A method for producing a modified protein composition by reaction with an oxidized phenol compound, the modified protein composition, and the nutritional uses of the modified protein composition.

Abstract

The present invention relates to a method for preparing a modified protein composition under conditions in which the free thiol groups of β-lactoglobulin are exposed to and modified by a reaction with an oxidized phenol compound. The resulting modified protein product (e.g., a protein-rich beverage product) has been found to have excellent performance, and in particular, it has been shown to reduce the level of unpleasant odors during sterilization heat treatment at a neutral pH and when such beverage products are consumed.

Description

[Technical Field]

[0001] The present invention relates to a method for preparing a modified protein composition under conditions in which the free thiol groups of β-lactoglobulin are exposed to and modified by a reaction with an oxidized phenol compound. The resulting modified protein product (e.g., a protein-rich beverage product) has been found to have excellent performance, and in particular, it has been shown to reduce the level of unpleasant odors during sterilization heat treatment at a neutral pH and when such beverage products are consumed. [Background technology]

[0002] Beverages rich in pH-neutral whey protein tend to develop an unpleasant odor similar to that of spoiled eggs during heat treatment. Since these beverages are typically bottled immediately after production, consumers are exposed to this unpleasant odor upon opening the bottle.

[0003] WO2021 / 198968A1 discloses a method for producing a beverage containing milk protein concentrate (MPC). The MPC is treated to enable heat homogenization of the beverage containing the MPC while reducing the generation of unpleasant odors (malodorous and / or eggy or sulfurous odors and / or tastes). Specifically, the MPC contains at least one type of whey protein, in which 50-100% of the whey protein is denatured, and in which its calcium content is reduced by approximately 5-20 percent by weight.

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

[0005] Poojary et al. studied the effects of adding green tea extract to skim milk powder before UHT (ultra-high temperature) treatment and investigated the formation of advanced glycation end products (AGEs) during long-term storage ("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, 2020D2).

[0006] Waqar et al. have disclosed the preparation and characterization of a complex of 4-methylbenzoquinone and β-lactoglobulin ("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, p. 133775). Waqar's literature does not disclose anything that would suggest a reduction in the generation of unpleasant odors due to chemical modification of β-lactoglobulin. [Overview of the project]

[0007] The inventors have noticed that the unpleasant odor of whey protein-rich beverages (often when the pH is neutral) is related to hydrogen sulfide generated from free thiol groups of cysteine ​​residues in whey protein during heat treatment.

[0008] The inventors have, surprisingly, further discovered that the above problems can be mitigated or avoided by reacting a β-lactoglobulin-containing protein with an oxidized phenol compound.

[0009] Thus, one aspect of the present invention relates to a method for preparing a protein composition containing modified β-lactoglobulin (BLG), the method comprising the following steps (a) to (d): (a) The process of providing the following sources; • A source containing one or more phenolic compounds, each containing at least two hydroxyl groups directly bonded to the same aromatic ring (PCA); and • Sources including BLG; (b) A step of optionally subjecting a portion of a supply containing one or more types of PCA to oxidation of a type that can convert PCA to quinone (PCA-type oxidation), thereby providing a supply containing one or more oxidized PCA; (c) A step of combining a portion of a source containing one or more PCA and / or a portion of a source containing one or more oxidized PCA with a source containing BLG and optionally further components for the purpose of providing a protein solution; Here, the protein solution is pH in the range of 6.5 to 9.5; and • BLG content of at least 0.2% w / w; • The molar ratio between the original amount of PCA used in the preparation of the protein solution and the BLG content of the protein solution, wherein the molar ratio is at least 0.1:1. Having; (d) Incubating the protein solution within a certain temperature range for a time sufficient to reduce the amount of free thiol groups in the protein solution to a maximum of 10 micromoles / g of protein; If the method does not include step (b), step (d) is a step of incubation under conditions that also include the use of an oxidation of a type capable of converting PCA to quinone; Preferably, the PCA type oxidation used in step (b) and / or step (d) is: The theoretical amount of quinone provided to the protein solution during the implementation of the method, the theoretical amount of quinone determined according to Analysis 1, and • BLG content of the protein solution, This is sufficient to make the molar ratio between them at least 0.1:1.

[0010] Another aspect of the present invention relates to a protein composition comprising modified BLG, which can be obtained by the method of the present invention.

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

[0012] A further aspect of the present invention relates to a heat treated beverage, preferably a heat sterilized beverage, having a pH of 5.5 to 8.5 and obtainable by the process of the present invention.

[0013] Another aspect of the present invention relates to a food ingredient comprising: · The solids of the protein composition of the present invention; and · One or more additional ingredients, preferably ingredients selected from: · Milk components, preferably non - oxidized milk components; · Plant - derived components; · Non - milk carbohydrate sources; · Flavoring and odor - masking agents; and / or · Sweeteners.

[0014] A further aspect of the present invention relates to the use of a protein composition comprising modified BLG, preferably for use as a food ingredient of a protein composition according to the present invention; Preferably for: · Improving the odor of a heat sterilized beverage having a pH in the range of 5.5 to 8.5; and / or · Reducing the level of an unpleasant odor similar to that of rotten eggs; and / or • To reduce H2S generation during manufacturing; and / or • To reduce the H2S content in the upper space of the container, This concerns its use; Here, the beverage preferably has a whey protein content of at least 3% w / w and is preferably heat-sterilized using indirect heat treatment. [Modes for carrying out the invention]

[0015] One aspect of the present invention relates to a method for preparing a protein composition containing modified β-lactoglobulin (BLG), the method comprising the following steps (a) to (d): (a) • A source containing one or more phenolic compounds, each containing at least two hydroxyl groups directly bonded to the same aromatic ring (PCA); and • Sources including BLG, A process to provide; (b) A step of optionally subjecting a portion of a source containing one or more types of PCA to an oxidation of a type that can convert PCA to quinone (called PCA-type oxidation), thereby providing a source containing one or more types of oxidized PCA; (c) A step of combining a portion of a source containing one or more PCA and / or a portion of a source containing one or more oxidized PCA with a source containing BLG and optionally further components for the purpose of providing a protein solution; Here, the protein solution is pH in the range of 6.5 to 9.5; and • BLG content of at least 0.2% w / w; • The molar ratio between the original amount of PCA used in the preparation of the protein solution and the BLG content of the protein solution, wherein the molar ratio is at least 0.1:1. Having; (d) Incubating the protein solution within a certain temperature range for a time sufficient to reduce the amount of free thiol groups in the protein solution to a maximum of 10 micromoles / g of protein; If the method does not include step (b), step (d) is a step of incubation under conditions that also include the use of an oxidation of a type capable of converting PCA to quinone; Preferably, the PCA type oxidation used in step (b) and / or step (d) is: The theoretical amount of quinone provided to the protein solution during the implementation of the method, the theoretical amount of quinone determined according to Analysis 1, and • BLG content of the protein solution, This is sufficient to make the molar ratio between them at least 0.1:1.

[0016] In relation to the present invention, the term "β-lactoglobulin" or BLG refers to BLG derived from mammalian species, e.g., native and / or glycosylated BLG containing native genetic variants. The term "BLG" also includes BLG produced by recombinant technology and / or microfermentation, e.g., based on genetically modified microorganisms or mammalian cells. In this specification, the terms "BLG" or "β-lactoglobulin" exclude denatured BLG and aggregated BLG. BLG content is measured according to analysis L of PCT International Patent Application Publication No. PCT / EP2022 / 078739.

[0017] In relation to the present invention, the term "phenol compound" has its usual meaning and refers to a molecular compound comprising an aromatic ring in which at least one of the six carbon atoms is directly bonded to a hydroxyl group. The other five carbon atoms of the aromatic ring may be individually bonded to various groups, including hydroxyl groups or hydrogen groups. The phenol compound also includes polyphenols.

[0018] In relation to the present invention, the term “phenol compound containing an aromatic ring to which at least two hydroxyl groups are directly bonded” (referred to as “PCA”) refers to a molecular compound containing an aromatic ring in which at least two carbon atoms within the ring are directly bonded to hydroxyl groups (e.g., catechol or epigallocatechin gallate (EGCG)). The other four carbon atoms of the aromatic ring may be individually bonded to various groups containing hydroxyl groups or hydrogen.

[0019] PCA preferably has the following chemical structural formula: [ka] Having; Here, at least two of R1, R2, R3, R4, R5, and R6 are hydroxyl groups.

[0020] Preferably, two or three of R1, R2, R3, R4, R5, and R6 are hydroxyl groups.

[0021] With respect to the present invention, the terms “PCA-to-quinone-converting type of oxidation” or “PCA-type oxidation” refer to an oxidation process that can oxidize PCA from a source containing one or more types of PCA to quinone. Such PCA-type oxidation typically involves electrochemical oxidation and / or the use of a chemical oxidizing agent.

[0022] With respect to the present invention, the expression “the PCA type oxidation used in step (b) and / or step (d) is sufficient to have a molar ratio of […]” is used to define the required level of PCA type oxidation in a manner that may apply to all embodiments of the method of the present invention. In this regard, “step (b) and / or step (d)” means that if the method includes PCA type oxidation occurring in both step (b) and step (d), the combined PCA type oxidation of both steps should be considered. However, if the method includes only PCA type oxidation in step (b), then only step (b) should be considered. Similarly, if the method includes only PCA type oxidation in step (d), then only step (d) should be considered.

[0023] As will be obvious to those skilled in the art, at least n X :n Y The molar ratio between component X and component Y that must be such that the molar ratio is at least n X to n Y This means that the value must be the result of dividing by n. X and n Y n 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 n X to n Y This means that the value obtained by dividing by must be at least 0.1. The same logic applies to weight ratios.

[0024] With respect to the present invention, the expression “theoretical amount of quinones supplied to the protein solution during the method” is a measure of the molar content of quinones produced when all PCA (including PCA converted to quinones) present in the protein solution of the method are oxidized under the same conditions and the same oxidation conditions used in the method, in the absence of protein or other sources of free thiols or amines. The “theoretical amount of quinones supplied to the protein solution during the method” is evaluated according to Analysis 1. For example, if the method of the present invention is based primarily on the oxidation of PCA (to provide a source containing one or more oxidized PCA) in step (b) prior to step (c), then the quinone content of a portion of the source containing one or more oxidized PCA used in step (c) is equal to the theoretical amount of quinones supplied to the protein solution during the method.

[0025] In some preferred embodiments of the present invention, a method for preparing a protein composition containing modified BLG further comprises the step (e) of drying a liquid material containing protein derived from the protein solution of step (d) incubating.

[0026] In some preferred embodiments of the present invention, the protein solution obtained in step (c) is: • The molar ratio between the original amount of PCA used in the preparation of the protein solution and the BLG content of the protein solution, having a molar ratio of at least 1:1; and The PCA-type oxidation used in step (b) and / or step (d) is preferably, The theoretical amount of quinone provided to the protein solution during the implementation of the method, the theoretical amount of quinone determined according to Analysis 1, and • BLG content of the protein solution, This is sufficient to make the molar ratio between them at least 1:1.

[0027] The term "source containing one or more PCA" refers to a composition used for the purpose of providing PCA for the protein solution of step (c) and / or the oxidizing aqueous solution of step (b). The term "part of a source containing one or more PCA" refers to the actual portion used in the preparation of the protein solution. In some preferred embodiments of the present invention, substantially all of a source containing one or more PCA is used.

[0028] When two or more PCA-containing compositions are used in the preparation of the protein solution in step (c) and / or the oxidizing aqueous solution in step (b), each of the PCA-containing components used is considered a secondary source that constitutes a source containing one or more PCA.

[0029] In some preferred embodiments of the present invention, the PCA comprises a flavonoid, preferably a flavanol or a flavanol ester. Preferred flavonoids include, for example, catechins and catechin derivatives (such as epicatechin, gallocatechin, epigallocatechin, 3-gallic acid catechin, 3-gallic acid epicatechin, 3-gallic acid gallocatechin, and 3-gallic acid epigallocatechin (EGCG)).

[0030] Another preferred embodiment of the present invention is that the PCA comprises a stilbenoide, preferably in the form of resveratrol.

[0031] PCA preferably includes caffeic acid, gallic acid, chlorogenic acid, catechol, 4-methylcatechol, catechin, epicatechin, gallocatechin, epigallocatechin, 3-gallic acid catechin, 3-gallic acid epicatechin, 3-gallic acid gallocatechin, 3-gallic acid epigallocatechin (EGCG), resveratrol, carnosic acid, carnosol, naringenin, or a mixture thereof.

[0032] Some preferred embodiments of the present invention are the following: Caffeic acid, gallic acid, chlorogenic acid, catechol, 4-methylcatechol, catechin, epicatechin, gallocatechin, epigallocatechin, 3-gallic acid catechin, 3-gallic acid epicatechin, 3-gallic acid gallocatechin, 3-gallic acid epigallocatechin (EGCG), resveratrol, carnosic acid, carnosol, naringenin, One or more of these PCA contributes at least 40 mol%, 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 total PCA in a supply containing one or more PCA.

[0033] Other preferred embodiments of the present invention are from the following group: Catechin, epicatechin, gallocatechin, epigallocatechin, 3-gallic acid catechin, 3-gallic acid epicatechin, 3-gallic acid gallocatechin, and 3-gallic acid epigallocatechin (EGCG), One or more of these PCA contributes at least 40 mol%, 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 total PCA in a supply containing one or more PCA.

[0034] Further preferred embodiments of the present invention are the following: Caffeic acid, gallic acid, chlorogenic acid, catechol, 4-methylcatechol, catechin, epicatechin, gallocatechin, epigallocatechin, 3-gallic acid catechin, 3-gallic acid epicatechin, 3-gallic acid gallocatechin, 3-gallic acid epigallocatechin (EGCG), resveratrol, carnosic acid, carnosol, naringenin, One or more of these PCA contributes at least 40 mol%, 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 total PCA in a supply containing one or more PCA.

[0035] Further preferred embodiments of the present invention include the following: Caffeic acid, gallic acid, and chlorogenic acid, One or more of these PCA contributes at least 40 mol%, 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 total PCA in a supply containing one or more PCA.

[0036] In another preferred embodiment of the present invention, epigallocatechin gallate (EGCG) contributes at least 40 mol%, 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 in a source containing one or more PCA.

[0037] In some preferred embodiments of the present invention, the molecular weight of PCA is 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.

[0038] In another preferred embodiment of the present invention, the molecular weight of PCA is 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.

[0039] In some preferred embodiments of the present invention, the molecular weight of PCA is in the range of 110 to 600 g / mol, more preferably in the range of 110 to 400 g / mol, even more preferably in the range of 110 to 350 g / mol, and more preferably in the range of 110 to 310 g / mol.

[0040] In some preferred embodiments of the present invention, the molecular weight of PCA is in the range of 110 to 250 g / mol, more preferably in the range of 110 to 230 g / mol, even more preferably in the range of 120 to 210 g / mol, and more preferably in the range of 120 to 200 g / mol.

[0041] In another preferred embodiment of the present invention, the molecular weight of PCA is in the range of 120 to 1000 g / mol, more preferably in the range of 250 to 700 g / mol, even more preferably in the range of 300 to 650 g / mol, and more preferably in the range of 350 to 600 g / mol.

[0042] In some preferred embodiments of the present invention, the PCA contains neither nitrogen nor sulfur.

[0043] In some preferred embodiments of the present invention, the PCA does not contain a carboxylic acid group.

[0044] In some preferred embodiments of the present invention, the water solubility of PCA is at least 8 mM, more preferably at least 12 mM, and most preferably at least 16 mM at 25°C.

[0045] In some preferred embodiments of the present invention, the source containing one or more PCA is selected from polyphenol extracts of herbs, polyphenol extracts of spices, polyphenol extracts of fruits, polyphenol extracts of berries, and mixtures thereof.

[0046] A supply containing one or more types of PCA is preferably: Polyphenol extracts from tea, more preferably from green tea; polyphenol extracts from coffee; polyphenol extracts from cocoa; polyphenol extracts from grapes; polyphenol extracts from rosemary; polyphenol extracts from lemon balm; polyphenol extracts from blackcurrant; single PCA isolates and mixtures thereof. It is often preferable to select from the group consisting of the following.

[0047] "Polyphenol extracts" are well known in the art and relate to extracts that target the polyphenol fraction of a given polyphenol source. In some preferred embodiments of the present invention, the polyphenol extract is an extract obtained with water. In other preferred embodiments of the present invention, the polyphenol extract is an extract obtained with water and ethanol.

[0048] In some preferred embodiments of the present invention, a supply containing one or more types of PCA contains PCA in an amount of 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, relative to the total solids of the supply containing one or more types of PCA.

[0049] A more preferred embodiment of the present invention is a single PCA isolate containing at least 25% w / w of a single PCA relative to the total solids of the supply containing one or more PCA, more preferably at least 40% w / w, even more preferably at least 60% w / w, and most preferably at least 80% w / w of a single PCA relative to the total solids of the supply containing one or more PCA.

[0050] It is preferable that a source containing one or more types of PCA does not contribute to the modified BLG protein composition by causing bitterness or altering the taste.

[0051] The term "BLG-containing source" refers to the composition used to provide BLG to the protein solution in step (c).

[0052] If two or more BLG-containing compositions are used to prepare the protein solution in step (c) and / or the oxidizing aqueous solution in step (b), each of the BLG-containing compositions used is considered a secondary source that constitutes a BLG-containing source.

[0053] In some preferred embodiments of the present invention, the BLG source protein is isolated from mammalian milk, preferably from ruminant milk (e.g., cattle, sheep, goats, buffalo, camels, llamas, donkeys, and / or deer). Protein isolated from cow (dairy cow) milk is particularly preferred. Therefore, the BLG and further whey protein are preferably bovine BLG and bovine whey protein. Alternatively, preferred proteins are proteins obtained by microbial fermentation during the production of recombinant BLG. If the BLG is recombinant BLG, it is preferable that the recombinant BLG has a sequence similar to or identical to that of BLG derived from a ruminant (e.g., cattle, sheep, goats, buffalo, camels, llamas, donkeys, and / or deer) source.

[0054] Preferably, the source containing BLG includes or consists of whey protein concentrate, whey protein isolate, whey protein concentrate, whey protein isolate, BLG isolate, or a combination thereof.

[0055] The source of BLG may be in liquid or powder form, and the secondary source may include a combination of one or more powders containing BLG and / or one or more liquids containing BLG.

[0056] The term "whey" refers to the liquid phase remaining after the casein in milk has been precipitated and removed. Casein precipitation may be carried out, for example, by acidifying the milk and / or using the enzyme rennet. Several types of whey exist; for example, "sweet whey," which is the whey product produced by the precipitation of casein with rennet, and "acidic whey" or "sour whey," which is the whey product produced by the acidic precipitation of casein. Acidic precipitation of casein may be carried out, for example, by adding food-grade acid or by bacterial culture.

[0057] The term "milk serum" refers to the liquid remaining after removing casein and milk fat globules from milk, for example, by microfiltration or ultrafiltration with a large pore size. Milk serum is also called "ideal whey."

[0058] In relation to the present invention, the term "whey protein" refers to proteins present in whey or milk serum. Whey protein may be a subset of protein molecular species present in whey or milk serum, or a single whey protein molecular species; or whey protein may be a complete set of protein molecular species present in whey and / or milk serum.

[0059] In some preferred embodiments of the present invention, the source protein containing BLG is isolated from bovine whey or milk serum.

[0060] Unfractionated whey protein typically contains α-lactalbumin (ALA), β-lactoglobulin (BLG), bovine serum albumin, immunoglobulins, osteopontin, lactoferrin, and lactoperoxidase. Whey protein derived from rennet-treated milk further contains casein macropeptides (CMP) in addition to other protein molecular species.

[0061] In relation to the present invention, the term "whey protein concentrate" (WPC) refers to a dry composition or aqueous composition containing 20-89% w / w of protein in total relative to the total solids.

[0062] WPC is preferably: 30-85% w / w of protein relative to total solids; BLG at 15-90% w / w relative to total protein; ALA at a rate of 4-50% w / w relative to total protein; and CMP at 0-40% w / w relative to the protein, Includes. Most preferably, WPC is: 70-85% w / w protein relative to total solids; BLG at 30-90% w / w relative to total protein; ALA at a rate of 4-35% w / w relative to total protein; and CMP at 0-25% w / w relative to the protein, Includes. WPCs based on whey protein typically contain little to no CMP (Chemical Powder).

[0063] The term "whey protein isolate" (WPI) refers to a dry or aqueous composition containing 86-100% w / w of protein relative to the total solids.

[0064] WPI is preferably: 86-99% w / w protein relative to total solids; BLG at 30-100% w / w relative to total protein; ALA at a concentration of 0-35% w / w relative to total protein; and CMP at 0-25% w / w relative to total protein, Includes.

[0065] Most preferably, WPI is: 90-99% w / w protein relative to total solids, BLG at 50-99% w / w relative to total protein, ALA at 0-35% w / w relative to total protein, and CMP at 0-25% w / w relative to total protein, Includes.

[0066] WPIs based on whey protein typically contain little to no CMP (Chemical Powder).

[0067] It is particularly preferable that the whey protein source be WPI (Whey Protein Isolate).

[0068] With respect to the present invention, the term "BLG isolate" refers to a composition containing BLG in an amount of at least 80% w / w of total solids, more preferably at least 90% w / w of total solids, and most preferably at least 95% w / w of total solids.

[0069] BLG isolates may be prepared, for example, by isolating BLG from whey or milk serum, preferably in accordance with WO2018115520A1, or by fermentation of genetically modified microorganisms or by recombinant technology using mammalian cells.

[0070] Sources containing BLG preferably have 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%.

[0071] A lower degree of protein denaturation is often preferred, and in some preferred embodiments of the present invention, the source containing BLG has a protein denaturation of up to 12%, more preferably up to 10%, even more preferably up to 8%, and most preferably up to 5%.

[0072] In some preferred embodiments of the present invention, the source containing BLG has a total fat content of up to 10% w / w, more preferably up to 8% w / w, and most preferably up to 6% w / w, relative to the total solids.

[0073] In another preferred embodiment of the present invention, the source containing BLG has a total fat content of up to 5% w / w, more preferably up to 2% w / w, even more preferably up to 0.5% w / w, and most preferably up to 0.1% w / w, relative to the total solids.

[0074] In a more preferred embodiment of the present invention, the source containing BLG has a total fat content in the range of 1 to 20% w / w relative to the total solids, more preferably in the range of 2 to 16% w / w relative to the total solids, even more preferably in the range of 3 to 12% w / w, and most preferably up to 4 to 10% w / w.

[0075] In some preferred embodiments of the present invention, the source containing BLG 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 the total solids.

[0076] In a more preferred embodiment of the present invention, the source containing BLG has a total carbohydrate content of up to 2% w / w, more preferably up to 1% w / w, more preferably up to 0.5% w / w, and most preferably up to 0.2% w / w, relative to the total solids.

[0077] In some preferred embodiments of the present invention, the method includes step (b), while in other preferred embodiments of the present invention, step (b) is optional in the sense that it involves oxidizing a portion of a supply containing one or more PCA before step (c).

[0078] Therefore, in some preferred embodiments of the present invention, the method for producing a protein composition containing modified BLG includes step (b) subjecting a portion of a source containing one or more PCA to oxidation that converts the PCA to quinone (also called PCA-type oxidation), thereby providing a source containing one or more oxidized PCA. It should be understood that the PCA-type oxidation must be capable of oxidizing the specific type of PCA identified in step (a) to quinone.

[0079] It should be noted that in some cases, oxidized PCA retains the functionality of quinones rather than PCA.

[0080] In some preferred embodiments of the present invention, the oxidation in step (b) includes preparing an oxidizing aqueous solution comprising a source containing one or more PCA, optionally a chemical oxidizing agent, and optionally one or more further components (such as water).

[0081] In relation to the present invention, the term "oxidizing aqueous solution" is used to refer to the aqueous solution in which PCA-type oxidation occurs in step (b).

[0082] The PCA content of the oxidizing aqueous solution is preferably at least 0.2 mM, more preferably at least 15 mM, and most preferably at least 50 mM.

[0083] In some preferred embodiments of the present invention, the PCA concentration of the oxidizing aqueous solution is 0.2 to 600 mM, more preferably 15 to 550 mM, and most preferably 50 to 500 mM.

[0084] For some types of PCA, it is advantageous that the oxidizing aqueous solution contains one or more amphiphilic modifiers that increase the solubility of the PCA. The one or more amphiphilic modifiers are preferably food-grade and / or pharmaceutical-grade amphiphilic modifiers. Preferred examples of amphiphilic modifiers include food-grade alcohols, such as ethanol, dimethyl sulfoxide (DMSO), polysorbates, sorbitan, fatty acid esters, and mixtures thereof.

[0085] Useful examples of polysorbates include, for example, those selected from the group consisting of polysorbates 20, 40, 60, and 80. Useful examples of sorbitan and fatty acid esters include, for example, those selected from the group consisting of sorbitan laurate monoester, sorbitan stearate monoester, sorbitan tristearate ester, and sorbitan oleate monoester.

[0086] In some preferred embodiments of the present invention, the pH of the oxidizing aqueous solution is in the range of 2 to 9.5, more preferably 6.5 to 9.5, even more preferably 7.1 to 9.0, even more preferably 7.3 to 8.7, and most preferably 7.4 to 8.5.

[0087] In this specification, the above pH values ​​refer to pH normalized to 25°C.

[0088] The inventors have found that it may be advantageous to carry out step (b) using an oxidizing aqueous solution with a pH in the range of 2 to 6.4, more preferably 3.0 to 6.0, even more preferably 3.5 to 5.5, and most preferably 4.0 to 5.0.

[0089] In some preferred embodiments of the present invention, the oxidizing aqueous solution comprises one or more further components selected from the group consisting of a food-acidible acid, a food-acidible base, water, an amphiphilic modifier, a chemical oxidizing agent, an oxidation catalyst, and combinations thereof.

[0090] When a source containing one or more PCA is provided in powder form, water is typically required as an additional ingredient.

[0091] If the PCA-type oxidation in step (b) is partially or completely based on a chemical oxidizing agent, then a chemical oxidizing agent is required in the oxidizing aqueous solution. A combination of multiple chemical oxidizing agents may also be used.

[0092] When pH adjustment is required, typically food-tolerant acids and / or food-tolerant bases are needed.

[0093] Useful examples of oxidation catalysts include one or more enzymes such as polyphenol oxidase, laccase, and / or tyrosinase.

[0094] In some preferred embodiments of the present invention, the oxidation of step (b) is carried out by contacting a portion of a supply containing one or more PCAs with a chemical oxidizing agent under conditions that at least a portion of the PCAs is converted to quinones. This oxidation is carried out in an oxidizing aqueous solution.

[0095] It is often preferable that the chemical oxidizing agent includes or consists of peroxides, ozone, dioxygen, or a combination thereof.

[0096] In another preferred embodiment of the present invention, the chemical oxidizing agent comprises or consists of hydrogen peroxide, benzoyl peroxide, or a combination thereof.

[0097] In a more preferred embodiment of the present invention, the chemical oxidizing agent comprises or consists of hydrogen peroxide, benzoyl peroxide, dioxygen, or a combination thereof.

[0098] In some preferred embodiments of the present invention, the oxidation of step (b) includes preparing an oxidizing aqueous solution comprising a portion of a source containing one or more PCA, the chemical oxidizing agent, and optionally one or more further components (such as water).

[0099] The PCA-type oxidation may also include enzymatically catalyzed oxidation, for example, enzymatically catalyzed oxidation involving enzymes such as polyphenol oxidase, laccase, and / or tyrosinase.

[0100] The dissolved dioxygen provided by the oxidizing aqueous solution used in step (b) and / or the liquid used to prepare the protein solution in step (c) may contribute to trace amounts of PCA oxidation, but this is typically insufficient to achieve the desired oxidation level required by the present invention.

[0101] It is generally preferable that the oxidizing aqueous solution does not contain proteins. Furthermore, it is preferable that the oxidizing aqueous solution does not contain molecules containing free thiol groups. In some preferred embodiments of the present invention, the oxidizing aqueous solution further does not contain molecules having amine groups.

[0102] In some preferred embodiments of the present invention, the oxidizing aqueous solution contains less than 5% w / w of protein by weight, more preferably up to 1% w / w, and even more preferably up to 0.1% of protein; and most preferably contains no protein at all.

[0103] In some preferred embodiments of the present invention, the PCA-type oxidation of step (b) includes electrochemically oxidizing a portion of a supply containing one or more PCAs under conditions that convert at least a portion of the PCAs into quinones.

[0104] In some preferred embodiments of the present invention, the oxidation in step (b) includes preparing an oxidizing aqueous solution comprising a portion of a supply containing one or more PCA and optionally one or more components (such as water).

[0105] Preferably, the electrochemical oxidation is carried out using a potential difference of -0.1 to 1.5 V, more preferably 0.0 to 1.2 V, and most preferably 0.0 to 0.7 V.

[0106] This potential difference, also called voltage, refers to the potential difference between the working electrode and the counter electrode used to perform the electrochemical oxidation of the PCA.

[0107] In some preferred embodiments of the present invention, the pH of the oxidizing aqueous solution is in the range of 6 to 9, and the electrochemical oxidation is carried out using a potential difference of -0.1 to 0.9 V, more preferably 0 to 0.8 V, and most preferably 0.2 to 0.7 V.

[0108] In another preferred embodiment of the present invention, the pH of the oxidizing aqueous solution is in the range of 3 to 5, and the electrochemical oxidation is carried out using a potential difference of 0.1 to 1.3 V, more preferably 0.2 to 0.9 V, and most preferably 0.3 to 0.7 V.

[0109] To determine the appropriate potential difference for electrochemical oxidation, it is advantageous to determine the oxidation potential of the PCA.

[0110] The required oxidation potential of the PCA may be determined, for example, by cyclic voltammetry, where the electrode potential is swept within a range of, for example, -0.2V to +1.2V. Since oxidation of the PCA begins at and / or near the surface of the working electrode, the current increases at the predetermined potential used. As the potential is increased, the current continues to increase until one or more peak currents are achieved, depleting the PCA oxidizable groups near the electrode.

[0111] The oxidation potential determined above at the peak current may be used for the electrochemical oxidation of the PCA. If two or more peaks are observed (for example, in the case of EGCG), the minimum applied potential difference that produces a peak current and results in the generation of an appropriate amount of quinone as measured by analysis 2 should be selected.

[0112] In some preferred embodiments of the present invention, electrochemical oxidation is performed by bulk electrolysis, while maintaining a constant potential current while monitoring the current decrease over time as the PCA is oxidized to its quinone form. Preferably, the PCA is quantitatively converted to its quinone form. The current may reach zero when the PCA is completely oxidized.

[0113] Shaking during bulk electrolysis is advantageous; for example, it is advantageous for the purpose of reducing diffusion restriction.

[0114] Bulk electrolysis may be carried out using a potentiostat and a two-electrode system, or preferably a three-electrode system including a potentiostat and one or more working electrodes, one or more counter electrodes, and a reference electrode.

[0115] The working electrode is preferably a cathode. Preferred examples of the working electrode include a glassy carbon electrode, a boron-doped diamond electrode, and a graphite electrode.

[0116] The counter electrode is preferably an anode. The counter electrode is also known as an auxiliary electrode. The preferred counter electrode is a platinum electrode, more preferably a platinum electrode selected from a coiled platinum electrode and a platinum flag electrode.

[0117] The reference electrode is preferably a true reference electrode, or more preferably a quasi-reference electrode, which is also known as a pseudo-reference electrode. A preferred example of a quasi-reference electrode is an Ag / AgCl electrode.

[0118] In some preferred embodiments of the present invention, the electrochemical oxidation is carried out under nitrogen.

[0119] The amount of quinone produced during electrochemical oxidation can be measured by Analysis 2. If Analysis 2 is unavailable, the quinone content can be measured using the GSH assay described in Example 2 as an alternative.

[0120] The PCA-type oxidation in step (b) is carried out partially or completely by electrochemical oxidation, and it is preferable that the oxidizing aqueous solution also contains an electrolyte. The oxidizing aqueous solution preferably contains an electrolyte, the electrolyte concentration of which is 0.01 to 0.5 M; more preferably 0.05 to 0.4 M, and most preferably 0.1 to 0.2 M. The electrolyte used is preferably food-grade, conductive, and capable of contributing to a stable pH. In some preferred embodiments of the present invention, the oxidizing aqueous solution contains one or more electrolytes selected from the group consisting of H2SO4, phosphoric acid, and acetic acid.

[0121] In some preferred embodiments of the present invention, the electrochemical oxidation is carried out in a temperature range of 2 to 80°C, more preferably 5 to 60°C, even more preferably 10 to 40°C, and most preferably 15 to 30°C.

[0122] In another preferred embodiment of the present invention, the electrochemical oxidation is carried out in a temperature range of 30 to 90°C, more preferably 40 to 90°C, even more preferably 50 to 90°C, and most preferably 60 to 90°C.

[0123] In a more preferred embodiment of the present invention, the electrochemical oxidation is carried out in a temperature range of 5 to 25°C, more preferably 10 to 25°C, even more preferably 15 to 25°C, and most preferably 18 to 25°C.

[0124] The duration of step (b) depends on the specific composition of the oxidizing aqueous solution and the conditions used for the PCA-type oxidation. When electrochemical oxidation is used, an electric current may be used to monitor the progress of oxidation. When the current approaches zero in the working potential difference, it is an indication of PCA depletion. The content of the generated quinone can be measured according to analysis 2.

[0125] In step (c) of the method, in order to provide a protein solution having a pH in the range of 6.5 to 9.5, a portion of a source containing one or more PCA and / or a portion of a source containing one or more oxidized PCA are combined with a source containing BLG and optionally further components.

[0126] 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), which is used to prepare the protein solution. In some preferred embodiments of the present invention, substantially the entire source containing one or more oxidized PCA is used.

[0127] In step (c), the term “a portion of a source containing one or more PCA” refers to one or more oxidized PCA used in the preparation of the protein solution, and concerns the actual portion of the source containing one or more PCA in step (a). In some preferred embodiments of the present invention, substantially the entirety of the source containing one or more PCA is used.

[0128] In some preferred embodiments of the present invention, the pH of the protein solution is in the range of 6.7 to 9.5, more preferably in the range of 7.1 to 9.0, even more preferably in the range of 7.3 to 8.7, and most preferably in the range of 7.4 to 8.5.

[0129] The inventors have found that the pH range specified herein for the protein solution tends to selectively modify the free thiol groups of BLG, thereby reducing the risk of undesirable reactions with the amine groups of BLG.

[0130] In some preferred embodiments of the present invention, the pH of the protein solution is in the range of 6.5 to 9.5, and its temperature is at least 20°C, more preferably at least 40°C, even more preferably at least 60°C, and most preferably at least 70°C.

[0131] In a more preferred embodiment of the present invention, the pH of the protein solution is in the range of 7.1 to 9.0, and its temperature is at least 20°C, more preferably at least 30°C, even more preferably at least 50°C, and most preferably at least 60°C.

[0132] In a particularly preferred embodiment of the present invention, the pH of the protein solution is in the range of 7.3 to 8.7, and its temperature is at least 20°C, more preferably at least 30°C, even more preferably at least 50°C, and most preferably at least 60°C.

[0133] In a more preferred embodiment of the present invention, the pH of the protein solution is in the range of 7.4 to 8.5, and its temperature is at least 35°C, more preferably at least 40°C, even more preferably at least 45°C, and most preferably at least 50°C.

[0134] Preferably, the pH of the protein solution is in the range of 6.5 to 9.5 and its temperature is 20 to 70°C; more preferably, the pH is in the range of 7.1 to 9.0 and its temperature is 20 to 60°C; even more preferably, the pH is in the range of 7.3 to 8.7 and its temperature is 20 to 60°C; and most preferably, the pH is in the range of 7.4 to 8.5 and its temperature is 35 to 50°C.

[0135] In other preferred embodiments of the present invention, the pH of the protein solution is in the range of 6.5 to 9.5 and the temperature is 3 to 90°C; more preferably, the pH is in the range of 7.1 to 9.0 and the temperature is 40 to 80°C; even more preferably, the pH is in the range of 7.3 to 8.7 and the temperature is 45 to 75°C; and most preferably, the pH is in the range of 7.4 to 8.5 and the temperature is 50 to 70°C.

[0136] In a more preferred embodiment of the present invention, the pH of the protein solution is in the range of 6.5 to 9.5 and the temperature is 5 to 90°C; more preferably, the pH is in the range of 7.1 to 9.0 and the temperature is 8 to 80°C; even more preferably, the pH is in the range of 7.3 to 8.7 and the temperature is 10 to 75°C; and most preferably, the pH is in the range of 7.4 to 8.5 and the temperature is 15 to 70°C.

[0137] In some preferred embodiments of the present invention, the BLG content of the protein solution is 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.

[0138] In a more preferred embodiment of the present invention, the BLG content of the protein solution is 0.5 to 30% w / w, more preferably 1 to 20% w / w, even more preferably 3 to 16% w / w, and most preferably 5 to 12% w / w.

[0139] In some preferred embodiments of the present invention, the BLG content of the protein solution is at least 30% w / w of total protein, more preferably at least 40% w / w of total protein, even more preferably at least 45% w / w of total protein, and most preferably at least 50% w / w of total protein.

[0140] In a more preferred embodiment of the present invention, the BLG content of the protein solution is 30-99% w / w relative to the total protein, more preferably 40-95% w / w relative to the total protein, even more preferably 45-90% w / w relative to the total protein, and most preferably 50-80% w / w.

[0141] In another preferred embodiment of the present invention, the BLG content of the protein solution is at least 60% w / w of total protein, more preferably at least 80% w / w of total protein, even more preferably at least 90% w / w of total protein, and most preferably at least 95% w / w of total protein.

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

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

[0144] In another preferred embodiment of the present invention, the BLG content of the protein solution is at least 60% w / w relative to the total solids, more preferably at least 80% w / w relative to the total solids, even more preferably at least 90% w / w relative to the total solids, and most preferably at least 95% w / w relative to the total solids.

[0145] In some preferred embodiments of the present invention, the total fat content of the protein solution is up to 10% w / w relative to the total solids, more preferably up to 8% w / w relative to the total solids, and most preferably up to 6% w / w.

[0146] In another preferred embodiment of the present invention, the total fat content of the protein solution is at most 5% w / w of total solids, more preferably at most 2% w / w of total solids, even more preferably at most 0.5% w / w, and most preferably at most 0.1% w / w.

[0147] In a more preferred embodiment of the present invention, the total fat content of the protein solution is in the range of 1 to 20% w / w relative to the total solids, more preferably in the range of 2 to 16% w / w relative to the total solids, even more preferably in the range of 3 to 12% w / w, and most preferably in the range of 4 to 10% w / w.

[0148] The inventors have found that an advantage of the present invention is that, compared to similar methods for thiol modification, it does not cause significant fatty acid fertilization. Therefore, the method of the present invention is suitable for mild BLG modification in fat-rich systems.

[0149] In some preferred embodiments of the present invention, the total carbohydrate content of the protein solution is up to 10% w / w of total solids, more preferably up to 8% w / w of total solids, even more preferably up to 6% w / w, and most preferably up to 5% w / w.

[0150] In a more preferred embodiment of the present invention, the total carbohydrate content of the protein solution is at most 2% w / w of total solids, more preferably at most 1% w / w of total solids, even more preferably at most 0.5% w / w, and most preferably at most 0.2% w / w.

[0151] In some preferred embodiments of the present invention, the protein solution is: • The original amount of PCA used to prepare the protein solution, and • BLG content of the protein solution, The molar ratio between:

[0152] The ratio is 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; Therefore, it is prepared to have such a molar ratio.

[0153] In another preferred embodiment of the present invention, the protein solution is: • The original amount of PCA used to prepare the protein solution, and • BLG content of the protein solution, The molar ratio between: The ratio is 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; Therefore, it is prepared to have such a molar ratio.

[0154] With respect to the present invention, the term “original amount of PCA used to prepare the protein solution” refers to the amount of a specific PCA described in step (a) used to prepare a portion of a source containing one or more PCA used to prepare the protein solution in step (c), and / or a portion of a source containing one or more oxidized PCA used to prepare the protein solution in step (c).

[0155] If step (c) uses only a portion of the source containing one or more types of oxidized PCA obtained in step (b), then "the original amount of PCA used to prepare the protein solution" refers to the amount of PCA used to prepare the portion of the source containing one or more types of oxidized PCA.

[0156] If step (c) uses only a portion of the supply source containing one or more types of PCA obtained in step (a), then "the original amount of PCA used to prepare the protein solution" refers to the amount of PCA used to prepare a portion of the supply source containing one or more types of PCA.

[0157] If step (c) uses both a portion of a source containing one or more types of PCA obtained in step (a) and a portion of a source containing one or more types of oxidized PCA obtained in step (b), then "the original amount of PCA used to prepare the protein solution" refers to the amount of PCA used to prepare the portion of the source containing one or more types of PCA and the portion of the source containing one or more types of oxidized PCA.

[0158] The PCA referred to in processes (b), (c), (d), and (e) is the PCA defined in process (a).

[0159] In some preferred embodiments of the present invention, the protein solution is: • The original amount of PCA used to prepare the protein solution, and • BLG content of the protein solution, The molar ratio between: The ratio is 0.1:1 to 1000:1, more preferably 0.2:1 to 500:1, even more preferably 0.3:1 to 200:1, and most preferably 0.4:1 to 100:1; It is prepared to achieve that molar ratio.

[0160] In a more preferred embodiment of the present invention, the protein solution is: • The original amount of PCA used to prepare the protein solution, and • BLG content of the protein solution, The molar ratio between: The ratio is 0.1:1 to 100:1, more preferably 0.1:1 to 50:1, even more preferably 0.1:1 to 20:1, and most preferably 0.1:1 to 10:1; It is prepared to achieve that molar ratio.

[0161] In a more preferred embodiment of the present invention, the protein solution is: • The original amount of PCA used to prepare the protein solution, and • BLG content of the protein solution, The molar ratio between: The ratio is 0.1:1 to 6:1, more preferably 0.2:1 to 5:1, even more preferably 0.3:1 to 4:1, and most preferably 0.4:1 to 3:1; It is prepared to achieve that molar ratio.

[0162] In some preferred embodiments of the present invention, the protein solution is: • The original amount of PCA used to prepare the protein solution, and • BLG content of the protein solution, The molar ratio between: The ratio is 1:1 to 1000:1, more preferably 2:1 to 500:1, even more preferably 5:1 to 200:1, and most preferably 10:1 to 100:1; It is prepared to achieve that molar ratio.

[0163] In a more preferred embodiment of the present invention, the protein solution is: • The original amount of PCA used to prepare the protein solution, and • BLG content of the protein solution, The molar ratio between: The ratio is 1:1 to 100:1, more preferably 1:1 to 50:1, even more preferably 1:1 to 20:1, and most preferably 1:1 to 10:1; It is prepared to achieve that molar ratio.

[0164] In a more preferred embodiment of the present invention, the protein solution is: • The original amount of PCA used to prepare the protein solution, and • BLG content of the protein solution, The molar ratio between: The ratio is 1:1 to 6:1, more preferably 1.1:1 to 5:1, even more preferably 1.3:1 to 4:1, and most preferably 1.5:1 to 3:1; It is prepared to achieve that molar ratio.

[0165] The protein solution is prepared by combining, and preferably by mixing, appropriate amounts of sources described herein to obtain a protein solution having the desired properties. Preferably, step (c) is carried out under conditions that do not lead to significant protein denaturation.

[0166] In particular, when steps (b), (c), and (d) are carried out as a continuous process, it is often preferable to carry out the mixing in step (c) by mixing within the piping.

[0167] In some preferred embodiments of the present invention, step (c) is carried out under conditions such that the protein denaturation of the BLG in the BLG-containing source is less than 15%, more preferably less than 10%, even more preferably less than 5%, and most preferably less than 2%.

[0168] The protein solution is preferably an aqueous solution. Preferably, at least 90% w / w of the non-solid matter, i.e., substances that do not contribute to the solid content of the protein solution, is water, more preferably at least 95% w / w, and most preferably at least 99% w / w is water.

[0169] The protein solution is preferably prepared at a temperature that does not cause protein denaturation, preferably in the range of 0 to 70°C, more preferably in the range of 5 to 65°C, even more preferably in the range of 10 to 60°C, and most preferably in the range of 20 to 55°C.

[0170] In another preferred embodiment of the present invention, the protein solution is prepared at a temperature range of 0 to 50°C, more preferably 2 to 45°C, even more preferably 5 to 40°C, and most preferably 10 to 35°C. These temperature ranges are often preferred when it is intended that dissolved dioxygen contributes to the majority of the PCA oxidation.

[0171] Preferably, the protein solution is prepared by mixing a portion of a source containing one or more PCA obtained in step (a), a source containing BLG obtained in step (a), and optionally further components.

[0172] In some preferred embodiments of the present invention, the method does not involve a step of oxidizing a portion of a supply containing one or more PCA prior to step (c); that is, the method does not involve step (b).

[0173] In another preferred embodiment of the present invention, the method includes step (b), i.e., a step of oxidizing a portion of a source containing one or more PCA before preparing the protein solution. The protein solution is prepared by mixing the portion of the source containing one or more oxidized PCA obtained in step (b) with a source containing BLG and optionally further components.

[0174] In most cases, it is preferable that most, if not all, of the PCA-type oxidation occurs in step (b).

[0175] In some preferred embodiments of the present invention, less than 20% of the PCA type oxidation occurs in step (d); more preferably up to 10% of the PCA type oxidation, even more preferably up to 5% of the PCA type oxidation, and most preferably up to 2% of the PCA type oxidation occurs in step (d).

[0176] Preferably, further components include water, one or more acids, one or more bases, one or more pH buffers, one or more oxidizing agents, or a mixture thereof.

[0177] In step (d) of the method, the protein solution is incubated over a certain temperature range for a sufficient amount of time to reduce the amount of free thiol groups in the protein solution to a maximum of 10 micromoles / g of protein. If the method does not include step (b), then step (d) includes PCA-type oxidation capable of converting PCA to quinone. This oxidation may be achieved, for example, by a chemical oxidizing agent added during the preparation of the protein solution and / or by electrochemical oxidation in step (d). In some preferred embodiments of the present invention, the method includes both step (b) and PCA-type oxidation in step (d).

[0178] With respect to the present invention, the term "incubating the protein solution" refers to the protein solution in step (d).

[0179] In some preferred embodiments of the present invention, the incubation reduces the amount of free thiols in the protein solution to a maximum of 9 micromoles / g of protein, more preferably to a maximum of 8 micromoles / g of protein, more preferably to a maximum of 5 micromoles / g of protein, even more preferably to a maximum of 3 micromoles / g of protein, and most preferably to a maximum of 2 micromoles / g of protein, and the incubation is carried out to achieve such a reduction.

[0180] In another preferred embodiment of the present invention, the incubation is carried out to achieve a reduction in the amount of free thiols in the protein solution to 0.001 to 10 micromoles / g of protein, more preferably 0.01 to 9 micromoles / g of protein, even more preferably 0.02 to 8 micromoles / g of protein, even more preferably 0.03 to 5 micromoles / g of protein, even more preferably 0.04 to 3 micromoles / g of protein, and most preferably 0.04 to 2 micromoles / g of protein.

[0181] In some preferred embodiments of the present invention, the pH of the protein solution to be incubated is in the range of 6.7 to 9.5, more preferably in the range of 7.1 to 9.0, even more preferably in the range of 7.3 to 8.7, and most preferably in the range of 7.4 to 8.5.

[0182] In some preferred embodiments of the present invention, during incubation in step (d), the protein solution is subjected to a pressure in the range of 1 to 1000 bar, more preferably in the range of 20 to 500 bar, even more preferably in the range of 30 to 300 bar, and most preferably in the range of 40 to 200 bar.

[0183] In some preferred embodiments of the present invention, during incubation in step (d), the temperature of the protein solution is in the range of 0 to 160°C, more preferably 10 to 155°C, even more preferably 15 to 150°C, and most preferably 20 to 145°C.

[0184] In another preferred embodiment of the present invention, the temperature of the protein solution to be incubated, i.e., the temperature of the protein solution in step (d), is in the range of 0 to 67°C, more preferably in the range of 10 to 65°C, even more preferably in the range of 15 to 60°C, and most preferably in the range of 20 to 55°C.

[0185] Preferably, the temperature of the protein solution to be incubated is in the range of 5 to 65°C, more preferably 10 to 65°C, even more preferably 30 to 60°C, and most preferably 40 to 55°C.

[0186] In other preferred embodiments, the temperature of the protein solution to be incubated is in the range of 60 to 98°C, more preferably 65 to 95°C, and most preferably 70 to 90°C.

[0187] In some embodiments of the present invention, the pH of the protein solution to be incubated is in the range of 6.5 to 7.0, and its temperature is in the range of 40 to 65°C; more preferably in the range of 45 to 65°C, even more preferably in the range of 50 to 65°C, and most preferably in the range of 55 to 65°C.

[0188] In another preferred embodiment of the present invention, the pH of the protein solution incubated in step (d) is in the range of 7.1 to 9.5, its temperature is in the range of 5 to 65°C, more preferably in the range of 10 to 65°C, even more preferably in the range of 30 to 60°C, and most preferably in the range of 40 to 55°C.

[0189] In a more preferred embodiment of the present invention, the pH of the protein solution incubated in step (d) is in the range of 8.5 to 9.5, its temperature is in the range of 0 to 65°C, more preferably in the range of 0 to 50°C, even more preferably in the range of 0 to 30°C, and most preferably in the range of 5 to 25°C.

[0190] The inventors have found that the pH of the protein solution incubated in step (d) is preferably in the range of 7.5 to 8.5, and its temperature is preferably in the range of 5 to 60°C, more preferably in the range of 10 to 60°C, even more preferably in the range of 15 to 60°C, and most preferably in the range of 20 to 60°C. These ranges appear to be favorable for both the selective modification of free thiols in BLG and a relatively fast reaction rate.

[0191] Furthermore, the inventors have found that the pH of the protein solution incubated in step (d) is particularly preferably in the range of 7.7 to 8.5, its temperature in the range of 25 to 55°C, more preferably in the range of 30 to 55°C, even more preferably in the range of 35 to 50°C, and most preferably in the range of 35 to 45°C. These ranges also appear to be favorable for both the selective modification of free thiols in BLG and a relatively fast reaction rate.

[0192] In a more preferred embodiment of the present invention, the pH of the protein solution incubated in step (d) is in the range of 7.5 to 8.5, and its temperature is in the range of 60 to 98°C, more preferably in the range of 65 to 95°C, and most preferably in the range of 70 to 90°C.

[0193] In a more preferred embodiment of the present invention, the pH of the protein solution incubated in step (d) is in the range of 7.5 to 8.5, and its temperature is in the range of 45 to 160°C, more preferably in the range of 50 to 155°C, and most preferably in the range of 55 to 150°C.

[0194] In some preferred embodiments of the present invention, the incubation time 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.

[0195] An advantage of this method is that it can be implemented with a relatively short incubation time. Therefore, in a more preferred embodiment of the present invention, the incubation time for step (d) is a maximum of 30 minutes, more preferably a maximum of 10 minutes, even more preferably a maximum of 5 minutes, and most preferably a maximum of 2 minutes.

[0196] The inventors have found that it is technically possible to further shorten the incubation period. Therefore, in a more preferred embodiment of the present invention, the incubation time in step (d) is a maximum of 100 seconds, more preferably a maximum of 60 seconds, even more preferably a maximum of 30 seconds, and most preferably a maximum of 20 seconds.

[0197] In some preferred embodiments of the present invention, the oxidation in step (d) includes contacting the PCA with a chemical oxidizing agent in the protein solution under conditions that convert at least a portion of the PCA into quinone. Furthermore, the chemical oxidizing agent may be added during the incubation in step (d).

[0198] One or more of the chemical oxidizing agents used in process (b) and / or process (d) must be capable of PCA-type oxidation in process (d) and / or process (b).

[0199] In a more preferred embodiment of the present invention, the chemical oxidizing agent comprises or consists of a peroxide, ozone, dioxygen, or a combination thereof.

[0200] In certain preferred embodiments of the present invention, the chemical oxidizing agent comprises or consists of hydrogen peroxide, benzoyl peroxide, or a combination thereof.

[0201] In some preferred embodiments of the present invention, the oxidation in step (d) includes electrochemically oxidizing the PCA in the protein solution under conditions that convert at least a portion of the PCA into quinone.

[0202] The potential difference in question is such that it enables the oxidation of PCA to quinone, and it is necessary to select a sufficiently high potential difference that allows for measurement in step (b) as described above.

[0203] Preferably, the electrochemical oxidation in step (d) is carried out using a potential difference of -0.1 to 1.5 V, and most preferably -0.05 to 0.7 V.

[0204] In some preferred embodiments of the present invention, the pH of the protein solution is in the range of 7.1 to 9.0, and the electrochemical oxidation is carried out using a potential difference of -0.1 to 0.9 V, more preferably -0.05 to 0.8 V, and most preferably 0 to 0.7 V.

[0205] In another preferred embodiment of the present invention, the pH of the protein solution is in the range of 7.3 to 8.7, and the electrochemical oxidation is carried out using a potential difference of -0.1 to 0.8 V, most preferably 0 to 0.7 V.

[0206] In a more preferred embodiment of the present invention, the pH of the protein solution is in the range of 7.4 to 8.5, and the electrochemical oxidation is carried out using a potential difference of -0.1 to 0.8 V, most preferably -0.05 to 0.7 V.

[0207] The features and embodiments of the electrochemical oxidation described for step (b) also apply to the electrochemical oxidation of step (d), except for the range of acidic pH shown for step (b).

[0208] In some preferred embodiments of the present invention, the temperature in the first stage of the incubation is in the range of 0 to 60°C, more preferably in the range of 10 to 50°C, and then in the second stage of the incubation, the protein solution to be incubated is heated to a temperature in the range of 70 to 160°C, more preferably in the range of 100 to 150°C.

[0209] When the incubated protein solution is to be used as a packaged instant beverage that needs to be filled into a suitable container immediately after step (d), using a high temperature in step (d) is particularly useful.

[0210] In some preferred embodiments of the present invention, step (d) further comprises adding additional PCA to the protein solution to be incubated.

[0211] Some preferred embodiments of the present invention: • The total amount of PCA supplied to the protein solution in steps (c) and (d), and • The content of BLG provided to the protein solution in steps (c) and (d), The molar ratio between them is: The range is 0.1:1 to 1000:1, more preferably 0.2:1 to 500:1, even more preferably 5:1 to 200:1, and most preferably 10:1 to 100:1.

[0212] In a more preferred embodiment: • The total amount of PCA supplied to the protein solution in steps (c) and (d), and • The content of BLG provided to the protein solution in steps (c) and (d), The molar ratio between them is: The range is 0.1:1 to 100:1, more preferably 0.1:1 to 50:1, even more preferably 0.1:1 to 20:1, and most preferably 0.1:1 to 10:1.

[0213] In a more preferred embodiment: • The total amount of PCA supplied to the protein solution in steps (c) and (d), and • The content of BLG provided to the protein solution in steps (c) and (d), The molar ratio between them is: The range is 0.1:1 to 6:1, more preferably 0.2:1 to 5:1, even more preferably 0.3:1 to 4:1, and most preferably 0.4:1 to 1.

[0214] In some preferred embodiments: • The total amount of PCA supplied to the protein solution in steps (c) and (d), and • The content of BLG provided to the protein solution in steps (c) and (d), The molar ratio between them is: The range is 1:1 to 1000:1, more preferably 2:1 to 500:1, even more preferably 5:1 to 200:1, and most preferably 10:1 to 100:1.

[0215] In a more preferred embodiment: • The total amount of PCA supplied to the protein solution in steps (c) and (d), and • The content of BLG provided to the protein solution in steps (c) and (d), The molar ratio between them is: The range is 1:1 to 100:1, more preferably 1:1 to 50:1, even more preferably 1:1 to 20:1, and most preferably 1:1 to 10:1.

[0216] A further preferred embodiment of the present invention is: • The total amount of PCA supplied to the protein solution in steps (c) and (d), and • The content of BLG provided to the protein solution in steps (c) and (d), The molar ratio between them is: The range is 1:1 to 6:1, more preferably 1.1:1 to 5:1, even more preferably 1.3:1 to 4:1, and most preferably 1.5:1 to 3:1.

[0217] In some preferred embodiments of the present invention, the PCA-type oxidation used in step (b) and / or step (d) is: The theoretical amount of quinone provided to the protein solution during the implementation of the method, the theoretical amount of quinone determined according to Analysis 1, and • BLG content of the protein solution, This is sufficient to make the molar ratio between them at least 0.1:1, more preferably at least 0.2:1.

[0218] A more preferred embodiment of the present invention is that the oxidation used in step (b) and / or step (d) is: The theoretical amount of quinone provided to the protein solution during the implementation of the method, the theoretical amount of quinone determined according to Analysis 1, and • BLG content of the protein solution, This is sufficient to set the molar ratio between and to 0.1:1 to 100:1, more preferably 0.1:1 to 50:1, even more preferably 0.2:1 to 15:1, and most preferably 0.2:1 to 5:1.

[0219] In a more preferred embodiment of the present invention, the oxidation used in step (b) and / or step (d) is: The theoretical amount of quinone provided to the protein solution during the implementation of the method, the theoretical amount of quinone determined according to Analysis 1, and • BLG content of the protein solution, This is sufficient to set the molar ratio between and to 0.1:1 to 5:1, more preferably 0.1:1 to 4:1, even more preferably 0.2:1 to 3:1, and most preferably 0.2:1 to 2:1.

[0220] In another preferred embodiment of the present invention, the PCA-type oxidation used in step (b) and / or step (d) is: The theoretical amount of quinone provided to the protein solution during the implementation of the method, the theoretical amount of quinone determined according to Analysis 1, and • BLG content of the protein solution, This is sufficient to make the molar ratio between them 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. A more preferred embodiment of the present invention is that the oxidation used in step (b) and / or step (d) is: The theoretical amount of quinone provided to the protein solution during the implementation of the method, the theoretical amount of quinone determined according to Analysis 1, and • BLG content of the protein solution, This is sufficient to set the molar ratio between and to 1:1 to 100:1, more preferably 1:1 to 50:1, even more preferably 1:1 to 15:1, and most preferably 1.5:1 to 5:1.

[0221] When the PCA oxidation occurs mainly or exclusively in step (b), the PCA type oxidation used in step (b) is: · The quinone content of a part of the source containing one or more oxidized PCAs used in the protein solution, and · The BLG content of the protein solution, It is often preferable that the molar ratio between them is 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.

[0222] This is the case, for example, when the PCA type oxidation occurring in step (d) accounts for at least 20% of the PCA type oxidation.

[0223] In the above preferred embodiments of the present invention, the oxidation used in step (b) is: · The quinone content of a part of the source containing one or more oxidized PCAs used in the protein solution, and · The BLG content of the protein solution, It is even more preferable that the molar ratio between them is sufficient to be 1:1 to 100:1, more preferably 1:1 to 50:1, even more preferably 1:1 to 15:1, and most preferably 1.5:1 to 5:1.

[0224] In some preferred embodiments of the present invention, the method does not include oxidizing a part of the source containing one or more PCAs before step (c); that is, it does not include step (b); and the PCA type oxidation is carried out by a chemical oxidant.

[0225] Alternatively, the preferred method is a method that does not include oxidizing a part of the source containing one or more PCAs before step (c); that is, a method that does not include step (b); and the oxidation of PCA is carried out by electrochemical oxidation.

[0226] In some preferred embodiments of the present invention, the method comprises step (b), in which a portion of the source containing one or more oxidized PCA obtained in step (b) is used to prepare the protein solution in step (c); and the oxidation of the PCA is carried out by a chemical oxidizing agent.

[0227] In a more preferred embodiment of the present invention, the method comprises step (b), wherein a portion of the source containing one or more oxidized PCA obtained in step (b) is used to prepare the protein solution in step (c); and the oxidation of the PCA is carried out by electrochemical oxidation.

[0228] In another preferred embodiment of the present invention, the method comprises oxidation of PCA in step (d), preferably oxidation of PCA by electrochemical oxidation in step (d).

[0229] If the PCA oxidation occurs primarily or exclusively in step (d), then the type of PCA oxidation used in step (d) is: The theoretical amount of quinone provided to the protein solution during the implementation of the method, the theoretical amount of quinone determined according to Analysis 1, and • BLG content of the protein solution, It is often preferable that the molar ratio between them be sufficient to be 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.

[0230] This is the case, for example, when the PCA-type oxidation occurring in step (d) accounts for at least 50% of the PCA-type oxidation in the method. Another preferred embodiment of the present invention is that the PCA-type oxidation used in step (d) is: The theoretical amount of quinone provided to the protein solution during the implementation of the method, the theoretical amount of quinone determined according to Analysis 1, and • BLG content of the protein solution, This is sufficient to set the molar ratio between them to 0.1:1 to 100:1, more preferably 0.1:1 to 50:1, even more preferably 0.2:1 to 15:1, and most preferably 0.2:1 to 5:1.

[0231] A further preferred embodiment of the present invention is that the PCA-type oxidation used in step (d) is: The theoretical amount of quinone provided to the protein solution during the implementation of the method, the theoretical amount of quinone determined according to Analysis 1, and • BLG content of the protein solution, This is sufficient to set the molar ratio between and to 0.1:1 to 5:1, more preferably 0.1:1 to 4:1, even more preferably 0.2:1 to 3:1, and most preferably 0.2:1 to 2:1.

[0232] In some preferred embodiments of the present invention, the PCA-type oxidation used in step (d) is: The theoretical amount of quinone provided to the protein solution during the implementation of the method, the theoretical amount of quinone determined according to Analysis 1, and • BLG content of the protein solution, This is sufficient to make the molar ratio between them 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.

[0233] In the above preferred embodiment of the present invention, the PCA-type oxidation used in step (d) is: The theoretical amount of quinone provided to the protein solution during the implementation of the method, the theoretical amount of quinone determined according to Analysis 1, and • BLG content of the protein solution, It is even more preferable that the molar ratio between them be sufficient to be 1:1 to 100:1, more preferably 1:1 to 50:1, even more preferably 1:1 to 15:1, and most preferably 1.5:1 to 5:1.

[0234] Step (d) may further include a step of reducing the content of unreacted PCA and oxidized PCA in the incubated protein solution. This may include, 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 carried out by diafiltration.

[0235] In some preferred embodiments of the present invention, the product obtained in step (d) is a protein concentrate of the incubated protein solution.

[0236] In some preferred embodiments of the present invention, the method includes a step (e) of drying a liquid material containing a protein derived from at least the incubated protein solution of step (d).

[0237] In other preferred embodiments of the present invention, the method includes a step (e) of drying a liquid material containing a solid derived from at least the incubated protein solution of step (d).

[0238] In some preferred embodiments of the present invention, the liquid raw material for drying contains, or consists of, the protein solution obtained in step (d) or its protein concentrate.

[0239] It is particularly preferred that the liquid raw material for drying is a protein concentrate of the protein solution obtained in step (d).

[0240] With respect to the present invention, the "protein concentrate" of the first liquid is a second liquid in which at least the protein originates from the first liquid, and which has a higher relative protein content to total solids compared to the first liquid. Preferably, substantially all of the solids in the protein concentrate originate from the first liquid. The "protein concentrate" of the first liquid is preferably prepared by ultrafiltration, nanofiltration, reverse osmosis, and / or evaporation. Ultrafiltration and / or nanofiltration of the protein concentrate may be carried out, for example, using diafiltration to wash away some of the small non-protein solids. The "protein concentrate" contains the same protein molecular species and preferably has the same weight % of whey protein molecular species as in the first liquid, based on total protein. Providing the protein concentrate may involve one or more pH adjustments.

[0241] Another preferred embodiment of the present invention is the incubated protein solution in step (d): · pH adjustment; · Concentration process; · Diafiltration; and • Heat treatment, The liquid raw material is prepared by subjecting it to one or more of the following.

[0242] In some preferred embodiments of the present invention, the preparation of the liquid raw material in step (e) includes subjecting the protein solution obtained in step (d), i.e., the incubated protein solution, to ultrafiltration / dialysis filtration, preferably to an extent sufficient to reduce the weight percent of free phenolic compounds based on total solids by at least 30%, more preferably at least 50%, even more preferably at least 80%, and most preferably at least 90%. Such ultrafiltration / dialysis filtration is preferably carried out using an ultrafiltration membrane that retains proteins but allows molecules having a molecular weight of less than 2000 Da to pass through during filtration.

[0243] In some preferred embodiments of the present invention, step (e) further includes pH adjustment to bring the pH of the liquid raw material to a range of 6.0 to 8.5, and most preferably 6.5 to 7.5.

[0244] Preferably, step (e) includes spray-drying the liquid raw material.

[0245] A useful example of spray drying is, for example, the one described in WO2018 / 115520A1.

[0246] The method of the present invention can be carried out as a batch process, a quasi-batch process, or a continuous process. A continuous process is particularly preferred.

[0247] In some preferred embodiments of the present invention, at least steps (b), (c), and (d) are carried out as a continuous process.

[0248] One aspect of the present invention relates to a protein composition containing modified BLG and having up to 10 micromoles of free thiol groups per gram of protein, wherein the protein composition can be obtained by the method of the present invention, and preferably the protein composition is: • Protein content of at least 30% w / w relative to total solids; • Tryptophan content of at least 0.7% w / w relative to total protein; • Methionine content of at least 0.3% w / w relative to total protein; • Maximum kynurenine content of 0.2 micrograms / mg of protein; Preferably, a fat content of up to 3% w / w relative to total solids; Preferably, the protein-bound sulfur content is in the range of 100 to 600 micromoles / g of protein; Preferably, the content of disulfide bond-forming protein-binding cysteine ​​residues is in the range of 150 to 400 micromoles / g of protein. It has one or more of the following types.

[0249] In some preferred embodiments of the present invention, the protein composition containing the modified BLG is the protein solution obtained in step (d), i.e., the incubated protein solution.

[0250] In another preferred embodiment of the present invention, the protein composition containing the modified BLG is a liquid raw material prepared in step (e).

[0251] In a more preferred embodiment of the present invention, the protein composition containing the modified BLG is a powder obtained in step (e). Therefore, it is often preferable that the protein composition containing the modified BLG can be obtained in steps (a), (c), (d), and (e) of the present method.

[0252] In some preferred embodiments of the present invention, the average molecular weight of the protein in the protein composition is in the range of 18 kDa to 10,000 kDa, more preferably in the range of 50 to 8,000 kDa, and most preferably in the range of 80 to 5,000 kDa.

[0253] In one particular preferred embodiment of the present invention, the average molecular weight of the protein in the protein composition is in the range of 18 kDa to 500 kDa, more preferably 18 to 100 kDa, and most preferably 18 to 40 kDa.

[0254] The pH of the protein composition is typically in the range of 5.5 to 9.5.

[0255] In some preferred embodiments of the present invention, the pH of the protein composition is in the range of 5.5 to 9.5, more preferably in the range of 6.0 to 8.5, even more preferably in the range of 6.2 to 8.0, and most preferably in the range of 6.5 to 7.5.

[0256] In some preferred embodiments of the present invention, the total protein content of the protein composition is at least 30% w / w of the total solids of the protein composition, more preferably at least 50% w / w of the total solids of the protein composition, even more preferably at least 75% w / w, and most preferably at least 85% w / w.

[0257] Preferably, the total protein content of the protein composition is in the range of 30 to 99% w / w relative to the total solids of the protein composition, more preferably in the range of 50 to 97% w / w relative to the total solids of the protein composition, even more preferably in the range of 75 to 96% w / w, and most preferably in the range of at least 85 to 95% w / w.

[0258] In some preferred embodiments of the present invention, the total fat content of the protein composition is up to 10% w / w of total solids, more preferably up to 8% w / w of total solids, and most preferably up to 6% w / w of total solids.

[0259] In another preferred embodiment of the present invention, the total fat content of the protein composition is at most 5% w / w of total solids, more preferably at most 2% w / w of total solids, even more preferably at most 0.5% w / w, and most preferably at most 0.1% w / w.

[0260] In a more preferred embodiment of the present invention, the total fat content of the protein composition is in the range of 1 to 20% w / w relative to the total solids, more preferably in the range of 2 to 16% w / w relative to the total solids, even more preferably in the range of 3 to 12% w / w, and most preferably in the range of up to 4 to 10% w / w.

[0261] Much lower fat levels are typically preferred, and the total fat content of the protein composition is often preferably up to 1% w / w of total solids, more preferably up to 0.5% w / w of total solids, even more preferably up to 0.2% w / w, and most preferably up to 0.1% w / w.

[0262] The protein composition may contain varying amounts of carbohydrates.

[0263] However, it is often preferable that the carbohydrate content of the protein composition be a maximum of 65% w / w relative to the total solids.

[0264] Much lower carbohydrate levels are typically preferred, and the carbohydrate content of the protein composition is often preferably up to 20% w / w of total solids, more preferably up to 8% w / w of total solids, even more preferably up to 2% w / w, and most preferably up to 0.2% w / w.

[0265] The ash content of the protein composition is preferably 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 the total solids.

[0266] In some preferred embodiments of the present invention, the ash content of the protein composition is 0.4 to 8% w / w of total solids, more preferably 0.5 to 6% w / w of total solids, even more preferably 0.5 to 5% w / w, and most preferably 0.6 to 4.0% w / w.

[0267] The combined magnesium and calcium content of the protein composition is preferably 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.

[0268] In some preferred embodiments of the present invention, the combined magnesium and calcium content of the protein composition is 0.01 to 1% w / w, more preferably 0.001 to 0.7% w / w, even more preferably 0.01 to 0.5% w / w, and most preferably 0.01 to 0.2% w / w relative to the total solids.

[0269] The inventors have found that a protein composition containing up to 15 micromoles of free thiol groups / gram of protein can reduce the level of unpleasant odor in a heat-treated whey protein beverage containing 3% whey protein compared to unmodified whey protein.

[0270] In some preferred embodiments of the present invention, the amount of free thiol groups in the protein composition is at most 15 micromoles / g of protein, more preferably at most 14 micromoles / g of protein, even more preferably at most 13 micromoles / g of protein, and most preferably at most 12 micromoles / g of protein.

[0271] In some preferred embodiments of the present invention, the amount of free thiol groups in the protein composition is 0.001 to 15 micromoles / g of protein, more preferably 0.01 to 14 micromoles / g of protein, even more preferably 0.01 to 13 micromoles / g of protein, and most preferably 0.01 to 12 micromoles / g of protein.

[0272] However, especially when the protein composition is used in a heat-treated high-protein beverage, for example, when it contains 6% or more whey protein, it is often preferable that the free thiol groups in the protein composition be at a lower level. Therefore, in some preferred embodiments of the present invention, the amount of free thiol groups in the protein composition is at most 10 micromoles / g of protein, more preferably at most 8 micromoles / g of protein, more preferably at most 5 micromoles / g of protein, even more preferably at most 3 micromoles / g of protein, and most preferably at most 2 micromoles / g of protein.

[0273] Preferably, the amount of free thiol groups in the protein composition is 0.01 to 10 micromoles / g of protein, more preferably 0.01 to 8 micromoles / g of protein, more preferably 0.01 to 5 micromoles / g of protein, even more preferably 0.01 to 3 micromoles / g of protein, and most preferably 0.01 to 2 micromoles / g of protein.

[0274] Much lower levels of free thiol groups are sometimes required, and in some preferred embodiments of the present invention, the amount of free thiol groups in the protein composition is at most 1 micromol / g of protein, more preferably at most 0.7 micromol / g of protein, even more preferably at most 0.5 micromol / g of protein, and most preferably at most 0.2 micromol / g of protein.

[0275] In some preferred embodiments of the present invention, the protein composition has a tryptophan content of at least 0.7% w / w, more preferably at least 0.8% w / w, even more preferably at least 0.9% w / w, and most preferably at least 1.0% w / w, relative to the total protein.

[0276] Preferably, the tryptophan content of the protein composition is 0.7 to 3% w / w of total protein, more preferably 0.8 to 2.6% w / w of total protein, even more preferably 0.9 to 2.4% w / w, and most preferably 1.0 to 2.2% w / w.

[0277] Alternatively, the preferred protein composition often has a tryptophan content of 0.7-3% w / w relative to the total protein, more preferably 0.8-3% w / w, even more preferably 0.9-3% w / w, and most preferably 1.0-3% w / w.

[0278] In some preferred embodiments of the present invention, the methionine content of the protein composition is at least 0.3% w / w of total protein, more preferably at least 0.4% w / w of total protein, even more preferably at least 0.5% w / w, and most preferably at least 0.6% w / w.

[0279] Preferably, the methionine content of the protein composition is 0.3 to 3.3% w / w of total protein, more preferably 0.4 to 3.2% w / w of total protein, even more preferably 0.5 to 3.2% w / w, and most preferably 0.6 to 3.2% w / w.

[0280] In many cases, it is preferable to increase the lower limit of methionine, and in some preferred embodiments of the present invention, the methionine content of the protein composition is 1.0 to 3.3% w / w relative to the total protein, more preferably 1.3 to 3.2% w / w, even more preferably 1.6 to 3.2% w / w, and most preferably 1.8 to 3.2% w / w relative to the total protein.

[0281] Preferably, the kynurenine content of the protein composition is at most 0.2 micrograms / mg of protein, more preferably at most 0.05 micrograms / mg of protein, even more preferably at most 0.01 micrograms / mg of protein, and most preferably at most 0.001 micrograms / mg of protein. It is particularly preferable that the protein composition does not contain detectable kynurenine.

[0282] Kynurenine content 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; July 2021).

[0283] While kynurenine is a useful marker of tryptophan oxidation, the inventors believe it is a partial cause of the yellowing of heat-sterilized protein beverages, due to excessive oxidation of the protein. Furthermore, this is undesirable from a health perspective.

[0284] Preferably, the protein-bound sulfur content of the protein composition is in the range of 100 to 600 micromoles / g of protein, more preferably in the range of 200 to 500 micromoles / g of protein, and most preferably in the range of 250 to 500 micromoles / g of protein.

[0285] Preferably, the content of disulfide bond-forming protein-binding cysteine ​​residues in the protein composition is in the range of 150 to 400 micromoles / g of protein, more preferably in the range of 160 to 350 micromoles / g of protein, and most preferably in the range of 170 to 300 micromoles / g of protein.

[0286] The inventors have found that the protein particle size of the protein composition is preferably 10,000 kDa or less, and that it is advantageous for the protein particle size to be small enough to avoid opacity in the case of clear beverage applications, and further to avoid an increase in viscosity and drying during the concentration of the protein.

[0287] In some preferred embodiments of the present invention, the weight-average molecular weight of the protein in the protein composition is in the range of 18 kDa to 10,000 kDa, more preferably in the range of 30 to 9,000 kDa, even more preferably in the range of 50 to 8,000 kDa, and most preferably in the range of 80 to 5,000 kDa.

[0288] Preferably, at least 60% w / w of the protein in the protein composition has a molecular weight of 18 kDa to 10,000 kDa; more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 99% w / w has a molecular weight of 18 kDa to 10,000 kDa.

[0289] More preferably, at least 60% w / w of the protein in the protein composition has a molecular weight of 50 kDa to 8000 kDa; more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 99% w / w has a molecular weight of 50 kDa to 8000 kDa.

[0290] More preferably, at least 60% w / w of the protein in the protein composition has a molecular weight of 80 kDa to 5000 kDa; more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 99% w / w has a molecular weight of 80 kDa to 5000 kDa.

[0291] In another preferred embodiment of the present invention, the weight-average molecular weight of the protein in the protein composition is in the range of 18 kDa to 200 kDa, more preferably in the range of 30 to 150 kDa, and most preferably in the range of 30 to 100 kDa.

[0292] The inventors have discovered that the smaller the weight-average molecular weight of the protein, the higher the total protein concentration can be achieved by concentration before spray drying (e.g., by ultrafiltration or nanofiltration).

[0293] Preferably, at least 60% w / w of the protein in the protein composition has a molecular weight of 18 kDa to 200 kDa; more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 99% w / w has a molecular weight of 18 kDa to 200 kDa.

[0294] More preferably, at least 60% w / w of the protein in the protein composition has a molecular weight of 18 kDa to 150 kDa; more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 99% w / w has a molecular weight of 18 kDa to 150 kDa.

[0295] More preferably, at least 60% w / w of the protein in the protein composition has a molecular weight of 18 kDa to 100 kDa; more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 99% w / w has a molecular weight of 18 kDa to 100 kDa.

[0296] The inventors have found that it may be advantageous for the majority of the proteins in the protein composition to have a molecular weight of at least 30 kDa, which is thought to be due to the dimerization of modified BLG.

[0297] Therefore, preferably, at least 60% w / w of the protein in the protein composition has a molecular weight of 30 kDa to 200 kDa; more preferably, at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 99% w / w has a molecular weight of 30 kDa to 200 kDa.

[0298] More preferably, at least 60% w / w of the protein in the protein composition has a molecular weight of 30 kDa to 150 kDa; more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 99% w / w has a molecular weight of 30 kDa to 150 kDa.

[0299] More preferably, at least 60% w / w of the protein in the protein composition has a molecular weight of 30 kDa to 100 kDa; more preferably at least 80% w / w, even more preferably at least 90% w / w, and most preferably at least 99% w / w has a molecular weight of 30 kDa to 100 kDa.

[0300] In some preferred embodiments of the present invention, the protein composition of the present invention can be obtained by the method described herein. In some preferred embodiments of the present invention, the protein composition is in liquid form, preferably an aqueous liquid. The protein composition in liquid form preferably has a solid content of 0.1 to 50% w / w, more preferably 1 to 35% w / w, even more preferably 5 to 30% w / w, and most preferably 10 to 30% w / w.

[0301] In some preferred embodiments of the present invention, the protein composition is in solid form, preferably in powder form, and preferably in powder form 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.

[0302] The portion of the protein composition and the oxidized whey protein solution that does not contribute to the solid content is preferably water.

[0303] The portion of the protein composition that does not contribute to the solid content preferably contains at least 80% w / w of water, more preferably at least 90% w / w of water, even more preferably at least 95% w / w of water, and more preferably at least 99% w / w of water.

[0304] In one particularly preferred embodiment of the present invention, the protein composition is: • Protein content of at least 86% w / w relative to total solids, and most preferably at least 90% w / w 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; • A maximum of 10 micromoles of free thiol groups / gram of protein, and most preferably a maximum of 5 micromoles of free thiol groups / gram of protein; • Tryptophan content of 0.7–3% w / w relative to total protein, and most preferably 1.0–3% w / w relative to total protein; • Methionine content of 0.3–3.3% w / w relative to total protein, and most preferably 1.3–3.2% w / w relative to total protein; • A kynurenine content of up to 0.2 micrograms / mg of protein, and most preferably up to 0.01 micrograms / mg of protein. It has.

[0305] In one particularly preferred embodiment of the present invention, the oxidized whey protein composition is preferably: • The protein-bound sulfur content is in the range of 100-600 micromoles / g of protein; and • The content of disulfide bond-forming protein-binding cysteine ​​residues is in the range of 150 to 400 micromoles / g of protein. Furthermore, in one particularly preferred embodiment of the present invention, the protein composition is preferably: • The protein-bound sulfur content is in the range of 100-600 micromoles / g of protein; and • The content of disulfide bond-forming protein-binding cysteine ​​residues is in the range of 150 to 400 micromoles / g of protein.

[0306] Furthermore, in the protein composition of the particularly preferred embodiment described above, it is often preferable 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 have a molecular weight of 30 kDa to 9000 kDa.

[0307] The pH of the protein composition of the particularly preferred embodiment described above is preferably in the range of 6.2 to 8.0, and most preferably 6.5 to 7.5.

[0308] In some preferred embodiments of the present invention, the protein composition is a sterile protein composition, preferably a packaged sterile protein composition. Preferably, it is in the form of a sterile liquid protein composition or a sterile powdered protein composition.

[0309] Another aspect of the present invention relates to a process for producing a heat-treated beverage, preferably a heat-sterilized beverage; The process includes the following steps (1) and (2): (1) A step of mixing the protein composition of the present invention with one or more further beverage components in order to obtain a liquid mixture having a pH of 5.5 to 8.5; and (2) Filling the liquid mixture into a suitable container; The process further includes at least one heat treatment step; Here, the liquid mixture is heat-treated, preferably heat-sterilized, before and / or after filling.

[0310] In some preferred embodiments of the present invention, the beverage mixture contains an amount of the protein composition sufficient to contribute at least 0.5% w / w of protein.

[0311] Therefore, a more specific aspect of the present invention relates to a process for producing a heat-treated beverage, preferably a heat-sterilized beverage, having a pH of 5.5 to 8.5, more preferably 6.5 to 7.5; The process includes the following steps (1) and (2): (1) A step of combining a protein composition as described herein with one or more further components in order to obtain a liquid mixture having a pH of 5.5 to 8.5, more preferably 6.5 to 7.5; Here, the liquid mixture is: • A sufficient amount of the protein composition to contribute at least 0.5% w / w of protein, and · Water, Includes; (2) The step of packaging the liquid mixture into a container, preferably a sterile container; Here, the liquid mixture is heat-treated, preferably heat-sterilized, before and / or after filling.

[0312] The protein compositions described herein are preferably only protein sources of food products or heat-sterilized beverages, and therefore only protein sources of liquid mixtures.

[0313] The inventors have discovered that it is advantageous to keep the free thiol group content of the liquid mixture low before heat treatment in order to prevent the generation of an unpleasant odor similar to that of rotten eggs.

[0314] Therefore, in some preferred embodiments of the present invention, before heat sterilization, the liquid mixture contains up to 60 micromoles of free thiol groups per 100g of liquid mixture, more preferably up to 40 micromoles of free thiol groups per 100g of liquid mixture, even more preferably up to 30 micromoles of free thiol groups per 100g of liquid mixture, and most preferably up to 30 micromoles of free thiol groups per 100g of liquid mixture.

[0315] In many cases, an even lower free thiol group content is required, and in some preferred embodiments of the present invention, before heat sterilization, the liquid mixture contains a maximum of 20 micromoles of free thiol groups per 100g of liquid mixture, more preferably a maximum of 15 micromoles of free thiol groups per 100g of liquid mixture, even more preferably a maximum of 10 micromoles of free thiol groups per 100g of liquid mixture, and most preferably a maximum of 5 micromoles of free thiol groups per 100g of liquid mixture.

[0316] Preferably, the liquid mixture contains protein in a total amount of 0.5 to 15% w / w relative to the weight of the liquid mixture, more preferably in a range of 1 to 10% w / w relative to the weight of the liquid mixture, even more preferably in a range of 2 to 9% w / w relative to the weight of the liquid mixture, and most preferably in a range of 3 to 8% w / w relative to the weight of the liquid mixture.

[0317] Alternatively, the preferred liquid mixture may contain protein in a total amount of 4 to 15% w / w relative to the weight of the liquid mixture, more preferably in a total amount of 5 to 14% w / w relative to the weight of the liquid mixture, even more preferably in a total amount of 6 to 13% w / w relative to the weight of the liquid mixture, and most preferably in a total amount of 8 to 12% w / w relative to the weight of the liquid mixture.

[0318] 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.

[0319] In many cases, an even higher contribution is preferable, and in some preferred embodiments of the present invention, the protein composition of the present invention provides a contribution equivalent to 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 at least 100% w / w of the total protein.

[0320] When using the protein composition in combination with other protein sources, it is preferable to use a source with a relatively low free thiol group content.

[0321] In some preferred embodiments of the present invention, the total protein content of the liquid mixture is at least 15% w / w relative to the total solids, more preferably at least 20% w / w, most preferably at least 25% w / w, and most preferably at least 30% w / w.

[0322] For example, if the beverage is intended to be a sports protein beverage, the total protein may contribute to a fairly large portion of the total solids. Therefore, in some preferred embodiments of the present invention, the total protein content of the liquid mixture is at least 80% w / w of the total solids, more preferably at least 90% w / w of the total solids, even more preferably at least 92% w / w, and most preferably at least 94% w / w.

[0323] The solid content of the liquid mixture is typically 0.5 to 50% w / w, more preferably 1 to 35% w / w, even more preferably 2 to 20% w / w, and most preferably 3 to 10% w / w.

[0324] The portion of the liquid mixture that does not contain solid matter preferably contains water. The water content of the portion of the liquid mixture that does not contain solid matter is preferably 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.

[0325] In some preferred embodiments of the present invention, the calorie content of the liquid mixture is up to 100 kcal / 100g, more preferably up to 80 kcal / 100g, even more preferably up to 70 kcal / 100g, and most preferably up to 60 kcal / 100g. Preferably, the calorie content of the liquid mixture may be 2 to 100 kcal / 100g, more preferably 4 to 80 kcal / 100g, even more preferably 8 to 70 kcal / 100g, and most preferably 12 to 60 kcal / 100g. These embodiments are preferred when the protein source is the primary energy source (e.g., for sports use).

[0326] In other preferred embodiments of the present invention, the calorie content of the liquid mixture is greater than 100 kcal / 100g; more preferably at least 120 kcal / 100g, even more preferably at least 140 kcal / 100g, and most preferably at least 150 kcal / 100g. Preferably, the calorie content of the liquid mixture may be 101 to 300 kcal / 100g, more preferably 120 to 280 kcal / 100g, even more preferably 140 to 270 kcal / 100g, and most preferably 150 to 260 kcal / 100g. These embodiments are preferred, for example, for clinical nutrition, when the protein source is associated with the actual amounts of carbohydrates and fats.

[0327] The compositional characteristics and preferences described on pages 65-81 of International Patent Publication PCT / EP2022 / 078739 for heat-treated beverages also apply equally to the liquid mixture.

[0328] The pH of the liquid mixture may be between slightly acidic and slightly alkaline.

[0329] For the production of beverages with a pH near neutral, it is particularly preferable that the pH of the liquid mixture be near neutral. In some preferred embodiments of the present invention, the pH of the liquid mixture is in the range of 5.5 to 8.0, more preferably in the range of 6.0 to 7.5, even more preferably in the range of 6.2 to 7.3, and most preferably in the range of 6.3 to 7.2.

[0330] In another preferred embodiment of the present invention, the pH of the liquid mixture is in the range of 6.0 to 7.5, more preferably in the range of 6.2 to 7.5, and most preferably in the range of 6.3 to 7.5.

[0331] In a more preferred embodiment of the present invention, the pH of the liquid mixture is in the range of 6.0 to 8.0, more preferably in the range of 6.6 to 7.7, even more preferably in the range of 6.7 to 7.6, and most preferably in the range of 6.8 to 7.5.

[0332] Generally, for adjusting the pH of the liquid mixture, any suitable food acid or food base may be used. Those skilled in the art will understand suitable means for adjusting the pH. Suitable food bases include sodium carbonate or potassium carbonate, sodium bicarbonate or potassium bicarbonate, or ammonium hydroxide. Alternatively, KOH or NaOH may be used for adjusting the pH. Examples of suitable food acids include citric acid, hydrochloric acid, malic acid, tartaric acid or phosphoric acid.

[0333] In some preferred embodiments of the present invention, at 20 °C and a shear rate of 300 s -1 the viscosity of the liquid mixture is at most 200 cP; more preferably, at 20 °C and a shear rate of 300 s -1 the viscosity is at most 100 cP, even more preferably, at 20 °C and a shear rate of 300 s -1 the viscosity is at most 50 cP, and most preferably, at 20 °C and a shear rate of 300 s -1 the viscosity is at most 20 cP.

[0334] The liquid mixture is typically prepared by mixing suitable components with the oxidized whey protein composition. When using powder components, it is often preferable to hydrate them before the heat treatment, and similarly, it may be preferable to homogenize the liquid mixture before the heat treatment.

[0335] In some preferred embodiments, the protein composition is provided in powder form, preferably mixed with water or an aqueous liquid and hydrated before the heat treatment.

[0336] In another preferred embodiment, the protein composition is provided in liquid form (e.g., the protein solution obtained in step (d) of the present method). The protein composition obtained in step (d) is then: • Mix with one or more additional ingredients necessary for the manufacture of the beverage; • To be subjected to homogenization at will; • Heat sterilize by heating it at a temperature in the range of 140-150°C for 1-10 seconds; To cool; and • Fill into suitable sterile containers and then seal them.

[0337] The package filling in step (2) may be any suitable package filling technology, and any suitable container may be used for package filling of the liquid mixture.

[0338] However, in a preferred embodiment of the present invention, the package filling in step (2) is aseptic package filling; that is, the liquid mixture is filled under sterile conditions. For example, the aseptic package filling may be carried out using an aseptic filling system, and preferably involves filling the liquid mixture into one or more aseptic containers.

[0339] Aseptic filling and sealing are particularly preferable if the liquid mixture is already sterile or has very low levels of microorganisms before filling.

[0340] Examples of useful containers include bottles, cartons, bricks, and / or bags.

[0341] In the heat treatment of this process, preferably, the liquid mixture is brought to a temperature of at least 70°C.

[0342] In some preferred embodiments of the present invention, the liquid mixture of step (1) is subjected to a heat treatment including at least pasteurization, and then subjected to the filling step of step (2).

[0343] In another embodiment of the process of the present invention, the filling liquid mixture of step (2) is subjected to a heat treatment including at least pasteurization.

[0344] In some preferred embodiments, the heat treatment includes heating the liquid mixture to a temperature in the range of 70 to 80°C.

[0345] In some preferred embodiments of the present invention, the temperature of the heat treatment is in the range of 70 to 80°C, preferably 70 to 79°C, more preferably 71 to 78°C, even more preferably 72 to 77°C, and most preferably 73 to 76°C (approximately 75°C, etc.).

[0346] Preferably, when carried out in a temperature range of 70-80°C, the heating time is 1 second to 60 minutes. The maximum exposure time is optimal at the lowest temperature within the above temperature range, and vice versa.

[0347] In other preferred embodiments, the heat treatment temperature is 70°C for at least 60 minutes, or preferably 75°C for at least 45 minutes, or preferably 80°C for at least 30 minutes, or preferably 85°C for at least 22 minutes, or preferably 90°C for at least 10 minutes.

[0348] In a particularly preferred embodiment of the present invention, the heat treatment is carried out at 70-78°C for 1 second to 30 minutes; more preferably at 71-77°C for 1 minute to 25 minutes; and even more preferably at 72-76°C for 2 minutes to 20 minutes.

[0349] In some preferred embodiments of the present invention, the heat treatment process includes heating at a temperature of 85°C to 95°C for 1 to 30 minutes.

[0350] For example, the temperature of the heat treatment may 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.

[0351] In some particularly preferred embodiments of the present invention, the heat treatment includes heating the liquid mixture at a temperature in the range of 100 to 160°C for a time sufficient to sterilize the liquid mixture. This preferably includes heating the liquid mixture at a temperature in the range of 120 to 155°C for a time sufficient to achieve sterility, typically 0.1 seconds to 10 minutes, and more preferably at 140 to 155°C for a time sufficient to achieve sterility, typically 0.1 to 30 seconds. This liquid heat treatment to sterilize the liquid is also called heat sterilization.

[0352] Another preferred heat treatment is UHT sterilization, which involves treating the material at a temperature typically in the range of 135–146°C for a sufficient time to achieve sterility, typically in the range of 1–10 seconds.

[0353] Alternatively, a preferred heat treatment includes processing at a temperature in the range of 145 to 180°C for a time sufficient to achieve sterility, typically in the range of 0.01 to 2 seconds, more preferably in the range of 150 to 180°C for a time in the range of 0.01 to 0.3 seconds.

[0354] The heat treatment may be carried out using equipment such as plate or tubular heat exchangers, scrape heat exchangers, or retort systems. Alternatively, direct heating with steam, such as direct steam injection, direct steam infusion, or spray cooking, may be used, which is particularly preferred for heat treatments exceeding 95°C. Furthermore, such direct steam heating is preferably used in combination with flash cooling. A preferred example of the implementation of spray cooking is described in WO2009113858A1; this reference is incorporated herein by reference for all purposes. Preferred examples of the implementation of direct steam injection and direct steam infusion are described in WO2009113858A1 and WO2010 / 085957A3; these references are incorporated herein by reference for all purposes. General aspects of high-temperature processing are described, for example, in "Thermal technologies in food processing" (ISBN 185573558X); this reference is incorporated herein by reference for all purposes.

[0355] In some preferred embodiments of the present invention, the heat treatment comprises or consists of retort heat treatment, preferably at a temperature of at least 80°C, more preferably at least 95°C, even more preferably at least 100°C, and most preferably at least 120°C, preferably for a time sufficient to sterilize the liquid of the treatment.

[0356] In another preferred embodiment of the present invention, the heat treatment comprises or consists of steam infusion or spray cooking, preferably at a temperature of at least 100°C, more preferably at least 120°C, even more preferably at least 130°C, and most preferably at least 140°C, preferably for a time sufficient to sterilize the liquid of the treatment.

[0357] Some preferred embodiments of the present invention combine pasteurization with physical microbial reduction.

[0358] Useful examples of physical microbial reduction include one or more of the following: sterilization filtration, ultraviolet light, high-pressure treatment, pulsed electric field treatment, and ultrasound.

[0359] In some preferred embodiments of the present invention, the heat treatment is a sterilization heat treatment, yielding a sterilization liquid mixture and therefore a sterilization beverage. Such sterilization may be achieved, for example, by a combination of sterilization filtration and pasteurization, or by performing a heat treatment at at least 100°C for a time sufficient to achieve sterilization.

[0360] Cooling the liquid mixture after heat treatment is beneficial. According to a preferred embodiment of the process of the present invention, after heat treatment, the heat-treated liquid mixture is cooled to preferably 0 to 70°C, preferably 0 to 60°C, more preferably 0 to 30°C, and most preferably 0 to 20°C.

[0361] If the liquid mixture is not sterilized by the heat treatment, the heat-treated liquid mixture is cooled after the heat treatment, preferably to 0-15°C, more preferably to 1-10°C, and most preferably to 1-5°C.

[0362] The cooling may be performed before or after the filling process.

[0363] The cooling typically includes flash cooling and / or conventional heat exchangers.

[0364] Flash cooling, at least partial cooling, is often preferable, especially after heat sterilization. Flash cooling typically removes some of the volatile compounds from the cooled liquid. Whey protein beverages with a pH in the range of 5.5 to 8.5 tend to develop unpleasant odors, particularly during heat treatment, and these odors are partially removed from the heat-treated liquid and released near the flash cooling system. This is a drawback because operators of the heat treatment system are exposed to the off-odors, which may also be associated with health problems.

[0365] The inventors have discovered that, advantageously, flash cooling of heat-treated beverages based on this protein composition results in a much lower, or sometimes no, release of such unpleasant odors.

[0366] The process of the present invention can be implemented as a batch process, a quasi-batch process, or a continuous process.

[0367] Another specific aspect of the present invention relates to a process for producing heat-treated beverages, preferably heat-sterilized beverages; For the purpose of obtaining the protein composition in liquid form, the method described herein: • Perform steps (a), (c), and (d); or • Perform steps (a), (b), (c), and (d), Includes,

[0368] Next, the protein composition, or a liquid raw material prepared from the protein composition, is packaged; The package filling is carried out according to step (2) of the process described above.

[0369] If the protein composition is to be used directly as a beverage, it is preferable that step (d) includes a heat sterilization treatment, that is, a heat treatment to sterilize the treated liquid.

[0370] Such heat treatments typically require heating the liquid to be treated at a temperature in the range of 100–160°C for a sufficient time to sterilize it. Suitable time / temperature combinations for such heat treatments are described herein.

[0371] Another aspect of the present invention relates to a beverage obtainable by the process of the present invention, which is heat-treated, preferably heat-sterilized, and has a pH of 5.5 to 8.5.

[0372] Another aspect of the present invention relates to a heat-treated, preferably heat-sterilized, beverage with a pH of 5.5 to 8.5, more preferably with a pH of 6.5 to 7.5, the beverage containing a sufficient amount of the protein composition of the present invention to contribute at least 0.5% w / w of protein, and preferably having an H2S content of up to 5 micromoles / L, more preferably up to 3 micromoles / L, even more preferably up to 1.0 micromoles / L, and most preferably up to 0.7 micromoles / L, 7 days after the production of the beverage.

[0373] A further aspect of the present invention relates to food ingredients including: • Solid matter of the protein composition of the present invention; and • One or more additional components, preferably selected from the following: • Milk components, preferably non-oxidized milk components; • Plant-derived ingredients; • Non-dairy carbohydrate source; • Flavoring and odor-masking agents; and / or • Sweeteners, such as sweetening sugars, polyols, and / or high-strength sweeteners.

[0374] Preferably, the sweetener includes one or more of the following: carbohydrate sweeteners, polyols, high-strength sweeteners, and combinations thereof.

[0375] In one embodiment of the present invention, the beverage contains at least one high-strength sweetener (HIS). The at least one HIS is preferably selected from the group consisting of aspartame, cyclamate, sucralose, acesulfame salt, neotame, saccharin, stevia extract, steviol glycoside (e.g., rebaudioside A), or a combination thereof.

[0376] In some embodiments of the present invention, it is particularly preferable that the sweetener contains or consists of one or more high-strength sweeteners.

[0377] HIS is present in both natural and artificial sweeteners and typically has a sweetness intensity at least 10 times that of sucrose.

[0378] When HIS is used, the total amount of HIS in the beverage is typically in the range of 0.001 to 2% w / w. Preferably, the total amount of HIS is in the range of 0.005 to 1% w / w. Most preferably, the total amount of HIS is in the range of 0.01 to 0.5% w / w.

[0379] The choice of sweetener may depend on the beverage being manufactured; for example, if it is desired that the sweetener does not contribute to energy, a high-strength sweetener (e.g., aspartame, acesulfame K, or sucralose) may be used in the beverage; on the other hand, for beverages with a natural profile, a natural sweetener (e.g., steviol glycosides, sorbitol, or sucrose) may be used.

[0380] It may be even more preferable that the sweetener contains one or more polyol sweeteners, or consists of the same.

[0381] Non-limiting examples of useful polyol sweeteners include reduced maltose, mannitol, lactitol, sorbitol, inositol, xylitol, slayitol, galactitol, or combinations thereof. When polyol sweeteners are used, the total amount of polyol sweetener in the beverage is typically in the range of 1-20% w / w. More preferably, the total amount of polyol sweetener in the beverage is in the range of 2-15% w / w. Even more preferably, the total amount of polyol sweetener in the beverage may be in the range of 4-10% w / w.

[0382] A further aspect of the present invention relates to the uses of protein compositions containing modified BLG, preferably the uses of protein compositions containing the modified BLG of the present invention as food ingredients; Preferably, a heat-sterilized beverage with a pH in the range of 5.5 to 8.5: • For the purpose of improving odor; and / or • To reduce the level of an unpleasant odor similar to that of rotten eggs; and / or • To reduce H2S generation during manufacturing; and / or • To reduce the H2S content in the upper space of the container,

[0383] This concerns its use; Here, the beverage preferably has a whey protein content of at least 3% w / w and is preferably heat-sterilized using indirect heat treatment.

[0384] Total protein, pH, viscosity, H2S content, total thiol group content and free thiol group content, amino acid content, amino acid oxidation, average molecular weight, intrinsic viscosity, sensory evaluation, intrinsic protein content, total fat content, lactose content, mineral composition, and turbidity are quantified or evaluated according to Analysis A-Q of International Patent Application Publication PCT / EP2022 / 078739; this reference is incorporated herein by reference for all purposes.

[0385] Preferred embodiments of the present invention are listed below in the following numbered embodiments. [Sequential numbering embodiment 1] A method for preparing a protein composition containing modified β-lactoglobulin (BLG); A method including the following steps (a) to (d): (a) • A source containing one or more phenolic compounds, each containing at least two hydroxyl groups directly bonded to the same aromatic ring (PCA); and • Sources including BLG, A process to provide; (b) A step of optionally subjecting a portion of a supply containing one or more types of PCA to oxidation of a type that can convert PCA to quinone, thereby providing a supply containing one or more oxidized PCA (PCA-type oxidation); (c) A step of combining a portion of a source containing one or more PCA and / or a portion of a source containing one or more oxidized PCA with a source containing BLG and optionally further components for the purpose of providing a protein solution; Here, the protein solution is pH in the range of 6.5 to 9.5; and • BLG content of at least 0.2% w / w; • The molar ratio between the original amount of PCA used in the preparation of the protein solution and the BLG content of the protein solution, wherein the molar ratio is at least 0.1:1. Having; (d) Incubating the protein solution within a certain temperature range for a period of time sufficient to reduce the amount of free thiol groups in the protein solution to a maximum of 10 micromoles / g of protein; If the method does not include step (b), step (d) is a step of incubation under conditions that also include the use of an oxidation of a type capable of converting PCA to quinone; Preferably, the PCA type oxidation used in step (b) and / or step (d) is: The theoretical amount of quinone provided to the protein solution during the implementation of the method, the theoretical amount of quinone determined according to Analysis 1, and • Amount of BLG in the protein solution, This is sufficient to make the molar ratio between them at least 0.1:1. [Sequential numbering embodiment 1.1] A method for preparing a protein composition containing modified β-lactoglobulin (BLG); A method including the following steps (a) to (d): (a) • A source containing one or more phenolic compounds, each containing at least two hydroxyl groups directly bonded to the same aromatic ring (PCA); and • Sources including BLG, A process to provide; (b) A step of optionally subjecting a portion of a supply containing one or more types of PCA to oxidation of a type that can convert PCA to quinone, thereby providing a supply containing one or more oxidized PCA (PCA-type oxidation); (c) A step of combining a portion of a source containing one or more PCA and / or a portion of a source containing one or more oxidized PCA with a source containing BLG and optionally further components for the purpose of providing a protein solution; Here, the protein solution is pH in the range of 6.5 to 9.5; and • BLG content of at least 0.2% w / w; • The molar ratio between the original amount of PCA used in the preparation of the protein solution and the BLG content of the protein solution, wherein the molar ratio is at least 1:1. Having; (d) Incubating the protein solution within a certain temperature range for a period of time sufficient to reduce the amount of free thiol groups in the protein solution to a maximum of 10 micromoles / g of protein; If the method does not include step (b), step (d) is a step of incubation under conditions that also include the use of an oxidation of a type capable of converting PCA to quinone; Preferably, the PCA type oxidation used in step (b) and / or step (d) is: The theoretical amount of quinone provided to the protein solution during the implementation of the method, the theoretical amount of quinone determined according to Analysis 1, and • Amount of BLG in the protein solution, This is sufficient to make the molar ratio between them at least 1:1. [Sequential Numbering Embodiment 2] A method according to sequential embodiment 1 or 1.1, further comprising the step (e) of drying a liquid material containing a protein derived from the incubated protein solution of at least step (d), method. [Sequential numbering embodiment 3] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the PCA includes flavonoids, preferably flavanols or flavanol esters, such as catechin, epicatechin, gallocatechin, epigallocatechin, 3-gallic acid catechin, 3-gallic acid epicatechin, 3-gallic acid gallocatechin, and 3-gallic acid epigallocatechin (EGCG). method. [Sequential numbering embodiment 4] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the PCA comprises a stilbenoide, preferably resveratrol. method. [Sequential numbering embodiment 5] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the PCA includes caffeic acid, gallic acid, chlorogenic acid, catechol, 4-methylcatechol, catechin, epicatechin, gallocatechin, epigallocatechin, 3-gallic acid catechin, 3-gallic acid epicatechin, 3-gallic acid gallocatechin, 3-gallic acid epigallocatechin (EGCG), resveratrol, carnosic acid, carnosol, naringenin, or a mixture thereof. method. [Sequential numbering embodiment 6] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the molecular weight of the PCA is a maximum of 600 g / mol, more preferably a maximum of 400 g / mol, even more preferably a maximum of 250 g / mol, and more preferably a maximum of 200 g / mol. method. [Sequential Numbering Embodiment 7] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the molecular weight of the PCA is in the range of 110 to 600 g / mol, more preferably in the range of 110 to 400 g / mol, even more preferably in the range of 110 to 350 g / mol, and more preferably in the range of 110 to 310 g / mol. method. [Sequential numbering embodiment 7.1] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the molecular weight of the PCA is in the range of 120 to 1000 g / mol, more preferably in the range of 250 to 700 g / mol, even more preferably in the range of 300 to 650 g / mol, and more preferably in the range of 350 to 600 g / mol. method. [Sequential numbering embodiment 8] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the PCA does not contain a carboxylic acid group. method. [Sequential numbering embodiment 9] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the water solubility of the PCA at 25°C is at least 8 mM, more preferably at least 12 mM, and most preferably at least 16 mM. method. [Sequential numbering embodiment 10] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the source containing one or more types of 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. method. [Sequential numbering embodiment 10.1] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the source containing one or more types of PCA is selected from the group consisting of tea polyphenol extract, more preferably green tea polyphenol extract; coffee polyphenol extract; cocoa polyphenol extract; grape polyphenol extract; rosemary polyphenol extract; lemon balm polyphenol extract; blackcurrant polyphenol extract; single PCA isolate; and mixtures thereof. method. [Sequential numbering embodiment 11] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the supply source containing one or more types of PCA contains at least 25% w / w of PCA relative to the total solids of the supply source containing one or more types of PCA, more preferably at least 40% w / w of PCA, even more preferably at least 60% w / w of PCA, and most preferably at least 80% w / w of PCA, method. [Sequential numbering embodiment 12] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the supply source containing one or more types of PCA is a single PCA isolate containing at least 25% w / w of a single PCA relative to the total solids of the supply source containing one or more types of PCA, more preferably a single PCA isolate containing at least 40% w / w of a single PCA, even more preferably at least 60% w / w of a single PCA, and most preferably at least 80% w / w of a single PCA relative to the total solids of the supply source containing one or more types of PCA. method. [Sequential numbering embodiment 13] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the source containing BLG includes or consists of whey protein concentrate, whey protein isolate, whey protein concentrate, whey protein isolate, BLG isolate, or a combination thereof. method. [Sequential numbering embodiment 14] A method according to any one embodiment of the aforementioned sequential numbering embodiment; The process includes step (b), which involves providing a portion of a source containing one or more types of PCA to an oxidation process capable of converting PCA to quinone, thereby providing a source containing one or more oxidized PCA. method. [Sequential numbering embodiment 15] A method according to sequential numbering embodiment 14; Here, the oxidation in step (b) involves preparing an oxidizing aqueous solution containing a portion of a supply containing one or more PCA, optionally a chemical oxidizing agent, and optionally one or more further components (such as water). method. [Sequential numbering embodiment 16] A method according to sequential numbering embodiment 15; Here, the PCA content of the oxidizing aqueous solution is at least 0.2 mM, more preferably at least 15 mM, and most preferably at least 50 mM. method. [Sequential numbering embodiment 17] A method according to sequential numbering embodiment 15 or 16; Here, the PCA concentration of the oxidizing aqueous solution is 0.2 to 600 mM, more preferably 15 to 550 mM, and most preferably 50 to 500 mM. method. [Sequential numbering embodiment 18] A method according to any one embodiment of sequential numbered embodiments 15 to 17; Here, the pH of the oxidizing aqueous solution is in the range of 2 to 9.5, more preferably in the range of 6.5 to 9.5, even more preferably in the range of 7.1 to 9.0, even more preferably in the range of 7.3 to 8.7, and most preferably in the range of 7.4 to 8.5. method. [Sequential numbering embodiment 19] A method according to any one embodiment of sequential numbered embodiments 15 to 17; Here, the pH of the oxidizing aqueous solution is in the range of 2 to 6.4, more preferably in the range of 3.0 to 6.0, even more preferably in the range of 3.5 to 5.5, and most preferably in the range of 4.0 to 5.0. method. [Sequential numbering embodiment 20] A method according to any one embodiment of sequentially numbered embodiments 14 to 17; Here, the oxidation in step (b) involves contacting a portion of a supply containing one or more types of PCA with a chemical oxidizing agent, under conditions that at least a portion of the PCA is converted into quinone. method. [Sequential numbering embodiment 21] A method according to sequential numbering embodiment 20; Here, the chemical oxidizing agent includes or consists of peroxides, ozone, dioxygen, or a combination thereof. method. [Sequential numbering embodiment 22] A method according to sequential numbering embodiment 20 or 21; Here, the chemical oxidizing agent includes or consists of hydrogen peroxide, benzoyl peroxide, or a combination thereof. method. [Sequential numbering embodiment 23] A method according to any one embodiment of sequentially numbered embodiments 14 to 22; Here, the oxidation in step (b) involves preparing an oxidizing aqueous solution comprising a portion of a supply containing one or more PCA, the chemical oxidizing agent, and optionally one or more further components (such as water). method. [Sequential numbering embodiment 24] A method according to any one embodiment of sequentially numbered embodiments 14 to 23; Here, the oxidation in step (b) involves electrochemically oxidizing a portion of a supply containing one or more types of PCA under conditions that at least a portion of the PCA is converted to quinone. method. [Sequential numbering embodiment 25] A method according to sequential numbering embodiment 24; Here, step (b) oxidation involves preparing an oxidizing aqueous solution containing a portion of a supply containing one or more PCA and optionally one or more components (such as water). method. [Sequential numbering embodiment 26] A method according to sequential numbering embodiment 24 or 25; Here, the electrochemical oxidation is carried out using a potential difference of -0.1 to 1.5 V, more preferably 0.0 to 1.2 V, and most preferably 0.0 to 0.7 V. method. [Sequential numbering embodiment 27] A method according to any one embodiment of sequential numbered embodiments 25 to 26; Here, the pH of the oxidizing aqueous solution is in the range of 6 to 9, and the electrochemical oxidation is carried out using a potential difference of -0.1 to 0.9 V, more preferably 0 to 0.8 V, and most preferably 0.2 to 0.7 V. method. [Sequential numbering embodiment 28] A method according to any one embodiment of sequential numbered embodiments 25 to 26; Here, the pH of the oxidizing aqueous solution is in the range of 3 to 5, and the electrochemical oxidation is carried out using a potential difference of 0.1 to 1.3 V, more preferably 0.2 to 0.9 V, and most preferably 0.3 to 0.7 V. method. [Sequential numbering embodiment 28.1] A method according to any one embodiment of sequentially numbered embodiments 24 to 28; Here, the electrochemical oxidation is carried out at a temperature in the range of 2 to 80°C, more preferably in the range of 5 to 60°C, even more preferably in the range of 10 to 40°C, and most preferably in the range of 15 to 30°C. method. [Sequential numbering embodiment 28.2] A method according to any one embodiment of sequentially numbered embodiments 24 to 28; Here, the electrochemical oxidation is carried out at a temperature in the range of 30 to 90°C, more preferably in the range of 40 to 90°C, even more preferably in the range of 50 to 90°C, and most preferably in the range of 60 to 90°C. method. [Sequential numbering embodiment 28.3] A method according to any one embodiment of sequentially numbered embodiments 24 to 28; Here, the electrochemical oxidation is carried out at a temperature in the range of 5 to 25°C, more preferably in the range of 10 to 25°C, even more preferably in the range of 15 to 25°C, and most preferably in the range of 18 to 25°C. method. [Sequential numbering embodiment 29] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the pH of the protein solution is in the range of 6.7 to 9.5, more preferably in the range of 7.1 to 9.0, even more preferably in the range of 7.3 to 8.7, and most preferably in the range of 7.4 to 8.5. method. [Sequential numbering embodiment 30] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the BLG content of the protein solution is 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. method. [Sequential numbering embodiment 31] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the BLG content of the protein solution is 0.5-30% w / w, more preferably 1-20% w / w, even more preferably 3-16% w / w, and most preferably 5-12% w / w. method. [Sequential numbering embodiment 32] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the BLG content of the protein solution is at least 30% w / w of total protein, more preferably at least 40% w / w of total protein, even more preferably at least 45% w / w of total protein, and most preferably at least 50% w / w of total protein. method. [Sequential numbering embodiment 33] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the BLG content of the protein solution is 30-99% w / w relative to the total protein, more preferably 40-95% w / w relative to the total protein, even more preferably 45-90% w / w relative to the total protein, and most preferably 50-80% w / w relative to the total protein. method. [Sequential numbering embodiment 34] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the BLG content of the protein solution is at least 60% w / w of the total protein, more preferably at least 80% w / w of the total protein, even more preferably at least 90% w / w of the total protein, and most preferably at least 95% w / w of the total protein. method. [Sequential numbering embodiment 35] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the BLG content of the protein solution is at least 30% w / w relative to the total solids, more preferably at least 40% w / w relative to the total solids, even more preferably at least 45% w / w relative to the total solids, and most preferably at least 50% w / w relative to the total solids. method. [Sequential numbering embodiment 36] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the BLG content of the protein solution is 30-99% w / w relative to the total solids, more preferably 40-95% w / w relative to the total solids, even more preferably 45-90% w / w relative to the total solids, and most preferably 50-80% w / w relative to the total solids. method. [Sequential numbering embodiment 37] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the BLG content of the protein solution is at least 60% w / w relative to the total solids, more preferably at least 80% w / w relative to the total solids, even more preferably at least 90% w / w relative to the total solids, and most preferably at least 95% w / w relative to the total solids. method. [Sequential numbering embodiment 38] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the total fat content of the protein solution is at most 5% w / w relative to the total solids, more preferably at most 2% w / w relative to the total solids, even more preferably at most 0.5% w / w relative to the total solids, and most preferably at most 0.1% w / w relative to the total solids. method. [Sequential numbering embodiment 39] A method according to any one embodiment of the aforementioned sequential numbering embodiment; A method wherein the total fat content of the protein solution is in the range of 1 to 20% w / w relative to the total solids, more preferably in the range of 2 to 16% w / w relative to the total solids, even more preferably in the range of 3 to 12% w / w, and most preferably in the range of 4 to 10% w / w. [Sequential numbering embodiment 40] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the total carbohydrate content of the protein solution is at most 10% w / w of the total solids, more preferably at most 8% w / w of the total solids, even more preferably at most 6% w / w, and most preferably at most 5% w / w. method. [Sequential numbering embodiment 41] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the total carbohydrate content of the protein solution is at most 2% w / w of the total solids, more preferably at most 1% w / w of the total solids, even more preferably at most 0.5% w / w, and most preferably at most 0.2% w / w. method. [Sequential numbering embodiment 42] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Now, in the protein solution of step (c): • The original amount of PCA used to prepare the protein solution, and • BLG content of the protein solution, The molar ratio between: The ratio is 0.1:1 to 100:1, more preferably 0.1:1 to 50:1, even more preferably 0.1:1 to 20:1, and most preferably 0.1:1 to 10:1. method. [Sequential numbering embodiment 42.1] A method according to any one embodiment of sequentially numbered embodiments 1 to 41; Here, in step (c) protein solution: • The original amount of PCA used to prepare the protein solution, and • BLG content of the protein solution, The molar ratio between: The ratio is 0.1:1 to 5:1, more preferably 0.1:1 to 4:1, even more preferably 0.2:1 to 3:1, and most preferably 0.3:1 to 2:1. method. [Sequential numbering embodiment 42.2] A method according to any one embodiment of sequentially numbered embodiments 1 to 41; Here in the protein solution: • The original amount of PCA used to prepare the protein solution, and • BLG content of the protein solution, The molar ratio between: The ratio is 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. method. [Sequential numbering embodiment 42.3] A method according to any one embodiment of sequentially numbered embodiments 1 to 41; Now, in the protein solution of step (c): • The original amount of PCA used to prepare the protein solution, and • BLG content of the protein solution, The molar ratio between: It is at least 1:1. method. [Sequential numbering embodiment 43] A method according to any one embodiment of sequentially numbered embodiments 1 to 41; Here in the protein solution: • The original amount of PCA used to prepare the protein solution, and • BLG content of the protein solution, The molar ratio between: The ratio is 1:1 to 1000:1, more preferably 2:1 to 500:1, even more preferably 5:1 to 200:1, and most preferably 10:1 to 100:1. method. [Sequential numbering embodiment 44] A method according to any one embodiment of sequentially numbered embodiments 1 to 41; Here in the protein solution: • The original amount of PCA used to prepare the protein solution, and • BLG content of the protein solution, The molar ratio between: The ratio is 1:1 to 100:1, more preferably 1:1 to 50:1, even more preferably 1:1 to 20:1, and most preferably 1:1 to 10:1. method. [Sequential numbering embodiment 44.1] A method according to any one embodiment of sequentially numbered embodiments 1 to 41; Here in the protein solution: • The original amount of PCA used to prepare the protein solution, and • BLG content of the protein solution, The molar ratio between: The ratio is 1:1 to 6:1, more preferably 1.1:1 to 5:1, even more preferably 1.3:1 to 4:1, and most preferably 1.5:1 to 3:1. method. [Sequential numbering embodiment 45] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the protein solution is prepared by mixing a portion of a source containing one or more PCA obtained in step (a), a source containing BLG obtained in step (a), and optionally further components. method. [Sequential numbering embodiment 46] A method according to any one embodiment of the aforementioned sequential numbered embodiments, wherein the method does not include a step of oxidizing a portion of a supply containing one or more types of PCA prior to step (c). method. [Sequential numbering embodiment 47] A method according to one of the sequentially numbered embodiments 1 to 45; Here, the method includes step (b); Here, the protein solution is prepared by mixing a portion of a source containing one or more oxidized PCA obtained in step (b) with a source containing BLG and optionally further components. method. [Sequential numbering embodiment 47.1] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the incubation reduces the amount of free thiols in the protein solution to a maximum of 9 micromoles / g of protein, more preferably to a maximum of 8 micromoles / g of protein, more preferably to a maximum of 5 micromoles / g of protein, even more preferably to a maximum of 3 micromoles / g of protein, and most preferably to a maximum of 2 micromoles / g of protein, or is carried out with the aim of achieving the above reduction. method. [Sequential numbering embodiment 48] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, during incubation, the protein solution is subjected to a pressure ranging from 1 to 1000 bar, more preferably in the range of 20 to 500 bar, even more preferably in the range of 30 to 300 bar, and most preferably in the range of 40 to 200 bar. method. [Sequential numbering embodiment 49] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the temperature of the protein solution during incubation is in the range of 0 to 160°C, more preferably in the range of 10 to 155°C, even more preferably in the range of 15 to 150°C, and most preferably in the range of 20 to 145°C. method. [Sequential numbering embodiment 49.1] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the protein solution is prepared such that its temperature is in the range of 0 to 50°C, more preferably 2 to 45°C, even more preferably 5 to 40°C, and most preferably 10 to 35°C. method. [Sequential numbering embodiment 50] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the incubation time is a maximum of 12 hours, more preferably a maximum of 6 hours, even more preferably a maximum of 3 hours, and most preferably a maximum of 1 hour. method. [Sequential numbering embodiment 51] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the incubation time is a maximum of 30 minutes, more preferably a maximum of 10 minutes, even more preferably a maximum of 5 minutes, and most preferably a maximum of 2 minutes. method. [Sequential numbering embodiment 52] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the oxidation in step (d) involves contacting the PCA with a chemical oxidizing agent under conditions that convert at least a portion of the PCA in the protein solution into quinone. method. [Sequential numbering embodiment 53] A method according to sequential numbering embodiment 52; Here, the chemical oxidizing agent includes or consists of peroxides, ozone, dioxygen, or a combination thereof. method. [Sequential numbering embodiment 54] A method according to sequential numbering embodiment 52 or 53; Here, the chemical oxidizing agent includes or consists of hydrogen peroxide, benzoyl peroxide, or a combination thereof. method. [Sequential numbering embodiment 55] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the oxidation in step (d) involves electrochemically oxidizing the PCA in the protein solution under conditions that convert at least a portion of the PCA into quinone. method. [Sequential numbering embodiment 56] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the electrochemical oxidation in step (d) is carried out using a potential difference of -0.1 to 1.5 V, and most preferably -0.05 to 0.7 V. method. [Sequential numbering embodiment 57] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the oxidation in step (d) comprises the protein solution having a pH in the range of 7.1 to 9.0, and the electrochemical oxidation is carried out using a potential difference of -0.1 to 0.9 V, more preferably -0.05 to 0.8 V, and most preferably 0 to 0.7 V. method. [Sequential numbering embodiment 57.1] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the oxidation in step (d) involves the protein solution having a pH in the range of 7.3 to 8.7, and the electrochemical oxidation is carried out using a potential difference of -0.1 to 0.8 V, most preferably 0 to 0.7 V. method. [Sequential numbering embodiment 57.2] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the oxidation in step (d) involves the protein solution having a pH in the range of 7.4 to 8.5, and the electrochemical oxidation is carried out using a potential difference of -0.1 to 0.8, most preferably -0.05 to 0.7 V. method. [Sequential numbering embodiment 58] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the temperature in the first stage of the incubation is in the range of 0 to 60°C, more preferably in the range of 10 to 50°C; And here, as the second step of the incubation, the protein solution to be incubated is heated to a temperature in the range of 70 to 160°C, more preferably in the range of 80 to 150°C. method. [Sequential numbering embodiment 59] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the oxidation used in step (b) and / or step (d) is: The theoretical amount of quinone provided to the protein solution during the implementation of the method, the theoretical amount of quinone determined according to Analysis 1, and • BLG content of the protein solution, This is sufficient to make the molar ratio between them 0.1:1 to 100:1, more preferably 0.1:1 to 50:1, even more preferably 0.2:1 to 15:1, and most preferably 0.2:1 to 5:1. method. [Sequential numbering embodiment 59.1] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the oxidation used in step (b) and / or step (d) is: The theoretical amount of quinone provided to the protein solution during the implementation of the method, the theoretical amount of quinone determined according to Analysis 1, and • BLG content of the protein solution, This is sufficient to make the molar ratio between them 0.1:1 to 5:1, more preferably 0.1:1 to 4:1, even more preferably 0.2:1 to 3:1, and most preferably 0.2:1 to 2:1. method. [Sequential numbering embodiment 59.2] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the oxidation used in step (d) is: The theoretical amount of quinone provided to the protein solution during the implementation of the method, the theoretical amount of quinone determined according to Analysis 1, and • BLG content of the protein solution, This is sufficient to make the molar ratio between them 0.1:1 to 100:1, more preferably 0.1:1 to 50:1, even more preferably 0.2:1 to 15:1, and most preferably 0.2:1 to 5:1. method. [Sequential numbering embodiment 59.3] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the oxidation used in step (d) is: The theoretical amount of quinone provided to the protein solution during the implementation of the method, the theoretical amount of quinone determined according to Analysis 1, and • BLG content of the protein solution, This is sufficient to make the molar ratio between them 0.1:1 to 5:1, more preferably 0.1:1 to 4:1, even more preferably 0.2:1 to 3:1, and most preferably 0.2:1 to 2:1. method. [Sequential numbering embodiment 59.4] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the oxidation used in step (b) and / or step (d) is: The theoretical amount of quinone provided to the protein solution during the implementation of the method, the theoretical amount of quinone determined according to Analysis 1, and • BLG content of the protein solution, This is sufficient to make the molar ratio between them at least 1:1. method. [Sequential numbering embodiment 59.5] A method according to one embodiment of sequentially numbered embodiments 1 to 58; Here, the PCA-type oxidation used in step (b) and / or step (d) is: The theoretical amount of quinone provided to the protein solution during the implementation of the method, the theoretical amount of quinone determined according to Analysis 1, and • Molar amount of BLG in the protein solution, This is sufficient to make the molar ratio between them 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. method. [Sequential numbering embodiment 60] A method according to one embodiment of sequentially numbered embodiments 1 to 58; Here, the oxidation used in step (b) and / or step (d) is: The theoretical amount of quinone provided to the protein solution during the implementation of the method, the theoretical amount of quinone determined according to Analysis 1, and • BLG content of the protein solution, This is sufficient to make the molar ratio between them 1:1 to 100:1, more preferably 1:1 to 50:1, even more preferably 1:1 to 15:1, and most preferably 1.5:1 to 5:1. method. [Sequential numbering embodiment 61] A method according to one embodiment of sequentially numbered embodiments 1 to 58; Here, the PCA type oxidation used in step (b) is: • Quinone content of a portion of a source containing one or more types of oxidized PCA used in the protein solution, and • Amount of BLG in the protein solution, This is sufficient to make the molar ratio between them 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. method. [Sequential numbering embodiment 62] A method according to one embodiment of sequentially numbered embodiments 1 to 58; Here, the PCA-type oxidation used in step (b) is: • Quinone content of a portion of a source containing one or more types of oxidized PCA used in the protein solution, and • Amount of BLG in the protein solution, This is sufficient to make the molar ratio between them 1:1 to 100:1, more preferably 1:1 to 50:1, even more preferably 1:1 to 15:1, and most preferably 1.5:1 to 5:1. method. [Sequential numbering embodiment 62] A method according to one embodiment of sequentially numbered embodiments 1 to 58; Here, the PCA-type oxidation used in step (d) is: • Quinone content of a portion of a source containing one or more types of oxidized PCA used in the protein solution, and • Amount of BLG in the protein solution, This is sufficient to make the molar ratio between them 1:1 to 100:1, more preferably 1:1 to 50:1, even more preferably 1:1 to 15:1, and most preferably 1.5:1 to 5:1. method. [Sequential numbering embodiment 63] A method according to any one embodiment of the aforementioned sequential numbering embodiment; The method here does not involve oxidizing a portion of a supply containing one or more types of PCA prior to step (c); and Here, the PCA-type oxidation is carried out by a chemical oxidizing agent. method. [Sequential numbering embodiment 64] A method according to any one embodiment of the aforementioned sequential numbering embodiment; The method here does not involve oxidizing a portion of a supply containing one or more types of PCA prior to step (c); and Here, the PCA-type oxidation is carried out by electrochemical oxidation. method. [Sequential numbering embodiment 65] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the method includes step (b); A portion of the source containing one or more types of oxidized PCA obtained in step (b) is used to prepare the protein solution in step (c); and Here, the PCA-type oxidation is carried out by a chemical oxidizing agent. method. [Sequential numbering embodiment 66] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the method includes step (b); A portion of the supply source containing one or more types of oxidized PCA obtained in process (b) Used to prepare the protein solution in step (c); and Here, the PCA-type oxidation is carried out by electrochemical oxidation. method. [Sequential numbering embodiment 67] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the method includes step (b); A portion of the supply source containing one or more types of oxidized PCA obtained in process (b) Used to prepare the protein solution in step (c); and Here, the PCA-type oxidation is carried out by both electrochemical oxidation and a chemical oxidizing agent. method. [Sequential numbering embodiment 68] A method according to any one embodiment of sequential numbered embodiments 2 to 67; The process includes at least step (d) drying a liquid material containing solids derived from the incubated protein solution, method. [Sequential numbering embodiment 69] A method according to any one embodiment of sequentially numbered embodiments 2 to 68; The liquid raw material to be dried here contains or consists of the protein solution or protein concentrate obtained in step (d). method. [Sequential numbering embodiment 70] A method according to any one embodiment of sequentially numbered embodiments 2 to 68; Now, the incubated protein solution from step (d): · pH adjustment process; · Concentration process; · Diafiltration process; and • Heat treatment process, The liquid raw material is prepared by subjecting it to one or more of the following steps. method. [Sequential numbering embodiment 71] A method according to any one embodiment of sequential numbered embodiments 2 to 70; A method wherein step (e) includes spray-drying the liquid raw material. [Sequential numbering embodiment 72] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the method is carried out as a batch process, a quasi-batch process, or a continuous process. method. [Sequential numbering embodiment 73] A method according to any one embodiment of the aforementioned sequential numbering embodiment; Here, the method is carried out as a continuous process; Alternatively, steps (b), (c), and (d) are carried out as a continuous process. method. [Sequential numbering embodiment 74] A protein composition comprising modified BLG, wherein the protein composition can be obtained by a method according to any one embodiment of the numbered embodiments, preferably the protein composition is: • Protein content of at least 30% w / w relative to total solids; • Up to 10 micromoles of free thiol groups per gram of protein; • Tryptophan content of at least 0.7% w / w relative to total protein; • Methionine content of at least 0.3% w / w relative to total protein; • Maximum kynurenine content of 0.2 micrograms / mg of protein; Preferably, a fat content of up to 3% w / w relative to total solids; Preferably, the protein-bound sulfur content is in the range of 100 to 600 micromoles / g of protein; Preferably, the content of disulfide bond-forming protein-binding cysteine ​​residues is in the range of 150 to 400 micromoles / g of protein. Having one or more of the following types Protein composition. [Sequential numbering embodiment 74.1] A modified whey protein composition according to sequential embodiment 74, wherein the modified whey protein composition has a protein content of at least 30% w / w relative to the total solids. [Sequential numbering embodiment 74.2] A modified whey protein composition according to sequential embodiment 74 or 74.1, wherein the modified whey protein composition has a maximum of 10 micromoles of free thiol groups per gram of protein. [Sequential numbering embodiment 74.3] A modified whey protein composition according to any one embodiment of sequentially numbered embodiments 74 to 74.2, wherein the modified whey protein composition has a tryptophan content of at least 0.7% w / w relative to the total protein. [Sequential numbering embodiment 74.4] A modified whey protein composition according to any one embodiment of sequentially numbered embodiments 74 to 74.3, wherein the modified whey protein composition has a methionine content of at least 0.3% w / w relative to the total protein. [Sequential numbering embodiment 74.5] A modified whey protein composition according to any one embodiment of sequentially numbered embodiments 74 to 74.4, wherein the modified whey protein composition has a kynurenine content of up to 0.2 micrograms / mg of protein. [Sequential numbering embodiment 74.6] A modified whey protein composition according to any one embodiment of sequentially numbered embodiments 74 to 74.5, wherein the modified whey protein composition has a fat content of up to 3% w / w relative to the total solids. [Sequential numbering embodiment 74.7] A modified whey protein composition according to any one embodiment of sequentially numbered embodiments 74 to 74.6, wherein the modified whey protein composition has a protein-bound sulfur content in the range of 100 to 600 micromoles / g of protein. [Sequential numbering embodiment 74.8] A modified whey protein composition according to any one embodiment of sequentially numbered embodiments 74 to 74.7, wherein the content of disulfide bond-forming protein-binding cysteine ​​residues is in the range of 150 to 400 micromoles / g of protein. [Sequential numbering embodiment 75] A modified whey protein composition according to any one embodiment of sequentially numbered embodiments 74 to 74.8; The average molecular weight of the protein is in the range of 18 kDa to 10,000 kDa, more preferably in the range of 50 to 8,000 kDa, and most preferably in the range of 80 to 5,000 kDa. Modified whey protein composition. [Sequential numbering embodiment 76] A protein composition according to any one embodiment of sequentially numbered embodiments 74 to 74.8; The average molecular weight of the protein is in the range of 18 kDa to 500 kDa, more preferably in the range of 18 to 100 kDa, and most preferably in the range of 18 to 40 kDa. Protein composition. [Sequential Numbering Embodiment 77] A process for producing a heat-treated beverage, preferably a heat-sterilized beverage; The process is: (1) A step of mixing a protein composition according to one or more of the sequentially numbered embodiments 74 to 76 with one or more further beverage components in order to obtain a liquid mixture with a pH of 5.5 to 8.5; and (2) A step of filling the liquid mixture into a suitable container, Includes; The process further includes at least one heat treatment step; Here, the liquid mixture is heat-treated before and / or after filling, preferably heat-sterilized. process. [Sequential numbering embodiment 78] A process according to sequential embodiment 77; Here, the liquid mixture contains a sufficient amount of protein composition to contribute at least 0.5% w / w of protein. process. [Sequential numbering embodiment 79] A heat-treated beverage, preferably a heat-sterilized beverage; A pH of 5.5 to 8.5, obtainable by a process according to sequentially numbered embodiment 77 or 78, Beverages. [Sequential numbering embodiment 80] A heat-treated beverage, preferably a heat-sterilized beverage; The protein composition comprises one or more embodiments of sequentially numbered embodiments 74 to 76, having a pH of 5.5 to 8.5, and more preferably 6.5 to 7.5; It contains a sufficient amount of protein composition to contribute at least 0.5% w / w of protein; And preferably, the H2S content 7 days after production is at most 5 micromol / L, more preferably at most 3 micromol / L, even more preferably at most 1.0 micromol / L, and most preferably at most 0.7 micromol / L. Beverages. [Sequential numbering embodiment 81] It is a food ingredient; • Solid protein composition according to one or more embodiments of sequentially numbered embodiments 74 to 76; and • One or more additional components, preferably selected from the following: • Milk components, preferably non-oxidized milk components; • Plant-derived ingredients; • Non-dairy carbohydrate source; • Flavoring and odor-masking agents; and / or Preferably, a sweetener comprising a sweet sugar, a polyol, a high-strength sweetener, or a combination thereof. Food ingredients that include... [Sequential numbering embodiment 82] Uses of the protein composition containing modified BLG, preferably as a food ingredient of the protein composition according to one or more embodiments from sequentially numbered embodiments 74 to 76; Preferably, a heat-sterilized beverage with a pH in the range of 5.5 to 8.5: • For the purpose of improving odor; and / or • To reduce the level of an unpleasant odor similar to that of rotten eggs; and / or • To reduce H2S generation during manufacturing; and / or • To reduce the H2S content in the upper space of the container, Its purpose is; Preferably, the beverage has a whey protein content of at least 3% w / w, and preferably, the beverage is heat-sterilized using indirect heat treatment. Purpose.

[0386] The present invention has been described above with reference to specific embodiments. However, other embodiments not described above are equally possible within the scope of the invention. Unless otherwise stated, different features and processes of various embodiments and aspects of the present invention can be combined in ways other than those described herein.

[0387] Examples Analysis 1: Determination of the theoretical quantity of quinone produced by the method. "Theoretical amount of quinone provided to the protein solution during the implementation of the method" is a measure of the molar content of quinone produced when all PCA (including PCA converted to quinone) 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 protein, other sources of free thiols, and other sources of amines.

[0388] For example, if the method of the present invention involves oxidizing the PCA before mixing it with a source containing BLG (for the purpose of providing a source containing one or more types of oxidized PCA), then the quinone content of the source containing one or more types of oxidized PCA is equal to the theoretical quinone content.

[0389] The quinone content is measured according to Analysis 2.

[0390] Analysis 2: Quantification of quinone content The quinone content of the liquid sample is quantified as follows. This quantification is based on measuring the reactive oxygen species generated by the reduction of quinone with NaBH4, and is determined by the conversion of the generated reactive oxygen species into the formazan dye of the colorimetric reagent 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT).

[0391] Stock solution NaH2PO4 buffer The NaH2PO4 buffer solution is a 0.1M sodium dihydrogen phosphate aqueous solution whose pH has been adjusted to 11.85 using NaOH.

[0392] INT The INT stock solution is prepared fresh as a solution containing 400 micromoles of INT in a NaH2PO4 buffered aqueous solution.

[0393] NaBH4 The stock solution of NaBH4 is prepared fresh as a solution containing 300 mmol of NaBH4 in a buffered aqueous solution of NaH2PO4.

[0394] See Quinon. The quinone reference solution is based on 2 mM 4-methylbenzoquinone (4MBQ) and is prepared by electrochemical oxidation of 4-methylcatechol (4MC) as described by Li et al. (Li, Y., Jongberg, S., Andersen, ML, Davies, MJ, & Lund, MN (2016)).

[0395] Quinone-induced protein modification "Kinetic preference for reaction of 1,2-benzoquinones with thiol groups in proteins," (Free Radical Biology and Medicine, 97, 148-157): A solution containing 2 mM 4MC was prepared in phosphate buffer solution (0.2 M, pH 4.5), and deoxygenated by passing nitrogen through it for 10 minutes. A cyclic voltammogram of the 4MC solution was obtained using a voltammetry analyzer CV-50W (BAS Co., Ltd.) equipped with a glassy carbon working electrode (3 mm in diameter, BAS Co., Ltd.), a platinum coil counter electrode (5 mm in diameter and 5 cm in length), and an Ag / AgCl (KCl, c=3 M) reference electrode (Metrohm, Switzerland). Bulk electrolysis was performed using the same CV-50W constant potential electrolytic apparatus under nitrogen with an initial potential of 460 mV for Ag / AgCl, and the initial concentration of the 4MC solution was 2 mM. The working electrode was a mesh glassy carbon tube (BAS Co., Ltd.) with a diameter of 3.5 cm and a length of 4.5 cm.

[0396] The quinone reference solution should be used within 30 minutes of preparation.

[0397] calibration Calibration curves are prepared using diluted standard solutions of the quinone reference solution, and are prepared in a concentration range of 0.001 to 4 micromoles by diluting the reference quinone solution with NaH2PO4 buffer.

[0398] The absorbance is plotted against the concentration of the reference quinone to obtain a calibration curve.

[0399] Measurement of quinones in a sample Dilute 10 microliters of the liquid sample to be analyzed by mixing it with 490 microliters of NaH2PO4 buffer.

[0400] In one well of a 96-well microplate, 50 microliters of diluted sample are mixed with 100 microliters of INT stock, followed by the addition of 50 microliters of NaBH4 stock. The microplate is then placed on a microplate reader and shaken for 30 seconds. After incubation for 2 minutes, the absorbance of the well is measured at 510 nm. The collected data is expressed as the average of three measurements using a quinone reference calibration curve.

[0401] Mixing, incubation, and measurement should be performed at room temperature (RT).

[0402] If the absorbance measurement exceeds the linear range of the analysis, a new sample should be prepared and subjected to appropriate dilution and measurement as described above.

[0403] calculation The quinone content of the liquid sample is calculated by comparing the measurement results with a calibration curve and converting the quinone content to the desired units. If the liquid sample is diluted before measurement, a correction for the quinone content related to the dilution is performed.

[0404] Example 1: Removal of free thiols from whey protein using oxidized phenol compounds The purpose of this experiment was to confirm the inventors' initial findings and to demonstrate a method for reducing, or even avoiding, the generation of off-odors during the heat treatment of pH-neutral beverages rich in whey protein by reacting whey protein, particularly BLG, with oxidized phenolic compounds.

[0405] Principle of reaction The inventors have discovered that dissolved dioxygen can oxidize PCA (such as EGCG) to quinone, and that the resulting quinone can react with the free thiol groups of BLG (provided the free thiol groups are adequately exposed). The inventors have further discovered that a combination of a well-ventilated reaction mixture (containing dissolved dioxygen) and sufficient upper space to accommodate ambient air (from which excess dioxygen can diffuse into the reaction mixture) can be used as a model system for the oxidation of PCA and the subsequent reaction between oxidized PCA and BLG.

[0406] Materials and methods material BLG-rich whey protein isolate powder (WPI-A) was prepared according to WO2018 / 115520A1. The properties of the above powder are shown in Table 1.

[0407] [Table 1]

[0408] 4-Methylcatechol (4MC), epigallocatechin gallate (EGCG), bovine serum albumin (BSA), and trifluoroacetic acid (TFA) were purchased from Thermo Scientific, Merck Life Science, and Merck. All reagents were analytical grade or of the highest possible purity. Distilled water (MQ water) was obtained using a Milli-Q purification device (Millipore, Bedford, MA).

[0409] The 2 ml HPLC vial was a 2 ml 32 x 11.6 mm injection vial (Catalog No.: ML33003VU, Mikrolab, Denmark).

[0410] Measurement of dissolved oxygen To evaluate oxygen consumption in a sample, an O2 microsensor (Unisense) was used to assess the consumption of dissolved oxygen in a PCA sample.

[0411] Measurements were performed according to the Unisense oxygen sensor user manual (revised March 2020). The O2 microsensor was calibrated at room temperature using vigorously agitated water samples and water through which N2 was passed until foamy; these correspond to acid concentration and oxygen concentration of 0 micromol / L in oxygen-saturated water, respectively.

[0412] Reaction between protein and PCA Three 100 mL solutions (each containing 4% w / w protein based on WPI-A powder) were prepared; preparation involved mixing the powder with MQ water and then hydrating the mixture at approximately 20°C for 1 hour with gentle stirring; the inventors then confirmed that no residual powder particles remained and the solution became clear.

[0413] The three solutions described above were adjusted to pH 7, 8, and 9, respectively; a minimum amount of 1 M NaOH was used for this pH adjustment. As shown in Table 1, sample WPI-A contains a very high level of BLG; specific absorption coefficient 0.96 L / g / cm -1 By measuring the absorbance of the solution at 278 nm using [the specified method], the inventors confirmed that the target concentration was within a 5% margin of error.

[0414] Using MQ water, a 32 mM 4MC stock solution (MW: 124.13 g / mol) or EGCG stock solution (MW: 458.372 g / mol) was prepared.

[0415] To prepare WPI-A4 to WPI-A15 solutions, protein solution (pH 7, 8, or 9), PCA (4MC or EGCG), and water were mixed to a final protein concentration of 3% w / w (1.6 mM); and the molar ratio of PPA:BLG was set to 2:1 (3.2 mM) or 5:1 (8 mM) as shown in Table 2.

[0416] A 2 mL sealed HPLC vial containing the sample was incubated at 50°C for 1 hour; the volume of the vial was approximately 40% liquid and approximately 60% upper space (ambient air). The inventors estimated that the combined amount of O2 present in the liquid and upper space was sufficient to oxidize substantially all of the PCA in the reaction mixture to quinone.

[0417] [Table 2]

[0418] Determination of free thiol groups, total thiol groups, and total protein The free thiol group content and total thiol group content of whey protein samples were quantified using the method described by Kurz et al. (2020) (the same instrument used by the present authors was used in Kurz et al.'s method). The free thiol (SH) content in the samples, along with the protein content measured by the total protein method described later, is typically expressed in micromoles / gram of protein. Kurz, F., Hengst, C., & Kulozik, U. (2020), "RP-HPLC method for simultaneous quantification of free and total thiol groups in native and heat aggregated whey proteins," MethodsX, 101112.

[0419] Determination of total protein content The total protein content (true protein) of the sample was measured as follows: (1) Measure the total nitrogen of the sample according to ISO 8968-1 / 2|IDF 020-1 / 2-milk. Measurement of Nitrogen Content - Part 1 / 2: Measurement of Nitrogen Content using the Kjeldahl Method. (2) Measure the non-protein nitrogen of the sample in accordance with ISO8968-4|IDF 020-4-Milk-. Nitrogen content measurement - Part 4: Measurement of non-protein nitrogen content. (3) Total protein: (m Total Nitrogen - m Non-protein Nitrogen) * 6.38 It is calculated as follows.

[0420] Gel permeation chromatography (GPC) analysis of molecular weight of whey protein species The molecular weights of protein species in whey protein samples were analyzed by size exclusion chromatography using a SEC-MALS-IV-RI HPLC system consisting essentially of a Thermo ISO.3100SD pump, a WPS-3000TSL autosampler, and a Refractomax 521 refractive index detector. This system was further equipped with a Wyatt miniDawn TREOS II light scattering detector and a WYATT VISCOSTAR online viscometer.

[0421] All samples were diluted with eluent (10 mM phosphate, 30 mM NaCl, pH 7.0) to a protein content of 1%, and 1 mL of the sample was centrifuged at 15,000 g for 15 minutes to remove large protein aggregates. Injection volume: 10 microliters of sample / supernatant. The samples were separated by injecting the eluent at a rate of 0.75 mL / min using 1 x Bio-SEC-5 guard + 2 x 300 Å BioSEC-5.

[0422] Data Analysis Weight-average molecular weight (MW) and number-average molecular weight (Mn) were determined using Astra software (v7.3.2.19) by analyzing refractive index analysis and light scattering signals, i.e., both monomers / oligomers and larger aggregated species, from a point in time prior to void volume determination. BSA was used as a standard to confirm proper calibration; the monomer peak showed a molecular weight of 64.4 kDa (98% of the theoretical value).

[0423] The sample recovery rate was calculated from the total integrated amount of eluted protein molecules relative to the protein content in the sample before GPC sample preparation. However, during the centrifugation process in GPC sample preparation, very large protein molecules may be removed.

[0424] With the exception of sample WPI-A3 (which showed a recovery rate of 42.6%, likely due to excessive aggregation), all other samples surprisingly showed high recovery rates of >80%, with most samples at 90-100%; this suggests that the vast majority of protein molecular species were included in the analysis.

[0425] LC-MS analysis of protein mass in protein PCA samples The protein PCA sample was diluted 30-fold with MQ water to a protein concentration of 0.1%. The above solution was filtered using a 0.22 micron PVDF filter and placed in HPLC vials (1.5 ml Short Thread Vial with 0.1 ml microinsert, 31 x 6 mm, clear glass (ML33119), and 32 x 11.6 mm, clear glass (ML33117)). Ten microliters of the solution were taken and injected into the HPLC. Proteins were separated using an Avantor® Ace® ultracore BIO 300A C4 HPLC column. Mobile phase A: H2O containing 0.1% TFA (v / v); Mobile phase B: Acetonitrile containing 0.08% TFA (v / v).

[0426] [Table 3]

[0427] Proteins were detected using a UV214nm detector and Thermo Scientific's Q-Exactive plus quadrupole orbitrap MS (positive polarity, scanning range 1000-3500 m / z, resolution 140,000, and temperature 500°C).

[0428] UV214nm data was analyzed using Thermo Scientific's FreeStyle software. The UV214nm chromatogram was integrated using the Genesis algorithm (cutoff peak size: 0.1% of the maximum peak; minimum peak signal-to-noise ratio: 2; and valley detection enabled). The injection peaks at the beginning and end of the above chromatogram (also observed in blank injection) were removed from the peak integral.

[0429] The protein masses of the peaks in the chromatogram were determined using high-resolution mass spectrometry data. Under the same conditions as UV peak integration, the total ion chromatogram was integrated using the genesis algorithm. Next, mass-to-charge ratio data was extracted from each peak, and the adducted ions were identified as H + The Xtract deconvolution process was used to deconvolve the data, with the charge range set to 5-50 and the minimum number of detectable charges set to 3.

[0430] UHT treatment (ultra-high temperature treatment) 1.0 mL of the sample was transferred to a 2 mL HPLC vial and crimped and sealed with an aluminum lid (Mikrolab ML 33032) using an electronic crimping tool (Thermo Scientific CRMA60180-ECRH11KI).

[0431] A sealed vial (at room temperature) was placed on an aluminum heating block (with a drilled hole sized to fit the dimensions of a 2 mL GC vial, as provided by the manufacturer) of a Mikrolab supertherm system (control unit ML 306228 and heating unit ML3062409, Mikrolab A / S, Denmark). The block was preheated to 160°C, and then the sample was placed on the block for 160 seconds. The temperature reached 100°C in approximately 40 seconds, approximately 120°C in 65 seconds, 140°C after 100 seconds of incubation, and 150°C after 160 seconds. After incubation on the heating block, the sample was transferred to an ice / water bath to quickly stop any reactions that could lead to the generation of an unpleasant odor. H2S was measured directly in the sealed vial according to the section "Quantitative Determination of H2S Using an H2S Sensor".

[0432] Determination of H2S using an H2S sensor The H2S level was measured using a microsensor (SULF-NPLR, needle type, Unisense A / S, Denmark) connected to a single-channel amplifier (Monometter-9514, Unisense A / S, Denmark). The acquired H2S signal was expressed in millivolts and can be used as an indicator of the H2S level in the sample; the above signal was recorded by the "LOGGER" software provided by Unisense A / S. The microsensor was calibrated using an H2S calibration kit supplied by the manufacturer (Calkit-H2S, Unisense A / S, Denmark). The maximum concentration of H2S in the calibration kit was diluted 10-fold according to Section 7 of the manual (November 2020 version, Unisense A / S). The above software can automatically convert the H2S concentration to micromolar units.

[0433] To measure the UHT samples, the samples were equilibrated at 20°C for 30 minutes, after which the sensor needle was inserted into the sample liquid phase through a silicon seal. This procedure was performed twice for each sample.

[0434] result The effect of the reaction between proteins and PCA on free thiols (SH) in proteins. The free thiol content of samples WPI-A1 to WPI-A15 was measured using the method described in the section "Determination of Free Thiol Groups, Total Thiol Groups, and Total Protein" above. The results are shown in Table 4.

[0435] The free thiol content (45-46.3 micromoles SH / g protein) was measured in samples WPI-A1 to WPI-A3 after incubation at 50°C for 1 hour without the addition of PCA.

[0436] Surprisingly, WPI-A4 incubated at pH 7.0 with 4MC:BLG = 2:1 showed a decrease in free thiol content (21.3%) within 1 hour of incubation compared to WPI-A1 (PCA-free). In WPI-A5 and WPI-A6, a significant decrease in free thiol content was observed at pH 8 (78.9%) and pH 9 (89.8%), respectively.

[0437] When the amount of 4MC in samples WPI-A7 to WPI-A9 was increased to 4MC:BLG=5:1, a similar pattern was observed, and the decrease in free thiols in samples WPI-A8 and WPI-A9 was 90% and 96%, respectively, compared to the sample without 4MC addition.

[0438] The inventors further investigated the ability of EGCG (which is abundant in green tea extract) to reduce free thiols in whey protein compositions.

[0439] Samples WPI-A10 to WPI-A12 were incubated with an EGCG:BLG molar ratio of 2:1. Compared to samples without EGCG (WPI-A2 and WPI-A3, respectively), samples WPI-A11 and WPI-A12 showed significant reductions of 95% and 100%, respectively.

[0440] Increasing the amount of EGCG to EGCG:BLG=5:1 resulted in a 99.6-100% decrease in free thiol content compared to WPI-A2 and WPI-A3.

[0441] The effect of the reaction between proteins and PCA on the molecular weight of protein species The molecular weights of protein molecules were analyzed by GPC using the method described above for samples WPI-A1 to WPI-A3 without PCA addition, and for samples WPI-A4 to WPI-A15 incubated with PCA at 50°C for 1 hour.

[0442] Table 4 shows the weight-average molecular weight of the samples, with estimated values ​​ranging from 26.5 to 278.3 kDa.

[0443] The maximum weight-average MW obtained for WPI-A3 was 278.3 kDa; the recovery rate was 42.6%. The inventors estimated that the much larger aggregates removed during sample preparation accounted for approximately 58% of the remaining protein.

[0444] As shown in Table 4, surprisingly, the inventors discovered that when PCA was added to a WPI sample, the MW estimate was consistently lower compared to a WPI sample without PCA at the same pH. This suggests that changing from a PCA-free sample to a PCA-added sample inhibits the thiol / disulfide exchange reaction and reduces aggregation during incubation.

[0445] By examining the chromatograms more closely and analyzing the molecular weight of individual peaks, the fraction of eluted proteins in the analysis of samples WPI-A1 to WPI-A15 was found to be primarily: Monomer > Dimer > Trimer > Tetramer molecular species > Higher-order aggregates, This suggests that the order should be followed.

[0446] GPC analysis revealed that the bulk amount of protein elutes as monomeric or dimeric molecular species; therefore, to evaluate the covalent bond of PPA to WPI-A, UV 214nm The inventors determined that it is preferable to use HPLC analysis and MS detection for intact protein in the above (see the section "LC-MS analysis of protein mass of protein PCA samples").

[0447] In BLG-containing samples at pH 8 (WPI-A5 and WPI-A8) and pH 9 (WPI-A6 and WPI-A9) with 4MC added, the inventors were able to correctly determine that the main morphology of the BLG protein was modified BLG, in which the mass of both the A-iso and B-iso forms of BLG was increased by 122 Da, corresponding to the mass of the quinone form of 4MC.

[0448] Furthermore, the inventors observed modified BLG variants in EGCG-containing samples at pH 8 and pH 9, where the mass of BLG increased by 456 Da, corresponding to the mass of EGCG quinone, in samples with EGCG:BLG=2:1 (WPI-A11 and WPI-A12) and EGCG:BLG=5:1 (WPI-A14 and WPI-A15).

[0449] Visual inspection of the sample Samples WPI-A1 to WPI-A3 remained completely transparent without discoloration during heat treatment. During heat treatment, the transparent color of the PCA-containing samples changed to a brownish hue in WPI-A4 to WPI-A9, and a faint brown / yellow discoloration in WPI-A10 to WPI-A15. The variation in color intensity between samples depended on both pH and PCA:BLG stoichiometric amounts, as shown in Table 4. Furthermore, in the presence of alkaline pH and amines (Tris buffer and folded BLG before heat treatment), 4MC was observed to turn red / pink. No particular red / pink discoloration was observed in the above samples.

[0450] [Table 4]

[0451] The effect of the reaction between protein and PCA on H2S production Sample WPI-A5 (4MC:BLG=2:1) ​​and reference sample WPI-A2 were adjusted to pH 7 and subjected to UHT treatment (ultra-high temperature treatment) (see the "UHT Treatment" section). The generated H2S (off-odor) was then quantified using the method described in the "Quantification of H2S using an H2S Sensor" section. The reference sample (WPI-A2) generated 7.9 micromoles of H2S, while the 4MC-treated sample (WPI-A5) contained only 1.5 micromoles of H2S; this suggests that the off-odor was reduced in the 4MC-treated sample.

[0452] conclusion The inventors have demonstrated that oxidation of PCA at pH 7–9 is sufficient to reduce or remove the amount of free thiols in BLG in whey protein solutions. They also found that at pH 7.0, the free thiol content can be significantly reduced by increasing the temperature and / or pressure.

[0453] Therefore, reacting BLG and other whey proteins with oxidized PCA is a possible means of reducing the free thiol group content of the proteins. The inventors have previously observed that proteins with a high free thiol group content generate an unpleasant odor when used in whey protein solutions or beverages and subjected to, for example, UHT (ultra-high temperature) treatment. Thus, the present invention provides a remarkable novel approach to reduce the generation of unpleasant odors during manufacturing and when consuming heat-treated, neutral pH whey protein-rich beverages.

[0454] Example 2: Dose response of 4MC subjected to electrochemical oxidation The purpose of this experiment was to demonstrate the possibility of using electrochemical oxidation to produce quinones from polyphenol solutions and the use of the produced quinones to reduce the amount of free thiols in whey protein.

[0455] Materials and methods protein source WPI-A, as described in Example 1, was used as the protein source.

[0456] Determination of quinone content An L-glutathione (GSH) assay was used to determine the concentration of the generated quinone. The principle of this assay is to measure the amount of unreacted GSH remaining after the quinone reacts with the free thiol of GSH using RP-HPLC. Therefore, the quinone content corresponds to the disappearance of GSH. The inventors found that this GSH assay is a good alternative to Analysis 2.

[0457] A 6 mM GSH solution was prepared in 5 mM phosphate buffer (pH 4.5). The GSH solution and quinone solution were mixed in a 1:1 ratio in an Eppendorf tube and reacted. This mixture was separated and quantified at 214 nm using the same free thiol assay method as in Example 1, compared with a standard curve of known GSH concentrations. The amount of quinone was then measured as the concentration of GSH lost by comparing it to a sample of GSH mixed 1:1 with electrolytic buffer (quinone-free).

[0458] Electrochemical oxidation of quinone 30 mL of 5 mM phosphate buffer (pH 4.5) and 200 mM NaCl solution were deoxygenated by blowing nitrogen gas into an electrolytic cell (Redox.me 50 mL two-compartment cell; Redoxme AB, Sweden). Whey protein / quinone samples and reference whey protein samples were incubated at 80°C for 30 minutes and then cooled.

[0459] Removal of excess quinones from the solution after reaction with proteins. Unreacted / excess 4MC and 4MBQ after reaction with protein were partially removed from the sample by ultrafiltration. 1 mL of the reaction sample was transferred to a 10 kDa cutoff Amicon Ultraspin filter and centrifuged at 3400 x g for 20 minutes using a swing bucket rotor that allowed for the recovery of 100 μL of retention solution. The retention solution was diluted to 1% protein with 5 mM phosphate buffer (pH 7) and collected, and each sample was transferred to an Eppendorf vial for further analysis. Our observations showed no signs of protein loss even with this conventional filtration method.

[0460] UHT (Ultra-High Temperature Treatment) For the measurement of H2S generated in the sample as a result of UHT (ultra-high temperature treatment), quantitative analysis was performed using an H2S sensor as described in Example 1.

[0461] The content of free thiol groups in the recovered holding liquid and after the UHT treatment (ultra-high temperature treatment) as described in Example 1 was measured.

[0462] result Cyclic voltammetry showed a typical "duck" pattern with a peak oxidation potential for 4MC at approximately +550mV; +550mV was then used to generate quinones by bulk electrolysis. GSH quinone assay revealed a 4MBQ yield of 27.7%; as shown in Table 5, the resulting 4MBQ:BLG ratios were 0.07:1 (sample WPI-A16) to 0.56:1 (sample WPI-A19), respectively.

[0463] After terminating the reaction by cooling and removing excess reagent, the free thiol content was analyzed as shown for WPI-A16 to WPI-A19 in Table 5. Surprisingly, even a small amount of added quinone solution was sufficient to significantly reduce the amount of free thiol in WPI-A16; it was also found that the amount of free thiol after UHT treatment was significantly reduced. In this way, the inventors discovered that by electrochemically oxidizing the 4MC solution to produce quinone and mixing it with BLG under conditions that allow it to react with free thiol, the amount of free thiol can be reduced to a level where the H2S detected by the H2S sensor is less than 1 μmol / L. This is in contrast to the highly undesirable level of 16.9 μmol / L measured after UHT treatment of the untreated WPI-A20 control. Surprisingly, the inventors observed that WPI-A16 was essentially colorless.

[0464] In WPI-A17 to WPI-A19, the greater the amount of quinone solution added, the lower the amount of residual thiols in the sample. Furthermore, UHT treatment (ultra-high temperature treatment) further reduced the free thiol content. As a result, the H2S level in these samples measured after UHT treatment was less than 1 μmol / L. A summary of the results of Example 2 is shown in Table 5.

[0465] [Table 5]

[0466] conclusion The inventors have shown that even when using surprisingly low stoichiometric amounts between quinone and BLG, it is possible to reduce the free thiol group content by generating quinone through electrochemical oxidation and subsequent mixing with whey protein.

[0467] The decrease in free thiol content resulted in low levels of H2S generated during UHT (ultra-high temperature) treatment of the modified protein sample. The inventors noted that the test sample contained only about 1% BLG. If the whey protein content were higher, the H2S level would likely increase significantly.

[0468] The reaction product obtained from the sample with the lowest amount of added quinone (WPI-A16) was found to be essentially colorless, while the sample reacted with a higher stoichiometric amount was slightly colored. Therefore, the inventors discovered that when it is necessary to prepare a protein source for producing colorless beverages, it is preferable to use polyphenols and quinones in amounts that can achieve the required reduction in free thiol levels, and in the lowest possible content.

[0469] The inventors have further discovered that it may be beneficial to perform a second heat treatment, for example in the form of UHT (ultra-high temperature) treatment, in order to further reduce the free thiol content in the whey protein solution during the incubation process. The inventors also point out that the UHT-treated whey protein solution (or its protein concentrate), as shown in Table 5, can be converted into protein powder by, for example, spray drying or other drying methods.

[0470] Example 3: Effects of controlling oxygen exposure The objective of these experiments was to demonstrate a method for controlling the oxidation of polyphenols to quinones by oxygen (O2) by controlling the accessible upper space volume.

[0471] Methods and Materials protein source These experiments used WPI-A from Example 1.

[0472] Sample preparation The following samples were prepared using WPI-A as the protein source.

[0473] Samples WPI-A21 and WPI-A22 A pH 8.0 stock solution containing 3% protein derived from WPI-A and 3.2 mM 4MC was prepared (with a molar ratio of 2:1 between 4MC and BLG). Sample WPI-A21 was prepared by placing 1 mL of the above stock solution into a 2 mL HPLC vial (see UHT treatment in Example 1), and the vial was crimped to reduce further exposure to ambient air. Sample WPI-A21 thus prepared consisted of 1 mL of aqueous sample and an upper space of 1 mL of ambient air containing dioxygen.

[0474] Sample WPI-A22 was prepared by placing 2 mL of the stock solution into a 2 mL HPLC vial and then sealing the vial as described above. Therefore, sample WPI-A22 did not contain any upper space.

[0475] Sample WPI-A23 One mL of the same stock solution as described above, without the addition of 4MC, was taken and placed in a 2 mL HPLC vial to prepare sample WPI-A23. The vial was then crimped to reduce further exposure to ambient air; the vial thus prepared contained an equal volume of aqueous phase and ambient air in the upper space.

[0476] WPI-A21 to WPI-A23 were incubated at 20°C for 2 days; these 2 days were sufficient time for quinone formation based on oxidation by oxygen (O2) and for oxygen (O2) to move from oxygen (O2)-available spaces.

[0477] The free thiol content of all samples was quantified using the method described in Example 1.

[0478] result

[0479] [Table 6]

[0480] The inventors previously discovered that a slightly alkaline pH is useful for exposing free thiols in BLG; they further found that incubating the sample and an equal volume of ambient air in the upper space for two days constitutes an effective means of blocking free thiols in the sample, which is evident from the decrease in free thiols (1.7 μmol / g protein) in sample WPI-A21 compared to 45 μmol / g protein in WPI-A23 incubated in the absence of PCA. Surprisingly, both WPI-A21 and WPI-A22 were essentially colorless solutions despite the WPI-A concentration used being 3%. The inventors surmise that the absence of discoloration is closely related to oxygen (O2) restriction; this oxygen restriction is thought to generate enough quinone to react with the free thiols in WPI-A21, but not to an oxygen level that would cause undesirable discoloration.

[0481] The inventors further discovered that completely filling the vial restricts access to ambient air, and thereby leaving no upper space, results in a milder reduction in the free thiol content of sample WPI-A22 to 19.1 μmol SH / protein g, which corresponds to approximately a 57% reduction in free thiols compared to WPI-A23. The inventors hypothesize that the free thiol blocking efficiency is inferior to that of WPI-A21 in reactions involving quinone formation by oxygen (O2) dispersed in the sample before the experiment, and the subsequent reaction with free thiols, when there is no upper space that can act as an oxygen (O2) reservoir. Therefore, the inventors found that increasing exposure to oxygen (O2) by increasing access to oxygen (O2) is beneficial; an example of increased access to oxygen (O2) is the wider (but still limited) upper space of ambient air in sample WPI-A22, in which the free thiol group content has been reduced to 1.7 μmol SH / protein g.

[0482] In addition to controlling exposure to oxygen (O2) through the accessible upper space, the inventors suggest that other means of controlling access to oxygen (O2), such as a pressurized or more concentrated oxygen (O2) atmosphere, may be available.

[0483] Furthermore, although the sample in this study was not stirred, stirring the sample may be beneficial in increasing the rate at which oxygen (O2) moves into the aqueous phase.

[0484] conclusion These results indicate that restricting access to oxygen (O2) reduces the likelihood of oxygen (O2)-mediated generation of quinones that can react with free thiols.

[0485] Therefore, the inventors conclude that when oxygen (O2) is the only oxidizing agent used for quinone production, it is advantageous to control and ensure sufficient access to oxygen (O2).

[0486] The inventors have found that the higher protein concentration used in the processing of this embodiment is advantageous for industrial implementation, and that according to the present invention, it is possible to process protein solutions with much higher protein concentrations.

[0487] The inventors further conclude that restricting oxygen (O2) access constitutes an attractive approach to reduce undesirable discoloration in the product.

[0488] Example 4: Direct heating under UHT-like conditions The objective of these experiments was to demonstrate the potential of using PCA to generate quinones and enable their reaction with free thiols under UHT-like heating conditions.

[0489] Materials and methods

[0490] These experiments used WPI-A from Example 1.

[0491] Sample preparation A stock solution containing 3% protein derived from WPI-A and 3.2 mM 4MC was prepared with a pH of 8.0 (the molar ratio between 4MC and BLG was 2:1).

[0492] Sample WPI-A24 was prepared by placing 1 mL of the above stock solution into a 2 mL HPLC vial and then sealing the vial using the method described in Example 3. Thus, sample WPI-A24 contained an equal volume of aqueous phase and ambient air in the upper space.

[0493] Sample WPI-A25 A 1 mL sample of the same stock solution as described above, without the addition of 4MC, was placed in a 2 mL HPLC vial to prepare sample WPI-A25, and the vial was crimped and sealed; therefore, the vial contained an equal volume of aqueous phase and ambient air in the upper space.

[0494] HPLC vials containing samples WPI-A24 and WPI-A25 were incubated for 160 seconds in an aluminum block preheated to 160°C, and then immediately cooled in an ice / water bath. Free thiols were then quantified using the method described in Example 1.

[0495] result

[0496] [Table 7]

[0497] The results shown in Table 6 indicate that heating sample WPI-A25 in the absence of quinone is insufficient to remove the free thiols generated by WPI-A. On the other hand, to completely remove the free thiols, sample WPI-A24, in which the molar ratio of 4MC:BLG is 2:1 and the volume of the upper space and aqueous phase are equal, was heated and investigated.

[0498] The inventors further observed by visual inspection that WPI-A24 is essentially colorless; this suggests that while it reduces the amount of free thiols, the O2 availability is sufficient to generate a sufficient amount of quinone to avoid the formation of colored products.

[0499] conclusion The inventors discovered that by combining pH 8 and a UHT-like treatment, with equivolute liquid samples and ambient air in the upper space, they could promote sufficient exposure of free thiols to 4MC-derived quinones, thereby enabling efficient free thiol blocking at high temperatures and in a short time.

[0500] The inventors further point out that the above-mentioned accessible upper space volume is equal to the upper space volume of sample WPI-A21 in Example 3, which ensures sufficient access to oxygen (O2) for the purpose of promoting quinone formation by oxygen (O2), and that by combining this with high temperature, more efficient free thiol blocking becomes possible in sample WPI-A24 compared to WPI-A25.

[0501] Example 5: In-situ formation of quinone and reaction of quinone with free thiol The aim of these experiments was to demonstrate the potential of using PCA to produce quinones in the presence of whey protein in a “single-step” modification method, which is considered advantageous for industrial processing.

[0502] method Cyclic voltammetry was performed to evaluate the degree and possibility of the electrochemical reaction described in Example 2. First, 30 mL of a 1% WPI-A solution prepared in 25 mM phosphate buffer (pH 7.5, 200 mM) was preheated in an aluminum block preheated to 60°C. The heating block was specially designed to fit and completely cover both the sides and bottom of the electrochemical cell of Example 2; this block was placed in a UHT (ultra-high temperature) heating unit as described in Example 1. Cyclic voltammetry was performed by applying -0.2 to +0.8 V using the electrode described in Example 2. Next, 30 mL of 25 mM phosphate buffer (pH 7.5, containing 200 mM NaCl) containing 1.09 mM 4MC was analyzed, as well as 30 mL of 1% WPI-A solution prepared with 25 mM phosphate buffer (pH 7.5, containing 200 mM NaCl) and 1.09 mM 4MC (4MC:BLG = 2:1).

[0503] For cyclic voltammetry measurements, 30 mL of a 1% WPI-A solution prepared in 25 mM phosphate buffer (pH 7.5, 200 mM) was preheated in an aluminum block preheated to 60°C. The heating block was specially designed to fit and completely cover both the sides and bottom of the electrochemical cell of Example 2; this block was placed in a UHT (ultra-high temperature) heating unit as described in Example 1.

[0504] Prior to the addition of 4MC and bulk electrolysis, 2 mL of WPI-A26 sample was set aside as a zero-reaction time sample, as shown in Table 7.

[0505] 250 μl of 33 mM 4MC was dissolved in nitrogen-degassed MQ water and held in a syringe to minimize exposure to oxygen; immediately after sampling WPI-A26, 4MC was injected from the syringe into the cell via an HPLC-connecting tube to achieve a 4MC:BLG ratio of 0.5. A potential of 0.45 V was applied to the cell to begin timing.

[0506] After bulk electrolysis for 5 minutes, 2 mL of WPI-A27 sample was taken, and then 250 μl of 33 mM 4MC was added; the 4MC:BLG molar ratio was set to 1.2:1 by the combined addition of 4MC.

[0507] After 10 minutes of bulk electrolysis, 2 mL of WPI-A28 sample was taken and the experiment was stopped.

[0508] All samples were cooled to 4°C immediately after sampling.

[0509] Unreacted reagents were removed using a spin filter as described in Example 2.

[0510] The free thiols in the spin filter holding solution of samples WPI-A26 to WPI-A28 were quantified using the method described in Example 1.

[0511] The holding solutions of WPI-A26, WPI-A27, and WPI-A28 were subjected to UHT-like heat treatment, and then H2S was measured using the method described in Example 1.

[0512] In another experiment, sample WPI-A29 (30 mL of 100 mM NaCl (pH 8.5) containing 5% WPI-A prepared from nitrogen-degassed MQ) was placed in an electrochemical cell and equilibrated to 60°C using the method described for sample WPI-A26. 4 MC was added to a final concentration of 5.4 mM (4 MC:BLG = 2:1), and bulk electrolysis was performed at 0.45 V for 20 minutes. The reaction was stopped by dilution with cooled 25 mM phosphate buffer (pH 7.0), resulting in a protein content of 1%. Unreacted reagents were removed using a spin filter as described in Example 2. Free thiols in the spin filter-held solution of sample WPI-A29 were quantified using the method described in Example 1.

[0513] result

[0514] [Table 8]

[0515] Cyclic voltammetry was performed at 60°C on a 1% WPI-A solution (pH 7.5) without 4MC, but it showed no signs of oxidation or reduction peaks, nor did it exhibit the characteristics of a "duck-type" voltammogram. In contrast, the inventors found that a duck-type voltammogram was obtained in the applied voltage range for quinone alone and quinone combined with protein. Therefore, it is considered that oxidation from 4MC to 4MBQ occurs at the above applied voltage. The oxidation potential was set to 0.45V and used in the test of Example 5.

[0516] As shown in Table 7, when WPI-A26 was analyzed before the addition of 4MC, the inventors initially observed that there were high levels of free thiols, and correspondingly, a high amount of H2S was generated during UHT treatment (ultra-high temperature treatment).

[0517] When 4MC was added to make the ratio 4MC:BLG = 0.5:1, the thiol content of sample WPI-A27 decreased to 23 μmol SH / g protein after 5 minutes of bulk electrolysis. The inventors noticed that the reaction mixture remained essentially colorless.

[0518] When combined with BLG in a ratio of 4MC:BLG=1.2:1, the thiol content of sample WPI-A28 further decreased to 6 μmol SH / g protein after 10 minutes of bulk electrolysis. Furthermore, in the UHT-treated (ultra-high temperature) sample, H2S was measured at less than 1 μmol / L as a result of the decrease in free thiol content.

[0519] The inventors further observed a slight discoloration in the WPI-A28 retention solution. The inventors had previously observed that strong discoloration could occur as a result of bulk electrolysis of 4MC at neutral pH in the absence of whey protein. In this study, the inventors observed a significant reduction in discoloration and no signs of undesirable side reactions during bulk electrolysis.

[0520] Sample WPI-A29 contained 5.4 mM 4MC (4MC:BLG molar ratio = 2:1) and was prepared with a higher protein content (5%) compared to other samples. After 20 minutes of bulk electrolysis, a significant decrease in free thiol content to 5.7 μmol SH / g protein was observed compared to treated WPI-A without 4MC (see, for example, WPI-A26). The inventors noted that approximately 9 mg / L (about 280 μM) of oxygen is soluble in water at 20°C, and this amount decreases with increasing temperature. Therefore, even if the starting solution is saturated with oxygen, which only corresponds to about 5% (5.4 mM) of the concentration of polyphenols present, the inventors used a nitrogen degassing source for the buffer in sample preparation to reduce potential oxidation by dissolved oxygen (O2).

[0521] In summary, these experiments: Possibility of using the direct electrochemical generation of quinones to facilitate the reaction between quinones and free thiols; Furthermore Possibility of using reactions with free thiols to reduce the electrochemical oxidation of polyphenols and the undesirable H2S formation during UHT (ultra-high temperature) treatment. This clearly demonstrates both of the above.

[0522] conclusion Electrochemical oxidation of quinones at a slightly alkaline pH and high temperature in the presence of whey protein constitutes a favorable approach to reduce the free thiol group content of whey protein solutions.

[0523] The inventors have discovered that by using relatively low doses of polyphenols in the presence of whey protein and by using direct oxidation of polyphenols, it is possible to keep the quinone concentration low in the reaction steps of this method. The inventors have discovered this approach to reduce or avoid discoloration caused by quinone side reactions. This approach is particularly advantageous for the preparation of protein sources that need to be used in colorless food applications, such as colorless high-protein beverages.

Claims

1. A method for preparing a protein composition containing modified β-lactoglobulin (BLG): (a) - A source containing one or more phenolic compounds, each containing at least two hydroxyl groups directly bonded to the same aromatic ring (PCA); and • Sources including BLG, A process to provide; (b) A step of optionally subjecting a portion of a source containing one or more types of PCA to an oxidation of a type that can convert PCA to quinone (called PCA-type oxidation), thereby providing a source containing one or more types of oxidized PCA; (c) A step of combining a portion of a source containing one or more PCA and / or a portion of a source containing one or more oxidized PCA with a source containing BLG and optionally further components, for the purpose of providing a protein solution; Here the protein solution pH in the range of 6.5 to 9.5; and • BLG content of at least 0.2% w / w; - The molar ratio between the original amount of PCA used in the preparation of the protein solution and the BLG content of the protein solution, wherein the molar ratio is at least 0.1:

1. Having, Process; (d) Incubating the protein solution within a certain temperature range for a period of time sufficient to reduce the amount of free thiol groups in the protein solution to a maximum of 10 micromoles / g of protein; If the method does not include step (b), step (d) is a step of incubation under conditions that also include the use of an oxidation of a type capable of converting PCA to quinone; including, method.

2. A method according to claim 1; Here, the PCA-type oxidation used in step (b) and / or step (d) is: - The theoretical amount of quinone provided to the protein solution during the implementation of the method, the theoretical amount of quinone determined according to analysis 1, and • The amount of BLG in the protein solution, This is sufficient to make the molar ratio between them at least 0.1:

1. method.

3. A method according to claim 1 or 2; Here, the PCA: Flavonoids, preferably flavanols or flavanol esters, such as catechin, epicatechin, gallocatechin, epigallocatechin, 3-gallic acid catechin, 3-gallic acid epicatechin, 3-gallic acid gallocatechin, and 3-gallic acid epigallocatechin (EGCG); and / or • Stilbenoides, preferably resveratrol, Methods that include...

4. A method according to any one of the above claims; Here's the PC in question: Caffeic acid, gallic acid, chlorogenic acid, catechol, 4-methylcatechol, catechin, epicatechin, gallocatechin, epigallocatechin, 3-gallic acid catechin, 3-gallic acid epicatechin, 3-gallic acid gallocatechin, 3-gallic acid epigallocatechin (EGCG), resveratrol, carnosic acid, carnosol, naringenin, or mixtures thereof. Including; and / or - Molecular weight in the range of 120 to 1000 g / mol, more preferably in the range of 250 to 700 g / mol, even more preferably in the range of 300 to 650 g / mol, and most preferably in the range of 350 to 600 g / mol. It has, and / or At 25°C, the water solubility is at least 8 mM, more preferably at least 12 mM, and most preferably at least 16 mM. has method.

5. A method according to any one of the above claims; Here, a supply source containing one or more types of PCA: • Polyphenol extracts from herbs, polyphenol extracts from spices, polyphenol extracts from fruits, polyphenol extracts from berries, and mixtures thereof. Selected from the group consisting of, and / or - Polyphenol extracts from tea, more preferably from green tea; polyphenol extracts from coffee; polyphenol extracts from cocoa; polyphenol extracts from grapes; polyphenol extracts from rosemary; polyphenol extracts from lemon balm; polyphenol extracts from blackcurrant; single PCA isolates and mixtures thereof. Selected from the group consisting of, method.

6. A method according to any one of the above claims; Here, the source containing BLG includes or consists of whey protein concentrate, whey protein isolate, whey protein concentrate, whey protein isolate, BLG isolate, or a combination thereof. method.

7. A method according to any one of the above claims; A method comprising step (b), that is, providing a portion of a source containing one or more types of PCA to an oxidation of a type capable of converting PCA to quinone, thereby providing a source containing one or more types of oxidized PCA.

8. A method according to any one of the above claims; Here the protein solution: pH in the range of 6.7 to 9.5, more preferably in the range of 7.1 to 9.0, even more preferably in the range of 7.3 to 8.7, and most preferably in the range of 7.4 to 8.5; - 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; - BLG content of 0.5 to 30% w / w, more preferably 1 to 20% w / w, even more preferably 3 to 16% w / w, and most preferably 5 to 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 - BLG content of 30-99% w / w relative to total protein, more preferably 40-95% w / w relative to total protein, even more preferably 45-90% w / w relative to total protein, and most preferably 50-80% w / w relative to total protein. A method comprising one or more of the above, more preferably all of the above.

9. A method according to any one of the above claims; Now, in the protein solution of step (c): - The original amount of PCA used to prepare the protein solution, and - BLG content of the protein solution, The molar ratio between: It is at least 1:

1. method.

10. A method according to any one of claims 1 to 8; Now, in the protein solution of step (c): - The original amount of PCA used to prepare the protein solution, and - BLG content of the protein solution, The molar ratio between: The ratio is 0.1:1 to 100:1, more preferably 0.1:1 to 50:1, even more preferably 0.1:1 to 20:1, and most preferably 0.1:1 to 10:

1. method.

11. A method according to any one of claims 1 to 8; Here in the protein solution: - The original amount of PCA used to prepare the protein solution, and - BLG content of the protein solution, The molar ratio between: The ratio is 0.1:1 to 5:1, more preferably 0.1:1 to 4:1, even more preferably 0.2:1 to 3:1, and most preferably 0.3:1 to 2:

1. method.

12. A method according to any one of claims 1 to 8; Here in the protein solution: - The original amount of PCA used to prepare the protein solution, and - BLG content of the protein solution, The molar ratio between: The ratio is 1:1 to 100:1, more preferably 1:1 to 50:1, even more preferably 1:1 to 20:1, and most preferably 1:1 to 10:

1. method.

13. A method according to any one of claims 1 to 12; Here, the oxidation used in step (b) and / or step (d) is: - The theoretical amount of quinone provided to the protein solution during the implementation of the method, the theoretical amount of quinone determined according to analysis 1, and - BLG content of the protein solution, This is sufficient to set the molar ratio between them to 0.1:1 to 100:1, more preferably 0.1:1 to 50:1, even more preferably 0.2:1 to 15:1, and most preferably 0.2:1 to 5:

1. method.

14. A method according to any one of claims 1 to 12; Here, the oxidation used in step (b) and / or step (d) is: - The theoretical amount of quinone provided to the protein solution during the implementation of the method, the theoretical amount of quinone determined according to analysis 1, and - BLG content of the protein solution, This is sufficient to set the molar ratio between them to 0.1:1 to 5:1, more preferably 0.1:1 to 4:1, even more preferably 0.2:1 to 3:1, and most preferably 0.2:1 to 2:

1. method.

15. A method according to any one of claims 1 to 12; Here, the oxidation used in step (b) and / or step (d) is: - The theoretical amount of quinone provided to the protein solution during the implementation of the method, the theoretical amount of quinone determined according to analysis 1, and - BLG content of the protein solution, This is sufficient to make the molar ratio between them at least 1:

1. method.

16. A method according to any one of claims 1 to 12; Here, the oxidation used in step (b) and / or step (d) is: - The theoretical amount of quinone provided to the protein solution during the implementation of the method, the theoretical amount of quinone determined according to analysis 1, and - BLG content of the protein solution, This is sufficient to set the molar ratio between them to 1:1 to 100:1, more preferably 1:1 to 50:1, even more preferably 1:1 to 15:1, and most preferably 1.5:1 to 5:

1. method.

17. A method according to any one of the above claims; Here, the method includes step (b); For the preparation of the protein solution in step (c), use a portion of the supply containing one or more types of oxidized PCA obtained in step (b); and Here, the PCA type oxidation is carried out by electrochemical oxidation. method.

18. A method according to any one aspect of the above claims; Here, the PCA-type oxidation used in step (b) is: - Quinone content of a portion of the supply source containing one or more types of oxidized PCA used in the protein solution, and • The amount of BLG in the protein solution, Sufficient to make the molar ratio between them 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. method.

19. A method according to any one of claims 1 to 16; Here, the PCA-type oxidation used in step (b) is: - Quinone content of a portion of the supply source containing one or more types of oxidized PCA used in the protein solution, and • The amount of BLG in the protein solution, This is sufficient to make the molar ratio between them 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. method.

20. A method according to any one of the above claims; The process further comprises the step (e) of drying a liquid material containing a protein derived from at least the incubated protein solution of step (d); Preferably, the liquid raw material to be dried here contains or consists of the protein solution or protein concentrate obtained in step (d). method.

21. A protein composition containing modified BLG and having a maximum of 10 micromoles of free thiol groups per gram of protein; The protein composition is obtainable by a method according to one or more of the above claims, preferably the protein composition is: • Protein content of at least 30% w / w relative to total solids; - Tryptophan content of at least 0.7% w / w relative to total protein, • Methionine content of at least 0.3% w / w relative to total protein; • Maximum kynurenine content of 0.2 micrograms / mg of protein; Preferably, a fat content of up to 3% w / w relative to total solids; Preferably, the content of protein-bound sulfur is in the range of 100 to 600 micromoles / g of protein; Preferably, the content of disulfide bond-forming protein-binding cysteine ​​residues is in the range of 150 to 400 micromoles / g of protein. Having one or more of the following types Protein composition.

22. A modified whey protein composition according to claim 21; Having a protein content of at least 30% w / w relative to total solids, Modified whey protein composition.

23. A 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, Modified whey protein composition.

24. A modified whey protein composition according to any one of claims 21 to 23; Having a methionine content of at least 0.3% w / w relative to total protein, Modified whey protein composition.

25. A modified whey protein composition according to any one of claims 21 to 24; It has a maximum kynurenine content of 0.2 micrograms / mg of protein. Modified whey protein composition.

26. A modified whey protein composition according to any one of claims 21 to 25; It has a fat content of up to 3% w / w relative to the total solids. Modified whey protein composition.

27. A modified whey protein composition according to any one of claims 21 to 26; Having a protein-bound sulfur content in the range of 100 to 600 micromoles / g of protein, Modified whey protein composition.

28. A modified whey protein composition according to any one of claims 21 to 26; The content of disulfide bond-forming protein-binding cysteine ​​residues is in the range of 150 to 400 micromoles / g of protein. Modified whey protein composition.

29. A modified whey protein composition according to any one of claims 21 to 27; The average molecular weight of the protein is: A range of 18 kDa to 10000 kDa, more preferably a range of 50 to 8000 kDa, and most preferably a range of 80 to 5000 kDa. That is, Modified whey protein composition.

30. A protein composition according to any one of claims 21 to 27; The average molecular weight of the protein is: The range is 18 kDa to 500 kDa, more preferably 18 to 100 kDa, and most preferably 18 to 40 kDa. Protein composition.

31. A process for producing a heat-treated beverage, preferably a heat-sterilized beverage; The process is: (1) A step of mixing a protein composition according to any one or more of claims 21 to 30 with one or more further beverage components in order to obtain a liquid mixture with a pH of 5.5 to 8.5; and (2) A step of filling the liquid mixture into a suitable container, Including; The process further includes at least one heat treatment step of heat-treating the liquid mixture before and / or after filling, and preferably heat-sterilizing it; Preferably, the liquid mixture contains an amount of the protein composition sufficient to contribute at least 0.5% w / w of protein. process.

32. A process for producing a heat-treated beverage, preferably a heat-sterilized beverage, according to claim 31; Here, the liquid mixture contains, before heat sterilization, a maximum of 60 micromoles of free thiol groups per 100 g of liquid mixture, more preferably a maximum of 40 micromoles of free thiol groups per 100 g of liquid mixture, even more preferably a maximum of 30 micromoles of free thiol groups per 100 g of liquid mixture, and most preferably a maximum of 30 micromoles of free thiol groups per 100 g of liquid mixture. process.

33. A process for producing a heat-treated beverage, preferably a heat-sterilized beverage, according to claim 31; Here, the liquid mixture contains, before heat sterilization, a maximum of 20 micromoles of free thiol groups per 100 g of liquid mixture, more preferably a maximum of 15 micromoles of free thiol groups per 100 g of liquid mixture, even more preferably a maximum of 10 micromoles of free thiol groups per 100 g of liquid mixture, and most preferably a maximum of 5 micromoles of free thiol groups per 100 g of liquid mixture. process.

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

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

36. A process for producing a heat-treated beverage, preferably a heat-sterilized beverage, according to any one of claims 31 to 35; Here, a protein composition according to any one or more of claims 21 to 30 is: Contributing to 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, process.

37. A process for producing a heat-treated beverage, preferably a heat-sterilized beverage, according to any one of claims 31 to 36; Here, a protein composition according to any one or more of claims 21 to 30 is: A component that 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. process.

38. A process for producing a heat-treated beverage, preferably a heat-sterilized beverage, according to any one of claims 31 to 37; Here, the total protein content of the liquid mixture is at least 15% w / w relative to the total solids, more preferably at least 20% w / w, most preferably at least 25% w / w, and most preferably at least 30% w / w. process.

39. A process for producing a heat-treated beverage, preferably a heat-sterilized beverage, according to any one of claims 31 to 38; Here, the total protein content of the liquid mixture is at least 80% w / w relative to the total solids, more preferably at least 90% w / w relative to the total solids, even more preferably at least 92% w / w, and most preferably at least 94% w / w. process.

40. A process for producing a heat-treated beverage, preferably a heat-sterilized beverage, according to any one of claims 31 to 39; Here, the solid content of the liquid mixture is 0.5 to 50% w / w, more preferably 1 to 35% w / w, even more preferably 2 to 20% w / w, and most preferably 3 to 10% w / w. process.

41. A process for producing a heat-treated beverage, preferably a heat-sterilized beverage, according to any one of claims 31 to 40; Here, the portion of the liquid mixture that does not constitute solid matter contains at least 80% w / w of water, more preferably at least 90% w / w of water, even more preferably at least 95% w / w of water, and more preferably at least 99% w / w of water. process.

42. A process for producing a heat-treated beverage, preferably a heat-sterilized beverage, according to any one of claims 31 to 41; Here, the calorie content of the liquid mixture is at most 100 kcal / 100g, more preferably at most 80 kcal / 100g, even more preferably at most 70 kcal / 100g, and most preferably at most 60 kcal / 100g. process.

43. A process for producing a heat-treated beverage, preferably a heat-sterilized beverage, according to any one of claims 31 to 41; Here, the calorie content of the liquid mixture is greater than 100 kcal / 100g; more preferably at least 120 kcal / 100g, even more preferably at least 140 kcal / 100g, and most preferably at least 150 kcal / 100g. process.

44. A heat-treated beverage, preferably a heat-sterilized beverage; A pH of 5.5 to 8.5 obtainable by a process according to any one or more of claims 30 to 43, Beverage.

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