drying liquid feed.

[0249] Useful examples of spray drying are described, for example, in WO 2018 / 115520 A1.

[0250] The method of the present invention can be implemented as a batch process, a semi-batch process, or a continuous process. A continuous process is particularly preferred.

[0251] In some preferred embodiments of the present invention, at least steps b), c), and d) are performed as a continuous process.

[0252] Aspects of the present invention relate to protein compositions comprising modified BLG and having at most 10 μmol free thiol groups / g protein, said protein compositions being obtainable by the method of the present invention, and preferably having one or more of the following: - Protein content of at least 30% w / w relative to total solids. - At least 0.7% w / w tryptophan content relative to total protein. - Methionine content of at least 0.3% w / w relative to total protein. - Kyrenine content of up to 0.2 μg / mg protein, - Preferably, the fat content is at most 3% w / w relative to the total solids. - Preferably, the content of protein-bound sulfur is in the range of 100-600 μmol / g protein. - Preferably, the content of cysteine ​​residues that form disulfide bonds in protein-bound proteins is in the range of 150-400 μmol / g protein.

[0253] In some preferred embodiments of the invention, the protein composition containing modified BLG is a protein solution obtained from step d), i.e., an incubated protein solution.

[0254] In other preferred embodiments of the invention, the protein composition comprising modified BLG is prepared as a liquid feed during step e).

[0255] In another preferred embodiment of the invention, the protein composition containing modified BLG is the powder of step e). Therefore, it is generally preferred that the protein composition containing modified BLG be obtained by steps a), c), d), and e) of this method.

[0256] In some preferred embodiments of the invention, the protein in the protein composition has an average molecular weight in the range of 18 kDa to 10,000 kDa, more preferably between 50 and 8,000 kDa, and most preferably between 80 and 5,000 kDa.

[0257] In a particularly preferred embodiment of the invention, the protein composition has an average molecular weight of protein in the range of 18 kDa to 500 kDa, more preferably between 18 kDa and 100 kDa, and most preferably between 18 kDa and 40 kDa.

[0258] Protein compositions typically have a pH in the range of 5.5–9.5.

[0259] 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.

[0260] 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 relative to the total solids of the protein composition, even more preferably at least 75% w / w relative to the total solids of the protein composition, and most preferably at least 85% w / w relative to the total solids of the protein composition.

[0261] Preferably, the protein composition has a total protein content in the range of 30% w / w-99% w / w, more preferably 50% w / w-97% w / w, even more preferably 75% w / w-96% w / w, and most preferably at least 85% w / w-95% w / w relative to the total solids of the protein composition.

[0262] In some preferred embodiments of the invention, the protein composition has a total fat content of up to 10% w / w relative to total solids, more preferably up to 8% w / w relative to total solids, and most preferably up to 6% w / w relative to total solids.

[0263] In other preferred embodiments of the invention, the protein composition has a total fat content of up to 5% w / w relative to total solids, more preferably up to 2% w / w relative to total solids, even more preferably up to 0.5% w / w relative to total solids, and most preferably up to 0.1% w / w relative to total solids.

[0264] In another preferred embodiment of the invention, the protein composition has a total fat content in the range of 1% w / w to 20% w / w relative to total solids, more preferably in the range of 2% w / w to 16% w / w relative to total solids, even more preferably in the range of 3% w / w to 12% w / w relative to total solids, and most preferably in the range of up to 4% w / w to 10% w / w relative to total solids.

[0265] Even lower levels of fat are generally preferred, and it is generally preferred that the protein composition has a total fat content of up to 1% w / w relative to total solids, more preferably up to 0.5% w / w relative to total solids, even more preferably up to 0.2% w / w relative to total solids, and most preferably up to 0.1% w / w relative to total solids.

[0266] Protein compositions can contain various amounts of carbohydrates.

[0267] However, it is generally preferred that the protein composition has a carbohydrate content of up to 65% w / w relative to the total solids.

[0268] Even lower levels of carbohydrates are generally preferred, and it is generally preferred that the protein composition has a carbohydrate content of up to 20% w / w relative to total solids, more preferably up to 8% w / w relative to total solids, even more preferably up to 2% w / w relative to total solids, and most preferably up to 0.2% w / w relative to total solids.

[0269] The protein composition preferably has an ash content of up to 8% w / w, more preferably up to 6% w / w, even more preferably up to 5% and most preferably up to 4.0% relative to total solids.

[0270] In some preferred embodiments of the invention, the protein composition has an ash content of 0.4% w / w-8% w / w, more preferably 0.5% w / w-6% w / w, even more preferably 0.5% w / w-5% w / w, and most preferably 0.6% w / w-4.0% w / w, relative to total solids.

[0271] The protein composition preferably has a combined content of magnesium and calcium of up to 1% w / w, more preferably up to 0.7% w / w, even more preferably up to 0.5%, and most preferably up to 0.2% relative to the total solids.

[0272] In some preferred embodiments of the invention, the protein composition has a combined content of magnesium and calcium of 0.01% w / w to 1% w / w relative to total solids, more preferably 0.001% w / w to 0.7% w / w relative to total solids, even more preferably 0.01% w / w to 0.5% w / w relative to total solids, and most preferably 0.01% w / w to 0.2% w / w relative to total solids.

[0273] The inventors have observed that in heat-treated whey protein beverages containing 3% whey protein, protein compositions containing even up to 15 μmol of free thiol groups / g protein can provide a reduced level of unpleasant odor compared to unmodified whey protein.

[0274] In some preferred embodiments of the invention, the protein composition comprises a free thiol group in an amount of up to 15 μmol / g protein, more preferably up to 14 μmol / g protein, even more preferably up to 13 μmol / g protein, and most preferably up to 12 μmol / g protein.

[0275] In some preferred embodiments of the invention, the protein composition comprises 0.001-15 μmol / g protein, more preferably 0.01-14 μmol / g protein, even more preferably 0.01-13 μmol / g protein, and most preferably 0.01-12 μmol / g protein of free thiol groups.

[0276] However, it is generally preferred, especially when the protein composition is intended for use in heat-treated high-protein beverages, such as heat-treated high-protein beverages containing 6% or more whey protein, that the protein composition contains a low level of free thiol groups. Therefore, in some preferred embodiments of the invention, the protein composition contains free thiol groups in amounts of up to 10 μmol / g protein, more preferably up to 8 μmol / g protein, more preferably up to 5 μmol / g protein, even more preferably up to 3 μmol / g protein, and most preferably up to 2 μmol / g protein.

[0277] Preferably, the protein composition contains 0.01-10 μmol / g protein, more preferably 0.01-8 μmol / g protein, even more preferably 0.01-5 μmol / g protein, even more preferably 0.01-3 μmol / g protein, and most preferably 0.01-2 μmol / g protein of free thiol groups.

[0278] Even lower levels of free thiol groups may be desired, and in some preferred embodiments of the invention, the protein composition contains free thiol groups in amounts of up to 1 μmol / g protein, more preferably up to 0.7 μmol / g protein, even more preferably up to 0.5 μmol / g protein, and most preferably up to 0.2 μmol / g protein.

[0279] In some preferred embodiments of the invention, the protein composition has a tryptophan content of at least 0.7% w / w relative to the total protein, more preferably at least 0.8% w / w relative to the total protein, even more preferably at least 0.9% w / w relative to the total protein, and most preferably at least 1.0% w / w relative to the total protein.

[0280] Preferably, the protein composition has a tryptophan content of 0.7% w / w-3% w / w relative to the total protein, more preferably 0.8% w / w-2.6% w / w relative to the total protein, even more preferably 0.9% w / w-2.4% w / w relative to the total protein, and most preferably 1.0% w / w-2.2% w / w relative to the total protein.

[0281] Optionally, but still preferred, the protein composition typically has a tryptophan content of 0.7% w / w-3% w / w relative to the total protein, more preferably 0.8% w / w-3% w / w relative to the total protein, even more preferably 0.9% w / w-3% w / w relative to the total protein, and most preferably 1.0% w / w-3% w / w relative to the total protein.

[0282] In some preferred embodiments of the invention, the protein composition has a methionine content of at least 0.3% w / w relative to the total protein, more preferably at least 0.4% w / w relative to the total protein, even more preferably at least 0.5% w / w relative to the total protein, and most preferably at least 0.6% w / w relative to the total protein.

[0283] Preferably, the protein composition has a methionine content of 0.3% w / w to 3.3% w / w relative to the total protein, more preferably 0.4% w / w to 3.2% w / w relative to the total protein, even more preferably 0.5% w / w to 3.2% w / w relative to the total protein, and most preferably 0.6% w / w to 3.2% w / w relative to the total protein.

[0284] The lower limit of the increase in methionine is generally preferred, and in some preferred embodiments of the invention, the protein composition has a methionine content of 1.0% w / w-3.3% w / w relative to the total protein, more preferably 1.3% w / w-3.2% w / w relative to the total protein, even more preferably 1.6% w / w-3.2% w / w relative to the total protein, and most preferably 1.8% w / w-3.2% w / w relative to the total protein.

[0285] Preferably, the protein composition has a kynurenine content of up to 0.2 μg / mg protein, more preferably up to 0.05 μg / mg protein, even more preferably up to 0.01 μg / mg protein, and most preferably up to 0.001 μg / mg protein. Particularly preferred is that the protein composition does not contain detectable kynurenine.

[0286] The content of kynurenine was quantified according to Poojary et al.; “Selective and sensitive UHPLC-ESI-Orbitrap MS method to quantify protein oxidation markers”; Talanta, Vol. 234, November 1, 2021 (available online in July 2021).

[0287] Kyrenine is a useful marker of tryptophan oxidation. The inventors believe that kyrenine is partly due to the yellow color produced in heat-sterilized protein drinks containing proteins that have undergone excessive oxidation. Furthermore, from a health perspective, kyrenine is undesirable.

[0288] Preferably, the protein composition has a protein-bound sulfur content in the range of 100-600 μmol / g protein, more preferably in the range of 200-500 μmol / g protein, and most preferably in the range of 250-500 μmol / g protein.

[0289] Preferably, the protein composition has a content of protein-bound cysteine ​​residues that form disulfide bonds in the range of 150-400 μmol / g protein, more preferably 160-350 μmol / g protein, and most preferably 170-300 μmol / g protein.

[0290] The inventors have found that it is advantageous for the protein composition to have a protein particle size of no more than 10,000 kDa, and preferably smaller, to avoid opacity in transparent beverage applications and further to avoid viscosity increase during the concentration and drying of the protein.

[0291] In some preferred embodiments of the invention, the protein composition has a protein weight-average molecular weight in the range of 18 kDa to 10,000 kDa, more preferably 30-9,000 kDa, even more preferably 50-8,000 kDa, and most preferably 80-5,000 kDa.

[0292] Preferably, at least 60% w / w, more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 99% w / w of the protein composition have a molecular weight between 18 kDa and 10,000 kDa.

[0293] More preferably, at least 60% w / w, more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 99% w / w of the protein composition have a molecular weight between 50 kDa and 8000 kDa.

[0294] Even more preferably, at least 60% w / w, more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 99% w / w of the protein composition have a molecular weight between 80 kDa and 5000 kDa.

[0295] In other preferred embodiments of the invention, the protein composition has a protein weight-average molecular weight in the range of 18 kDa to 200 kDa, more preferably between 30 kDa and 150 kDa, and most preferably between 30 kDa and 100 kDa.

[0296] The inventors have discovered that the smaller the weight-average molecular weight of the protein, the higher the total protein concentration is possible during concentration, such as by ultrafiltration or nanofiltration, prior to spray drying.

[0297] Preferably, at least 60% w / w, more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 99% w / w of the protein composition have a molecular weight between 18 kDa and 200 kDa.

[0298] More preferably, at least 60% w / w, more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 99% w / w of the protein composition have a molecular weight between 18 kDa and 150 kDa.

[0299] Even more preferably, at least 60% w / w, more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 99% w / w of the protein composition have a molecular weight between 18 kDa and 100 kDa.

[0300] The inventors have observed that it may be beneficial for the important proteins in the protein composition to have a molecular weight of at least 30 kDa, possibly due to the dimerization of the modified BLG.

[0301] Therefore, preferably, at least 60% w / w, more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 99% w / w of the protein composition have a molecular weight between 30 kDa and 200 kDa.

[0302] More preferably, at least 60% w / w, more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 99% w / w of the protein composition have a molecular weight between 30 kDa and 150 kDa.

[0303] Even more preferably, at least 60% w / w, more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 99% w / w of the protein composition have a molecular weight between 30 kDa and 100 kDa.

[0304] In some preferred embodiments of the invention, the protein compositions of the invention can be obtained by the methods described herein.

[0305] In some preferred embodiments of the invention, the protein composition is in liquid form, and preferably in aqueous liquid form. The liquid protein composition preferably has a solids content of 0.1% w / w-50% w / w, more preferably 1% w / w-35% w / w, even more preferably 5% w / w-30% w / w, and most preferably 10% w / w-30% w / w.

[0306] In some preferred embodiments of the invention, the protein composition is in solid form, and preferably in powder form, which has preferably been prepared by spray drying. The protein composition in powder form preferably has a solid 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.

[0307] The portion of the protein composition and oxidized whey protein solution that does not contribute to the solids content is preferably water.

[0308] The portion of the protein composition that does not contribute to the solids content preferably contains at least 80% w / w, more preferably at least 90% w / w, even more preferably at least 95% w / w, and even more preferably at least 99% w / w of water.

[0309] In a particularly preferred embodiment of the invention, the protein composition has: - Protein content of at least 86% w / w relative to total solids, and most preferably at least 90% relative to total solids. - Fat content of up to 1% w / w and most preferably up to 0.2% w / w relative to total solids. - Up to 10 μmol free thiol groups / g protein, and most preferably up to 5 μmol free thiol groups / g protein. - Tryptophan content relative to 0.7% w / w-3% w / w of total protein, and most preferably 1.0% w / w-3% w / w of total protein. - Methionine content of 0.3% w / w to 3.3% w / w relative to total protein, and most preferably 1.3% w / w to 3.2% w / w relative to total protein. - The kynurenine content is up to 0.2 μg / mg of protein, and most preferably up to 0.01 μg / mg of protein.

[0310] In the particularly preferred embodiments mentioned above, the oxidized whey protein composition preferably has: - The content of protein-bound sulfur in the range of 100-600 μmol / g protein, and - The content of cysteine ​​residues that form disulfide bonds in protein, within the range of 150-400 μmol / g protein.

[0311] Furthermore, in the particularly preferred embodiments of the present invention described above, the protein composition preferably comprises: - The content of protein-bound sulfur in the range of 100-600 μmol / g protein, and - The content of cysteine ​​residues that form disulfide bonds in protein, within the range of 150-400 μmol / g protein.

[0312] Furthermore, it is generally preferred that at least 60% w / w, more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 99% w / w of the protein composition of the particularly preferred embodiments mentioned above have a molecular weight between 30 kDa and 9000 kDa.

[0313] The pH of the particularly preferred embodiments of the protein composition mentioned above is preferably in the range of 6.2-8.0 and most preferably 6.5-7.5.

[0314] In some preferred embodiments of the invention, the protein composition is a sterile protein composition, and preferably, a packaged sterile protein composition. Preferably, the protein composition is in the form of a sterile liquid protein composition or a sterile powdered protein composition.

[0315] Another aspect of the present invention relates to a process for producing a heat-treated, preferably heat-sterilized, beverage, the process comprising the following steps: 1) The protein composition of the present invention is mixed with one or more other beverage ingredients to obtain a liquid mixture having a pH of 5.5-8.5, and 2) Fill the liquid mixture into a suitable container. The process further includes at least one heat treatment step, wherein the liquid mixture is heat-treated before and / or after filling, and preferably heat-sterilized.

[0316] In some preferred embodiments of the invention, the beverage mixture contains a protein composition sufficient to contribute at least 0.5% w / w protein.

[0317] Therefore, a more specific aspect of the present invention relates to a process for producing a heat-treated and preferably heat-sterilized beverage having a pH of 5.5-8.5, more preferably 6.5-7.5, comprising: 1) Combining the protein composition as described herein with one or more additional ingredients to obtain a liquid mixture having a pH of 5.5-8.5, more preferably 6.5-7.5, and comprising: - A protein composition sufficient to contribute at least 0.5% w / w protein, and - water, 2) The liquid mixture is packaged in a container, preferably a sterile container, and The liquid mixture is heat-treated before and / or after packaging, and preferably heat-sterilized.

[0318] The protein composition described herein is preferably the sole protein source for food products or heat-sterilized beverages, and therefore also the sole protein source for liquid mixtures.

[0319] The inventors have discovered that it is advantageous to keep the content of free thiol groups in the liquid mixture low before heat treatment to prevent the formation of an unpleasant odor similar to rotten eggs.

[0320] Therefore, in some preferred embodiments of the invention, prior to heat sterilization, the liquid mixture contains up to 60 μmol free thiol groups / 100 g liquid mixture, more preferably up to 40 μmol free thiol groups / 100 g liquid mixture, even more preferably up to 30 μmol free thiol groups / 100 g liquid mixture, and most preferably up to 30 μmol free thiol groups / 100 g liquid mixture.

[0321] Typically, even lower levels of free thiol groups are required, and in some preferred embodiments of the invention, prior to heat sterilization, the liquid mixture contains up to 20 μmol free thiol groups / 100 g liquid mixture, more preferably up to 15 μmol free thiol groups / 100 g liquid mixture, even more preferably up to 10 μmol free thiol groups / 100 g liquid mixture, and most preferably up to 5 μmol free thiol groups / 100 g liquid mixture.

[0322] The liquid mixture preferably contains a total amount of protein in the range of 0.5% w / w-15% w / w, more preferably 1% w / w-10% w / w, even more preferably 2% w / w-9% w / w, and most preferably 3% w / w-8% w / w, relative to the weight of the liquid mixture.

[0323] Optionally, but preferably, the liquid mixture may contain a total amount of protein in the range of 4% w / w-15% w / w, more preferably 5% w / w-14% w / w, even more preferably 6% w / w-13% w / w, and most preferably 8% w / w-12% w / w, relative to the weight of the liquid mixture.

[0324] The protein composition of the present invention preferably contributes 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.

[0325] Even higher contributions are generally preferred, and in some preferred embodiments of the invention, the protein composition of the invention contributes 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.

[0326] If the protein composition is used in combination with other protein sources, it is preferable to use a source with a relatively low content of free thiol groups.

[0327] In some preferred embodiments of the invention, the liquid mixture contains total protein in an amount of at least 15% w / w, 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.

[0328] For example, when a beverage is intended as a sports protein drink, total protein can contribute an even larger portion of the total solids. Therefore, in some preferred embodiments of the invention, the liquid mixture contains at least 80% w / w, more preferably at least 90% w / w, even more preferably at least 92% w / w, and most preferably at least 94% w / w, of total protein relative to the total solids.

[0329] The liquid mixture typically has a solids content of 0.5% w / w-50% w / w, more preferably 1% w / w-35% w / w, even more preferably 2% w / w-20% w / w, and most preferably 3% w / w-10% w / w.

[0330] The portion of the liquid mixture that does not consist of solids preferably contains water. The portion of the liquid mixture that does not consist of solids preferably contains at least 80% w / w, more preferably at least 90% w / w, even more preferably at least 95% w / w, and even more preferably at least 99% w / w, of water.

[0331] In some preferred embodiments of the invention, the liquid mixture has a calorie content of up to 100 kcal / 100 g, more preferably up to 80 kcal / 100 g, even more preferably up to 70 kcal / 100 g, and most preferably up to 60 kcal / 100 g. Preferably, the liquid mixture may have a calorie content of 2-100 kcal / 100 g, more preferably 4-80 kcal / 100 g, even more preferably 8-70 kcal / 100 g, and most preferably 12-60 kcal / 100 g. These embodiments are preferred for sports applications, for example, where the protein source is the primary energy source.

[0332] In other preferred embodiments of the invention, the liquid mixture has a caloric content greater than 100 kcal / 100 g, more preferably at least 120 kcal / 100 g, even more preferably at least 140 kcal / 100 g, and most preferably at least 150 kcal / 100 g. Preferably, the liquid mixture may have a caloric content of 101-300 kcal / 100 g, more preferably 120-280 kcal / 100 g, even more preferably 140-270 kcal / 100 g, and most preferably 150-260 kcal / 100 g. These embodiments are preferred for, for example, clinical nutrition in which the protein source is accompanied by a large amount of carbohydrates and fats.

[0333] The compositional characteristics and preferences described in the context of heat-treated beverages as described on pages 65-81 of PCT application number PCT / EP2022 / 078739 also apply to liquid mixtures.

[0334] The pH of a liquid mixture can range from slightly acidic to slightly alkaline.

[0335] Liquid mixtures with near-neutral pH are particularly preferred for the production of near-neutral pH beverages. In some preferred embodiments of the invention, the liquid mixture has a pH in the range of 5.5-8.0, more preferably 6.0-7.5, even more preferably 6.2-7.3, and most preferably 6.3-7.2.

[0336] In other preferred embodiments of the invention, the liquid mixture has a pH in the range of 6.0-7.5, more preferably 6.2-7.5, and most preferably 6.3-7.5.

[0337] In another preferred embodiment of the invention, the liquid mixture has a pH in the range of 6.0-8.0, more preferably 6.6-7.7, even more preferably 6.7-7.6, and most preferably 6.8-7.5.

[0338] Generally, the pH of a liquid mixture can be adjusted using any suitable food acid or food base. Those skilled in the art will recognize suitable means for adjusting pH. Suitable food bases include sodium carbonate or potassium carbonate, sodium bicarbonate or potassium bicarbonate, or ammonium hydroxide. Alternatively, KOH or NaOH can be used to adjust the pH. Suitable food acids include, for example, citric acid, hydrochloric acid, malic acid, tartaric acid, or phosphoric acid.

[0339] In some preferred embodiments of the invention, the liquid mixture has a temperature of 20°C and a temperature of 300 s. -1 The shear rate is at most 200 cP, more preferably at 20°C and at 300 s. -1 The shear rate is at most 100 cP, or even more preferably at 20°C and 300 s. -1 The shear rate is at most 50 cP and is most preferably at 20°C and 300 s. -1 The shear rate is at most 20 cP for viscosity.

[0340] Liquid mixtures are typically prepared by mixing appropriate components with an oxidized whey protein composition. If powdered components are used, it is generally preferred to allow these components to hydrate prior to heat treatment, and similarly, if preferable, to homogenize the liquid mixture prior to heat treatment.

[0341] In some preferred embodiments, the protein composition is provided in powder form, and preferably in the form of a mixture of water or an aqueous liquid, allowing for hydration prior to heat treatment.

[0342] In other preferred embodiments, the protein composition is provided in liquid form, such as the protein solution obtained from step d) of the method of the present invention. The protein composition obtained from step d) is then: - Mixed with one or more other ingredients required for the production of the beverage. - Optional homogenization - It undergoes heat sterilization by heating it to a temperature in the range of 140℃-150℃ for 1-10 seconds. - being cooled, and - It is filled into a suitable sterile container, which is then sealed.

[0343] The packaging in step 2) can be any suitable packaging technology, and any suitable container can be used to package the liquid mixture.

[0344] However, in a preferred embodiment of the invention, the packaging in step 2) is aseptic packaging, i.e., the liquid mixture is packaged under aseptic conditions. For example, aseptic packaging can be performed using an aseptic filling system, and preferably involves filling the liquid mixture into one or more aseptic containers.

[0345] Aseptic filling and sealing are particularly preferred if the liquid mixture is already sterile or has very low levels of microorganisms before filling.

[0346] Examples of useful containers are bottles, cartons, bricks, and / or bags.

[0347] The heat treatment in this process preferably involves subjecting the liquid mixture to a temperature of at least 70°C.

[0348] In some preferred embodiments of the process of the present invention, the liquid mixture of step 1) undergoes a heat treatment including at least pasteurization, and is then packaged in step 2).

[0349] In another embodiment of the process of the present invention, the packaged liquid mixture of step 2) is subjected to a heat treatment including at least pasteurization.

[0350] In some preferred embodiments, heat treatment involves heating the liquid mixture to a temperature in the range of 70°C to 80°C.

[0351] In some preferred embodiments of the invention, the heat treatment temperature is in the range of 70°C-80°C, preferably in the range of 70°C-79°C, more preferably in the range of 71°C-78°C, even more preferably in the range of 72°C-77°C, and most preferably in the range of 73°C-76°C, such as about 75°C.

[0352] Preferably, when the heat treatment is carried out in a temperature range of 70°C to 80°C, the duration is from 1 second to 60 minutes. The maximum exposure time is best suited to the lowest temperature in the temperature range, and vice versa.

[0353] In other preferred embodiments, the heat treatment temperature is maintained at 70°C for at least 60 minutes, or preferably at 75°C for at least 45 minutes, or preferably at 80°C for at least 30 minutes, or preferably at 85°C for at least 22 minutes, or preferably at 90°C for at least 10 minutes.

[0354] In a particularly preferred embodiment of the invention, the heat treatment is provided at 70°C-78°C for 1 second to 30 minutes, more preferably at 71°C-77°C for 1 minute to 25 minutes, and even more preferably at 72°C-76°C for 2 minutes to 20 minutes.

[0355] In some preferred embodiments of the invention, the heat treatment process involves heating to a temperature of 85°C-95°C for 1 minute to 30 minutes.

[0356] For example, the heat treatment temperature can be at least 81°C, preferably at least 91°C, preferably at least 95°C, more preferably at least 100°C, even more preferably at least 120°C, and most preferably at least 140°C.

[0357] In some particularly preferred embodiments of the invention, the heat treatment involves heating the liquid mixture to a temperature in the range of 100°C to 160°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°C to 155°C for a duration sufficient to achieve sterility, typically 0.1 seconds to 10 minutes, and more preferably to a temperature in the range of 140°C to 155°C for a duration sufficient to achieve sterility, typically 0.1 seconds to 30 seconds. The heat treatment of the liquid to sterilize it is also referred to as heat sterilization.

[0358] Another preferred heat treatment is sterilization UHT type treatment, which typically involves a temperature in the range of 135°C to 146°C and a duration sufficient to achieve sterility, typically in the range of 1 to 10 seconds.

[0359] Optionally, but still preferred, the heat treatment may involve a temperature in the range of 145°C to 180°C and a duration sufficient to achieve sterility, typically in the range of 0.01 seconds to 2 seconds, and more preferably in the range of 150°C to 180°C and a duration in the range of 0.01 seconds to 0.3 seconds.

[0360] The implementation of heat treatment may involve the use of equipment such as plate or tubular heat exchangers, scraped heat exchangers, or distillation systems. Optionally, and particularly preferred for heat treatment above 95°C, direct steam-based heating may be employed, such as direct steam injection, direct steam delivery, or spray cooking. Furthermore, such direct steam-based heating is preferably used in conjunction with flash cooling. Suitable examples of implementing spray cooking can be found in WO2009113858A1, which is incorporated herein by reference for all purposes. Suitable examples of implementing direct steam injection and direct steam delivery can be found in WO2009113858A1 and WO2010 / 085957 A3, which are incorporated herein by reference for all purposes. General aspects of high-temperature processing can be found, for example, in "Thermal technologies in food processing" ISBN 185573558 X, which is incorporated herein by reference for all purposes.

[0361] In some preferred embodiments of the invention, the heat treatment involves or even consists of the following: dry distillation heat treatment, preferably at a temperature of at least 80°C, more preferably at at least 95°C, even more preferably at least 100°C, and most preferably at least 120°C, and preferably for a duration sufficient to sterilize the treated liquid.

[0362] 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°C and more preferably at at least 120°C, even more preferably at least 130°C and most preferably at least 140°C, and preferably for a duration sufficient to sterilize the treated liquid.

[0363] In some preferred embodiments of the invention, pasteurization is combined with physical microbial reduction.

[0364] Useful examples of physical microbial reduction include one or more of bacterial filtration, UV radiation, high-pressure treatment, pulsed electric field treatment, and ultrasound.

[0365] In some preferred embodiments of the invention, the heat treatment is a sterilization heat treatment, and thus produces a sterile liquid mixture, and thus a sterile beverage. Such sterilization can be achieved, for example, by combining bacterial filtration and pasteurization, or by heat treatment at at least 100°C for a duration sufficient to achieve sterilization.

[0366] It is advantageous to subject the liquid mixture to cooling after heat treatment. According to a preferred embodiment of the process of the invention, after heat treatment, the heat-treated liquid mixture is cooled to preferably 0°C to 70°C, preferably 0°C to 60°C, even more preferably 0°C to 30°C, and most preferably 0°C to 20°C.

[0367] If the heat treatment does not sterilize the liquid mixture, then after the heat treatment, the heat-treated liquid mixture is preferably cooled to 0°C to 15°C, more preferably 1°C to 10°C, and most preferably 1°C to 5°C.

[0368] Cooling can occur before or after the filling step.

[0369] Cooling typically involves flash cooling and / or conventional heat exchangers.

[0370] At least partial cooling via flash evaporation is generally preferred, especially after heat treatment followed by heat sterilization. Flash evaporation typically removes some volatile compounds from the cooled liquid. Whey protein beverages with a pH in the range of 5.5–8.5 are particularly prone to developing unpleasant odors during heat treatment, and these unpleasant odors are partially removed from the heat-treated liquid and released near the flash evaporation system. This is a disadvantage because it exposes personnel operating the heat treatment system to irritating odors and can also be associated with health problems.

[0371] The inventors have discovered that, advantageously, the flash cooling of beverages heat-treated with the protein compositions of the present invention releases far less of this unpleasant odor and sometimes even none at all.

[0372] The process of the present invention can be implemented as a batch process, a semi-batch process, and a continuous process.

[0373] Another specific aspect of the invention relates to a process for producing heat-treated, and preferably heat-sterilized, beverages, the process comprising the steps of the method described herein: - Perform steps a), c), and d), or - Proceed to steps a), b), c), and d). To obtain a protein composition in liquid form; and subsequently package the protein composition or a liquid feed prepared from the protein composition, said packaging being according to step 2 of the process described above.

[0374] If the protein composition is to be used directly as a beverage, it is preferable that step d) involves heat sterilization, i.e., heat treatment to sterilize the treated liquid.

[0375] Such heat treatment typically requires heating the liquid to a temperature in the range of 100°C–160°C for a duration sufficient to sterilize the liquid. This article describes suitable time / temperature combinations for applications such as heat treatment.

[0376] Another aspect of the invention relates to a heat-treated, preferably heat-sterilized beverage having a pH of 5.5-8.5, which can be obtained by the process of the invention.

[0377] Another aspect of the invention relates to a heat-treated, preferably heat-sterilized beverage having a pH of 5.5-8.5, more preferably 6.5-7.5, comprising a protein composition of the invention sufficient to contribute at least 0.5% w / w protein, and preferably having an H2S content of up to 5 μmol / L, more preferably up to 3 μmol / L, even more preferably up to 1.0 μmol / L, and most preferably up to 0.7 μmol / L 7 days after production.

[0378] Another aspect of the present invention relates to a food ingredient comprising: - The solid form of the protein composition of the present invention, and - One or more additional ingredients, preferably selected from: - Dairy ingredients, preferably unoxidized dairy ingredients. - Plant-based ingredients, - Non-dairy carbohydrate sources, - Flavorings, and / or - Sweeteners, such as those in the form of sweet carbohydrates, polyols, and / or high-intensity sweeteners.

[0379] Preferably, the sweetener comprises one or more of carbohydrate sweeteners, polyols, high-intensity sweeteners, and combinations thereof.

[0380] In one embodiment of the invention, the beverage contains at least one high-intensity sweetener (HIS). The at least one HIS is preferably selected from the group consisting of aspartame, cyclamate, sucralose, acesulfame potassium, neotame, saccharin, stevia extract, steviol glycosides such as, for example, steviol disaccharide A, or combinations thereof.

[0381] In some embodiments of the invention, it is particularly preferred that the sweetener comprises one or more high-intensity sweeteners or is composed of one or more high-intensity sweeteners.

[0382] HIS has been found in both natural and artificial sweeteners and typically has a sweetness intensity that is at least 10 times that of sucrose.

[0383] If used, the total HIS content of the beverage is typically in the range of 0.001% w / w to 2% w / w. Preferably, the total HIS content is in the range of 0.005% w / w to 1% w / w. Most preferably, the total HIS content is in the range of 0.01% w / w to 0.5% w / w.

[0384] The choice of sweetener can depend on the beverage being produced. For example, high-intensity sweeteners (e.g., aspartame, acesulfame potassium, or sucralose) can be used in beverages where no energy contribution from the sweetener is required, while for beverages with a natural profile, natural sweeteners (e.g., steviol glycosides, sorbitol, or sucrose) can be used.

[0385] Furthermore, it is preferable that the sweetener comprises or is composed of one or more polyol sweeteners. 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 sweeteners in the beverage is typically in the range of 1% w / w to 20% w / w. More preferably, the total amount of polyol sweeteners in the beverage is in the range of 2% w / w to 15% w / w. Even more preferably, the total amount of polyol sweeteners can be in the range of 4% w / w to 10% w / w.

[0386] Another aspect of the present invention relates to a protein composition containing modified BLG in a heat-sterilized beverage, and preferably to the use of the protein composition containing modified BLG of the present invention as a food ingredient, preferably for: - Improves odor, and / or - Reduces unpleasant odor levels similar to rotten eggs, and / or - Reduce H2S generation during production, and / or - Reduce the H2S content in the headspace of the container. The heat-sterilized beverage has a pH in the range of 5.5-8.5, preferably has a whey protein content of at least 3% w / w, and is preferably heat-sterilized using indirect heat treatment.

[0387] The total protein, pH, viscosity, H2S content, total thiol and free thiol content, amino acid content, amino acid oxidation, average molecular weight, intrinsic viscosity, sensory evaluation, natural protein content, total fat content, lactose content, mineral composition, and turbidity were quantified or determined according to Analysis A-Analysis Q of PCT application number PCT / EP2022 / 078739, which is incorporated herein by reference for all purposes.

[0388] Preferred embodiments of the invention are described in the following numbered embodiments.

[0389] Scheme 1. A method for producing a protein composition comprising modified BLG, the method comprising the following steps: a) Provide: - A source comprising one or more phenolic compounds (PCA), said one or more phenolic compounds comprising at least two hydroxyl groups directly bonded to the same aromatic ring, and - Sources containing BLG (BoG) b) Optionally, a portion of the source containing one or more PCA undergoes a type of oxidation (PCA-type oxidation) capable of converting PCA into quinone, thereby providing a source of PCA containing one or more oxidations. c) Combining a portion of the source containing one or more PCA and / or a portion of the source containing one or more oxidized PCA with a source containing BLG and optional additional components to provide a protein solution having: - pH in the range of 6.5-9.5, and - At least 0.2% w / w BLG content, - A molar ratio of at least 0.1:1 between the following: - The original amount of PCA used to prepare the protein solution, and - The BLG content of the protein solution, d) Incubate the protein solution at a temperature sufficient to reduce the amount of free thiol groups in the protein solution to at most 10 μmol / g protein for a duration sufficient to reduce the amount of free thiol groups in the protein solution to at most 10 μmol / g protein, provided that if the method does not include step b), then step d) also involves applying a type of oxidation capable of converting PCA to quinone. Preferably, the PCA-type oxidation applied during step b) and / or step d) is sufficient to produce a molar ratio of at least 0.1:1 among the following: - Based on the theoretical amount of quinone supplied to the protein solution during the method determined by Analysis 1, and - The amount of BLG in the protein solution.

[0390] Implementation scheme 1.1. A method for producing a protein composition comprising modified BLG, the method comprising the following steps: a) Provide: - A source comprising one or more phenolic compounds (PCA), said one or more phenolic compounds comprising at least two hydroxyl groups directly bonded to the same aromatic ring, and - Sources containing BLG (BoG) b) Optionally, a portion of the source containing one or more PCA undergoes a type of oxidation (PCA-type oxidation) capable of converting PCA into quinone, thereby providing a source of PCA containing one or more oxidations. c) Combining a portion of the source containing one or more PCA and / or a portion of the source containing one or more oxidized PCA with a source containing BLG and optional additional components to provide a protein solution having: - pH in the range of 6.5-9.5, and - At least 0.2% w / w BLG content, - A molar ratio of at least 1:1 among the following: - The original amount of PCA used to prepare the protein solution, and - The BLG content of the protein solution, d) Incubate the protein solution at a temperature sufficient to reduce the amount of free thiol groups in the protein solution to at most 10 μmol / g protein for a duration sufficient to reduce the amount of free thiol groups in the protein solution to at most 10 μmol / g protein, provided that if the method does not include step b), then step d) also involves applying a type of oxidation capable of converting PCA to quinone. Preferably, the PCA-type oxidation applied during step b) and / or step d) is sufficient to produce a molar ratio of at least 1:1 among the following: - Based on the theoretical amount of quinone supplied to the protein solution during the method determined by Analysis 1, and - The amount of BLG in the protein solution.

[0391] Scheme 2. The method according to Scheme 1 or 1.1 further includes step e): drying the liquid feed, said liquid feed containing at least the protein derived from the incubated protein solution of step d).

[0392] Scheme 3 of the numbered embodiments. The method according to any one of the aforementioned numbered embodiments, wherein the PCA comprises flavonoids, preferably flavanols or flavanol esters, such as catechin, epicatechin, gallocatechin, epigallocatechin, catechin 3-gallate, epicatechin 3-gallate, gallocatechin 3-gallate and epigallocatechin 3-gallate (EGCG).

[0393] Scheme 4. The method according to any one of the aforementioned schemes, wherein the PCA comprises a stilbene compound, preferably resveratrol.

[0394] Scheme 5 of the numbered embodiments. The method according to any one of the preceding numbered embodiments, wherein the PCA comprises caffeic acid, gallic acid, chlorogenic acid, catechin, 4-methylcatechin, catechin, epicatechin, gallocatechin, epigallocatechin, catechin 3-gallate, epicatechin 3-gallate, gallocatechin 3-gallate, epigallocatechin 3-gallate (EGCG), resveratrol, sarsaparilla acid, sarsaparillaol, naringenin, or mixtures thereof.

[0395] Scheme 6. The method according to any one of the aforementioned schemes, wherein the PCA has a molecular weight of up to 600 g / mol, more preferably up to 400 g / mol, even more preferably up to 250 g / mol and more preferably up to 200 g / mol.

[0396] Scheme 7. The method according to any one of the aforementioned schemes, wherein the PCA has a molecular weight in the range of 110 g / mol-600 g / mol, more preferably 110 g / mol-400 g / mol, even more preferably 110 g / mol-350 g / mol, and even more preferably 110 g / mol-310 g / mol.

[0397] Scheme 7.1. The method according to any one of the aforementioned schemes, wherein the PCA has a molecular weight in the range of 120 g / mol-1000 g / mol, more preferably 250 g / mol-700 g / mol, even more preferably 300 g / mol-650 g / mol and more preferably 350 g / mol-600 g / mol.

[0398] Implementation scheme 8. The method according to any one of the aforementioned implementation schemes, wherein the PCA does not contain a carboxylic acid group.

[0399] Scheme 9. The method according to any one of the aforementioned schemes, wherein the PCA has a water solubility of at least 8 mM, more preferably at least 12 mM and most preferably at least 16 mM at 25°C.

[0400] Scheme 10 of the numbered embodiments. The method according to any one of the preceding numbered embodiments, wherein the source containing one or more PCA is selected from the group consisting of: polyphenol extracts from herbs, polyphenol extracts from spices, polyphenol extracts from fruits, polyphenol extracts from berries, and mixtures thereof.

[0401] Scheme 10.1. The method according to any one of the aforementioned schemes, wherein the source comprising one or more PCA is selected from the group consisting of: polyphenol extracts of tea, more preferably polyphenol extracts of green tea; polyphenol extracts of coffee; polyphenol extracts of cocoa; polyphenol extracts of grapes; polyphenol extracts of rosemary; polyphenol extracts of lemon mint; polyphenol extracts of blackcurrant; single PCA isolates, and mixtures thereof.

[0402] Embodiment 11 of the numbered scheme. The method according to any one of the preceding numbered embodiments, wherein the source of one or more PCA comprises at least 25% w / w of PCA relative to the total solids of the source containing one or more PCA, more preferably at least 40% w / w of the total solids of the source containing one or more PCA, even more preferably at least 60% w / w of the total solids of the source containing one or more PCA, and most preferably at least 80% w / w of the total solids of the source containing one or more PCA.

[0403] Scheme 12. The method according to any one of the aforementioned schemes, wherein the source of one or more PCA is a single PCA isolate, the single PCA isolate comprising at least 25% w / w, more preferably at least 40% w / w, even more preferably at least 60% w / w, and most preferably at least 80% w / w of the amount of single PCA relative to the total solids of the source containing one or more PCA.

[0404] Implementation scheme 13. The method according to any one of the aforementioned implementation schemes, wherein the source containing BLG comprises or is composed of: whey protein concentrate, whey protein isolate, milk serum protein concentrate, milk serum protein isolate, BLG isolate, or a combination thereof.

[0405] Scheme 14. The method according to any one of the aforementioned schemes, comprising step b): subjecting a portion of the source containing one or more PCA to an oxidation of a type capable of converting PCA into quinone, thereby providing a source of PCA containing one or more oxidations.

[0406] Implementation Scheme 15. The method according to Implementation Scheme 14, wherein the oxidation in step b) involves preparing an oxidizing aqueous solution comprising a portion of a source containing one or more PCA, an optional chemical oxidant, and an optional one or more other components such as water.

[0407] Embodiment 16. The method according to Embodiment 15, wherein the oxidizing aqueous solution has a PCA content of at least 0.2 mM, more preferably at least 15 mM, and most preferably at least 50 mM.

[0408] Scheme 17. The method according to Scheme 15 or 16, wherein the oxidizing aqueous solution has a PCA concentration of 0.2 mM-600 mM, more preferably 15 mM-550 mM, and most preferably 50 mM-500 mM.

[0409] Scheme 18. The method according to any one of Schemes 15-17, wherein 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.

[0410] Scheme 19. The method according to any one of Schemes 15-17, wherein the oxidizing aqueous solution has a pH in the range of 2-6.4, more preferably 3.0-6.0, even more preferably 3.5-5.5 and most preferably 4.0-5.0.

[0411] Scheme 20. The method according to any one of Schemes 14-17, wherein the oxidation in step b) involves contacting a portion of a source containing one or more PCA with a chemical oxidant under conditions that convert at least some of the PCA into quinone compounds.

[0412] Scheme 21. The method according to Scheme 20, wherein the chemical oxidant comprises or is composed of peroxide, ozone, hydrogen peroxide, or a combination thereof.

[0413] Scheme 22. The method according to Scheme 20 or 21, wherein the chemical oxidant comprises or is composed of hydrogen peroxide, benzoyl peroxide, or a combination thereof.

[0414] Scheme 23. The method according to any one of Schemes 14-22, wherein the oxidation in step b) involves preparing an oxidizing aqueous solution comprising a portion of a source containing one or more PCA, a chemical oxidant, and optionally one or more other components such as water.

[0415] Scheme 24. The method according to any one of Schemes 14-23, wherein the oxidation in step b) involves electrochemical oxidation of a portion of a source containing one or more PCA compounds under conditions that convert at least some of the PCA into quinone compounds.

[0416] Implementation scheme 25. The method according to implementation scheme 24, wherein the oxidation in step b) involves preparing an oxidizing aqueous solution, the oxidizing aqueous solution comprising a portion of a source containing one or more PCA and optionally one or more components such as water.

[0417] Scheme 26. The method according to Scheme 24 or 25, wherein the electrochemical oxidation is operated using a potential difference of -0.1 V to 1.5 V, more preferably 0.0 V to 1.2 V, and most preferably 0.0 V to 0.7 V.

[0418] Scheme 27. The method according to any one of Schemes 25-26, wherein 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 V to 0.9 V, even more preferably 0 V to 0.8 V, and most preferably 0.2 V to 0.7 V.

[0419] Embodiment 28. The method according to any one of embodiments 25-26, wherein the oxidizing aqueous solution has a pH in the range of 3-5, and the electrochemical oxidation is operated using a potential difference of 0.1 V-1.3 V, more preferably 0.2 V-0.9 V, and most preferably 0.3 V-0.7 V.

[0420] Scheme 28.1. The method according to any one of Schemes 24-28, wherein the electrochemical oxidation is carried out at a temperature in the range of 2°C-80°C, more preferably 5°C-60°C, even more preferably 10°C-40°C, and most preferably in the range of 15°C-30°C.

[0421] Scheme 28.2. The method according to any one of Schemes 24-28, wherein the electrochemical oxidation is carried out at a temperature in the range of 30°C-90°C, more preferably 40°C-90°C, even more preferably 50°C-90°C, and most preferably in the range of 60°C-90°C.

[0422] Scheme 28.3. The method according to any one of Schemes 24-28, wherein the electrochemical oxidation is carried out at a temperature in the range of 5°C-25°C, more preferably 10°C-25°C, even more preferably 15°C-25°C, and most preferably in the range of 18°C-25°C.

[0423] Scheme 29. The method according to any one of the aforementioned schemes, wherein the protein solution has a pH in the range of 6.7-9.5, even more preferably 7.1-9.0, even more preferably 7.3-8.7 and most preferably 7.4-8.5.

[0424] Scheme 30. The method according to any one of the aforementioned schemes, 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.

[0425] Scheme 31. The method according to any one of the aforementioned schemes, wherein the protein solution has a BLG content of 0.5% w / w-30% w / w, more preferably 1% w / w-20% w / w, even more preferably 3% w / w-16% w / w, and most preferably 5% w / w-12% w / w.

[0426] Scheme 32. The method according to any one of the aforementioned schemes, wherein the protein solution has a BLG content of at least 30% w / w, more preferably at least 40% w / w, even more preferably at least 45% w / w, and most preferably at least 50% w / w, relative to the total protein.

[0427] Scheme 33. The method according to any one of the aforementioned schemes, wherein the protein solution has a BLG content of 30% w / w-99% w / w, more preferably 40% w / w-95% w / w, even more preferably 45% w / w-90% w / w, and most preferably 50% w / w-80% w / w, relative to the total protein.

[0428] Scheme 34. The method according to any one of the aforementioned schemes, wherein the protein solution has a BLG content of at least 60% w / w, more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 95% w / w, relative to the total protein.

[0429] Scheme 35. The method according to any one of the aforementioned schemes, wherein the protein solution has a BLG content of at least 30% w / w, more preferably at least 40% w / w, even more preferably at least 45% w / w, and most preferably at least 50% w / w, relative to total solids.

[0430] Scheme 36. The method according to any one of the aforementioned schemes, wherein the protein solution has a BLG content of 30% w / w-99% w / w, more preferably 40% w / w-95% w / w, even more preferably 45% w / w-90% w / w, and most preferably 50% w / w-80% w / w, relative to total solids.

[0431] Scheme 37. The method according to any one of the aforementioned schemes, wherein the protein solution has a BLG content of at least 60% w / w, more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 95% w / w, relative to total solids.

[0432] Scheme 38. The method according to any one of the aforementioned schemes, wherein the protein solution has a total fat content of up to 5% w / w relative to total solids, more preferably up to 2% w / w relative to total solids, even more preferably up to 0.5% w / w relative to total solids, and most preferably up to 0.1% w / w relative to total solids.

[0433] Scheme 39. The method according to any one of the aforementioned schemes, wherein the protein solution has a total fat content in the range of 1% w / w to 20% w / w relative to total solids, more preferably in the range of 2% w / w to 16% w / w relative to total solids, even more preferably in the range of 3% w / w to 12% w / w relative to total solids, and most preferably in the range of 4% w / w to 10% w / w relative to total solids.

[0434] Scheme 40. The method according to any one of the aforementioned schemes, wherein the protein solution has a total carbohydrate content of up to 10% w / w, more preferably up to 8% w / w, even more preferably up to 6% w / w, and most preferably up to 5% w / w, relative to total solids.

[0435] Scheme 41. The method according to any one of the aforementioned schemes, wherein the protein solution has a total carbohydrate content of up to 2% w / w relative to total solids, more preferably up to 1% w / w relative to total solids, even more preferably up to 0.5% w / w relative to total solids, and most preferably up to 0.2% w / w relative to total solids.

[0436] Scheme 42. The method according to any one of the preceding schemes, wherein the protein solution in step c) has a molar ratio between 0.1:1 and 100:1, more preferably 0.1:1 and 50:1, even more preferably 0.1:1 and 20:1, and most preferably 0.1:1 and 10:1: - The original amount of PCA used to prepare the protein solution, and - The BLG content of the protein solution.

[0437] Scheme 42.1. The method according to any one of Schemes 1-41, wherein the protein solution in step c) has a molar ratio 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: - The original amount of PCA used to prepare the protein solution, and - The BLG content of the protein solution.

[0438] Embodiment 42.2. The method according to any one of embodiments 1-41, wherein the protein solution has a molar ratio 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 among the following: - The original amount of PCA used to prepare the protein solution, and - The BLG content of the protein solution.

[0439] Scheme 42.3. The method according to any one of claims 1-41, wherein the protein solution in step c) has a molar ratio of at least 1:1 among the following: - The original amount of PCA used to prepare the protein solution, and - The BLG content of the protein solution.

[0440] Embodiment 43. The method according to any one of embodiments 1-41, wherein the protein solution has a molar ratio between 1:1-1000:1, more preferably 2:1-500:1, even more preferably 5:1-200:1, and most preferably 10:1-100:1: - The original amount of PCA used to prepare the protein solution, and - The BLG content of the protein solution.

[0441] Embodiment 44. The method according to any one of embodiments 1-41, wherein the protein solution has a molar ratio 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: - The original amount of PCA used to prepare the protein solution, and - The BLG content of the protein solution.

[0442] Scheme 44.1. The method according to any one of Schemes 1-41, wherein the protein solution has a molar ratio 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: - The original amount of PCA used to prepare the protein solution, and - The BLG content of the protein solution.

[0443] Scheme 45. The method according to any one of the aforementioned schemes, wherein the protein solution is prepared by mixing a portion of a source containing one or more PCA obtained from step a), a source containing BLG obtained from step a), and optional additional components.

[0444] Scheme 46. The method according to any one of the preceding schemes, wherein the method does not include the step of oxidizing a portion of a source containing one or more PCA prior to step c).

[0445] Scheme 47. The method according to any one of Schemes 1-45, wherein the method includes step b), and wherein the protein solution is prepared by mixing a portion of a source containing one or more oxidized PCA obtained from step b) with a source containing BLG and optional additional components.

[0446] Scheme 47.1. The method according to any one of the aforementioned schemes, wherein the incubation reduces the amount of free thiol in the protein solution to at most 9 μmol / g protein, more preferably at most 8 μmol / g protein, more preferably at most 5 μmol / g protein, even more preferably at most 3 μmol / g protein and most preferably at most 2 μmol / g protein, or the incubation is performed to reduce the amount of free thiol in the protein solution to at most 9 μmol / g protein, more preferably at most 8 μmol / g protein, more preferably at most 5 μmol / g protein, even more preferably at most 3 μmol / g protein and most preferably at most 2 μmol / g protein.

[0447] Scheme 48. The method according to any one of the aforementioned schemes, wherein the protein solution is subjected to a pressure in the range of 1-1000 bar, more preferably 20-500 bar, even more preferably 30-300 bar, and most preferably 40-200 bar during the incubation.

[0448] Scheme 49. The method according to any one of the aforementioned schemes, wherein the protein solution has a temperature in the range of 0°C-160°C, more preferably 10°C-155°C, even more preferably 15°C-150°C, and most preferably 20°C-145°C during the incubation.

[0449] Scheme 49.1. The method according to any one of the aforementioned schemes, wherein the protein solution is prepared at a temperature in the range of 0°C-50°C, more preferably in the range of 2°C-45°C, even more preferably in the range of 5°C-40°C, and most preferably in the range of 10°C-35°C.

[0450] Scheme 50 of the numbered embodiments. The method according to any one of the aforementioned numbered embodiments, wherein the incubation duration 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.

[0451] Scheme 51. The method according to any one of the aforementioned schemes, wherein the incubation duration 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.

[0452] Scheme 52. The method according to any one of the aforementioned schemes, wherein the oxidation in step d) involves contacting the PCA with a chemical oxidant in the protein solution under conditions that convert at least some of the PCA into quinone compounds.

[0453] Scheme 53. The method according to Scheme 52, wherein the chemical oxidant comprises or is composed of peroxide, ozone, hydrogen peroxide, or a combination thereof.

[0454] Embodiment 54. The method according to Embodiment 52 or 53, wherein the chemical oxidant comprises or is composed of hydrogen peroxide, benzoyl peroxide, or a combination thereof.

[0455] Scheme 55. The method according to any one of the aforementioned schemes, wherein the oxidation in step d) involves electrochemical oxidation of the PCA in the protein solution under conditions that convert at least some of the PCA into quinone compounds.

[0456] Scheme 56. The method according to any one of the aforementioned schemes, wherein the electrochemical oxidation of step d) is performed using a potential difference of -0.1 V to 1.5 V and most preferably -0.05 V to 0.7 V.

[0457] Scheme 57. The method according to any one of the aforementioned schemes, wherein the oxidation in step d) involves a protein solution having a pH in the range of 7.1-9.0, and is performed using a potential difference of -0.1 V to 0.9 V, more preferably -0.05 V to 0.8 V, and most preferably 0 V to 0.7 V.

[0458] Scheme 57.1. The method according to any one of the aforementioned schemes, wherein the oxidation in step d) involves a protein solution having a pH in the range of 7.3-8.7, and is performed using a potential difference of -0.1 V to 0.8 V and most preferably 0 V to 0.7 V.

[0459] Scheme 57.2. The method according to any one of the aforementioned schemes, wherein the oxidation in step d) involves a protein solution having a pH in the range of 7.4-8.5, and is performed using a potential difference of -0.1 V to 0.8 V and most preferably -0.05 V to 0.7 V.

[0460] Scheme 58. The method according to any one of the aforementioned schemes, wherein the temperature of the first stage of incubation is in the range of 0°C-60°C, more preferably in the range of 10°C-50°C, and wherein the incubated protein solution is subsequently heated in the second stage of incubation to a temperature in the range of 70°C-160°C, more preferably in the range of 80°C-150°C.

[0461] Scheme 59. The method according to any one of the preceding schemes, wherein the oxidation applied during step b) and / or step d) is sufficient to produce a molar ratio 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: - Based on the theoretical amount of quinone supplied to the protein solution during the method determined by Analysis 1, and - The BLG content of the protein solution.

[0462] Scheme 59.1. The method according to any one of the aforementioned schemes, wherein the oxidation applied during step b) and / or step d) is sufficient to produce a molar ratio 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: - Based on the theoretical amount of quinone supplied to the protein solution during the method determined by Analysis 1, and - The BLG content of the protein solution.

[0463] Scheme 59.2. The method according to any one of the aforementioned schemes, wherein the oxidation applied during step d) is sufficient to produce a molar ratio between 0.1:1 and 100:1, more preferably 0.1:1 and 50:1, even more preferably 0.2:1 and 15:1, and most preferably 0.2:1 and 5:1: - Based on the theoretical amount of quinone supplied to the protein solution during the method determined by Analysis 1, and - The BLG content of the protein solution.

[0464] Scheme 59.3. The method according to any one of the aforementioned schemes, wherein the oxidation applied during step d) is sufficient to produce a molar ratio 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: - Based on the theoretical amount of quinone supplied to the protein solution during the method determined by Analysis 1, and - The BLG content of the protein solution.

[0465] Scheme 59.4. The method according to any one of the preceding schemes, wherein the oxidation applied during step b) and / or step d) is sufficient to produce a molar ratio of at least 1:1 among: - Based on the theoretical amount of quinone supplied to the protein solution during the method determined by Analysis 1, and - The BLG content of the protein solution.

[0466] Embodiment 59.5. The method according to any one of embodiments 1-58, wherein the PCA-type oxidation applied during step b) and / or step d) is sufficient to produce a molar ratio 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 among the following: - Based on the theoretical amount of quinone supplied to the protein solution during the method determined by Analysis 1, and - The molar amount of BLG in the protein solution.

[0467] Scheme 60. The method according to any one of Schemes 1-58, wherein the oxidation applied during step b) and / or step d) is sufficient to produce a molar ratio 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: - Based on the theoretical amount of quinone supplied to the protein solution during the method determined by Analysis 1, and - The BLG content of the protein solution.

[0468] Scheme 61. The method according to any one of Schemes 1-58, wherein the PCA-type oxidation applied during step b) is sufficient to produce a molar ratio 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 among the following: - The quinone content of the portion of the source containing one or more oxidized PCA used in the protein solution, and - The amount of BLG in the protein solution.

[0469] Scheme 62. The method according to any one of Schemes 1-58, wherein the PCA-type oxidation applied during step b) is sufficient to produce a molar ratio 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: - The quinone content of the portion of the source containing one or more oxidized PCA used in the protein solution, and - The amount of BLG in the protein solution.

[0470] Scheme 62. The method according to any one of Schemes 1-58, wherein the PCA-type oxidation applied during step d) is sufficient to produce a molar ratio 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: - The quinone content of the portion of the source containing one or more oxidized PCA used in the protein solution, and - The amount of BLG in the protein solution.

[0471] Scheme 63, numbered. The method according to any one of the aforementioned schemes, numbered: - The method described therein does not involve oxidizing a portion of a source containing one or more PCA prior to step c), and - The PCA-type oxidation mentioned above is carried out by a chemical oxidant.

[0472] Scheme 64, numbered. The method according to any one of the aforementioned schemes, numbered: - The method described therein does not involve oxidizing a portion of a source containing one or more PCA prior to step c), and The PCA-type oxidation is carried out via electrochemical oxidation.

[0473] Scheme 65. The method according to any one of the aforementioned schemes: The method includes step b), and a portion of the source containing one or more oxidized PCA obtained from step b) is used to prepare the protein solution of step c). - The PCA-type oxidation mentioned above is carried out by a chemical oxidant.

[0474] Scheme 66. The method according to any one of the aforementioned schemes: The method includes step b), and a portion of the source containing one or more oxidized PCA obtained from step b) is used to prepare the protein solution of step c). The PCA-type oxidation is carried out via electrochemical oxidation.

[0475] Scheme 67. The method according to any one of the aforementioned schemes: The method includes step b), and a portion of the source containing one or more oxidized PCA obtained from step b) is used to prepare the protein solution of step c). - The PCA-type oxidation described herein is carried out through both electrochemical oxidation and chemical oxidants.

[0476] Scheme 68. The method according to any one of Schemes 2-67, comprising step e): drying the liquid feed, said liquid feed comprising at least a solid derived from the incubated protein solution of step d).

[0477] Scheme 69. The method according to any one of Schemes 2-68, wherein the liquid feed for drying comprises a protein solution or a protein concentrate thereof obtained from step d), or even consists of a protein solution or a protein concentrate thereof obtained from step d).

[0478] Scheme 70. The method according to any one of Schemes 2-68, wherein the liquid feed is prepared by subjecting the incubated protein solution of step d) to one or more of the following: - pH adjustment, - Concentration steps, - Percolation, and - Heat treatment.

[0479] Scheme 71. The method according to any one of Schemes 2-70, wherein step e) involves spray drying the liquid feed.

[0480] Scheme 72. The method according to any one of the aforementioned schemes, wherein the method is implemented as a batch process, a semi-batch process or a continuous process.

[0481] Scheme 73. The method according to any one of the aforementioned schemes, wherein the method is performed as a continuous process, or wherein at least steps b), c) and d) are performed as a continuous process.

[0482] Embodiment 74 of the numbered scheme. A protein composition comprising modified BLG, the protein composition being obtainable by means of one or more of the methods described in the preceding embodiments of the numbered scheme, the protein composition preferably having one or more of the following: - Protein content of at least 30% w / w relative to total solids. - Up to 10 μmol of free thiol groups / g protein, - At least 0.7% w / w tryptophan content relative to total protein. - Methionine content of at least 0.3% w / w relative to total protein. - Kyrenine content of up to 0.2 μg / mg protein, - Preferably, the fat content is at most 3% w / w relative to the total solids. - Preferably, the content of protein-bound sulfur is in the range of 100-600 μmol / g protein. - Preferably, the content of cysteine ​​residues that form disulfide bonds in protein-bound proteins is in the range of 150-400 μmol / g protein.

[0483] Embodiment 74.1. The modified whey protein composition according to Embodiment 74 has a protein content of at least 30% w / w relative to total solids.

[0484] Scheme 74.2. The modified whey protein composition according to Scheme 74 or 74.1 has up to 10 μmol free thiol groups / g protein.

[0485] Scheme 74.3. The modified whey protein composition according to any one of Schemes 74-74.2 has a tryptophan content of at least 0.7% w / w relative to the total protein.

[0486] Scheme 74.4. The modified whey protein composition according to any one of Schemes 74-74.3 has a methionine content of at least 0.3% w / w relative to the total protein.

[0487] Scheme 74.5. The modified whey protein composition according to any one of Schemes 74-74.4 has a kynurenine content of up to 0.2 μg / mg protein.

[0488] Scheme 74.6. A modified whey protein composition according to any one of Schemes 74-74.5, having a fat content of up to 3% w / w relative to total solids.

[0489] Scheme 74.7. The modified whey protein composition according to any one of Schemes 74-74.6 has a protein-bound sulfur content in the range of 100-600 μmol / g protein.

[0490] Scheme 74.8. A modified whey protein composition according to any one of Schemes 74-74.7, having a content of protein-bound disulfide-bonded cysteine ​​residues in the range of 150-400 μmol / g protein.

[0491] Embodiment 75. A modified whey protein composition according to any one of embodiments 74-74.8, having an average molecular weight of the protein in the range of 18 kDa to 10000 kDa, more preferably between 50 kDa and 8000 kDa, and most preferably between 80 kDa and 5000 kDa.

[0492] Embodiment 76. The protein composition according to any one of embodiments 74-74.8, having an average molecular weight of the protein in the range of 18 kDa to 500 kDa, more preferably between 18 kDa and 100 kDa, and most preferably between 18 kDa and 40 kDa.

[0493] Scheme 77. A process for producing a heat-treated, preferably heat-sterilized, beverage, the process comprising the following steps: 1) Mixing the protein composition according to one or more of embodiments 74-76 with one or more additional beverage ingredients to obtain a liquid mixture having a pH of 5.5-8.5, and 2) Fill the liquid mixture into a suitable container. The process further includes at least one heat treatment step, wherein the liquid mixture is heat-treated, preferably heat-sterilized, before and / or after filling.

[0494] Embodiment 78. The process according to Embodiment 77, wherein the liquid mixture comprises the protein composition in an amount sufficient to contribute at least 0.5% w / w protein.

[0495] Scheme 79. A heat-treated, preferably heat-sterilized beverage having a pH of 5.5-8.5, obtainable by means of the method according to Scheme 77 or 78.

[0496] Embodiment 80. A heat-treated, preferably heat-sterilized beverage having a pH of 5.5-8.5 and more preferably 6.5-7.5, said beverage containing a protein composition according to one or more of embodiments 74-76 that contributes at least 0.5% w / w of protein, and preferably having an H2S content of up to 5 μmol / L, more preferably up to 3 μmol / L, even more preferably up to 1.0 μmol / L, and most preferably up to 0.7 μmol / L 7 days after production.

[0497] Implementation scheme number 81. A food ingredient comprising: - A solid of the protein composition according to one or more of embodiments 74-76, and - One or more additional ingredients, preferably selected from: - Dairy ingredients, preferably unoxidized dairy ingredients. - Plant-based ingredients, - Non-dairy carbohydrate sources, - Flavorings, and / or - Sweeteners, preferably comprising carbohydrate sweeteners, polyols, high-intensity sweeteners, or combinations thereof.

[0498] Scheme 82. Use of a heat-sterilized beverage containing a protein composition of modified BLG, preferably a protein composition according to one or more of Schemes 74-76, as a food ingredient, preferably for: - Improves odor, and / or - Reduces unpleasant odor levels similar to rotten eggs, and / or - Reduce H2S generation during production, and / or - Reduce the H2S content in the headspace of the container. The heat-sterilized beverage has a pH in the range of 5.5-8.5, preferably has a whey protein content of at least 3% w / w, and is preferably heat-sterilized using indirect heat treatment.

[0499] The invention has been described above with reference to specific embodiments. However, other embodiments besides those described above are also possible within the scope of the invention. Unless otherwise stated, various features and steps of the various embodiments and aspects of the invention can be combined in ways other than those described herein. Example

[0500] Analysis 1: Determination of the theoretical amount of quinone provided during the method "The theoretical amount of quinone supplied to the protein solution during the method" is a measure of the molar content of quinone compounds formed under the same conditions and the same oxidation conditions used in the method, but in the absence of a source of protein or other free thiols or free amines, all PCA present in the protein solution of the method (including PCA that has been converted to quinone compounds) is oxidized.

[0501] For example, if the method of the present invention is based on the oxidation of PCA before mixing with a source containing BLG (to provide a source containing one or more oxidized PCA), then the content of quinone compounds in the source containing one or more oxidized PCA is equal to the theoretical quinone content.

[0502] The content of quinone compounds was determined based on analysis 2.

[0503] Analysis 2: Quantitative analysis of quinone content The following describes the quantitative determination of quinone content in liquid samples. Quantification is based on measuring the reactive oxygen species generated by the reduction of quinones with NaBH4, which converts the colorimetric reagent 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazonium chloride (INT) into formazan dye.

[0504] Stock solution: NaH2PO4 buffer: NaH2PO4 buffer is an aqueous solution of 0.1 M sodium dihydrogen phosphate adjusted to pH 11.85 using NaOH.

[0505] INT: The INT stock solution was freshly prepared as a solution of 400 μmol of INT in an aqueous solution of NaH2PO4 buffer.

[0506] NaBH4: The NaBH4 stock solution was freshly prepared as a solution of 300 mmol NaBH4 in an aqueous solution of NaH2PO4 buffer.

[0507] Quinone reference: The quinone reference solution was prepared based on 2 mM 4-methylbenzoquinone (4MBQ) via the electrochemical oxidation of 4-methylcatechol (4MC), as described by Li et al. (Li, Y., Jongberg, S., Andersen, ML, Davies, MJ, & Lund, MN (2016). Quinone-induced protein modifications: Kineticpreference for reaction of 1,2-benzoquinones with thiol groups in proteins. Free Radical Biology and Medicine, 97, 148-157). A 2 mM 4MC solution was prepared in phosphate buffer (0.2 M, pH 4.5) and deoxygenated by purging with nitrogen for 10 minutes. Cyclic voltammetry of the 4MC solution was obtained using a CV-50W voltammetry analyzer (BAS Co., Ltd.) with a glassy carbon working electrode (3 mm diameter, BAS Co., Ltd.), a platinum coil counter electrode (5 mm diameter and 5 cm length), and an Ag / AgCl (KCl, c=3 M) reference electrode (Metrohm, Switzerland). Bulk electrolysis was performed under nitrogen using the same CV-50W potentiostat at an initial potential of 460 mV relative to Ag / AgCl, with an initial concentration of 2 mM for the 4MC solution. The working electrode was a mesh glassy carbon nanotube (BAS Co., Ltd.) with a diameter of 3.5 cm and a length of 4.5 cm.

[0508] The quinone reference solution should be used within 30 minutes of preparation.

[0509] calibration A calibration curve was prepared by diluting the reference quinone solution with NaH2PO4 buffer and using a working solution of the quinone reference solution in the concentration range of 0.001–4 μmol.

[0510] A calibration curve is obtained by plotting absorbance relative to the concentration of a reference quinone.

[0511] Measurement of quinones in samples Dilute and mix 10 μL of the liquid sample to be analyzed with 490 μL of NaH2PO4 buffer.

[0512] 50 μL of diluted sample was mixed with 100 μL of INT stock solution in the wells of a 96-well microplate, followed by the addition of 50 μL of NaBH4 stock solution. The microplate was transferred to a microplate reader and agitated for 30 s. The microplate was then allowed to incubate for 2 min, after which the absorbance of the wells was read at 510 nm. The collected data are presented as the average of three measurements using a quinone reference calibration curve.

[0513] Mixing, incubation, and readout are performed at room temperature (RT).

[0514] If the absorbance reading is above the linear range of the analysis, collect a new sample, dilute it appropriately, and perform the measurement as described above.

[0515] calculate The quinone content of a liquid sample is calculated by comparing the results from the measurement with a calibration curve and converting the quinone content to the desired units. If the liquid sample was diluted before measurement, the quinone content compensates for the dilution.

[0516] Example 1: Elimination of free thiols in whey protein by using oxidized phenolic compounds The purpose of this experiment is to confirm the inventors’ initial findings and to demonstrate how off-flavors can be reduced and even avoided during heat treatment of pH-neutral whey protein-rich beverages by reacting whey proteins, particularly BLG, with oxidized phenolic compounds.

[0517] Reaction principle The inventors have learned that dissolved hydrogen peroxide can oxidize PCA, such as EGCG, to quinone compounds, which can then react again with the free thiol groups of BLG if the free thiol groups are properly exposed. Furthermore, the inventors have found that a combination of a well-aerated reaction mixture (containing dissolved hydrogen peroxide) and sufficient atmospheric headspace (from which additional hydrogen peroxide can diffuse into the reaction mixture) can be used as a model system for the oxidation of PCA and the subsequent reaction between the oxidized PCA and BLG.

[0518] Materials and methods Material A BLG-rich whey protein isolate powder (WPI-A) was prepared according to WO 2018 / 115520 A1. The properties of this powder are described in Table 1.

[0519] Table 1: Composition of WPI-A

[0520] 4-Methylcatechol (4MC), epigallocatechin gallate (EGCG), bovine serum albumin (BSA), and trifluoroacetic acid (TFA) were purchased from Thermo Scientific, Thermo Scientific, Merck Life Science, and Merck. All reagents were of analytical grade or the highest possible purity. Distilled water (MQ water) was obtained using a Milli-Q purification system (Millipore, Bedford, MA).

[0521] The 2 mL HPLC vial is a 2 mL 32x11.6 mm injection vial (part number ML33003VU) from Mikrolab in Denmark.

[0522] Measurement of dissolved hydrogen peroxide The O2 microsensor (Unisense) was used to assess the consumption of dissolved oxygen in PCA samples to evaluate the oxygen consumption in the samples.

[0523] Measurements were performed according to the Unisense Oxygen Sensor User Manual (March 2020 revised edition). The O2 microsensor was calibrated using a vigorously stirred water sample and water bubbled with N2, corresponding to the oxygen concentration at RT in oxygen-saturated water and 0 μmol / L, respectively.

[0524] Reaction between protein and PCA Three 100 mL solutions, each containing 4% w / w protein based on WPI-A powder, were prepared by mixing the powder with MQ water and then allowing the mixture to hydrate at approximately 20°C with gentle stirring for 1 h. After this, the inventors observed that no powder particles remained and the solutions became transparent.

[0525] The solutions were adjusted to pH 7, pH 8, and pH 9 using a minimum amount of 1M NaOH. As described in Table 1, the WPI-A samples contained very high levels of BLG, and the inventors confirmed that this was achieved by using a specific extinction coefficient of 0.96 L / g / cm. -1 The absorbance of the solution was measured at 278 nm, and the target concentration was achieved within 5% error.

[0526] Prepare 32 mM stock solutions of 4MC (MW 124.13 g / mol) or EGCG (MW 458.372 g / mol) in MQ water.

[0527] The protein solution (pH 7, pH 8, or pH 9), PCA (4MC or EGCG), and water were mixed to a final protein concentration of 3% w / w (1.6 mM) and a PPA:BLG mol:mol ratio of 2:1 (3.2 mM) or 5:1 (8 mM) to produce WPI-A4 to WPI-A15 solutions as shown in Table 2.

[0528] The sample was incubated in a sealed 2 mL HPLC vial at 50°C for 1 h, the vial containing approximately 40% liquid and approximately 60% headspace (atmospheric air) by volume. The inventors estimated that the combined amount of O2 present in the liquid and headspace was sufficient to oxidize substantially all of the PCA in the reaction mixture to the quinone form.

[0529] Table 2: pH and PCA content / type of the samples.

[0530]

[0531] Determination of free thiol groups, total thiol groups, and total protein. The content of free thiol groups and total thiol groups in whey protein samples was quantified using the method described by Kurz et al. (2020) and the same equipment used by the authors. The content of free thiols (SH) in the samples was typically reported as μmol / g protein, where the protein content was determined by the total protein method described below.

[0532] Kurz, F., Hengst, C., & Kulozik, U. (2020). RP-HPLC method forsimultaneous quantification of free and total thiol groups in native and heataggregated whey proteins. MethodsX, 101112.

[0533] Determination of total protein content The total protein content (true protein) of a sample is determined by the following: 1) Determine the total nitrogen of the sample according to the following: ISO 8968-1 / 2|IDF 020-1 / 2- Milk - Determination of nitrogen content - Part 1 / 2: Determination of nitrogen content using the Kjeldahl method.

[0534] 2) Determine the non-protein nitrogen content of the sample according to the following: ISO 8968-4 | IDF 020-4 - Milk - Determination of nitrogen content - Part 4: Determination of non-protein nitrogen content.

[0535] 3) Calculate the total protein content as (m 总氮 - m 非蛋白氮 )*6.38.

[0536] Gel permeation chromatography (GPC) for analyzing the molecular weight of whey proteins The molecular weight of proteins in whey protein samples was analyzed using the SEC-MALS-IV-RI HPLC system via size exclusion chromatography. This system primarily consists of a Thermo ISO.3100SD pump, a WPS-3000TSL autosampler, and a Refractomax 521 refractive index detector. The system is also equipped with a Wyatt miniDawn TREOS II light scattering detector and a Wyatt Viscostar online viscometer.

[0537] All samples were diluted to 1% protein in elution buffer (10 mM phosphate, 30 mM NaCl, pH 7.0), and each sample (1 mL) was centrifuged at 15,000 g for 15 min to remove large protein aggregates. Injection volume: 10 μL sample / supernatant. Samples were separated in elution buffer at 0.75 mL / min using 1x Bio-SEC-5 guard + 2x 300Å BioSec-5.

[0538] Data Analysis: Weight-average molecular weight (MW) and number-average molecular weight (Mn) were determined using Astra software (v7.3.2.19) by analyzing the refractive index and light scattering signal before the void volume (i.e., both monomers, oligomers, and larger aggregates). BSA was used as a standard to verify correct calibration and showed a molecular weight of 64.4 kDa for the monomer peak (98% of the theoretical value).

[0539] Sample recovery is calculated as the integral of the eluted protein material relative to the total protein content in the sample before GPC sample preparation, where the centrifugation step can remove very large amounts of protein material.

[0540] Except for sample WPI-A3 (which showed a recovery of 42.6%, presumably due to excessive aggregation), all samples showed surprisingly high quality recoveries of >80%, with most samples even reaching 90%-100%, indicating that the vast majority of protein matter was included in the analysis.

[0541] LC-MS analysis of protein quality in protein-PCA samples The protein-PCA sample was diluted 30-fold with MQ water to a protein concentration of 0.1%. The solution was filtered into HPLC vials (1.5 mL Short Thread Vial, 32 mm × 11.6 mm, clear glass (ML33117) with 0.1 mL Micro-Insert, 31 mm × 6 mm, clear glass (ML33119)) using a 0.22 μm PVDF filter. 10 μL of the solution was injected into the HPLC system, where the protein was separated using an Avantor™ Ace™ ultracore BIO300A C4 HPLC column.

[0542] Mobile phase A: H2O containing 0.1% TFA (v / v) Mobile phase B: Acetonitrile containing 0.08% TFA (v / v).

[0543] Table 3: HPLC gradient

[0544] Use the following to detect proteins: UV 214nm The detector and Thermo Scientific Q-Exactiveplus, in which Orbitrap MS is positively polarized, has a scan range of 1000 m / z–3500 m / z, a resolution of 140,000, and a temperature of 500 °C.

[0545] UV 214nm Data was analyzed using FreeStyle software from Thermo Scientific. The Genesis algorithm was used to analyze UV ​​data. 214nm The chromatogram is integrated with a cutoff peak size of 0.1% of the highest peak, a minimum peak signal-to-noise ratio of 2, and valley detection enabled. Injection peaks at the beginning and end of the chromatogram, which are also present in the blank injection, are removed from the integrated peaks.

[0546] High-resolution mass spectrometry data were used to determine the protein mass of peaks in the chromatogram. The total ion chromatogram was integrated using the Genesis algorithm under the same conditions as the UV peak integration. Mass-to-charge ratio (MTR) data were then extracted from each peak, and the MTR data were deconvolved using the Xtract deconvolution feature, where H... + As an adduct ion, its charge range is 5-50, and the minimum detectable charge number is 3.

[0547] UHT processing Transfer 1.0 mL of sample to a 2 mL HPLC vial and seal it using an aluminum cap (Mikrolab ML 33032) and an electronic crimper (Thermo Scientific CRMA60180-ECRH11KI).

[0548] The sealed vial (at room temperature) was transferred to the aluminum heating block of the Mikrolab ultra-high temperature system (control unit ML 306228 and heating unit ML3062409, Mikrolab A / S, Denmark), which had holes drilled by the manufacturer to match the dimensions of the 2 mL GC vials. The block was preheated to 160°C, and the sample was held in the block for 160 seconds. The temperature reached 100°C in approximately 40 seconds, approximately 120°C in 65 seconds, and 140°C after 100 seconds of incubation, and 150°C after 160 seconds. After incubation in the heating block, the sample was transferred to an ice-water bath to rapidly quench the reaction that resulted in an unpleasant odor. (According to the section "...") Quantification of H2 S using a H 2 S sensor ( use H 2 S sensor for H 2 Quantitative analysis of S H2S was measured directly in a sealed vial.

[0549] Quantitative analysis of H2S using an H2S sensor The H2S level was measured using a microsensor (SULF-NPLR, needle type, Unisense A / S, Denmark), which was connected to a single-channel amplifier (Monometter-9514, Unisense A / S, Denmark). The resulting H2S signal, expressed in millivolts, was used as an indicator of the H2S level in the sample and recorded in the software "LOGGER" provided by Unisense A / S. The microsensor was calibrated using an H2S calibration kit (Calkit-H2S, Unisense A / S, Denmark) provided by the manufacturer. Following Section 7 of the manual (November 2020 edition, Unisense A / S), the highest concentration of H2S in the calibration kit was further diluted 10-fold. The H2S concentration could be automatically converted to μmol by the software.

[0550] To measure samples from UHT, the samples were equilibrated at 20°C for 30 minutes, and the sensor needle was inserted into the liquid phase of the sample after piercing the silicon seal. Two measurements were performed on each sample.

[0551] result The effect of the reaction between protein and PCA on the free thiols (SH) of the protein. The free thiol content in samples WPI-A1 to WPI-A15 is as described above in the section " Free thiol groups and total thiols Determination of functional groups and total protein The measurements were performed as described. The results are shown in Table 4.

[0552] Without the addition of PCA, after incubation at 50°C for 1 hour, the free thiol content of 45-46.3 μmol SH / g protein was measured in samples WPI-A1 to WPI-A3.

[0553] Surprisingly, WPI-A4 incubated with 2:1 4MC:BLG at pH 7.0 showed a 21.3% reduction in free thiol content relative to WPI-A1 (without PCA) over a 1-h incubation period. Even greater reductions in free thiol content were observed in WPI-A5 and WPI-A6 at pH 8 (78.9%) and pH 9 (89.8%), respectively.

[0554] Increasing the 4MC dose in samples WPI-A7 to WPI-A9 to a 5:1 4MC:BLG ratio showed a similar pattern, with free thiols in samples WPI-A8 and WPI-A9 reduced by 90% and 96%, respectively, compared to the samples without added 4MC.

[0555] The inventors further investigated the ability of EGCG, which is abundant in green tea extract, to reduce free thiols in whey protein compositions.

[0556] Samples WPI-A10 to WPI-A12 were incubated at a 2:1 EGCG:BLG molar ratio. Significant reductions of 95% and 100% were observed in samples WPI-A11 and WPI-A12, respectively, compared to samples without EGCG (WPI-A2 and WPI-A3, respectively).

[0557] Increasing the EGCG dosage to 5:1 EGCG:BLG resulted in a 99.6%-100% reduction in free thiol content relative to WPI-A2 and WPI-A3.

[0558] The effect of the reaction between protein and PCA on the molecular weight of protein. As described above, the molecular weight of protein substances in samples WPI-A1 to WPI-A3 without added PCA and samples WPI-A4 to WPI-A15 incubated with PCA at 50°C for 1 h were analyzed by GPC.

[0559] Table 4 shows the weight-average molecular weight of the samples and the estimated weight-average molecular weight from 26.5 kDa to 278.3 kDa.

[0560] The largest weight-average molecular weight (MW) was observed in WPI-A3, at 278.3 kDa, with a recovery rate of 42.6%. The inventors anticipate that even larger aggregates removed during sample preparation account for approximately 58% of the remaining protein.

[0561] As shown in Table 4, the inventors surprisingly found that adding PCA to the WPI samples consistently resulted in smaller MW estimates compared to WPI samples with a matching pH but without added PCA. This indicates that modification with added PCA reduces aggregation during incubation by blocking the thiol-disulfide exchange reaction.

[0562] A closer examination of the chromatograms and analysis of the molecular weight of individual peaks revealed that when analyzing samples WPI-A1 to WPI-A15, the eluted protein fractions primarily followed the order of monomers > dimers > trimers > tetramers > higher-order aggregates.

[0563] Because a large amount of protein was found to elute as monomers or dimers in GPC analysis, the inventors discovered that using UV-sensitive polymers for intact proteins... 214nm HPLC analysis and MS detection are appropriate to assess the covalent binding of PPA to WPI-A (see section "..."). LC-MS analysis of protein quality in protein-PCA samples ”).

[0564] The inventors were able to successfully determine that in BLG-containing samples with 4MC at pH 8 (WPI-A5 and WPI-A8) and pH 9 (WPI-A6 and WPI-A9), the dominant form of BLG protein was a modified form of BLG, with an increase in mass of 122 Da relative to both BLG A and BLG B isoforms, which corresponds to the mass of the quinone form of 4MC.

[0565] Furthermore, the inventors observed modified variants of BLG in samples with 2:1 EGCG:BLG (WPI-A11 and WPI-A12) and 5:1 EGCG:BLG (WPI-A14 and WPI-A15), with an increase in mass of 456 Da, which corresponds to the mass of EGCG quinone in EGCG-containing samples at pH 8 and pH 9.

[0566] Visual inspection of samples Samples WPI-A1 to WPI-A3 showed no color change during heat treatment and remained completely transparent. In samples containing PCA, significant color changes were observed during heat treatment, with WPI-A4 to WPI-A9 being brown and WPI-A10 to WPI-A15 being pale brown / yellow. The color intensity variations between samples depended on both pH and the PCA:BLG stoichiometry, as shown in Table 4. Furthermore, at alkaline pH and in the presence of amine (Tris buffer and folded BLG before heat treatment), a red / pink color was observed in 4MC. Notably, no red / pink color was observed in the samples.

[0567] Table 4: Residual free thiols, molecular weight, and visual appearance after incubation with oxidized PCA. The percentages shown represent the level of reduction in free thiols relative to WPI samples (samples WPI-A1 to WPI-A3) without added PCA at the same pH.

[0568]

[0569] The effect of the reaction between proteins and PCA on H2S production The pH of sample WPI-A5 (2:1 4MC:BLG) and reference sample WPI-A2 were adjusted to pH 7 and subjected to UHT treatment (see section "..."). UHT processing (This is followed by the chapter ""). Use H 2 S sensor for H 2 Quantitative analysis of S The generated H2S (odor) was quantified. The reference (WPI-A2) generated 7.9 μmol H2S, while only 1.5 μmol H2S was present in the 4MC-treated sample (WPI-A5), indicating that the odor was reduced in the 4MC-treated sample.

[0570] in conclusion The inventors have demonstrated that oxidation of PCA at pH 7 to pH 9 is sufficient to reduce or even eliminate the amount of free thiols in BLG in whey protein solutions. The inventors have also observed that, by increasing temperature and / or pressure, even lower free thiols levels can be obtained at pH 7.0.

[0571] Therefore, reacting BLG and other whey proteins with oxidized PCA constitutes a feasible approach to reduce the content of free thiol groups in proteins. The inventors have previously observed that proteins with high free thiol group content cause unpleasant odors when used in whey protein solutions or beverages undergoing, for example, UHT treatment. Therefore, the present invention provides a novel and surprising method for reducing the generation of unpleasant odors during the production and consumption of heat-treated, pH-neutral, whey protein-rich beverages.

[0572] Example 2: Dose response of 4MC undergoing electrochemical oxidation The purpose of this experiment is to document the possibility of using electrochemical oxidation to generate quinone compounds from polyphenol solutions and using the generated quinone compounds to reduce the amount of free thiols in whey proteins.

[0573] Materials and methods Protein source: WPI-A, as described in Example 1, is used as a protein source.

[0574] Determination of quinone content: To determine the concentration of quinones generated, L-glutathione (GSH) was used for assay. The principle of this assay is that the quinones react with free thiols in the GSH, and then the amount of unreacted GSH remaining is determined using RP-HPLC. Therefore, the quinone content corresponds to the loss of GSH. The inventors have found this GSH assay to be a good alternative to Analysis 2.

[0575] A 6 mM GSH solution was prepared in 5 mM phosphate buffer at pH 4.5. The GSH and quinone solution were mixed 1:1 in an Eppendorf tube for the reaction, and the mixture was separated and quantified at 214 nm relative to a standard curve of known GSH concentration using the same method used to determine the free thiol concentration in Example 1. The amount of quinone was then determined as the concentration of GSH lost compared to a GSH sample mixed 1:1 with an electrolyte buffer (quinone-free).

[0576] Electrochemical oxidation of quinone compounds: Deoxygenation was performed in 30 mL of 5 mM phosphate buffer (pH 4.5) and 200 mM NaCl solution by purging with nitrogen in an electrolytic cell (Redoxme 50 mL dual-chamber electrolytic cell; Redoxme AB, Sweden). The whey protein-quinone sample and a reference whey protein sample were incubated at 80 °C for 30 minutes and then cooled.

[0577] Removal of excess quinone solution after reaction with proteins: Unreacted / excess 4MC and 4MBQ after reaction with proteins were partially removed from the sample by ultrafiltration: 1 mL of the reacted sample was transferred to an Amicon ultraspin filter with a 10 kDa cutoff and centrifuged at 3400 x g for 20 min in a oscillating bucket rotor, allowing a collection of 100 μL of lysate. The lysate was recovered by diluting it to 1% protein using 5 mM phosphate buffer (pH 7) and transferring each sample to an Eppendorf vial for further analysis. Using this filtration procedure, the inventors observed no signs of protein loss.

[0578] UHT processing: The UHT processing and measurement of H2S generated in the sample are quantified using an H2S sensor, as described in Example 1.

[0579] The content of free thiol groups was measured in the recovered residue and after UHT treatment, as described in Example 1.

[0580] result: Cyclic voltammetry showed a typical "duck" shape, with the peak oxidation potential of 4MC at approximately +550 mV, after which 4MC was used to generate quinone compounds during bulk electrolysis. A yield of 27.7% 4MBQ was determined by GSH quinone determination, producing 4MBQ:BLG ratios ranging from 0.07:1 (sample WPI-A16) to 0.56:1 (sample WPI-A19), as given in Table 5.

[0581] After the reaction was terminated by cooling and excess reagent was removed, the free thiol content was analyzed as shown in Table 5 for WPI-A16 to WPI-A19. Surprisingly, even a small amount of added quinone solution was sufficient to significantly reduce the amount of free thiol in WPI-A16, and an even greater reduction was observed after UHT treatment. Therefore, the inventors found that the electrochemical oxidation of the 4MC solution to form quinone compounds, and the mixing of the quinone compounds with BLG under conditions allowing reaction with free thiols, can reduce the amount of free thiol to a level producing less than 1 μmol / L H2S, as detected by an H2S sensor. This contrasts sharply with the highly undesirable level of 16.9 μmol / L measured after UHT treatment in the untreated WPI-A20 control. Surprisingly, the inventors also observed that WPI-A16 was substantially colorless.

[0582] The addition of larger amounts of quinone solution in WPI-A17 to WPI-A19 reduced the residual thiol content in the samples to even lower levels, and UHT treatment further reduced the free thiol content. Therefore, after UHT treatment, H2S levels below 1 μmol / L were measured in these samples. The results of Example 2 are summarized in Table 5.

[0583] Table 5. Results obtained in Example 2.

[0584]

[0585] in conclusion: The inventors have documented that it is feasible to reduce the content of free thiol groups by generating quinone compounds through electrochemical oxidation and then mixing them with whey protein, even when using surprisingly low stoichiometry between the quinone compounds and BLG.

[0586] The reduced free thiol content resulted in low levels of H2S during UHT treatment of the modified protein samples. The inventors noted that the tested samples contained only 1% BLG. Higher whey protein content leads to significantly higher levels of H2S.

[0587] It was found that the reaction products produced by the sample with the lowest added amount of quinone (WPI-A16) were substantially colorless, while the samples based on the reaction with higher stoichiometry were slightly colored. Therefore, if the purpose is to prepare a protein source for colorless beverages, the inventors found that using the lowest possible amounts of polyphenols and quinones is preferable to achieve the desired level of free thiol reduction.

[0588] The inventors have also observed that performing a second heat treatment, such as UHT treatment, to further reduce the free thiol content in the whey protein solution during the incubation step can be beneficial. The inventors further point out that UHT-treated whey protein solutions, such as those in Table 5 (or their protein concentrates), can be converted into protein powder by, for example, spray drying or other drying methods.

[0589] Example 3: The Effects of Controlling Oxygen Exposure The aim of these experiments was to document how the O2-based oxidation of polyphenols into quinones could be controlled by manipulating the available headspace volume.

[0590] Methods and Materials Protein source: These experiments used WPI-A from Example 1.

[0591] Sample preparation: The following samples were prepared using WPI-A as the protein source.

[0592] Samples WPI-A21 and WPI-A22: Stock solutions containing 3% protein from WPI-A, 3.2 mM 4MC, and a pH of 8.0 were prepared (with a 2:1 molar ratio of 4MC to BLG). Sample WPI-A21 was prepared by transferring 1 mL of the stock solution to a 2 mL HPLC vial (see UHT treatment in Example 1), which was crimped and sealed to minimize further exposure to atmospheric air. Thus, sample WPI-A21 consisted of 1 mL of aqueous sample and 1 mL of headspace containing atmospheric air including hydrogen peroxide.

[0593] Sample WPI-A22 was prepared by transferring 2 mL of the stock solution into a 2 mL HPLC vial and then sealing the vial as described above. Therefore, sample WPI-A22 does not contain headspace.

[0594] Sample WPI-A23: Sample WPI-A23 was prepared by transferring 1 mL of a stock solution similar to the stock solution described above but without the addition of 4MC into a 2 mL HPLC vial. The HPLC vial was crimp-sealed to reduce further exposure to atmospheric air and thus contained an equal volume of aqueous phase and atmospheric air headspace.

[0595] WPI-A21 to WPI-A23 were incubated at 20°C for 2 days to allow sufficient time for the production of quinone compounds via O2-based oxidation and, where applicable, O2 transfer from the headspace.

[0596] As described in Example 1, the free thiol content of all samples was quantified.

[0597] result Table 6. Results obtained in Example 3.

[0598]

[0599] *) Top space relative to the total volume of the vial The inventors have previously discovered that a slightly alkaline pH is useful for exposing free thiols in BLG, and have also found that an extended incubation period of 2 days with an equal volume of sample in topspace with atmospheric air constitutes an effective means of blocking free thiols in the sample, as demonstrated below: Free thiols in sample WPI-A21 were reduced by 1.7 μmol / g protein compared to 45 μmol / g protein WPI-A23 incubated in the absence of PCA. Surprisingly, both WPI-A21 and WPI-A22 were essentially colorless, despite using a 3% WPI-A concentration. The inventors hypothesize that the lack of color production is closely related to O2 limitation, which allows sufficient quinones to react with the free thiols in WPI-A21, but not exceeding the O2 level that would lead to undesirable color production.

[0600] Furthermore, the inventors discovered that by completely filling the vial and thus leaving no headspace to restrict access to atmospheric air, the free thiol content in sample WPI-A22 was more moderately reduced to 19.1 μmol SH / g protein, corresponding to a reduction of approximately 57% in free thiols compared to WPI-A23. The inventors hypothesize that only O2 dispersed in the sample prior to the experiment allows for the formation of some quinones, which then react with the free thiols; however, the absence of headspace to act as a reservoir for O2 results in a lower blocking efficiency for free thiols compared to WPI-A21. Therefore, the inventors found it beneficial to increase O2 exposure by increasing O2 accessibility, exemplified by the larger (but still limited) atmospheric headspace of sample WPI-A22, in which the free thiol group content was reduced to 1.7 μmol SH / g protein.

[0601] In addition to controlling O2 exposure through accessible headspace, the inventors also suggest other means of controlling O2 accessibility, such as pressurization or an atmosphere with a higher concentration of O2.

[0602] Furthermore, although the samples in this study were not stirred, it may be beneficial to stir the samples to increase the rate of O2 transfer to the aqueous phase.

[0603] in conclusion: These results demonstrate that restricted access to O2 reduces the likelihood of O2-based generation of quinone compounds that can react with free thiols.

[0604] Therefore, the inventors conclude that when O2 is the only oxidant used for the generation of quinone compounds, it is advantageous to control and ensure the availability of sufficient O2.

[0605] The inventors have discovered that the higher protein concentration used during the processing in this embodiment is advantageous for industrial implementation, and that protein solutions with even higher protein concentrations can be processed according to the present invention.

[0606] The inventors further conclude that restricting O2 accessibility constitutes an attractive method for reducing the generation of unwanted colors in products.

[0607] Example 4: Direct heating under UHT-like conditions The purpose of these experiments was to document the possibility of forming quinone compounds using PCA and reacting them with free thiols under UHT-like heating conditions.

[0608] Materials and methods These experiments used WPI-A from Example 1.

[0609] Sample preparation: Prepare a stock solution containing 3% protein from WPI-A, 3.2 mM 4MC, and pH 8.0 (with a molar ratio of 2:1 between 4MC and BLG).

[0610] Sample WPI-A24 was prepared by transferring 1 mL of the stock solution into a 2 mL HPLC vial and subsequently sealing the vial as described in Example 3. Therefore, sample WPI-A24 contains equal volumes of aqueous phase and atmospheric air headspace.

[0611] Sample WPI-A25: Sample WPI-A25 was prepared by transferring 1 mL of a stock solution similar to the stock solution described above but without the addition of 4MC into a 2 mL HPLC vial, which was press-sealed and therefore also contained an equal volume of aqueous phase and atmospheric air headspace.

[0612] The HPLC vials containing samples WPI-A24 and WPI-A25 were incubated in an aluminum block preheated to 160°C for 160 seconds and immediately cooled in an ice-water bath. The free thiols were then quantified as described in Example 1.

[0613] result Table 6. Results obtained in Example 4.

[0614]

[0615] *) Top space relative to the total volume of the vial The results presented in Table 6 confirm that heating sample WPI-A25 in the absence of quinone compounds was insufficient to eliminate the free thiols supplied by WPI-A. Conversely, heating sample WPI-A24 containing a 2:1 4MC:BLG molar ratio and equal volumes of headspace and aqueous phase completely eliminated the free thiols.

[0616] The inventors further observed through visual inspection that WPI-A24 is essentially colorless, indicating that O2 availability is sufficient to generate enough quinone compounds to reduce the amount of free thiols, but avoids the formation of colored products.

[0617] in conclusion The inventors have discovered that combining pH 8 and a UHT-like treatment to promote full exposure of free thiols to quinone compounds derived from 4MC, and using equal volumes of liquid sample and atmospheric air headspace, allows for effective blocking of free thiols at high temperatures for short periods.

[0618] The inventors also noted that the accessible headspace volume is equal to that of sample WPI-A21 in Example 3, to ensure sufficient accessibility to O2 to promote O2-based quinone formation, which, combined with high temperature, allows for effective blocking of free thiols in sample WPI-A24 compared to WPI-A25.

[0619] Example 5: In-situ formation and reaction of quinone compounds with free thiols The aim of these experiments was to document the potential for the formation of quinone compounds using PCA in the presence of whey protein during a “one-step” modification process that is considered favorable for industrial processing.

[0620] method Cyclic voltammetry was performed to assess the extent and potential of the electrochemical reaction as described in Example 2. First, 30 mL of a 1% WPI-A solution in 200 mM 25 mM phosphate buffer (pH 7.5) was preheated to 60°C within an aluminum block. The heating block was specifically designed to fit and completely span the sides and bottom of the electrochemical electrolyzer of Example 2 and was placed within the heating unit for UHT treatment as described in Example 1. Cyclic voltammetry was performed using the electrodes described in Example 2, with a voltage applied from -0.2 V to +0.8 V. Next, 30 mL of 1.09 mM 4MC in 200 mM NaCl (pH 7.5) was analyzed, and finally, 30 mL of a 1% WPI-A solution in 200 mM NaCl and 1.09 mM 4MC (2:1 4MC:BLG) was analyzed.

[0621] Based on cyclic voltammetry measurements, 30 mL of a 1% WPI-A solution in 25 mM phosphate buffer (pH 7.5, 200 mM) was preheated to 60°C inside an aluminum block. The heating block was specially designed to fit and completely span the sides and bottom of the electrochemical electrolyzer of Example 2 and was placed in the heating unit for UHT treatment as described in Example 1.

[0622] Two mL of WPI-A26 sample was removed before adding 4MC and bulk electrolysis to establish zero reaction time, as shown in Table 7.

[0623] Immediately after sampling WPI-A26, 250 μL of 33 mM 4MC dissolved in nitrogen-degassed MQ water and kept in a syringe to minimize oxygen exposure was injected into the electrolyzer via an HPLC connector to achieve a 4MC:BLG ratio of 0.5. A potential of 0.45 V was applied to the electrolyzer and timing was started.

[0624] After 5 min of bulk electrolysis, 2 mL of WPI-A27 sample was taken and then 250 μL of 33 mM 4MC was added to achieve a 4MC:BLG molar ratio of 1.2:1.

[0625] After 10 minutes of bulk electrolysis, take 2 mL of WPI-A28 sample and stop the experiment.

[0626] All samples were cooled to 4°C immediately after sampling.

[0627] Unreacted reagents are removed by using a rotary filter as described in Example 2.

[0628] As described in Example 1, the free thiols in the rotary filter residues of samples WPI-A26 to WPI-A28 were determined.

[0629] The residues of WPI-A26, WPI-A27, and WPI-A28 were subjected to a UHT-like heat treatment and H2S was determined, as described in Example 1.

[0630] In a separate experiment, as described for sample WPI-A26, sample WPI-A29 (30 mL 5% WPI-A in 100 mM NaCl, pH 8.5, prepared from nitrogen-degassed MQ) was equilibrated in an electrochemical electrolyzer at 60 °C. 4 MC was added to a final concentration of 5.4 mM (2:1 4 MC: BLG), and bulk electrolysis was performed at 0.45 V for 20 minutes. The reaction was quenched by diluting to 1% protein content using cold 25 mM phosphate pH 7.0 buffer. Unreacted reagents were removed using a rotary filter as described in Example 2. Free thiols in the rotary filter residue of sample WPI-A29 were determined as described in Example 1.

[0631] result Table 7. Results obtained in Example 5.

[0632]

[0633] Without the addition of 4MC, cyclic voltammetry performed at 60°C on a 1% WPI-A solution at pH 7.5 showed no signs of oxidation or reduction peaks, but instead exhibited a characteristic "duck-shaped" pattern. Conversely, the inventors observed that the applied voltage range did indeed result in a duck-shaped voltammogram when analyzing quinones alone and quinones in combination with proteins. Therefore, the applied voltage was considered to mediate the oxidation of 4MC to 4MBQ. An oxidation potential of 0.45 V was determined using the experiments in Example 5.

[0634] As shown in Table 7, when analyzing WPI-A26 before the addition of 4MC, the inventors initially observed high levels of free thiols and correspondingly high H2S formation after UHT treatment.

[0635] After 5 minutes of bulk electrolysis with 4MC added to a 0.5:1 4MC:BLG ratio, the thiol content of sample WPI-A27 decreased to 23 μmol SH / g protein. The inventors noted that the reaction mixture remained substantially colorless.

[0636] After 10 minutes of bulk electrolysis with the addition of a 1.2:1 4MC:BLG mixture, the thiol content of sample WPI-A28 was further reduced to 6 μmol SH / g protein. Furthermore, due to the reduced free thiol content, less than 1 μmol / L of H2S was measured in the UHT-treated sample.

[0637] Furthermore, the inventors observed only a faint color production in the residue of WPI-A28. The inventors had previously observed that bulk electrolysis of 4MC at neutral pH in the absence of whey protein could result in significant color production. In this study, the inventors observed a significant reduction in color production, and no signs of unwanted side reactions were observed during bulk electrolysis.

[0638] The resulting sample, WPI-A29, had a higher protein content (5%) and 5.4 mM 4MC (2:14MC:BLG molar ratio) than other samples. After 20 minutes of bulk electrolysis, a significant decrease in free thiol content was observed to 5.7 μmol SH / g protein compared to WPI-A treated without the addition of 4MC (see, for example, WPI-A26). The inventors noted that approximately 9 mg / L (~280 μM) of oxygen is soluble in water at 20 °C, and this amount decreases with increasing temperature. Therefore, although the inventors used a nitrogen-washed buffer source for sample preparation to reduce possible oxidation by dissolved O2, even oxygen saturation in the initial solution corresponded only to ~5% (5.4 mM) of the present polyphenol concentration.

[0639] In summary, these experiments clearly demonstrate both the feasibility of using the direct electrochemical generation of quinones to facilitate their reaction with free thiols, and the potential of using the electrochemical oxidation of polyphenols and their reaction with free thiols to reduce the unwanted H2S generation during UHT treatment.

[0640] in conclusion: Electrochemical oxidation of quinone compounds in the presence of whey protein at a slightly alkaline pH and elevated temperature constitutes an advantageous method for reducing the content of free thiol groups in whey protein solutions.

[0641] The inventors have discovered that by using relatively low doses of polyphenols and by directly oxidizing the polyphenols in the presence of whey protein, the concentration of quinone compounds can be kept low during the reaction steps of the method of the present invention. The inventors have also found that this method reduces or even avoids color formation caused by quinone side reactions. This method is particularly advantageous for preparing protein sources that will be used in colorless food applications, such as colorless high-protein beverages.

Claims

1. A method of producing a protein composition comprising modified beta-lactoglobulin (BLG), said method comprising the steps of: a) providing: a source comprising one or more phenolic compounds (PCA) comprising at least two hydroxyl groups directly bound to the same aromatic ring, and a source comprising BLG, b) optionally subjecting a part of the source comprising one or more PCA to a type of oxidation capable of converting PCA to quinone (referred to as PCA-type oxidation), thereby providing a source comprising one or more oxidized PCA, c) combining a part of the source comprising one or more PCA and / or a part of the source comprising one or more oxidized PCA with the source comprising BLG and optional further ingredients to provide a protein solution, said protein solution having: a pH in the range of 6.5-9.5, and a BLG content of at least 0.2% w / w, a molar ratio between: the original amount of PCA used to make the protein solution, and the content of BLG of the protein solution, of at least 0.1 : 1, d) incubating the protein solution in a temperature range and for a duration sufficient to reduce the amount of free thiol groups of the protein solution to at most 10 pmol / g protein, with the proviso that if the method does not comprise step b), step d) also involves applying a type of oxidation capable of converting PCA to quinone.

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

1.

3. The method according to claim 1 or 2, wherein the PCA comprises: a flavonoid, preferably a flavanol or a flavanol ester, such as catechin, epicatechin, gallocatechin, epigallocatechin, catechin 3-gallate, epicatechin 3-gallate, gallocatechin 3-gallate, and epigallocatechin 3-gallate (EGCG), and / or a stilbenoid, preferably resveratrol.

4. The method according to any one of the preceding claims, wherein the PCA: comprises caffeic acid, gallic acid, chlorogenic acid, catechol, 4-methylcatechol, 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, and / or ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ having a molecular weight in the range of 120 g / mol - 1000 g / mol, more preferably 250 g / mol - 700 g / mol, even more preferably 300 g / mol - 650 g / mol and most preferably 350 g / mol - 600 g / mol, and / or having a water solubility of at least 8 mM at 25°C, more preferably at least 12 mM and most preferably at least 16 mM.

5. The method according to any one of the preceding claims, wherein the source comprising one or more PCAs: is selected from the group consisting of polyphenol extract from herbs, polyphenol extract from spices, polyphenol extract from fruits, polyphenol extract from berries and mixtures thereof, and / or is selected from the group consisting of polyphenol extract of tea, more preferably polyphenol extract of green tea; polyphenol extract of coffee; polyphenol extract of cocoa; polyphenol extract of grape; polyphenol extract of rosemary; polyphenol extract of lemon balm; polyphenol extract of black currant; single PCA isolate, and mixtures thereof.

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

7. The method according to any one of the preceding claims, comprising step b) subjecting a part of the source comprising one or more PCAs to a type of oxidation capable of converting PCAs to quinones, thereby providing a source comprising one or more oxidized PCAs.

8. The method according to any one of the preceding claims, wherein the protein solution has one or more of, and more preferably all of, the following: a pH in the range of 6.7 - 9.5, even more preferably 7.1 - 9.0, even more preferably 7.3 - 8.7 and most preferably 7.4 - 8.5, 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% w / w - 30% w / w, more preferably 1% w / w - 20% w / w, even more preferably 3% w / w - 16% w / w and most preferably 5% w / w - 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% w / w - 99% w / w relative to total protein, more preferably 40% w / w - 95% w / w relative to total protein, even more preferably 45% w / w - 90% w / w relative to total protein and most preferably 50% w / w - 80% w / w relative to total protein.

9. The method according to any of the preceding claims, wherein the protein solution of step c) has a molar 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 1 :

1.

10. The method according to any of claims 1-8, wherein the protein solution of step c) has a molar ratio between: the original amount of PCA used for preparing the protein solution, and the content of BLG of the protein solution, of 0.1 : 1 - 100: 1, more preferred 0.1 : 1 - 50: 1, even more preferred 0.1 : 1 - 20: 1 and most preferred 0.1 : 1 - 10:

1.

11. The method according to any of claims 1-8, wherein the protein solution has a molar ratio between: the original amount of PCA used for preparing the protein solution, and the content of BLG of the protein solution, of 0.1 : 1 - 5: 1, more preferred 0.1 : 1 - 4: 1, even more preferred 0.2: 1 - 3: 1 and most preferred 0.3: 1 - 2:

1.

12. The method according to any of claims 1-8, wherein the protein solution has a molar ratio between: the original amount of PCA used for preparing the protein solution, and the content of BLG of the protein solution, of 1 : 1 - 100: 1, more preferred 1 : 1 - 50: 1, even more preferred 1 : 1 - 20: 1 and most preferred 1 : 1 - 10:

1.

13. The method according to any of claims 1-12, wherein the oxidation applied during step b) and / or step d) is sufficient to result in a molar ratio between: the theoretical amount of quinone provided to the protein solution during the method determined according to analysis 1, and the content of BLG of the protein solution, of 0.1 : 1 - 100: 1, more preferred 0.1 : 1 - 50: 1, even more preferred 0.2: 1 - 15: 1 and most preferred 0.2: 1 - 5:

1.

14. The method according to any of claims 1-12, wherein the oxidation applied during step b) and / or step d) is sufficient to result in a molar ratio between: the theoretical amount of quinone provided to the protein solution during the method determined according to analysis 1, and the content of BLG of the protein solution, of 0.1 : 1 - 5: 1, more preferred 0.1 : 1 - 4: 1, even more preferred 0.2: 1 - 3: 1 and most preferred 0.2: 1 - 2:

1.

15. The method according to any of claims 1-12, wherein the oxidation applied during step b) and / or step d) is sufficient to result in a molar ratio between: the theoretical amount of quinone provided to the protein solution during the method determined according to analysis 1, and the content of BLG of the protein solution, of at least 1 :

1.

16. The method according to any one of claims 1-12, wherein the oxidation applied during step b) and / or step d) is sufficient to yield a molar ratio between: the quinone content of the part of the source comprising one or more oxidized PCAs used in the protein solution, and the amount of BLG of the protein solution, of between 1 : 1-100: 1, more preferably 1 : 1-50: 1, even more preferably 1 : 1-15: 1, most preferably 1.5: 1-5:

1. the theoretical amount of quinone provided to the protein solution during the method as determined according to analysis 1, and the content of BLG of the protein solution.

17. The method according to any one of the preceding claims: wherein the method comprises step b), and the part of the source comprising one or more oxidized PCAs obtained from step b) is used for preparing the protein solution of step c), and wherein the PCA-type oxidation is carried out by electrochemical oxidation.

18. The method according to any one of the preceding claims, wherein the PCA-type oxidation applied during step b) is sufficient to yield a molar ratio between: the quinone content of the part of the source comprising one or more oxidized PCAs used in the protein solution, and the amount of BLG of the protein solution, 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.

19. The method according to any one of claims 1-16, wherein the PCA-type oxidation applied during step b) is sufficient to yield a molar ratio between: the quinone content of the part of the source comprising one or more oxidized PCAs used in the protein solution, and the amount of BLG of the protein solution, 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.

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

21. A protein composition comprising modified BLG and having at most 10 pmol free thiol groups per g protein, obtainable by the method according to one or more of the preceding claims, said 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 pg / mg protein, preferably a fat content of at most 3% w / w relative to total solids, preferably a content of protein-bound sulphur in the range of 100-600 pmol / g protein, Preferably, the content of protein-bound cysteine residues forming disulfide bonds is in the range of 150-400 pmol / g protein.

22. The modified whey protein composition according to claim 21, having a protein content of at least 30% w / w relative to total solids.

23. The modified whey protein composition according to claim 21 or 22, having a tryptophan content of at least 0.7% w / w relative to total protein.

24. The modified whey protein composition according to any one of claims 21 - 23, having a methionine content of at least 0.3% w / w relative to total protein.

25. The modified whey protein composition according to any one of claims 21 - 24, having a kynurenine content of at most 0.2 pg / mg protein.

26. The modified whey protein composition according to any one of claims 21 - 25, having a fat content of at most 3% w / w relative to total solids.

27. The modified whey protein composition according to any one of claims 21 - 26, having a content of protein-bound sulphur in the range of 100-600 pmol / g protein.

28. The modified whey protein composition according to any one of claims 21 - 26, having a content of protein-bound cysteine residues forming disulfide bonds in the range of 150-400 pmol / g protein.

29. The modified whey protein composition according to any one of claims 21 - 27, having an average molecular weight of the protein in the range of 18 kDa to 10000 kDa, more preferably between 50 kDa - 8000 kDa and most preferably between 80 kDa - 5000 kDa.

30. The protein composition according to any one of claims 21 - 27, having an average molecular weight of the protein in the range of 18 kDa to 500 kDa, more preferably between 18 kDa - 100 kDa and most preferably between 18 kDa - 40 kDa.

31. A process for producing a heat-treated, preferably heat-sterilized beverage, the process comprising the steps of: 1) mixing a protein composition according to one or more of claims 21 - 30 with one or more additional beverage ingredients to obtain a liquid mixture having a pH of 5.5-8.5, and 2) filling the liquid mixture into a suitable container, the process further 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 at least 0.5% w / w protein.

32. The process for producing a heat-treated, preferably heat-sterilized beverage according to claim 31, wherein the liquid mixture comprises at most 60 pmol free thiol groups per 100 g of liquid mixture, more preferably at most 40 pmol free thiol groups per 100 g of liquid mixture, even more preferably at most 30 pmol free thiol groups per 100 g of liquid mixture and most preferably at most 30 pmol free thiol groups per 100 g of liquid mixture prior to the heat-sterilization.

33. The process for producing a heat-treated, preferably heat-sterilized beverage according to claim 31, wherein the liquid mixture comprises at most 20 pmol free thiol groups per 100 g of liquid mixture, more preferably at most 15 pmol free thiol groups per 100 g of liquid mixture, even more preferably at most 10 pmol free thiol groups per 100 g of liquid mixture and most preferably at most 5 pmol free thiol groups per 100 g of liquid mixture prior to the heat-sterilization.

34. The process for producing a heat-treated, preferably heat-sterilized beverage according to any one of claims 31 -33, wherein the liquid mixture comprises a total amount of protein in the range of 0.5% w / w to 15% w / w relative to the weight of the liquid mixture, more preferably 1 % w / w to 10% w / w relative to the weight of the liquid mixture, even more preferably 2% w / w to 9% w / w relative to the weight of the liquid mixture and most preferably 3% w / w to 8% w / w relative to the weight of the liquid mixture.

35. The process for producing a heat-treated, preferably heat-sterilized beverage according to any one of claims 31 -34, wherein the liquid mixture comprises a total amount of protein in the range of 4% w / w to 15% w / w relative to the weight of the liquid mixture, more preferably 5% w / w to 14% w / w relative to the weight of the liquid mixture, even more preferably 6% w / w to 13% w / w relative to the weight of the liquid mixture and most preferably 8% w / w to 12% w / w relative to the weight of the liquid mixture.

36. The process for producing a heat-treated, preferably heat-sterilized beverage according to any one of claims 31 -35, wherein the protein composition according to one or more of claims 21 -30 contributes 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.

37. Process for the production of a heat-treated, preferably heat-sterilized beverage according to any one of claims 31-36, wherein the protein composition according to one or more of claims 21-30 contributes at least 90% w / w of the total protein, 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 of the liquid mixture.

38. Process for the production of a heat-treated, preferably heat-sterilized beverage according to any one of claims 31-37, 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 relative to total solids and most preferably at least 25% w / w relative to total solids and most preferably at least 30% w / w relative to total solids.

39. Process for the production of a heat-treated, preferably heat-sterilized beverage according to any one of claims 31-38, 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 relative to total solids, even more preferably at least 92% w / w relative to total solids and most preferably at least 94% w / w relative to total solids.

40. Process for the production of a heat-treated, preferably heat-sterilized beverage according to any one of claims 31-39, wherein the liquid mixture has a solids content of 0.5% w / w - 50% w / w, more preferably 1% w / w - 35% w / w, even more preferably 2% w / w - 20% w / w and most preferably 3% w / w - 10% w / w.

41. Process for the production of a heat-treated, preferably heat-sterilized beverage according to any one of claims 31-40, wherein the portion of the liquid mixture that does not consist 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.

42. Process for the production of a heat-treated, preferably heat-sterilized beverage according to any one of claims 31-41, wherein the liquid mixture has a caloric content of at most 100 kcal / 100 g, more preferably at most 80 kcal / 100 g, even more preferably at most 70 kcal / 100 g and most preferably at most 60 kcal / 100 g.

43. Process for the production of a heat-treated, preferably heat-sterilized beverage according to any one of claims 31-41, wherein the liquid mixture has a caloric content of more than 100 kcal / 100 g, more preferably at least 120 kcal / 100 g, even more preferably at least 140 kcal / 100 g and most preferably at least 150 kcal / 100 g.

44. A heat treated, preferably heat sterilized beverage having a pH of 5.5-8.5, obtainable by the process of one or more of claims 30-43.

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