Whey protein hydrolysate, its uses, method of preparation thereof, and carbonated beverage comprising said hydrolysate.

Abstract

Palatable extensively hydrolyzed whey protein hydrolysates. The present invention relates to novel whey protein hydrolysates having a high degree of hydrolysis, being palatable and with low turbidity without being subjected to ultrafiltration. The invention also relates to methods of preparing novel whey protein hydrolysates, uses of the novel whey protein hydrolysates, and food products comprising these novel whey protein hydrolysates.

Description

1 / 86 Whey protein hydrolysate, its uses, method of preparation thereof, and carbonated beverage comprising said hydrolysate. Field of invention

[001] The present invention relates to novel whey protein hydrolysates that have a high degree of hydrolysis, are palatable, and have low turbidity without being subjected to ultrafiltration. The invention also relates to methods of preparing novel whey protein hydrolysates, uses of the novel whey protein hydrolysates, and food products comprising these novel whey protein hydrolysates. Background of the invention

[002] The use of whey protein hydrolysates as ingredients in various food products is well known. Whey protein hydrolysates are commonly prepared by hydrolyzing a whey protein substance, such as a whey protein isolate or whey protein concentrate, with a food-grade proteolytic and / or peptidolytic preparation to a desired degree of hydrolysis. In some situations, it is desirable to prepare a whey protein hydrolysate with a high degree of hydrolysis, for example, a degree of hydrolysis of 15% or above, for example 20-30%, to prepare whey protein hydrolysates with low antigenicity or hydrolysates that are well absorbed in the intestine.However, existing whey protein hydrolysates have the problem that when hydrolysis becomes too extensive and a high degree of hydrolysis is achieved, the whey protein hydrolysate has an unpleasant bitter taste and is therefore not suitable for use in food products or beverages in large quantities. Petition 870230043920, dated 05 / 25 / 2023, page 10 / 101 2 / 86

[003] WO 02 / 19 837 A1 discloses a process for preparing a whey protein hydrolysate from a whey protein isolate (WPI) substrate with improved flavor, functionality and ACE inhibitory properties. WO 02 / 19 837 A1 discusses the problems of bitter tastes in whey protein hydrolysates and solves the problem of bitter tastes by controlling enzymatic hydrolysis so that hydrolysis is stopped when the degree of hydrolysis is at most 10%, such as 3-10%.

[004] Therefore, a whey protein hydrolysate with a high degree of hydrolysis (a degree of hydrolysis above 15%) and therefore low antigenicity and improved absorption properties, but at the same time not having an unpleasant bitter taste would be advantageous. Summary of the invention

[005] The inventors of the present invention have surprisingly discovered that by using specific combinations of enzymes for enzymatic hydrolysis of whey protein, whey protein hydrolysates are obtained with a high degree of hydrolysis, while the whey protein hydrolysates have an acceptable taste and no bitter taste or at least an acceptable level of bitter compounds.

[006] Thus, an object of the present invention relates to a method of preparing a whey protein hydrolysate with a degree of hydrolysis of at least 15% and wherein a 4% w / w protein solution has a bitterness score corresponding to a 0.08% w / v caffeine solution or less, without the whey protein hydrolysate being subjected to any bitterness reduction treatments.

[007] Preferably, the method of the present invention relates to a method of preparing a whey protein hydrolysate that Petition 870230043920, dated 05 / 25 / 2023, page 11 / 101 3 / 86 has low turbidity and is therefore clear in appearance, without any ultrafiltration step.

[008] In particular, it is an objective of the present invention to provide a whey protein hydrolysate that solves the aforementioned problems of the prior art with an unpleasant bitter taste when the degree of hydrolysis is high.

[009] Thus, one aspect of the invention relates to a method of preparing a whey protein hydrolysate comprising: a) provide a whey protein solution comprising whey protein in an amount of at least 50% by weight based on total solids content, b) subjecting the whey protein solution to enzymatic hydrolysis, wherein the enzymatic hydrolysis is performed using any of the following enzyme combinations: i. Comprising at least one serine endopeptidase from Bacillus, at least one serine endopeptidase from Aspergillus, and at least one trypsin-like protease ii. Comprising at least one serine endopeptidase from Bacillus, at least one serine endopeptidase from Aspergillus, and at least one leucilaminopeptidase from Aspergillus iii. Comprising at least one bacillolysin from Bacillus amyloliquefaciens, at least bromelain, and at least one leucilaminopeptidase from Aspergillus c) to stop enzymatic hydrolysis by inactivating the enzymes when the degree of hydrolysis (DH) is 15% or more in order to obtain a whey protein hydrolysate. [0 10] Another aspect of the present invention relates to a Petition 870230043920, dated 05 / 25 / 2023, page 12 / 101 4 / 86 hydrolyzed whey protein comprising: - free amino acids and peptides, and - have a degree of hydrolysis of at least 15%, and - peptides with a molecular weight of 2500 Da or more in an amount of 25% by weight or less of the total amount of peptides, and - free amino acids in an amount of 15% by weight or less of the total amino acid content in the hydrolysate, and wherein the whey protein hydrolysate in a 4% w / w protein solution has a bitterness score corresponding to a 0.08% w / v or less caffeine solution.

[011] Another aspect of the present invention is to provide a food product comprising whey protein hydrolysate according to the invention.

[012] Yet another aspect of the present invention is to provide a beverage comprising whey protein hydrolysate according to the invention, wherein the whey protein hydrolysate is present in the beverage in an amount corresponding to 2 to 25% by weight of hydrolyzed whey protein.

[013] Yet another aspect of the invention is the use of whey protein hydrolysate according to the invention as a food ingredient. Brief description of the figures

[014] Figure 1A shows the nephelometric turbidity (NTU) at different protein concentrations for sample 16 (a whey protein hydrolysate according to the invention made by enzymatic hydrolysis of a WPI), sample 14 (whey protein hydrolysate according to the invention made by enzymatic hydrolysis of a whey protein concentrate (WPC)) and sample 13 (a whey protein hydrolysate of Petition 870230043920, dated 05 / 25 / 2023, page 13 / 101 5 / 86 reference that is not prepared using a combination of enzymes of the invention, but the whey protein hydrolysate is subjected to ultrafiltration).

[015] Figure 1 B shows the nephelometric turbidity (NTU) at different protein concentrations for sample 16 (a whey protein hydrolysate according to the invention made by enzymatic hydrolysis of a WPI). Standard deviations are given.

[016] Figure 1C shows the nephelometric turbidity (NTU) at different protein concentrations for sample 14 (whey protein hydrolysate according to the invention made by enzymatic hydrolysis of a WPC). Standard deviations are given.

[017] Figure 2 shows samples of different protein concentrations from samples 13, 14, and 16. The protein concentration (w / w) is from left to right: 8%, 6.4%, 4.8%, 3.2%, and 1.8%. A) shows sample 16, B) shows sample 14, and C) shows sample 13.

[018] Figure 3 shows the bitterness score of different caffeine concentrations and the bitterness score of samples 13, 14 and 16.

[019] Figure 4 shows a spiderweb representation of the taste and mouthfeel profile of samples 13, 15 and 16. The significance level of the difference between the attributes with the highest and lowest scores is indicated by *** and is 99.9% and with P <0.001 (ANNOVA analysis).

[020] Figure 5 shows the percentage of peptides between 7 and 19 amino acids in the 7-10 amino acid and 11-19 amino acid ranges when analyzed using size exclusion chromatography (SEC) or LC-MS / MS (MS).

[021] Figure 6 shows phenylalanine comprising peptides shown as the percentage of all alpha-lactalbumin, beta peptides Petition 870230043920, dated 05 / 25 / 2023, page 14 / 101 6 / 86 lactoglobulin and beta-casein. The smallest peptides analyzed were 5 amino acids.

[022] Figure 7 shows the percentage by number of peptides of 5-19 amino acids of alpha-lactalbumin, beta-lactoglobulin and beta-casein.

[023] Figure 8 shows beverage samples from left to right: a beverage prepared without heat treatment, a beverage prepared with direct UHT treatment at 143 °C for 6 seconds, a beverage prepared with indirect UHT treatment for 6 seconds, and a beverage prepared with pasteurization at 90 °C for 6.5 minutes.

[024] Figure 9 shows images of SDS-page gels used to determine the amount of undegraded BSA in different samples of whey protein hydrolysates.

[025] Figure 10 shows the correlation between carbonation and pH of a beverage.

[026] Figure 11 shows the pH of different samples comprising whey protein hydrolysates and carbonates with 2.5 volumes of carbon dioxide per volume of composition.

[027] Figure 12 shows the pH and turbidity during heating of a sample comprising an 8% solution of carbonated sample 16 protein with 2.5 volumes of CO2 per volume of solution.

[028] The present invention will now be described in more detail below. Detailed description of the invention Definitions

[029] Before discussing the present invention in more detail, the following terms and conventions will first be defined:

[030] All references to singular features or limitations of the present invention shall include the plural feature or limitation. Petition 870230043920, dated 05 / 25 / 2023, page 15 / 101 7 / 86 corresponding, and vice versa, unless otherwise specified or clearly implied otherwise by the context in which the reference is made.

[031] All percentages referred to in this invention are percentages by weight, unless otherwise indicated. In addition, the terms “by weight of dry matter” and “based on dry matter” refer to the same concept and are used interchangeably.

[032] The term “w / w” as in, for example, 1% w / w refers to a composition comprising 1% by weight of a compound.

[033] The term “palatable” refers to having a taste good enough to eat and / or drink, that is, having an acceptable or satisfactory taste for the human consumer.

[034] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by a person skilled in the art. Whey protein solution:

[035] In the context of the present invention, the term “solution” as in “whey protein solution” encompasses compositions containing a combination of liquid and solid compounds or semi-solid particles, such as, for example, protein particles. A “solution” can therefore be a suspension or even a paste. However, the “whey protein solution” is preferably pumpable and the amount of liquid in the whey protein solution is preferably 70-98%, more preferably 80-96%. The liquid used for the whey protein solution is normally water.

[036] The whey protein solution will normally comprise protein in an amount of 2% by weight or more of the whey protein solution. In one embodiment of the invention, the whey protein solution Petition 870230043920, dated 05 / 25 / 2023, page 16 / 101 8 / 86 of milk comprises protein in the range of 2 to 20% by weight of the whey protein solution. Preferably, the whey protein solution comprises protein in an amount in the range of 5 to 15% by weight of the whey protein solution.

[037] The whey protein solution used in hydrolysis is obtained by dispersing a composition comprising whey protein in a liquid, such as water. Preferably, the whey protein solution is made by mixing any one of a whey protein concentrate, a whey protein concentrate, a whey protein isolate and / or a whey protein isolate with water. Thus, in one embodiment of the present invention, the whey protein solution comprises a whey protein concentrate, a whey protein concentrate, a whey protein isolate and / or a whey protein isolate.

[038] The whey protein solution of the present invention comprises whey protein in an amount of at least 50% based on the total solids content. If the whey protein content is less than 50% of the total solids content, the overall molecular composition (ratios between proteins, carbohydrates, lipids and minerals) will be different, the enzymes may behave differently and therefore the product obtained will be different.

[039] In addition to whey protein, whey protein solution may contain other proteins in small amounts, for example casein.

[040] Whey protein solution typically comprises other components in addition to protein. Whey protein solution may comprise other components that are normally Petition 870230043920, dated 05 / 25 / 2023, page 17 / 101 9 / 86 found in whey or lactose, such as, for example, minerals, carbohydrates and / or lipids. Alternatively or additionally, the whey protein solution may comprise components that are not native to whey or lactose. However, such non-native milk components must be suitable and safe for use in food production.

[041] The lower the protein content, based on total solids content, in the whey protein solution, the higher the amounts of lipids, carbohydrates (mainly lactose) and other proteins besides whey protein.

[042] The whey protein solution may, for example, comprise carbohydrates, such as, for example, lactose, oligosaccharides and / or lactose hydrolysis products (i.e., glucose and galactose). The whey protein solution may, for example, comprise carbohydrates in the range of 0 to 10% by weight based on the total solids content.

[043] Whey protein isolate (WPI) and whey protein isolate (SPI) contain very low amounts of carbohydrates, such as lactose. Therefore, when WPI or SPI is used to prepare whey protein solution, the carbohydrate content in the whey protein solution is in the range of 0 to 1% by weight based on the total solids content. If whey protein concentrate (WPC) or whey protein concentrate (SPC) is used to prepare whey protein solution, the amount of carbohydrates in the whey protein solution is preferably in the range of 2 to 8% by weight based on the total solids content.

[044] Whey protein solution may also comprise lipids, for example, in the form of triglycerides and / or other types of lipids, such as phospholipids. Petition 870230043920, dated 05 / 25 / 2023, page 18 / 101 10 / 86

[045] In the context of the present invention, the terms fat and lipid have the same meaning and can be used interchangeably.

[046] The whey protein solution according to the present invention shall comprise whey protein in an amount of at least 50% based on the total solids content. If the protein content in the whey protein solution is less than 50% of the total solids content, the whey protein hydrolysate obtained after hydrolysis may not have the characteristics that define whey protein hydrolysate according to the present invention. That is, no unpleasant bitter taste in a 4% protein solution, a degree of hydrolysis above 15%, free amino acids in an amount of 15% by weight or less, and peptides with a molecular weight of 2500 Da or more in an amount of 25% by weight or less of the total amount of peptides.

[047] A whey protein solution with a whey protein content below 50% based on the total solids content will comprise a high amount of minerals, fat, and carbohydrates. It is undesirable to make a whey protein hydrolysate with a whey protein content below 50% by weight of the solids content and high amounts of minerals, fats, and carbohydrates. Without being limited by any theory, the inventors of the present invention believe that minerals can affect the activity of some enzymes. This also applies to lipids and carbohydrates. High amounts of lipids, minerals, and carbohydrates can also affect the taste and turbidity of the resulting whey protein hydrolysate.

[048] Preferably, the whey protein solution comprises whey protein in an amount of at least 60% by weight based on total solids content, such as at least 70% by weight based on total solids content, even more preferably at least 80%. Petition 870230043920, dated 05 / 25 / 2023, page 19 / 101 11 / 86 by weight based on total solids content. In a more preferred embodiment of the present invention, the whey protein solution comprises whey protein in an amount of at least 85% by weight based on total solids content, most preferably the whey protein solution comprises whey protein in an amount of at least 90% by weight based on total solids content.

[049] The protein present in the whey protein solution must be primarily whey proteins. However, small amounts of other proteins, such as, for example, casein, may be present. In one embodiment of the present invention, the whey protein solution therefore comprises whey protein in an amount of 90% by weight or more based on the total amount of protein. Preferably, the whey protein solution comprises whey protein in an amount of 95% by weight or more based on the total amount of protein.Therefore, in further embodiments of the present invention, the whey protein solution comprises a maximum of 10% by weight of casein or other non-whey protein based on the total amount of protein, preferably a maximum of 5% by weight, more preferably a maximum of 3% by weight of casein or other non-whey protein based on the total amount of protein.

[050] If the fat content in the whey protein solution is high, this will influence the clarity and taste of the resulting protein hydrolysate. Therefore, in one embodiment of the invention, the whey protein solution comprises lipids in an amount of no more than 10% by weight based on the total solids content, such as no more than 8% by weight based on the total solids content, even more preferably the whey protein solution comprises lipids in an amount of no more than 6% by weight with Petition 870230043920, dated 05 / 25 / 2023, page 20 / 101 12 / 86 based on total solids content.

[051] If a whey protein concentrate is used to prepare the whey protein solution, the lipid / fat content will be approximately 6-8% by weight of the total solids content. If a whey protein isolate, on the other hand, is used to prepare the whey protein solution, the whey protein solution is essentially fat-free.

[052] In a preferred embodiment of the invention, the whey protein solution is essentially fat-free. By the term "essentially fat-free" it is understood that the lipid content in the whey protein solution is less than 1% by weight based on the total solids content, preferably less than 0.5% by weight and even more preferably less than 0.1% by weight based on the total solids content.

[053] If the fat content in the whey protein solution is low, such as at most 0.5% based on the total solids content, the whey protein hydrolysate prepared according to the method of the present invention will have a clear appearance. Preferably, the lipid content in the whey protein solution is less than 0.3% by weight of the total solids content, and more preferably less than 0.2% lipids by weight based on the total solids content.

[054] In a preferred embodiment of the invention, whey protein hydrolysate is obtained using a whey protein solution from a whey protein isolate and / or a lactose protein isolate. When a whey protein isolate and / or a lactose protein isolate is used for hydrolysis, the fat content will be low (below 0.5%). This makes it possible to prepare a whey protein hydrolysate that, in addition to having a high degree of hydrolysis (DH> 15%) and a good taste, also Petition 870230043920, dated 05 / 25 / 2023, page 21 / 101 13 / 86 has a clear appearance, without any ultrafiltration step. Preferably, the whey protein solution is a whey protein isolate or whey protein isolate mixed in water. Whey protein:

[055] In one aspect of the present invention, whey protein hydrolysate is obtained by hydrolysis of a solution comprising whey protein.

[056] In the context of the present invention, the term “whey protein” refers to the protein found in whey or lactose. The whey protein in the whey protein solution may be a subset of protein species found in whey or lactose, or it may be the complete set of protein species found in whey and / or lactose. Whey protein is a mixture of globular proteins isolated from whey, the liquid material created as a byproduct of cheese production. Whey proteins are proteins present in the serum phase of milk or curdled milk. Milk serum phase proteins are, in addition to whey proteins, also referred to as lactose proteins.

[057] The term “lactose” refers to the liquid that remains when casein and milk fat globules are removed from milk, for example, by microfiltration or large pore ultrafiltration. Lactose may also be referred to as “ideal whey”.

[058] The term “whey protein” or “whey protein” refers to the protein that is present in whey.

[059] The term “whey” refers to the liquid supernatant that remains after the casein in milk has been precipitated and removed. Casein precipitation can, for example, be achieved by acidifying milk and / or by using Petition 870230043920, dated 05 / 25 / 2023, p. 22 / 101 14 / 86 of rennet enzyme.

[060] There are several types of whey, such as sweet whey, sour whey, and casein whey.

[061] The whey protein present in the whey protein solution of the present invention may be derived from different whey sources, for example, casein whey, acid whey or sweet whey.

[062] In a preferred embodiment of the invention, the whey proteins in the whey protein solution are sweet whey. Sweet whey mainly comprises the proteins beta-lactoglobulin (BLG), alpha-lactalbumin (ALA), and casein macropeptide (CMP). However, sweet whey may comprise other proteins such as immunoglobulins, osteopontin, lactoferrin, and fat globule membrane proteins. CMP is not present in casein whey or acid whey. In one embodiment of the invention, the whey proteins in the whey protein solution are sweet whey in which the CMP has been totally or partially removed. This may be referred to as modified sweet whey. The removal of CMP from sweet whey results in a protein material with threonine and tryptophan contents that are closer to human milk.

[063] In the context of the present invention, the term “beta-lactoglobulin” may also be referred to as BLG. The terms may be used interchangeably and refer to BLG from mammalian species. In addition, the term alpha-lactalbumin may, in the context of the present invention, be referred to as ALA and is related to alpha-lactalbumin from mammalian species.

[064] The term sweet whey, as used in the present invention, refers to the liquid remaining after milk has been curdled and strained during Petition 870230043920, dated 05 / 25 / 2023, p. 23 / 101 15 / 86 The manufacture of rennet-type cheeses. “Sweet whey” is obtained during the production of hard rennet-type cheeses, such as Cheddar or Swiss cheese. Sweet whey is obtained by adding rennet enzymes to a milk composition, which cleaves kappa-casein into para-kappa-casein and caseinomacropeptide (CMP), thus destabilizing the casein micelles and causing casein precipitation. The liquid surrounding the precipitated casein with rennet is referred to as sweet whey. The pH value of sweet whey can vary between 5.2 and 6.7.

[065] Sweet whey is a product of cheese production comprising about 10-15% protein by weight and about 75-80% lactose. Sweet whey proteins are primarily whey proteins, but small amounts of casein may also be present. Whey proteins include beta-lactoglobulin (about 55-65%), alpha-lactalbumin (about 18-25%), bovine serum albumin, immunoglobulins, caseinomacropeptides (CMP), osteopontin, lactoferrin, and milk fat globule membrane proteins.

[066] The term “casein whey” (sometimes also referred to as sour whey or acid whey) relates to whey, which is obtained from the production of casein / caseinate. In the context of the present invention, casein whey is not the same as acid whey. Casein whey is the whey fraction obtained after the separation of casein / caseinates by microfiltration. Casein whey does not contain CMP.

[067] The term acid whey is used for whey obtained during the production of acid-type cheeses, such as cottage cheese and quark. In the preparation of acid-type cheeses, casein is removed from the milk by acid precipitation, that is, reducing the pH value of the milk to a pH below Petition 870230043920, dated 05 / 25 / 2023, page 24 / 101 16 / 86 4.6, which is the isoelectric point of casein and causes the casein micelles to disintegrate and precipitate. The pH is often reduced to a range of 3.8 to 4.6. The liquid surrounding the acid-precipitated casein is often referred to as acid whey and does not contain CMP.

[068] In one embodiment of the invention, the whey protein used in the whey protein solution is not acid whey or casein whey.

[069] The whey protein used in the whey protein solution in the present invention may be a whey protein concentrate (WPC), a whey protein concentrate (SPC), a whey protein isolate (WPI), or a whey protein isolate (SPI). The difference between a whey protein concentrate and a whey protein isolate is the composition of the product, particularly the protein content. Whey protein isolates are purer than concentrates, and other non-protein components have been partially removed to isolate the whey protein. Thus, a whey protein isolate has a higher percentage of protein and may be pure enough to be virtually free of lactose, carbohydrate, fat, and cholesterol.

[070] In the present context, the terms whey protein concentrate (WPC) and whey protein concentrate (SPC) encompass dry and liquid compositions of whey protein. The protein content in a WPC and SPC used in the present invention is not less than 50% by weight based on total solids content. However, a whey protein concentrate may comprise higher amounts of whey protein, for example 80% by weight of whey protein based on dry matter content. The dry portion of liquid whey is obtained by removing sufficient non-protein constituents from the whey so that the product Petition 870230043920, dated 05 / 25 / 2023, page 25 / 101 17 / 86 dry matter should not be less than 50% by weight of whey protein.

[071] A WPC or SPC used in the present invention typically comprises: 50-89% protein by weight relative to total solids content; 15-70% by weight of BLG in relation to the total protein content; 8-50% ALA by weight relative to total protein content; 0-40% by weight of CMP in relation to the total protein content.

[072] Alternatively, but also preferable, is a WPC or an SPC comprising: 50-89% protein by weight relative to total solids content; 15-80% by weight of BLG in relation to the total protein content; 4-50% by weight of ALA in relation to the total protein content; 0-40% by weight of CMP in relation to the total protein content.

[073] Preferably, a WPC or an SPC comprises: 50-89% protein by weight relative to total solids content; 15-80% by weight of BLG in relation to the total protein content; 4-50% by weight of ALA in relation to the total protein content; 0-40% by weight of CMP in relation to the total protein content.

[074] More preferably, a WPC or an SPC comprises: 70-89% protein by weight relative to total solids content; 30-80% by weight of BLG in relation to the total protein content; 4-35% by weight of ALA in relation to the total protein content; 0-25% by weight of CMP in relation to the total protein content.

[075] The terms “whey protein isolate” and “serum protein isolate” refer to dry or liquid compositions, which are generally considered to be nearly lactose- and cholesterol-free and have a whey protein content of at least 90% by weight based on total solids content. Petition 870230043920, dated 05 / 25 / 2023, page 26 / 101 18 / 86 A whey protein isolate may, for example, comprise 92% by weight of whey protein or higher based on total solids content. Preferably, WPI and SPI comprise 90-100% by weight of protein based on total solids content, such as 92-99% by weight of protein based on total solids content.

[076] A WPI or an SPI may preferably comprise: 90-100% protein by weight relative to total solids content; 15-70% by weight of BLG in relation to the total protein content; 8-50% ALA by weight relative to total protein content; 0-40% by weight of CMP in relation to the total protein content.

[077] Alternatively, but also preferably, a WPI or an SPI may comprise: 90-100% protein by weight relative to total solids content; 30-80% by weight of BLG in relation to the total protein content; 4-35% by weight of ALA in relation to the total protein content; 0-25% by weight of CMP in relation to the total protein content.

[078] Preferably, a WPI may preferably comprise: 90-100% by weight of protein in relation to total solids content; 60-70% by weight of BLG in relation to the total protein content; 10-20% by weight of ALA in relation to the total protein content; 10-20% by weight of CMP in relation to the total protein content.

[079] In one embodiment of the invention, the whey protein solution used in the preparation of whey protein hydrolysate according to the invention comprises a total amount of whey protein in the range of 50-98% by weight of dry matter, such as 70-97% by weight, preferably 72-95% by weight, even more preferably in the range of 75-95% by weight of dry matter. Petition 870230043920, dated 05 / 25 / 2023, page 27 / 101 19 / 86

[080] Any suitable whey protein source can be used to prepare the whey protein solution according to the present invention. The whey proteins used in the whey protein solution according to the present invention are preferably whey proteins from mammals, such as, for example, cow, sheep, goat, buffalo, camel, llama, mare, horse and / or deer milk. In some preferred embodiments of the invention, the whey proteins are derived from bovine (cow) milk.

[081] It is preferable that the whey protein solution be a demineralized whey protein solution. It is preferable that the mineral content in the whey protein solution be low, since low concentrations of minerals such as sodium, calcium, potassium, magnesium, and phosphate are preferable in protein hydrolysates from a nutrition and health perspective. Furthermore, minerals including, for example, sodium and calcium can interact with whey proteins and affect the product turbidity, aggregation behavior, and heat tolerance of whey protein hydrolysates. At high concentrations, some minerals, especially sodium, calcium, and zinc, can inhibit or promote the proteolytic activity of some proteases, and thus, the presence of high concentrations of these ions can alter the concerted cleavage pattern of proteases.Thus, in one embodiment of the invention, the mineral content in the whey protein solution is 10% or less based on the total solids content, more preferably 8% or less. In the context of the present invention, the term minerals refers to the ash content. The terms mineral and ash can be used interchangeably and refer to the same concepts. Therefore, referring to the mineral content of the whey protein solution should be understood as the ash content of the whey protein solution. Petition 870230043920, dated 05 / 25 / 2023, p. 28 / 101 20 / 86 milk.

[082] In one embodiment of the invention, the whey protein solution comprises 30% or more by weight of BLG based on the total protein content, such as 40% or more by weight of BLG. More preferably, the whey protein solution comprises 50% or more by weight of BLG based on the total protein content, even more preferably the whey protein solution comprises BLG in an amount of 55% or more by weight based on the total protein content. In another embodiment of the invention, the whey protein solution comprises BLG in an amount in the range of 30 to 95% by weight of BLG based on the total protein content, such as 40 to 90% by weight of BLG based on the total protein content, even more preferably 45 to 80% by weight based on the total protein content.

[083] In the context of the present invention, the term “whey” relates to the liquid composition that remains when casein is removed from milk. Casein can, for example, be removed by microfiltration, yielding a liquid permeate that is free or essentially free of micellar casein but contains the native whey proteins. This liquid permeate is sometimes referred to as ideal whey, whey, or lactosorum.

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

[085] Step b) in the method according to the present invention relates to subjecting the whey protein solution to enzymatic hydrolysis, wherein the enzymatic hydrolysis is carried out using any of the following enzyme combinations: i. a combination of enzymes comprising at least one serine Petition 870230043920, dated 05 / 25 / 2023, page 29 / 101 21 / 86 endopeptidase from Bacillus, at least one serine endopeptidase from Aspergillus, and at least one trypsin-like protease; ii. a combination of enzymes comprising at least one serine endopeptidase from Bacillus, at least one serine endopeptidase from Aspergillus, and at least one leucyl-aminopeptidase from Aspergillus; iii. an enzyme combination comprising at least one bacillolysin from Bacillus amyloliquefaciens, at least one leucilaminopeptidase from Aspergillus, and at least bromelain.

[086] The inventors of the present invention have surprisingly discovered that enzymatic hydrolysis of a whey protein solution by adding any of the three enzyme combinations mentioned will result in whey protein hydrolysates with a degree of hydrolysis above 15%, a free amino acid content of 15% or less, and where the whey protein hydrolysate has an acceptable taste and a low content of bitter peptides. The inventors have found that whey protein hydrolysates prepared by hydrolysis with the enzyme combinations mentioned i. to iii. do not have a bitter taste in a 4% w / w protein solution.

[087] In step c) of the present invention, enzymatic hydrolysis is stopped by inactivating the enzymes when the degree of hydrolysis (DH) is 15% or more to obtain a whey protein hydrolysate. The degree of hydrolysis (DH) is defined as the percentage of peptide bonds in the original proteins that have been cleaved by hydrolysis.

[088] In one embodiment of the invention, the Aspergillus serine endopeptidase is a serine endopeptidase from Aspergillus oryzae and / or Aspergillus flavus. The Aspergillus serine endopeptidase is preferably a subtilisin-like serine endopeptidase (EC 3.4.21). Petition 870230043920, dated 05 / 25 / 2023, page 30 / 101 22 / 86

[089] In one embodiment of the invention, the enzyme combination i) further comprises a Bacillus metalloendopeptidase, such as bacillolysin. Therefore, in one embodiment, the enzyme combination i) comprises: - at least one Bacillus serine endopeptidase, at least one Bacillus metalloendopeptidase, at least one Aspergillus serine endopeptidase, and at least one trypsin-like protease.

[090] In another embodiment of the invention, the enzyme combination ii) may further comprise a Bacillus metalloendopeptidase, such as bacillolysin. Therefore, in one embodiment, the enzyme combination ii) comprises: - at least one serine endopeptidase from Bacillus, at least one metalloendopeptidase from Bacillus, at least one serine endopeptidase from Aspergillus, and at least one leucyl-aminopeptidase from Aspergillus.

[091] In one embodiment of the invention, the leucil-aminopeptidase from Aspergillus is from Aspergillus oryzae.

[092] In one embodiment, the Bacillus serine endopeptidase is subtilisin. Subtilisin is preferentially from Bacillus licheniformis.

[093] In another embodiment of the invention, the metalloendopeptidase is bacillolysin. The bacillolysin is preferably from Bacillus, and more preferably from Bacillus amyloliquefaciens. In a preferred embodiment of the invention, the bacillolysin is not from Bacillus subtilis.

[094] In the context of the present invention, the term “trypsin-like protease” refers to a protease of microbial origin. Preferably, the microbial trypsin-like protease is from Fusarium sp., in particular Fusarium oxsporum. Therefore, the term “trypsin-like protease”, for example, does not include pancreatin that is not of microbial origin. By Petition 870230043920, dated 05 / 25 / 2023, page 31 / 101 23 / 86 On the contrary, pancreatin is a mixture of enzymes derived from the pancreas that, for example, includes trypsin, chymotrypsin, amylase, and lipase. Furthermore, the term "trypsin-like protease" should not be confused with "trypsin".

[095] In yet another form, bromelain is from Ananas comosus. Bromelain is a cysteine ​​endopeptidase.

[096] In one embodiment of the invention, the enzymatic hydrolysis in step b) is carried out using any of the following enzyme combinations: i) a combination of enzymes comprising at least one serine endopeptidase from Bacillus, at least one serine endopeptidase from Aspergillus, and at least one trypsin-like protease, ii) a combination of enzymes comprising at least one serine endopeptidase from Bacillus, at least one serine endopeptidase from Aspergillus, and at least one leucyl-aminopeptidase from Aspergillus, iii) a combination of enzymes comprising at least one combination of a serine endopeptidase and metalloendopeptidase from Bacillus, at least one serine endoprotease from Aspergillus, and at least one leucyl-aminopeptidase from Aspergillus, iv) a combination of enzymes comprising at least one bacillolysin from Bacillus amyloliquefaciens, at least bromelain, and at least one leucyl-aminopeptidase from Aspergillus.

[097] In a preferred embodiment of the invention, the enzymatic hydrolysis in step b) is carried out using any of the following enzyme combinations: i) a combination of enzymes comprising at least one serine endopeptidase from a Bacillus species, at least one serine endopeptidase from Aspergillus oryzae, and at least one protease similar to Petition 870230043920, dated 05 / 25 / 2023, page 32 / 101 24 / 86 trypsin of microbial origin, ii) a combination of enzymes comprising at least one subtilisin from a Bacillus species, at least one serine endopeptidase from Aspergillus oryzae, and at least one leucil-aminopeptidase from Aspergillus oryzae, iii) a combination of enzymes comprising at least one combination of bacillolysin and subtilisin from a Bacillus species, at least one serine endoprotease from Aspergillus oryzae, and at least one leucil-aminopeptidase from Aspergillus oryzae, iv) a combination of enzymes comprising at least one bacillolysin from Bacillus amyloliquefaciens, at least one bromelain from Ananas comosus, and at least one leucil-aminopeptidase from Aspergillus oryzae.

[098] In preferred embodiments of the invention, the enzyme combinations are: i) a combination of enzymes comprising at least one enzyme from group EC 3.4.21.62, at least one other enzyme from group EC 3.4.21, and at least one other enzyme from group EC 3.4.21.4, ii) a combination of enzymes comprising at least one enzyme from group EC 3.4.21.62, at least one other enzyme from group EC 3.4.21 and at least one other enzyme from group EC 3.4.11, iii) a combination of enzymes comprising at least one enzyme from group EC 3.4.21.62, another enzyme from group EC 3.4.24.28, at least one other enzyme from group EC 3.4.21 and at least one other enzyme from group EC 3.4.11, iv) a combination of enzymes comprising at least one enzyme from group EC 3.4.24.28, at least one other enzyme from group EC 3.4.22.32 and at least one other enzyme from EC group 3.4.11.

[099] In another preferred embodiment of the invention, combinations of Petition 870230043920, dated 05 / 25 / 2023, page 33 / 101 25 / 86 enzymes are: i) comprising at least one serine endopeptidase from Bacillus licheniformis, at least one serine endopeptidase from Aspergillus oryzae, and at least one trypsin-like protease from Fusarium oxysporum, optionally also bacillolysin from Bacillus amyloliquefaciens, ii) comprising at least one serine endopeptidase from Bacillus licheniformis, at least one serine endopeptidase from Aspergillus oryzae and at least one leucilaminopeptidase from Aspergillus oryzae, iii) comprising at least one serine endopeptidase from Bacillus licheniformis, one bacillolysin from Bacillus amyloliquefaciens, at least one serine endopeptidase from Aspergillus oryzae and at least one leucilaminopeptidase from Aspergillus oryzae, iv) comprising at least one bacillolysin from Bacillus amyloliquefaciens, at least bromelain from Ananas comosus and at least one leucyl aminopeptidase from Aspergillus oryzae.

[100] The serine endopeptidase of Bacillus licheniformis is preferentially subtilisin.

[101] In another preferred embodiment of the invention, the enzyme combinations are: i) comprising at least one Bacillus serine endopeptidase (EC 3.4.21.62), at least one Aspergillus serine endopeptidase (EC 3.4.21), and at least one trypsin-like protease (EC 3.4.21.4), ii) comprising at least one Bacillus serine endopeptidase (EC 3.4.21.62), at least one Aspergillus serine endopeptidase (EC 3.4.21), and at least one Aspergillus leucyl-aminopeptidase (EC 3.4.11), iii) comprising at least one Bacillus serine endopeptidase (EC 3.4.21.62), one bacillolysin (EC 3.4.24.28), and at least one serine Petition 870230043920, dated 05 / 25 / 2023, page 34 / 101 26 / 86 Aspergillus endopeptidase (EC 3.4.21.63) and at least one Aspergillus leucilaminopeptidase (EC 3.4.11), iv) comprising at least one Bacillus amyloliquefaciens bacillolysin (EC 3.4.24.28), at least bromelain (EC 3.4.22.32), and at least one Aspergillus leucilaminopeptidase (EC 3.4.11).

[102] The enzyme combinations i) to iv) used in the present method for preparing a whey protein hydrolysate may comprise other enzymes besides the primary enzymes mentioned. By the term “primary enzymes” is meant the enzymes that are most abundant in the preparations. In the following list, the Uniprot accession numbers are given in parentheses to identify proteases noted with a specific name.For example, enzyme combinations may comprise one or more enzymes with high sequence identity (95-100%) to enzymes selected from the group consisting of peptide hydrolase (A0A364MDR7), subtilase family protein (I8A6W5), fungallysin metallopeptidase M36 (A0A2P2H013), neutral protease 2 (A0A364MH70), aspergillopepsin-1 (B8NLY9), leucilaminopeptidase A (Q2U1F3), leucilaminopeptidase 2 (Q2ULM2), dipeptidylpeptidase 4 (Q2UH35), dipeptidyl peptidase 5 (Q9Y8E3), neutral protease 1 (Q2U1G7), neutral protease 2 (P46076), alkaline protease 1 (P12547), and prolyl family protein. oligopeptidase (B8NBM3).

[103] In one aspect of the invention, the primary enzymes of the enzyme combination i) are a Bacillus serine endopeptidase, an Aspergillus serine endopeptidase, and a trypsin-like protease. The Bacillus serine endopeptidase is preferably subtilisin, and the Aspergillus serine endopeptidase is preferably a protein of the subtilase family. In one embodiment of the invention, the enzyme combination i) may further comprise one or more aminopeptidases (such as peptide hydrolase and leucyl-aminopeptidase), Petition 870230043920, dated 05 / 25 / 2023, page 35 / 101 27 / 86 metalloendopeptidase (such as bacillolysin and fungallysin metallopeptidase, neutral protease), prolyl oligopeptidase family protein, and aspergillopepsin-1 (aspartic endopeptidase). It is expected that at least 80% of the enzymes present in enzyme combination i) will be Bacillus serine endopeptidase, Aspergillus serine endopeptidase, and trypsin-like protease. An example of a preparation comprising a Bacillus serine endopeptidase is Protamex (Novozymes A / S). Protamex also contains bacillolysin. Examples of preparations comprising an Aspergillus serine endopeptidase are Promod 782 (Biocatalysts Ltd) and Protease A Amano 2 SD (Amano Enzyme Ltd), where the primary enzyme is an Aspergillus ozyzae serine endopeptidase.Promod 782 and Protease A Amano 2 SD also comprise the enzymes peptide hydrolase, leucyl-aminopeptidase, fungallysin metallopeptidase M36, prolyl oligopeptidase family protein, neutral protease 2 and aspergylopepsin-1, and the primary enzymes are serine endopeptidases. Trypsin-like protease can, for example, be supplied by Formea ​​TL 1200 BG (Novozymes A / S).

[104] In one aspect of the invention, the enzyme combination ii) comprises Bacillus serine endopeptidase, Aspergillus serine endopeptidase, and Aspergillus leucyl-aminopeptidase as the primary enzymes. The Bacillus serine endopeptidase is preferably subtilisin, and the Aspergillus serine endopeptidase is preferably a protein of the subtilase family and an alkaline protease. The Aspergillus leucyl-aminopeptidases may be, for example, one or more peptide hydrolases, leucyl-aminopeptidase A, and leucyl-aminopeptidase 2. In one embodiment of the invention, the enzyme combination ii) may further comprise one or more of the metalloendopeptidases: fungallysin metallopeptidase M36, neutral protease 1, and neutral protease 2. The enzyme combination ii) may also comprise aspergillopepsin-1 (endopeptidase Petition 870230043920, dated 05 / 25 / 2023, page 36 / 101 28 / 86 aspartic acid), dipeptidyl peptidase 4 and dipeptidyl peptidase 5. It is expected that at least 80% of the enzymes present in enzyme combination ii) will be Bacillus serine endopeptidase, Aspergillus serine endopeptidase and Aspergillus leucilaminopeptidase. An example of a preparation comprising a Bacillus serine endopeptidase is Alcalase (Novozymes A / S) comprising subtilisin. Examples of preparations comprising an Aspergillus serine endopeptidase are Protease A Amano 2 SD and Promod 782, where the primary enzyme is an Aspergillus ozyzae serine endopeptidase. Promod 782 and Protease A Amano 2 SD also comprise the enzymes peptide hydrolase, leucilaminopeptidase, fungallysin metallopeptidase M36, prolyl oligopeptidase family protein, neutral protease 2, Aspergillopepsin-1, and the primary enzymes are serine endopeptidases.An example of a preparation comprising Aspergillus leucyl-aminopeptidase is Flavourzyme Conc BG (Novozymes A / S), where the primary enzyme is a leucyl-aminopeptidase. Flavourzyme Conc BG also comprises leucyl-aminopeptidase A, leucyl-aminopeptidase 2, dipeptidylpeptidase 4, dipeptidylpeptidase 5, neutral protease 1, neutral protease 2, alkaline protease 1, and the primary enzyme is leucyl-aminopeptidase.

[105] In one aspect of the invention, the enzyme combination iii) comprises Bacillus serine endopeptidase, Bacillus metalloendopeptidase, Aspergillus serine endopeptidase and an Aspergillus leucyl-aminopeptidase as the primary enzymes. The Bacillus serine endopeptidase is preferably subtilisin, the Bacillus metalloendopeptidase is preferably bacillolysin and the Aspergillus serine endopeptidase is preferably a protein of the subtilase family and an alkaline protease. The Aspergillus leucyl-aminopeptidases may be, for example, one or more peptide hydrolases, leucyl-aminopeptidase A and leucyl-aminopeptidase 2. In a Petition 870230043920, dated 05 / 25 / 2023, page 37 / 101 29 / 86 embodiment of the invention, the enzyme combination iii) may further comprise one or more of the metalloendopeptidases; fungallysin metallopeptidase M36, neutral protease 1 and neutral protease 2. The enzyme combination ii) may also comprise aspergillopepsin-1 (aspartic endopeptidase), dipeptidylpeptidase 4 and dipeptidyl peptidase 5. At least 80% of the enzymes present in the enzyme combination iii) are Bacillus serine endopeptidase, bacillolysin, Aspergillus serine endopeptidase and an Aspergillus leucyl-aminopeptidase. Examples of preparations comprising Bacillus serine endopeptidase (subtilisin) and bacillolysin are Promod 950L (Biocatalysts Ltd) and Protamex. Examples of preparations comprising an Aspergillus serine endopeptidase are Protease A Amano 2 SD and Promod 782.Promod 782 and Protease A Amano 2 SD also comprise the enzymes peptide hydrolase, leucil-aminopeptidase, fungallysin metallopeptidase M36, prolyl oligopeptidase family protein, neutral protease 2, and aspergillopepsin-1, and the primary enzymes are serine endopeptidases. An example of a preparation comprising Aspergillus leucil-aminopeptidase is Flavourzyme conc BG where the primary enzyme is a leucil-aminopeptidase. Flavourzyme conc BG also comprises the enzymes leucil-aminopeptidase A, leucil-aminopeptidase 2, dipeptidylpeptidase 4, dipeptidyl peptidase 5, neutral protease 1, neutral protease 2, and alkaline protease 1, and the primary enzyme is leucil-aminopeptidase.

[106] In one aspect of the invention, the enzyme combination iv) comprises bacillolysin from Bacillus amyloliquefaciens, bromelain and leucilaminopeptidase from Aspergillus as the primary enzymes. The leucilaminopeptidases from Aspergillus may be, for example, one or more peptide hydrolases, leucil-aminopeptidase A and leucil-aminopeptidase 2. In one embodiment of the invention, the enzyme combination iv) may further comprise Petition 870230043920, dated 05 / 25 / 2023, p. 38 / 101 30 / 86 one or more of the metalloendopeptidases; fungallysin metallopeptidase M36, neutral protease 1 and neutral protease 2. Enzyme combination ii) may also comprise dipeptidylpeptidase 4, dipeptidyl peptidase 5 and alkaline protease (Aspergillus serine protease). At least 80% of the enzymes present in enzyme combination iv) are expected to be bacillolysins from Bacillus amyloliquefaciens, bromelain and leucyl-aminopeptidase from Aspergillus. An example of a preparation comprising bacillolysin from Bacillus amyloliquefaciens is Neutrase. An example of bromelain is Promod 523 MDP, while an example of leucyl-aminopeptidase from Aspergillus is Flavourzyme conc BG.

[107] The method for preparing whey protein hydrolysates of the present invention should not be limited by the amount of enzymes added in the hydrolysis step, since the amount of enzyme added depends on the type of enzyme and the enzyme activity. However, as a guideline, enzymatic hydrolysis is carried out with a combination of enzymes, where the total amount of enzymes is in the range of 0.05 to 10 g per 100 g of protein, such as 0.1 to 7.5 g of enzyme per 100 g of protein. Preferably, the amount of enzyme is in the range of 0.2 to 5.0 g per 100 g of protein.

[108] The ratio between the three different enzymes in combinations i. to iii. may, for example, be in the range of 1-10:1-10:1-10, as well as in the range of 1-8:1-8:1-8. However, the present invention should not be limited to the amount of enzyme added, since this will be dependent on the activity of the enzymes used.

[109] The enzymatic hydrolysis performed in step b) of the present invention is preferably carried out at a temperature in the range of 40 °C to 75 °C, such as 40 °C to 70 °C. The enzymatic hydrolysis should be carried out at a temperature at which the enzymes have optimal activity. In one embodiment Petition 870230043920, dated 05 / 25 / 2023, p. 39 / 101 31 / 86 preferably, enzymatic hydrolysis is carried out at a temperature in the range of 45 °C to 65 °C.

[110] The time period for enzymatic hydrolysis before hydrolysis is stopped in step c) depends on the quantity and activity of the enzymes used. Hydrolysis is continued until the degree of hydrolysis is 15% or more. Enzymatic hydrolysis in step b) is preferably carried out in a time period in the range of 3 hours to 20 hours, such as 3.5 hours to 15 hours, preferably 4 hours to 10 hours and even more preferably 4 hours to 7 hours.

[111] The whey protein solution should preferably have a pH in the range of 6 to 9 during enzymatic hydrolysis. In a preferred embodiment, the pH during enzymatic hydrolysis in step b) is 6.5 to 8.0. In this pH range, the enzymes have more activity and therefore cleave the proteins into peptides and free amino acids more efficiently. Additionally, in this pH range aggregation is avoided, both during the hydrolysis process and also during the heat treatment to inactivate the enzymes.

[112] In step c) of the method for preparing a whey protein hydrolysate according to the present invention, enzymatic hydrolysis is stopped by inactivating the enzymes. In the context of the present invention, the term inactivation refers to the irreversible inactivation of the enzyme. Enzyme inactivation must be irreversible so that the enzymes do not become active under other conditions.

[113] Hydrolysis is stopped when the degree of hydrolysis is at least 15%, such as at least 18%, preferably at least 20%. In one embodiment of the invention, hydrolysis is stopped at step c) when the degree of hydrolysis is in the range of 15% to 35%, preferably 17% to 30%, and even more preferably 18% to 28%. Petition 870230043920, dated 05 / 25 / 2023, p. 40 / 101 32 / 86

[114] The inactivation of enzymes in step c) and therefore the interruption of hydrolysis can be done by any method known in the art. For example, enzyme inactivation by changing the temperature to a temperature at which the enzymes are inactive and denatured. Enzyme inactivation and denaturation can also be done by changing the pH of the solution to a pH at which the enzymes are inactive.

[115] Therefore, in one embodiment of the invention, the inactivation of the enzymes in step c) is by heating the whey protein solution with added enzymes to a temperature of at least 80 °C. Enzyme inactivation is preferably by heating to a temperature of 80 °C to 130 °C, such as 85 °C to 125 °C, even more preferably 90 °C to 120 °C. Enzyme inactivation in step c) by heating can be, for example, by heating to a high temperature for a short period of time, such as heating to a temperature of 110 °C to 130 °C for 10 to 30 seconds. Alternatively, enzyme inactivation in step c) can be by heating to a relatively low temperature, but for a longer period of time. This may involve heating from 80 °C to 90 °C for 5 to 10 minutes.

[116] In another embodiment of the present invention, the irreversible inactivation of enzymes in step c) comprises increasing or decreasing the pH of the whey protein solution with added enzymes, i.e., the whey protein hydrolysate, to a pH at which the enzymes are inactive. In one embodiment of the invention, the pH is increased to a pH of 10 or above. In another embodiment, the pH is decreased to a pH of 4 or below.

[117] In a preferred embodiment of the invention, the method does not comprise any ultrafiltration step of the whey protein hydrolysate obtained in step c). It was surprising to the inventors of the present Petition 870230043920, dated 05 / 25 / 2023, page 41 / 101 33 / 86 invention that the enzymatic hydrolysis of a whey protein solution with a low amount of lipids, i.e., a WPI or SPI, with the specified enzyme combinations resulted in whey protein hydrolysates with a palatable taste and that were also clear in appearance, without any ultrafiltration steps involved.

[118] The whey protein hydrolysates known in the art can be divided into ultrafiltered and non-ultrafiltered hydrolysates. The known non-ultrafiltered protein hydrolysates will have an obscure or cloudy appearance, whereas ultrafiltered protein hydrolysates are generally clear in appearance.

[119] In the context of the present invention, the term ultrafiltration refers to membrane filtration with a membrane having a cutoff in the range of 1500 Da to 50000 Da, preferably 2000 Da to 20000 Da.

[120] During ultrafiltration of protein hydrolysates, fat, intact protein, as well as some of the larger peptides, are retained by the ultrafiltration membrane and retained in the retentate, while free amino acids, smaller peptides and minerals are in the ultrafiltration permeate.

[121] In one embodiment of the present invention, the whey protein solution is prepared as a WPI and SPI solution with a low lipid content. It was surprising to the inventors of the present invention that the preparation of a whey protein hydrolysate with the enzyme combinations of the present invention resulted in whey protein hydrolysates with a high degree of hydrolysis, a good taste with low bitterness, and a clear appearance.

[122] However, the low lipid content is not the only explanation for why the inventors of the present invention were able to prepare clear protein hydrolysates. The inventors of the present invention discovered Petition 870230043920, dated 05 / 25 / 2023, page 42 / 101 34 / 86 Surprisingly, when a low-lipid whey protein solution was subjected to enzymatic hydrolysis with any of the enzyme combinations mentioned, it was possible to prepare a whey protein hydrolysate with a degree of hydrolysis above 15%, without a bitter taste in a 4% protein solution and with a clear appearance. To achieve a clear appearance, it was necessary to use a low-lipid whey protein solution. However, the low lipid content was not the only reason why clear hydrolysates were obtained. It was surprisingly discovered by the inventors of the present invention that hydrolysis with specific enzyme combinations leads to clear hydrolysates.In comparative tests, it was observed that other low-lipid whey protein hydrolysates, but in which hydrolysis was performed with enzymes other than those used in the method of the present invention, did not result in whey protein hydrolysates with a clear appearance and low bitterness.

[123] The whey protein hydrolysate obtained by the method of the present invention may preferably be concentrated and / or dried. Therefore, in one embodiment of the invention, the method comprises a step d) of concentrating and / or drying the whey protein hydrolysate obtained in step c). The concentration may be, for example, by one or more of the following operations: nanofiltration, reverse osmosis filtration and evaporation.

[124] In another embodiment of the invention, the drying step involves one or more of the following operations: spray drying, freeze-drying and centrifugal drying, rotary drying and / or fluidized bed drying may also be used. Whey protein hydrolysate

[125] In one aspect, the present invention relates to a Petition 870230043920, dated 05 / 25 / 2023, page 43 / 101 35 / 86 hydrolyzed whey protein comprising: - free amino acids and peptides, and - having a degree of hydrolysis of at least 15%, and - peptides having a molecular weight of 2500 Da or more in an amount of 25% by weight or less of the total amount of peptides, and - free amino acids in an amount of 15% by weight or less of the total amino acid content in the hydrolysate, and wherein the whey protein hydrolysate in a 4% protein solution has a bitterness score corresponding to a solution of 0.08% w / v or less of caffeine.

[126] In one aspect of the invention, the degree of hydrolysis of the whey protein hydrolysate of the invention is at least 15%. An objective of the present invention was to make a whey protein hydrolysate with a high degree of hydrolysis, which without any ultrafiltration step would not have an unpleasant bitter taste. It is well known that peptides are responsible for the bitter taste in many hydrolysates. In general, extensive hydrolysis yielding hydrolysates with a high degree of hydrolysis is expected to result in hydrolysates with a bitter taste. To reduce the bitter taste of extensively hydrolyzed protein hydrolysates, the hydrolysates can be treated with activated charcoal which can be removed again along with the bitter-tasting peptides. Extensively hydrolyzed proteins may be desirable because the peptides have other functionalities besides the intact protein.For example, peptides can withstand heat treatments better than intact whey protein.

[127] However, the inventors of the present invention have surprisingly discovered a method of preparing a whey protein hydrolysate with a high degree of hydrolysis that does not have a bitter taste. Petition 870230043920, dated 05 / 25 / 2023, page 44 / 101 36 / 86 unpleasant, although the whey protein hydrolysate has not been subjected to any additional treatments to remove the bitter-tasting peptides.

[128] In a preferred embodiment of the invention, the degree of hydrolysis of the whey protein hydrolysate is at least 18%, even more preferably the degree of hydrolysis of the whey protein hydrolysate is at least 20%.

[129] In another embodiment of the present invention, the whey protein hydrolysate according to the invention has a degree of hydrolysis of 15 to 35%, such as 18 to 30%, preferably 18 to 28% and even more preferably 20 to 25%.

[130] Whey protein hydrolysate may comprise free amino acids and, if free amino acids are present, they shall be present in an amount of 15% by weight or less of the total amino acid content in the hydrolysate. Preferably, the free amino acid content is 12% by weight or less of the total amino acid content. By the term “total amino acid content” in the context of the present invention means the total amount of amino acids present, including free amino acids and amino acids bound in peptides and proteins.

[131] In some embodiments of the invention, the free amino acid content in whey protein hydrolysate is no more than 15% by weight of the total amino acid content, such as no more than 13% by weight of the total amino acid content, preferably no more than 10% by weight of the total amino acid content, even more preferably, the free amino acid content is no more than 8% of the total amino acid content.

[132] In other embodiments of the invention, the whey protein hydrolysate comprises free amino acids in an amount of 2 to 15% in Petition 870230043920, dated 05 / 25 / 2023, page 45 / 101 37 / 86 by weight of the total amino acid content in the hydrolysate, preferably free amino acids in an amount of 4 to 13% by weight of the total amino acid content in the hydrolysate.

[133] The inventors of the present invention have discovered that the peptides in the whey protein hydrolysate of the invention comprise peptides with a molecular weight of 2500 Da or more in an amount of 25% by weight or less of the total amount of peptides. Preferably, the whey protein hydrolysate comprises peptides with a molecular weight of 2500 Da or more in an amount in the range of 8 to 25% by weight, even more preferably 10 to 20% by weight.

[134] In one embodiment of the invention, the whey protein hydrolysate of the invention comprises peptides with a molecular weight of 375 Da or less in an amount of at least 10% by weight. The peptides with a molecular weight of 375 Da or less may, for example, be present in the whey protein hydrolysate in an amount in the range of 10 to 25% by weight.

[135] The inventors of the present invention have found that the whey protein hydrolysates of the invention have a reduced bitter taste compared to known whey protein hydrolysates with a high degree of hydrolysis, even if those have been subjected to membrane filtration with an ultrafiltration membrane and / or subjected to treatment with activated carbon.

[136] The bitterness of whey protein hydrolysates was compared to the bitterness of caffeine, and whey protein hydrolysates in a 4% w / w protein solution have a less bitter taste than the taste of a 0.08% w / v caffeine solution.

[137] Therefore, the bitterness score of protein hydrolysates of Petition 870230043920, dated 05 / 25 / 2023, page 46 / 101 38 / 86 whey in a 4% w / w protein solution corresponds to a bitterness score of 0.08% w / v caffeine or less. Preferably, the bitterness score of the whey protein hydrolysates in a 4% w / w protein solution of the invention corresponds to a bitterness score of 0.07% w / v caffeine or less, even more preferably to a bitterness score of 0.065% caffeine or less. Most preferably, the bitterness score of the whey protein hydrolysates in a 4% w / w protein solution of the invention corresponds to a bitterness score of 0.060% w / v.

[138] In a further embodiment of the invention, the whey protein hydrolysate has a nephelometric turbidity (NTU) of 100 or less in a 4% w / w protein solution. It is a further embodiment of the invention to obtain a whey protein hydrolysate that, in addition to having a high degree of hydrolysis and an acceptable taste, is also clear in appearance. A clear and good-tasting whey protein hydrolysate is desired, since it can be used, for example, in drinks, gels and shakes and have an improved appeal to the consumer.

[139] If the measured nephelometric turbidity is less than 100 NTU in a 4% w / w protein solution, the sample is perceived as transparent. If the nephelometric turbidity is above 100 NTU in a 4% protein solution, the measured whey protein hydrolysate is perceived as not being transparent. If the nephelometric turbidity is below 40 NTU, the solution is perceived as clear. However, a whey protein hydrolysate with turbidity between 40 and 100 NTU may be transparent (but unclear or cloudy). In the context of the present invention, the term transparent refers to a solution that allows some light to pass through so that objects behind the solution can be seen, i.e., it is possible Petition 870230043920, dated 05 / 25 / 2023, p. 47 / 101 39 / 86 see through the solution. The term clear refers to a solution that is colorless, and therefore it is possible to see through the solution without anything limiting its translucency. Therefore, a solution can be transparent but not clear.

[140] In another embodiment of the invention, the whey protein hydrolysate has a nephelometric turbidity (NTU) of 80 or less in a 4% w / w protein solution, such as a nephelometric turbidity (NTU) of 60 or less in a 4% w / w protein solution, preferably a nephelometric turbidity (NTU) of 50 or less in a 4% w / w protein solution, even more preferably a nephelometric turbidity (NTU) of 40 or less in a 4% w / w protein solution.

[141] In another embodiment of the invention, the whey protein hydrolysate has antioxidant activity. Preferably, the antioxidant activity of the whey protein hydrolysate is measured as having a 54 to 60 elimination percentage in a 1.5% by weight protein solution.

[142] Whey protein hydrolysate may contain other ingredients besides protein, for example, carbohydrates, lipids and minerals.

[143] In one embodiment of the invention, the whey protein hydrolysate comprises lipids in an amount of 8% by weight or less based on the total solids content, such as 6% by weight or less based on the total solids content. In another embodiment, the whey protein hydrolysate comprises lipids in an amount of 1% by weight or less based on the total solids content, such as a lipid content of 0.5% by weight or less of the total solids content.

[144] Whey protein hydrolysate may also contain minerals such as potassium, sodium and calcium.

[145] In one embodiment of the invention, whey protein hydrolysate Petition 870230043920, dated 05 / 25 / 2023, page 48 / 101 40 / 86 of milk contains potassium in an amount of 3.0% by weight or less.

[146] In another embodiment of the invention, the whey protein hydrolysate comprises sodium in an amount of 2% by weight or less.

[147] In yet another embodiment of the invention, the whey protein hydrolysate comprises citric acid in an amount in the range of 4 to 10 g per kg of solids content of the whey protein hydrolysate.

[148] In one embodiment of the invention, the whey protein hydrolysate comprises intact or undegraded bovine serum albumin (BSA) in an amount of 0.5 to 2% by weight based on total protein content.

[149] In a preferred embodiment of the invention, the whey protein hydrolysate is in the form of a dry composition, such as a powder or granules.

[150] In another embodiment, whey protein hydrolysate is a liquid composition. Food products:

[151] In one aspect, the present invention relates to providing a food product comprising whey protein hydrolysate according to the invention.

[152] The food product may, for example, be any product selected from the dairy group, including a drink, a shake, a gel, a food bar, a concentrate or a liquid shot.

[153] In preferred modalities, the food product is selected from the group of a protein drink, a protein serving, a protein shake, a protein gel or a protein bar. The food product may also be an infant formula or other infant nutrition products.

[154] If the food product is in liquid form, such as a drink, shake, gel or shot, the food product may comprise Petition 870230043920, dated 05 / 25 / 2023, page 49 / 101 41 / 86 Whey protein hydrolysate according to the invention. The whey protein hydrolysate is preferably present in the beverage in an amount corresponding to 2 to 25% by weight of hydrolyzed whey protein, preferably 3 to 20% by weight of hydrolyzed whey protein, such as 3 to 15% by weight of hydrolyzed whey protein, even more preferably forming 3 to 10% by weight of hydrolyzed whey protein.

[155] If the food product is a bar, such as a protein bar, the food product comprises hydrolyzed whey protein according to the invention in an amount corresponding to 2 to 30% by weight of hydrolyzed whey protein. Preferably, the amount of hydrolyzed whey protein according to the invention is present in a bar in an amount corresponding to 3 to 20% by weight of hydrolyzed whey protein, such as 4 to 15% by weight. If the hydrolyzed whey protein according to the invention is used in a food bar, such as for example a protein bar, the hydrolyzed whey protein can be used as a softener. It is well known that increasing concentrations of hydrolyzed protein in protein bars have a softening effect and prevent the bars from hardening during long-term storage.

[156] In a further aspect, the invention relates to providing a beverage comprising hydrolyzed whey protein according to the invention in an amount corresponding to 2 to 20% by weight of hydrolyzed whey protein. The beverage may be, for example, a protein drink which in addition to protein comprises carbohydrates, vitamins and minerals.

[157] In a preferred embodiment of the invention, the beverage has a pH Petition 870230043920, dated 05 / 25 / 2023, page 50 / 101 42 / 86 neutral, that is, a pH in the range of 6.5 to 8.0 in a 4% protein solution at 22 °C.

[158] In one embodiment of the invention, the whey protein hydrolysate according to the invention can be used as an ingredient in the preparation of a carbonated beverage. Therefore, the food product of the invention comprising the whey protein hydrolysate according to the invention is a carbonated beverage.

[159] A further embodiment of the invention relates to a carbonated beverage comprising the whey protein hydrolysate of the invention.

[160] In one embodiment, the carbonated beverage comprises whey protein hydrolysate in an amount corresponding to 2 to 10% by weight. The carbonated beverage may also comprise carbohydrates and, if carbohydrates are present, it is in an amount of 5% by weight or less. The carbonated beverage preferably does not contain fat.

[161] In one embodiment, the amount of carbonation in a carbonated beverage comprising the whey protein hydrolysate of the invention ranges from 0.1 volumes of carbonation (per volume of liquid present in the beverage) to 4 volumes of carbonation. More typically, the amount of carbonation ranges from about 1.6 volumes to about 3.5 volumes, with the most typical concentration ranging from about 1.7 volumes to about 3.0 volumes, and the amount of carbonation is most preferably in the range of 2.0 to 3.0 volumes. By carbonation in the context of the present invention is meant adding carbon dioxide to a mixture of beverage ingredients in an amount sufficient to obtain a carbonated protein beverage where the amount of carbonation present in the beverage ranges from Petition 870230043920, dated 05 / 25 / 2023, p. 51 / 101 43 / 86 0.1 volumes to 4 volumes per volume of liquid mixture.

[162] In some embodiments of the method, carbon dioxide is added in the form of sterile carbonate water. In other embodiments, sterile carbon dioxide is bubbled through the liquid mixture until the desired amount of carbon dioxide is present.

[163] Carbonation increases the acidity of a beverage. The more carbon dioxide is added to a beverage, the lower the pH value of the beverage. However, the inventors of the present invention have found that the addition of carbon dioxide will decrease the pH of the beverage to a minimum of pH 5.5-6.0 at a temperature of 5 °C. The addition of about 2.5-3.0 volumes of carbon dioxide per volume of beverage resulted in a reduction of the pH of the beverage to about pH 6.0 at a temperature of 5 °C, while the addition of about 4 volumes of carbon dioxide per volume of beverage resulted in a reduction of the pH of the beverage to about pH 5.5 at a temperature of 5 °C. Therefore, in one embodiment of the invention, the carbonated beverage comprising the whey protein hydrolysate of the invention has, at a temperature of 5 °C, a pH of at least 5.5. The pH will typically be in the range of 5.5 to 8.25, as well as in the range of 5.5 to 7.0, preferably a pH in the range of 5.8 to 6.5.A carbonated beverage with a pH above 5.5 is preferable.

[164] The carbonated beverage comprising the whey protein hydrolysate of the invention can, for example, be heat-treated, such as, for example, pasteurized or autoclaved. The inventors of the present invention have found that the turbidity of a carbonated beverage comprising the whey protein hydrolysate of the invention does not change after heat treatment at temperatures up to 120 °C for an extended period of time, such as, for example, 20 minutes.

[165] In a further embodiment of the invention, a carbonated beverage Petition 870230043920, dated 05 / 25 / 2023, page 52 / 101 44 / 86 may comprise whey protein hydrolysate according to the invention in combination with a non-hydrolyzed whey protein isolate.

[166] The whey protein hydrolysate of the invention can also be used in the preparation of a protein shot, a protein shake or a protein gel. The protein shot, shake or gel may, in addition to hydrolyzed whey protein in an amount of 2 to 20% by weight, comprise carbohydrates, vitamins and minerals. The pH of the protein shot, protein shake or protein gel is preferably neutral, i.e., a pH in the range of 6.5 to 8.0.

[167] In other respects, the invention relates to the use of whey protein hydrolysates according to the invention as a food ingredient. The whey protein hydrolysate can be added to any type of food product as a food ingredient. Preferably, the whey protein hydrolysate of the invention is used as a food ingredient in the preparation of hot or cold beverages.

[168] In one embodiment of the invention, whey protein hydrolysate is used as a food ingredient in the preparation of stable UHT beverages with a pH in the range of 6.5 to 8.5.

[169] In another embodiment of the invention, whey protein hydrolysate is used as a food ingredient in the preparation of beverages used for sports nutrition. In the context of the present invention, the term sports nutrition refers to adequate nutrition in conjunction with exercise or training, i.e., for building muscle mass.

[170] In yet another embodiment of the invention, whey protein hydrolysate is used as a food ingredient in the preparation of clinical beverages. In the context of the present invention, the term clinical beverage refers to Petition 870230043920, dated 05 / 25 / 2023, p. 53 / 101 45 / 86 beverages with a clinical or medical indication. For example, a clinical beverage may have a health-related effect. A clinical beverage is typically used by hospitalized or elderly people with nutritional difficulties or individuals who require pre-digested protein to recover from a medical condition. For example, a clinical beverage may be a beverage used by individuals suffering from malnutrition or malabsorption. The clinical beverage may also be for people suffering from gastrointestinal diseases. In the context of the present context, the term "clinical beverage" and "medicinal beverage" have the same meaning.

[171] A clinical beverage may, for example, comprise the whey protein hydrolysate of the invention in an amount corresponding to the beverage comprising from 2 to 20% by weight of hydrolyzed whey protein. The clinical beverage may comprise carbohydrates in an amount of 5 to 50% by weight of the beverage. The amount of carbohydrates may, for example, be in the range of 10 to 40% by weight, such as from 15 to 35% by weight. The clinical beverage may also contain fat. For example, the fat content in the clinical beverage may be in the range of 2 to 30% by weight, such as from 3 to 20% by weight, more preferably from 3 to 18% by weight.

[172] In one example, the clinical beverage comprises hydrolyzed whey protein according to the invention corresponding to an amount of 4-10% by weight, 3-15% by weight of fat and 10-35% by weight of carbohydrates.

[173] The clinical beverage preferably has a neutral pH value, that is, a pH in the range of 6.5 to 8.0.

[174] The clinical drink may be in the form of a clear drink, a milk-based drink, enteral nutrition, or in the form of a powder to be reconstituted into a liquid. Petition 870230043920, dated 05 / 25 / 2023, page 54 / 101 46 / 86

[175] In one embodiment, the whey protein hydrolysate of the invention can also be used in the preparation of a juice-type beverage. The juice-type beverage preferably comprises hydrolyzed whey protein according to the invention in an amount corresponding to 410% by weight of protein, 0-1% by weight of fat and 15-35% by weight of carbohydrates.

[176] If the clinical beverage is in the form of parenteral nutrition, it may comprise from 4 to 15% by weight of hydrolyzed whey protein according to the invention, about 5-35% by weight of carbohydrates and about 3 to 15% by weight of fat.

[177] The whey protein hydrolysate according to the present invention can also be used in infant nutrition, such as in infant formulas. In the context of the present invention, the term infant formula refers to any type of infant formula, including follow-on formula, growth formula and preterm formula.

[178] If the whey protein hydrolysate according to the present invention is used in an infant formula, the protein content in the infant formula is in the range of 1.6 to 5.0 g / 100 kcal. In addition to the whey protein hydrolysate, the infant formula may also contain carbohydrates such as lactose, oligosaccharides, lipids, vitamins and minerals.

[179] The whey protein hydrolysate according to the present invention can also be used in the preparation of other infant nutrition besides infant formulas, for example in smoothies, porridges and the like.

[180] The whey protein hydrolysate of the invention can also be used in the preparation of an emulsion. An emulsion will typically be prepared by reconstituting a whey protein hydrolysate powder in a liquid, for example, water or milk and fats. The hydrolysate of Petition 870230043920, dated 05 / 25 / 2023, page 55 / 101 47 / 86 Whey protein will have an emulsifying effect on water and fats. The powder will typically comprise whey protein hydrolysate in an amount corresponding to 5 to 15% by weight of protein. After reconstitution, the emulsion comprises proteins in an amount of 2 to 4% by weight.

[181] Protein hydrolysis causes changes in the protein, such as an increase in the number of charged groups, a decrease in average molecular weight, and exposure of reactive groups. These are the factors that influence the emulsion-forming and stabilizing abilities of protein hydrolysates. The whey protein hydrolysates of the present invention can be used as emulsifying agents, stabilizers, etc., by combining them with other ingredients.

[182] The whey protein hydrolysates of the invention can also be used in bakery products, for example in biscuits, cookies and crackers.

[183] ​​In other respects, the invention relates to the use of whey protein hydrolysate according to the invention as an antioxidant. The inventors of the present invention have surprisingly discovered that the whey protein hydrolysate of the invention has an antioxidant effect. Therefore, whey protein hydrolysate can be used in nutritional compositions as a source of antioxidant peptides.

[184] Therefore, the present invention relates to a whey protein hydrolysate of the invention with an antioxidant effect. More particularly, the present invention relates to a whey protein hydrolysate with an antioxidant effect defined by a 54 to 60% elimination percentage in a solution comprising 1.5% by weight of protein. A Petition 870230043920, dated 05 / 25 / 2023, page 56 / 101 48 / 86 The percentage of elimination is measured by the DPPH (2,2-diphenyl-1-picrylhydrazyl hydrate) assay. The percentage of elimination is calculated as 100x (A0As) / Ao, where Ao is the absorption in the absence of sample, and As is the absorbance in the presence of sample.

[185] It should be noted that the embodiments and features described in the context of one aspect of the present invention also apply to other aspects of the invention.

[186] All patent and non-patent references cited in this application are incorporated herein by reference in their entirety.

[187] The invention will now be described in more detail by the following non-limiting examples. Examples Example 1: Analysis methods Example 1.1: Determination of the degree of hydrolysis (DH)

[188] The degree of hydrolysis (DH) is defined as the percentage of peptide bonds cleaved by hydrolysis, see equation (1) below. The DH value gives information about the number of peptides formed which is related to the number of peptide bonds available.

[189] The DH of whey protein hydrolysates was measured as described in Adler-Nissen, J. Determination of the degree of hydrolysis of food protein hydrolysates by trinitrobenzenesulfonic acid. J. Agric. Food Chem. 27, 1256-1262 (1979).

[190] and Nielsen, PM, Petersen, D. & Dambmann, C. Improved method for determining food protein degree of hydrolysis. J. Food Sci. 66, 642-646 (2001). In equation (1), h represents the number of peptide bonds cleaved and htotal represents the total number of peptide bonds available. Thus, DH gives the percentage of peptide bonds cleaved. Petition 870230043920, dated 05 / 25 / 2023, page 57 / 101 49 / 86 Equation (1): DH = (number of free amino terminals) / (total number of available peptide bonds)-100% = h / htotar100%

[191] The free alpha-amino groups formed after hydrolysis react with ophthalaldehyde (OPA) and form a yellow complex that absorbs light at 340 nm and can therefore be measured spectrophotometrically. Based on the color formation, the DH can be calculated.

[192] The hydrolysates were resuspended in water at appropriate concentrations (0.03-0.08% protein) and 2 volumes were reacted for 2 minutes at 25 °C with 15 volumes of OPA reagent (100 mM Na2B4O7, 0.1% sodium dodecyl sulfate, 6 mM DL-dithioure ... Agricultural and Food Chemistry, 1979 27 (6) 1256] which give the same answer as the OPA method described. The factors used for the whey protein hydrolysates were a = 1, b = 0.4, htotal = 8.8. Example 1.2: Determination of turbidity

[193] Nephelometric turbidity of whey protein hydrolysates is used as a measure of clarity and transparency. If the measured nephelometric turbidity is less than 100 NTU in a 4% w / w protein solution, the sample is perceived as transparent. Furthermore, if the measured nephelometric turbidity is less than 40 NTU in a 4% w / w protein solution, the sample is perceived as clear.

[194] When measuring nephelometric turbidity, a sample is diluted in 5 different protein concentrations, 1.8%, 3.2%, 4.8%, 6.4% and 8% and the turbidity Petition 870230043920, dated 05 / 25 / 2023, page 58 / 101 50 / 86 nephelometric density is measured with a Merck Turbiquant 3000 IR. Example 1.3: Determination of total protein

[195] The total protein content (protein equivalents) of a sample is determined by: 1) Determination of total nitrogen in the sample according to ISO 89681 / 2IIDF 020-1 / 2-Milk - Determination of nitrogen content - Part 172: Determination of nitrogen content by the Kjeldahl method. 2) Calculating the total amount of protein as: Nx6.38. Example 1.4: Determination of total amino acid content

[196] Analysis of amino acid composition gives detailed information about protein hydrolysate as a source of amino acid supplementation.

[197] The total amino acid content was measured using the ISO 13903:2005, EU 152 / 2009 method. A sample was hydrolyzed in aqueous hydrochloric acid to break the peptide bonds in the sample. After hydrolysis, the pH of the sample was adjusted, it was made to volume, and filtered. The amino acids were separated in an amino acid analyzer, and detection was performed using post-column derivatization with ninhydrin reagent and measured at 440 and 570 nm. For quantification, a 1-point calibration was used. For quality assurance, an internal standard was analyzed in each run.

[198] Cysteine ​​and methionine must be oxidized before analysis in the amino acid analyzer. Samples were oxidized with hydrogen peroxide and formic acid at cold temperature, followed by acid hydrolysis with aqueous hydrochloric acid. The oxidation process oxidizes methionine and cysteine, preventing loss during hydrolysis. After hydrolysis, the sample was analyzed as described above. Quantification of total tryptophan: Tryptophan was analyzed by another method, as tryptophan cannot be quantified in the same way as other amino acids because it is destroyed during hydrolysis. Petition 870230043920, dated 05 / 25 / 2023, page 59 / 101 51 / 86 acidic proteins. Instead, the sample was hydrolyzed by alkaline treatment and quantified by HPLC analysis. The results for each of the amino acids are normalized to the total amount of amino acids found: [g / 100 g of amino acids]. Example 1.5: Determination of free amino acid content

[199] Free amino acids in whey protein hydrolysates are determined by the methods of R. Schuster, Determination of Amino Acids in Biological, Pharmaceutical, Plant and Food Samples by Automated Precolumn Derivatization and HPLC, Journal of Chromatography, 431:271-284 (1988) and Henderson, JW, Ricker, RD Bidlingmeyer, BA, Woodward, C., Rapid, Accurate, Sensitive, and Reproducible HPLC Analysis of Amino Acids, Amino Acid Analysis Using Zorbax Eclipse-AAA Columns and the Agilent 1100 HPLC, Agilent Publication, 2000.

[200] Free amino acids are determined by extracting amino acids in an aqueous or acidic solution. Samples can be deproteinized by molecular weight filtration. Samples are analyzed by HPLC after pre-injection derivatization. Primary amino acids are derivatized with o-phthalaldehyde and secondary amino acids are derivatized with fluorenylmethyl chloroformate before injection. The results are given as: [mg of free amino acids / 100 g of whey protein hydrolysate powder] Example 1.6: Method for determining peptide distribution in whey protein hydrolysates

[201] Size exclusion chromatography (SEC) was used to analyze the molecular weight distribution of peptides in whey protein hydrolysate. SEC is used to separate polymer-like molecules by size. A mixture of components of different sizes, here Petition 870230043920, dated 05 / 25 / 2023, pages 60 / 101 52 / 86 peptides can be separated by SEC. The elution time depends on the molecule size. The smaller the molecule, the longer the elution time.

[202] Samples were dissolved in the mobile phase at a concentration of 0.5% w / v. Before injection, the sample was filtered through a 0.45 µm filter. Chromatographic separation was performed on three TSK G2000 SWXL columns (125 Å, 5 µm, 7.5 mm x 300 mm) connected in series. A 0.0375 M phosphate buffer, 0.375 M ammonium chloride, 0.1% trifluoroacetic acid (TFA), and 25% acetonitrile (CH3CN) was used as the mobile phase with a flow rate of 0.7 mL per minute. Peptide detection was performed using a UV detector measuring at 214 nm. Based on retention time, the peptide distribution is divided according to size, and the relative quantity is given according to molecular weight. Example 2: Screening of enzyme combinations

[203] A total of 55 different enzyme combinations were tested for use in the enzymatic hydrolysis of whey proteins. The whey protein hydrolysates obtained were analyzed for clarity, bitterness, degree of hydrolysis, and peptide composition. Hydrolysis tests were performed on a 0.5 liter scale.

[204] The enzymes used to test different enzyme combinations were serine endopeptidases from Bacillus (EC 3.4.21.62), serine endopeptidases from Aspergillus (EC 3.4.21), trypsin-like protease of microbial origin (EC 3.4.21.4), aminopeptidase from Aspergillus (EC 3.4.11), metalloendopeptidase from Bacillus amyloliquefaciens (EC 3.4.24.28), endoprotease from Ananas comosus (Bromelain) (EC 3.4.22.32), a proline-specific endopeptidase from Aspergillus niger (EC: 3.4.21.26), an aminopeptidase preparation from Aspergillus oryzae (EC 3.4.11), a metalloendopeptidase preparation from Geobacillus stearothermophilus (EC 3.4.24) and an endopeptidase preparation Petition 870230043920, dated 05 / 25 / 2023, pp. 61 / 101 53 / 86 of Bacillus amyloliquefaciens (EC 3.4).

[205] The enzyme preparations used for the experiment were: Protamex (Novozymes A / S) comprising a serine endopeptidase from Bacillus sp. (subtilisin) and bacillolysin. Protease A Amano 2 SD (Amano Enzymes Ltd.), comprising serine endopeptidase from Apergillus oryzae. Promod 782 MDP (Biocatalysts Ltd.), which comprises serine endopeptidases from Aspergillus sp. Formea ​​TL 1200 BG (Novozymes A / S), comprising a trypsin-like protease of microbial origin. Promod 950L (Biocatalysts Ltd.), comprising serine endopeptidases from Bacillus sp. (subtilisin) and bacillolysin. Flavourzyme conc BG (Novozymes A / S), comprising leucilaminopeptidases from Aspergillus oryzae. Alcalase AF 2,4 L (Novozymes A / S), comprising serine endopeptidases (subtilisin) from Bacillus licheniformis. Neutrase conc BG (Novozymes A / S), comprising metalloendopeptidases (bacilolysin) from Bacillus amyloliquefaciens. Promod 523 MDP (Bromelain) (Biocatalysts Ltd.), comprising cysteine ​​endopeptidases from Ananas comosus. Maxipro PSP (DSM), proline-specific endopeptidase from Aspergillus niger. Flavorpro 766 (Biocatalyst Ltd.), comprising aminopeptidases from Aspergillus oryzae. Thermoase PC10F (Amano Enzymes Ltd.), comprising metalloendopeptidase from Geobacillus stearothermophilus. Protin NY100 (Amano Enzymes Ltd.), comprising endopeptidases of Petition 870230043920, dated 05 / 25 / 2023, pp. 62 / 101 54 / 86 Bacillus amyloliquefaciens.

[206] 55 enzyme combinations were used in the hydrolysis of whey protein. As a substrate for hydrolysis, a whey protein isolate (WPI) solution (Lacprodan DI-9224 from Arla Food Ingredients) was used. The WPI solution for all experiments had a protein concentration of 8% by weight. The hydrolysis reaction was carried out at 50 °C, with pH-stat at pH = 7 and a reaction time of 6 hours after the addition of the first enzyme. After 6 hours of hydrolysis, the hydrolysis was stopped by heating to 99 °C with a holding time of 90 seconds to inactivate the enzymes. The amount of enzymes used is mentioned in Table 1 as the amount of enzyme in grams per 100 grams of protein. The product of each hydrolysis assay was lyophilized and used for further analyses, including clarity (in the form of turbidity measurements), degree of hydrolysis, and taste evaluation and scoring. The results are given in Table 1 below: NEU: refers to Neutrase; FZ: refers to Flavourzyme conc BG; Alca: refers to Alcalase AF 2.4 L; PA: refers to Amano 2 SD Protease A; FTL: refers to Formea ​​TL 1200 BG; PM782: refers to Promod 782 MDP; FP766: refers to Flavorpro 766; MP PSP: refers to Maxipro PSP; THER: refers to Thermoase PC10F; PRO: refers to Protin NY100; PM950: refers to Promod 950L; PTM: refers to Protamex.

[207] The enzymes used for the assays shown in Table 1 were Petition 870230043920, dated 05 / 25 / 2023, pp. 63 / 101 55 / 86 were selected from a large number of enzymes based on 96-well scale assays. These studies included hydrolysis with different enzyme combinations and visual scoring of apparent clarity and stability to heat and hydrolysis (SDS-PAGE). To allow for pH-stat hydrolysis and larger quantities of material for analysis, the combinations presented in Table 1 were made on a 500 ml scale, as described above. The enzymes presented in Table 1 were therefore not randomly selected. Some of the enzyme combinations in Table 1 were repeated at different doses (e.g., combination 1-3).

[208] The term “visual after inactivity”. In Table 1 it refers to visual after inactivation”. Table 1 Demonstration ID 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Enz 1 (dose) PT M 0.15 6 PT M 0.31 2 PT M 0.62 5 PTM 0.62 5 PA 0.31 2 PA 0.31 2 0.312 PA PT M 0.31 2 PT M 0.31 2 PM 950 3.12 5 Alca 1.05 PA 0.31 2 PA 0.31 2 THE R 0.3 12 Enz 2 (dose) PA 0.15 6 PA 0.31 2 PA 0.62 5 PM7 82 0.62 5 PA 0.31 2 PA 0.31 2 PA 0.31 2 AP 0.31 2 FTL 0.31 2 FTL 0.31 2 PA 0.3 12 Enz 3 (dose) FTL 0.15 6 FTL 0.31 2 FTL 0.62 5 FTL 0.62 5 FTL 0.31 2 FZ 0.31 2 FP766 0.312 FZ 0.31 2 FZ 0.31 2 FZ 0.31 2 FZ 0.31 2 FZ 0.31 2 FP7 66 0.31 2 FZ 0.3 12 Display inactive. clar or clar or clar or claro clar or turv or turvo turv or turv or clar or clar or clar or clar or turv or DH-% 22.1 31.5 31.5 Petition 870230043920, 05 / 25 / 2023, pág. 64 / 101 56 / 86 Gosto amarg or 4% 4% 4% >4% 4% 4% 4% 8% 8% 8% 8% 4% 4% 8% >2500 From (5%) 36.4 28.6 20.9 17.6 34.5 27.7 41 21.1 33.7 11.7 9.6 17.8 21,2 20, 7 Sample ID 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Enz 1 PRO PM PM PM PM PM PM PM Neu Neu Neu Neu Neu Neu (dos 0.31 782 782 782 782 782 782 782 0.21 0.21 0.21 0.21 0.21 0.2 e) 2 0.62 5 0.62 5 0.62 5 0.62 5 0.62 5 0.62 5 0.62 5 9 9 9 9 9 19 Enz 2 PA FTL FTL FTL FTL FTL FTL FTL PM Alca PM PM PM PM (dos 0.31 0.62 0.62 0.62 0.62 0.62 0.62 0.62 523 6.25 950 523 523 523 e) 2 5 4 5 5 5 5 5 0.62 5 6.25 0.62 5 0.62 5 0.6 25 Enz 3 FZ FZ FP7 FZ FP FZ FZ FZ PM PM PM MP MP (dos 0.31 0.62 66 0.62 766 0.62 0.62 0.62 782 782 782 PSP CPP e) 2 5 0.62 5 5 0.62 5 5 5 5 0.62 5 0.62 5 0.62 5 6.25 6.25 Inactive visual. turbid gel clear turbid clear turbid white clear turbid turbid clear gel clear DH- % 22.6 16.6 34.7 18.8 52.4 55.4 54.5 18.7 24.5 21.7 14.7 16.3 15.3 Taste bitter 8% N / AN / A 4% 8% 4% 4% 4% 4% 2% 4% 4% 4% 8% >250 0 Da (5%) 31.9 36.0 40.5 15.8 27.6 19.2 16.5 17.7 26.9 5.4 14.6 26.8 20.8 24.6 Petition 870230043920, dated 05 / 25 / 2023, pages 65 / 101 57 / 86 Sample ID 29 30 31 32 33 34 35 36 37 38 39 40 41 42 Enz 1 Neu Neu Neu Neu Neu PM PM PM PM PT PT PT PM Neu (dos 0.21 0.21 0.12 0.21 0.21 523 523 523 523 MMM 523 0.21 e) 9 9 9 9 9 0.62 5 0.62 5 0.62 5 0.62 5 0.62 5 0.62 5 0.62 5 0.62 5 9 Enz 2 PM PM PM MP PM PM Alca PM PT PM PM MP Neu PM (of the 782 523 523 PSP 523 144 6.25 950 M 523 782 PSP 0.21 523 e) 0.62 5 0.62 5 0.62 5 6.25 0.62 5 0.62 5 6.25 0.62 5 0.62 5 0.62 5 6.25 9 0.62 5 Enz 3 FZ FZ FTL PM FP7 PM FP7 PM PM Neu Neu Neu FZ FZ (from 0.12 0.12 .062 782 66 782 82 782 782 0.21 0.21 0.21 .062 .062 and) 5 5 5 0.62 5 0.62 5 0.62 5 0.6 5 0.62 5 0.62 5 9 9 9 5 5 Visua turv clar turv clar clar clar clar bra bra clar turv turv clar clar clar clar la inactive. oooooo nco nco oooooo DH- % 25.4 25.6 18.8 20.5 19.6 15.8 24.4 22.5 18.8 17.9 20.8 14.5 22.8 22.4 Bitter taste 8% 8% 8% 8% 8% 8% 2% 4% 8% 4% 4% 2% N / AN / A >250 0 Da (5%) 26.7 15.3 14.1 15.8 17.8 20.8 3.5 8.5 24.3 17.3 27.3 48.9 19.3 35.2 Sample ID 43 44 45 46 47 48 49 50 51 52 53 54 55 Enz 1 Neu Neu PTM PTM PM5 Neu Neu Neu PTM Neu Neu Neu PTM (dos 0.21 0.21 0.62 0.62 23 0.21 0.21 0.21 0.62 0.21 0.21 0.21 0.62 e) 9 9 5 5 0.62 5 9 9 9 5 9 9 9 5 Petition 870230043920, dated 05 / 25 / 2023, pages 66 / 101 58 / 86 Enz 2 PTM MPP Neu Neu Neu PM7 PTM MPP Neu PM5 PM7 MPP Neu (dos 0.62 SP 0.21 0.21 0.21 82 0.62 SP 0.21 23 82 SP 0.21 e) 5 9 9 9 0.62 5 5 6.25 9 0.62 5 0.62 5 6.25 9 Enz 3 FZ FZ FZ PM7 FP76 FP76 FP76 FP76 FP76 PTM PTM PTM MPP (dos .062 .062 .062 82 6 6 6 6 6 0.62 0.62 0.62 SP e) 5 5 5 0.62 5 0.62 5 0.62 5 0.62 5 0.62 5 0.62 5 5 5 5 6.25 Display inactive. clear cloudy cloudy cloudy clear cloudy clear clear clear cloudy cloudy clear clear DH- % 19.9 17.7 17.9 8.1 8.1 8.2 8.1 16.3 17.3 18.3 23.6 14.9 15.5 Taste bitter 4% 4% 4% 4% 8% 8% 4% 8% 4% 8% 6% 8% 8% >250 0 Da (5%) 28.7 42.6 34.3 28.7 22.1 26.6 33.6 45.3 37.0 15.3 26.7 43.4 39.7

[209] The data were evaluated for each whey protein hydrolysate. For a positive evaluation, the following conditions must be met: - clear appearance after enzyme inactivation; - to have a high degree of hydrolysis above 15% and, preferably, above 20%; - It does not have a bitter taste in a 4% w / w protein solution; - 25% by weight or less of the peptides must have a molecular weight above 2500 Da.

[210] Samples should be clear after 90 seconds at 99 °C, as this is indicative of UHT stability (stable and clear after treatment with a 4% solution at 143 °C for 6 seconds). Petition 870230043920, dated 05 / 25 / 2023, pp. 67 / 101 59 / 86

[211] Therefore, it can be seen from the tables above that 4 of the 55 enzyme combinations, namely the samples referred to as samples 4, 10, 11 and 30, met the conditions. Sample 4 was obtained by hydrolysis with a combination of the enzymes Bacillus serine endopeptidase, Aspergillus serine endopeptidase and trypsin-like protease. Sample 10 was obtained by hydrolysis with a combination of the enzymes Bacillus serine endopeptidase, Bacillus metalloendopeptidase, Aspergillus serine endopeptidase, and Aspergillus leucyl-aminopeptidase. Sample 11 was obtained by hydrolysis with Bacillus serine endopeptidase, Aspergillus serine endopeptidase, and Aspergillus leucyl-aminopeptidase. Sample 30 was obtained by hydrolysis with bacillolysin from Bacillus amyloliquefaciens, bromelain, and leucyl-aminopeptide from Aspergillus. Example 3: Further analysis of whey protein hydrolysates according to the invention.

[212] The four whey protein hydrolysates from Example 2 that met the conditions for clarity, taste, degree of hydrolysis, and peptide distribution were subsequently analyzed and referred to as samples 1-4 (S1-S4) in the table below. Samples 5-12 (S5-S12) are whey protein hydrolysates from hydrolysis with other enzyme combinations. Sample 13 (S13) is a spray-dried WPI hydrolysate obtained by hydrolysis of sweet whey with a combination of Bacillus licheniformis subtilisin (Alcalase) and Bacillus amyloliquefaciens bacillolysin (Neutrase), followed by ultrafiltration, activated carbon treatment, and microfiltration for activated carbon removal (as described in WO 1993 / 024020A1). The degree of hydrolysis is approximately 25%. Sample 14 (S14) is prepared using the same enzyme combination as sample 4 (enzyme combination of the invention), but where a WPC is used as a substrate for hydrolysis.Samples 15 and 16 (S15-S16) are. Petition 870230043920, dated 05 / 25 / 2023, pages 68 / 101 60 / 86 replicates of sample 4. Samples 15 and 16 are WPI-based hydrolysates prepared without UF filtration using the same conditions as sample 4.

[213] Whey protein hydrolysates made by enzymatic hydrolysis with different enzyme combinations were again analyzed for clarity, degree of hydrolysis, and peptide content with a molecular weight above 2500 Da and peptide content with a molecular weight below 375 Da. The clarity (turbidity) of the samples was determined by measurements according to example 1.2, and nephelometric turbidity should be less than 40 NTU for the sample to be perceived as clear and less than 100 NTU for the sample to be perceived as transparent. A sample was classified as bitter if a bitter taste was perceived with 4% protein or less.

[214] The degree of hydrolysis was measured by the method described in Example 1.1, while bitterness was measured by tasting the sample at different concentrations (2%, 4%, 8% protein concentration). Peptide distribution was measured as described in Example 1.5. The result is shown in Table 2: Table 2: Origin m Combination of enzymes Clear (nephelometric turbidity at 4% w / w protein less than 40 NTU) Bitterness as a percentage of protein DH (%) >2500 Da (%) <375 Da (%) S1 WPI Bacillus serine endopeptidase Aspergillus serine endopeptidase Leucil-aminopeptidase Yes 8 31.5 11.7 20.4 Petition 870230043920, dated 05 / 25 / 2023, pp. 69 / 101 61 / 86 S2 WPI Serine endopeptidase from Bacillus Serine endopeptidase from Aspergillus Trypsin-like protease Yes >4 31.5 17.6 13.5 S3 WPI Serine endopeptidase from Bacillus Serine endopeptidase from Aspergillus Leucil-aminopeptidase Yes 8 22.1 9.6 22.2 S4 WPI Bacillolysin from Bacillus amyloliquefaciens Bromelain Leucil-aminopeptidase Yes 8 25.6 15.3 14.4 S5 WPI Serine endopeptidase from Aspergillus Trypsin-like protease Leucil-aminopeptidase No. 8 18.9 27.6 8.7 S6 WPI Bacillolysin from Bacillus amyloliquefaciens Serine endopeptidase from Bacillus Serine endopeptidase from Aspergillus No. 2 24.6 5.4 21 Petition 870230043920, dated 05 / 25 / 2023, pp. 70 / 101 62 / 86 S7 WPI Bromelain Serine endopeptidase of Bacillus Metaloendopeptidase of Bacillus Serine endopeptidase of Aspergillus Sim 8 18.9 24.5 9.9 S8 WPI Bacillus amyloliquefaciens Bacilolysin Serina endopeptidase Serina endopeptidase of Aspergillus, N 1416° 13.8 S9 WPI Bacilolysin of Bacillus amyloliquefaciens Serine endopeptidase of Aspergillus Leucil-aminopeptidase N° 8 23.4 26.7 13.8 S10 WPI Bacilolysin of Bacillus amyloliquefaciens Prolyl endopeptidase Serine endopeptidase of Aspergillus 8 20.5 28.4 24.3 S11 WPI Bromelain Serine endopeptidase of Bacillus Serine endopeptidase of Aspergillus N° 2 24.4 1.9 54.5 Petition 870230043920, of 25 / 05 / 2023, p. 71 / 101 63 / 86 S12 WPI Bromelain Serine endopeptidase from Bacillus Serine endopeptidase from Aspergillus No. 4 22.5 8.5 16.6 S13 WPC Serine endopeptidase from Bacillus Bacillolysin from Bacillus amyloliquefaciens No. >2 23.0 7.5 18.9 S14 WPC Bacillus Bacillolysin amyloliquefaciens Bromelain Leucyl-aminopeptidase No. 8 25.4 23.2 15 S15 WPI Bacillolysin from Bacillus amyloliquefaciens Bromelain Leucyl-aminopeptidase Yes 8 23.0 23.2 12.7 S16 WPI Bacillolysin from Bacillus amyloliquefaciens Bromelain Leucylaminopeptidase Yes 8 23.0 17.9 13.5

[215] Thus, from Table 2, it is shown that the use of specific enzyme combinations according to the present invention results in whey protein hydrolysates that 1) have a degree of hydrolysis above 20%, 2) less than 25% of the peptides have a molecular weight of 2500 Da or above and 3) do not have a bitter taste at a protein concentration of 4% or less.

[216] Furthermore, Table 2 shows that if a WPI is used for protein hydrolysis, the use of the specific enzyme combinations of the invention results in whey protein hydrolysates that are clear in appearance. Table 2 also shows that the use of specific enzyme combinations in Petition 870230043920, dated 05 / 25 / 2023, pp. 72 / 101 64 / 86 Preparation of whey protein hydrolysates where a WPC is used as a substrate results in a whey protein hydrolysate with a degree of hydrolysis above 20%, less than 25% of the peptides have a molecular weight of 2500 Da or above, and it does not have a bitter taste at a protein concentration of 4% or lower. However, whey protein hydrolysates prepared using a WPC as a substrate are not clear in appearance (because of the lipids present in the WPC).

[217] In a preferred embodiment of the invention, a WPI is used as the substrate for protein hydrolysis to obtain clear hydrolysates. Example 4: Turbidity analysis

[218] Nephelometric turbidity at different protein concentrations of samples 1 to 16 from Example 3 was subsequently analyzed. Samples with turbidity less than 100 NTU were considered clear and samples with turbidity less than 40 were considered clear. Table 3 below shows the turbidities of the hydrolysates mentioned as samples 1-12 in Example 3 measured at different protein concentrations. The protein concentrations are 1.8% protein, 3.2% protein, 4.8% protein, 6.4% protein, and 8% protein. Protein concentration is percent by weight.

[219] Whey protein hydrolysate powders were hydrated for at least 30 minutes at the indicated concentrations before turbidity was measured (n = 3). Table 3: Turbidity (NTU) of test samples at 1.8, 3.2, 4.8, 6.4 and 8% protein. Protein content (% by weight) 1.8 3.2 4.8 6.4 8 Sample 1 9.26 18.69 29.05 34.14 43.48 Petition 870230043920, dated 05 / 25 / 2023, pp. 73 / 101 65 / 86 Sample 2 14.46 26.86 34.38 42.45 50.15 Sample 3 10.93 21.08 30.48 41.29 48.56 Sample 4 11.58 23.78 33.82 43.73 63.41 Sample 5 129.42 213.08 269.67 292.63 305.15 Sample 6 177.24 366.35 620.99 827.23 921.99 Sample 7 12.4 21.5 31.31 37.11 42.04 Sample 8 86.94 186.66 254.45 336.05 399.52 Sample 9 Sample 10: 68.41 121.46 182.88 236.2 263.59 Sample 11: 16.5 31.52 46.38 58.8 67.4 Sample 12: 154.82 318.38 511.59 740.01 972.62 Sample 114.53 228.26 334.15 435.53 575.48

[220] Therefore, it is shown in table 3 that the specific enzyme combinations according to the invention have a turbidity of less than 100 NTU at concentrations of 8% protein.

[221] In Figure 1AC, the measured turbids of samples 13, 14, and 16 are shown. Figure 1A shows the turbidity of the whey protein hydrolysates from samples 13, 14, and 16 and shows the difference in turbidity. In Figure 1A, it is shown that a whey protein hydrolysate (not of the invention, sample 13) subjected to ultrafiltration has a very low turbidity (below 1 NTU) and is the clearest hydrolysate. The hydrolysate prepared according to the method according to the present invention (sample 16) using one of the specific enzyme combinations has a turbidity below 100 NTU at a protein concentration of 8% and is therefore transparent. Furthermore, the hydrolysate of the invention has a turbidity below 40 NTU at a protein concentration of 4% and is therefore perceived as clear at a protein concentration of 4%. Conversely, the whey protein hydrolysate prepared according to the invention using a WPC as a substrate (sample Petition 870230043920, dated 05 / 25 / 2023, pp. 74 / 101 66 / 86 14) has a turbidity above 1000 NTU and is therefore perceived as unclear.

[222] Figure 1B shows more clearly that the turbidity of sample 16 is less than 40 NTU at a protein concentration of 5% or less.

[223] Figure 1C shows the turbidity of sample 14. It is shown that even at very low concentrations (about 2%), the turbidity is above 2,000 NTU. Sample 14 is perceived as very unclear and not transparent.

[224] Figure 2 AC includes images of samples 13, 14, and 16 to visually show clear versus unclear samples. Samples from left to right were prepared with 8%, 6.4%, 4.8%, 3.2%, and 1.8% protein.

[225] Figure 2A shows that sample 16 is visually clear and transparent at 8%, 6.4%, 4.8%, 3.2% and 1.8% protein.

[226] Figure 2B shows sample 14, and it is shown that sample 14 is not clear and is not transparent, even at the lowest protein concentration of 1.8%.

[227] Figure 2C shows sample 13, and it is shown that sample 13 is visually clear and transparent at all concentrations. Example 5: Evaluating bitterness in relation to caffeine.

[228] In Example 5, the bitterness of a whey protein hydrolysate according to the present invention is being analyzed in comparison with a whey protein hydrolysate treated with activated charcoal and filtered with UF made by enzymatic hydrolysis of WPI with enzymes other than those in the present invention (hydrolysis with a subtilisin from Bacillus licheniformis (Alcalase) and a bacillolysin from Bacillus amyloliquefaciens (Neutrase)).

[229] A sensory evaluation was performed to compare the taste of the samples Petition 870230043920, dated 05 / 25 / 2023, pages 75 / 101 67 / 86 13, 15 and 16 of example 3.

[230] A sensory panel was trained to detect and quantify bitterness using a caffeine solution as a reference. The training involved panelists first receiving reference samples, including solutions composed of increasing concentrations of caffeine. Panelists were trained to assign bitterness scores to unknown solutions based on a 15 cm scale. The reference samples consisted of 3 caffeine solutions containing 0.025%, 0.05%, and 0.1% caffeine, respectively. After evaluating the reference samples, panelists tasted the hydrolysate samples 3 times in a 4% by weight protein solution in a random order and rated the bitterness intensity of the test solutions based on the bitterness in the reference solutions.The reference solution containing 0.025% caffeine was perceived as not being bitter, and the reference solution containing 0.1% caffeine was considered bitter (score of 13 on a 15 cm bitterness scale). The sensory panel consisted of 7 panelists who participated in the evaluation.

[231] In addition, a sensory profile was performed in accordance with the international standard Quantitative Descriptive Profile ISO 13299: 2016 2nd ed. 5.5, Annex F1-F6 and H3 using the panel. Seven trained assessors participated in the assessment. The assessment was performed in 3 repetitions. The response scale used was a continuous line scale (15 cm). Samples of approximately 2 ml were served at room temperature. Red light was used during the assessment.

[232] The bitterness assessed by the sensory panel with reference to caffeine bitterness is shown in Figure 3, where the bitterness score of the test samples is plotted against the caffeine concentration. The bitterness of the 3 different caffeine concentrations and the bitterness scores of samples 13, 15 and 16 from example 3 are shown in Figure 3. Petition 870230043920, dated 05 / 25 / 2023, pp. 76 / 101 68 / 86

[233] Figure 3 shows that sample 13 (WPI hydrolysate, but not with the enzyme combination of the invention, but the hydrolysate is ultrafiltered and treated with activated carbon) has the highest relative bitterness of 0.095%. Sample 15 (WPI hydrolysate using the enzyme combination of the invention, without ultrafiltration and activated carbon treatment) has the lowest relative bitterness of the 3 product samples with a relative bitterness of 0.054%, but was very close to the hydrolysate in sample 16 (similar to sample 15) which has a relative bitterness of 0.061% relative to caffeine.

[234] Therefore, the taste of the palatable whey protein hydrolysates prepared according to the invention (sample 15 and sample 16) was perceived to be less bitter than 0.08% caffeine and less bitter than sample 13. Example 6: Taste profile

[235] An example was made to evaluate the taste profile of whey protein hydrolysates in samples 13, 15 and 16. The taste profile was made by a trained panel and 5 attributes were used to identify the differences between the samples focusing on odor, palate and taste.

[236] The data were analyzed to identify significant differences between the samples for each attribute. A statistical evaluation of the data is shown in Table 4. In addition, a multiple range test was used to identify the differences between the samples. In Table 4, the samples with the same letter are not significantly different. Table 4: Sensory scores Cheese_O *** Broth_O *** Astringent_M F *** Umami_T *** Bitter_T *** Sample 15 5.33a 4.89a 5.79a 7.15a 7.53a Sample 1.4b 1.14b 10.71b 1.54b 12.3b Petition 870230043920, dated 05 / 25 / 2023, pp. 77 / 101 69 / 86 Sample 13: 4.1a, 7.58C, 5.96a, 8.49a, 8.11a *** p <0.001 Duncan's test: Samples with different letters for an attribute are significantly different at the 95% level (p <0.05).

[237] Therefore, the bitterness score of samples 15 and 16 is lower than the bitterness score of sample 13.

[238] The data mentioned in Table 4 can be represented as a spiderweb representation, see Figure 4. The spiderweb shows the attributes of Odor (O), Palate (MF), and Taste (T). On the periphery of the graph, the 'high' intensity of each sensory attribute is shown, and the 'low' intensity is in the center of the graph. Each product has a different label, which is shown below the figure. As an example, see the data for Bitter_T, where *** indicates that the data from sample 13 and samples 15 and 16 are significantly different (p <0.001).

[239] Based on the spiderweb representation of the taste profile, it is shown that sample 13 is very different from samples 15 and 16 in taste profile and, most importantly, samples 15 and 16 have low bitterness. Example 7: LC-MS / MS analysis and peptide cleavage pattern

[240] Peptides in liquid samples can be identified by peptide mass spectrometry analysis and database search. Samples 1, 2, 3, 13, 15, and 16 were analyzed by LC-MS / MS to identify the peptide sequences and the proteins from which they were derived. The peptides present in the respective samples were dissolved in water and injected into a Dionex nano-LC system for MS / MS analysis on a Bruker Maxis Impact QTOF mass spectrometer. The search with the acquired MS / MS spectra was performed on a customized database containing sequences of bovine-derived proteins. The overall results of Petition 870230043920, dated 05 / 25 / 2023, pp. 78 / 101 70 / 86 analyses are illustrated in Table 5. Table 5: Proteins identified by LC-MS / MS Coverage (%) Sample 1 Sample 2 Sample 3 Sample 13 Sample 15 Sample 16 Beta-lactoglobulin 79.8 80.9 82.6 74.2 82.6 Beta-casein 79.9 77.7 79.5 71.4 64.7 62.5 Kappacasein 29.5 38.9 32.6 30.5 36.3 33.2 GDCA* molecule 1 46.4 65.4 34.6 48.4 56.2 40.5 Osteopontin 40.6 30.9 36.7 39.9 33.8 29.9 Alpha-lactalbumin 42.3 54.2 58.5 47.2 53.5 62 Alpha-S1-casein 49.1 53.7 41.1 36 41.1 36.4 BSA** 18.9 15 21.3 10.2 8.4 10.5 Alpha-S2-casein 27 33.3 20.7 23.9 19.4 11.3 Total without identified peptides 776 917 709 631 595 615 *Glycosylation-dependent cell adhesion molecule 1 **Bovine serum albumin

[241] The data in Table 5 showed that the analyses provided high sequence coverage for the most prevalent proteins in the samples, including beta-lactoglobulin, alpha-lactalbumin, and beta-casein. Furthermore, it is Petition 870230043920, dated 05 / 25 / 2023, pp. 79 / 101 71 / 86 indicates the number of peptides identified in each sample. As the datasets were of the expected quality for LC-MS / MS analysis, they were valid for a more detailed analysis (example 9). Example 8: LS-MS / MS analysis of bitter peptides

[242] First, it was confirmed that the LC-MS / MS data from example 7 matched the data obtained by SEC (size exclusion chromatography) analysis comparing the peptide distributions. Here, the SEC data were converted into percentage numbers (the number of peptides of a given molecular weight) and plotted for each hydrolysate along with the same data calculated based on LC-MS / MS (see figure 5). The LC-MS / MS data included only beta-lactoglobulin peptides, while the SEC analysis accounted for all proteins in the samples.

[243] From figure 5, it is shown that the percentage of beta-lactoglobulin-derived peptides in the 7-10 amino acid range and in the 1119 amino acid range of the number of beta-lactoglobulin-derived peptides in 719 amino acids were similar when using LC-MS / MS and SEC.

[244] Thus, the data shown in Figure 5 show that the number of peptides in different amino acid length ranges in the LC-MS / MS dataset can be used to extract quantitative information about the relative distribution of peptides, since the data were comparable to SEC data and the SEC method is quantitative. Furthermore, Figure 5 shows that sample 13 (outside the invention) had more peptides with a smaller peptide size than the hydrolysates according to the invention.

[245] The amount of detectable peptides comprising phenylalanine as a percentage of the total number of peptides was identified and analyzed in samples 1, 2, 3, 13, 15 and 16 by MS-LC / MS. The result is shown in figure 6. Petition 870230043920, dated 05 / 25 / 2023, pages 80 / 101 72 / 86

[246] Without being limited by any theory, the inventors of the present invention believe that the presence of phenylalanine in peptides is correlated with bitterness, and the bitterness of phenylalanine can be increased when its amino or carboxy terminal is blocked by a peptide bond to another amino acid residue. For example, the phenylalanine residue in the bitter peptide YPFPGPIPN identified in a bitter whey protein hydrolysate has been suggested as a primary determinant of bitterness (Liu. X. Jiang. D. and Peterson. DG Identification of Bitter Peptides in whey protein hydrolysate. J. Agric. Food Chem. 2014. 62: 5719-5725).

[247] Figure 6 shows the phenylalanine content in peptides as a percentage of the total number of peptides originating from beta-lactoglobulin, alpha-lactalbumin, and beta-casein. The percentage of peptides comprising phenylalanine was lower for the less bitter hydrolysates in peptide sizes of less than 9 amino acid residues. Thus, for the whey protein hydrolysate according to the invention in samples 1 and 3, phenylalanine is present in peptides with a larger peptide size. The whey protein hydrolysates according to the invention represented by samples 2, 15, and 16 also have a low percentage of phenylalanine comprising peptides, but, in addition, samples 2, 15, and 16 also exhibited a lower percentage of phenylalanine bound to peptides overall. This indicates that there may be more phenylalanine present as free amino acids in samples 2, 15, and 16 than in samples 1, 3, and 13.Therefore, an indication of non-bitter whey protein hydrolysates according to the present invention is that a large percentage of phenylalanine is present in larger peptides or as free amino acids.

[248] Figure 7 shows the percentage of peptides from 5-19 Petition 870230043920, dated 05 / 25 / 2023, pages 81 / 101 73 / 86 amino acids from beta-lactoglobulin, alpha-lactalbumin and beta-casein.

[249] Figure 7 shows that sample 13 (outside the invention) comprises more smaller peptides (5-9 amino acids) than the other five whey protein hydrolysates. Without being limited by any theory, the inventors of the present invention believe that the higher phenylalanine content comprising smaller peptide sizes in the hydrolysate in sample 13 may be the reason for the greater bitterness in sample 13 than in samples 1, 2, 3, 15 and 16 which are hydrolysates according to the invention. Example 9: SEC size distribution data

[250] The size of the peptides in samples 1, 2, 3, 13, 15 and 16 was analyzed by Size Exclusion Chromatography (SEC). The results are shown in Table 6 below: Table 6: Data on the size distribution of SEC, DH, and free amino acid (FAA) content for whey protein hydrolysates in samples 1, 2, 3, 13, 15, and 16. Sample 1 Sample 2 Sample 3 Sample 13 Sample 15 Sample 16 <375 Da (%) 20.4 13.5 22.2 16.1 13 13.5 375-750 Da (%) 20.1 24.5 24.2 35.9 20.4 22.9 750-1250 Da (%) 18.2 15.3 20.6 24.6 17 18.8 1250-2500 Da (%) 29.5 29.1 23.3 21.4 27.3 26.9 >2500 Da (%) 11.7 17.6 9.6 2.1 22.4 17.9 DH (%) 31.5 22.1 31.5 27.7 23.3 25.4 FAA (mg / 100 13825 4404 12241 413 7266 5936 Petition 870230043920, dated 05 / 25 / 2023, pages 82 / 101 74 / 86 g of protein (Nx6.38)

[251] Size exclusion chromatography data are not accurately accounted for by free amino acids, since the data were acquired by measurements at 214 nm, where peptide bonds are expected to be primarily absorbed.

[252] Therefore, the free amino acid content was measured by a different method (see example 10). Compared with the reference whey protein hydrolysate in sample 13, the whey protein hydrolysates according to the present invention have a higher free amino acid content. However, the content of small peptides is higher in the reference whey protein hydrolysate (sample 13) than in the whey protein hydrolysates of the invention. For example, the peptide content being 750 Da or less in sample 13 is more than 50%. Conversely, the peptide content in the whey protein hydrolysates of the present invention with a size of 750 Da or less is less than 40%. Furthermore, sample 13 comprises fewer peptides being 2500 Da or above than the whey protein hydrolysates of the invention.

[253] When these data are compared with the data from example 8, it becomes obvious that the low overall phenylalanine content in beta-lactoglobulin, alpha-lactalbumin, and beta-casein-derived peptides from samples 2, 15, and 16 is the result of these phenylalanine residues being released from the peptides in the free amino acid fraction. Therefore, one method for reducing bitterness in hydrolysates could be to identify enzyme combinations that specifically concentrate phenylalanine in the free amino acid fraction of the total hydrolysate. Example 10: Measured free amino acid content Petition 870230043920, dated 05 / 25 / 2023, pp. 83 / 101 75 / 86

[254] The free amino acid content in the whey protein hydrolysates of the present invention was measured and compared with a reference whey protein hydrolysate (outside the invention). The result is shown in Table 7 below. Table 7: Free amino acid content (mg / 100 g of protein (Nx6.38)) Sample 1 Sample 2 Sample 3 Sample 13 Sample 15 Sample 16 Aspartic acid 113.51 <10 93.24 <10 32.56 17.70 Threonine 898.44 178.05 850.42 22.72 282.30 249.70 Serine 397.09 57.51 374.84 17.34 173.36 117.33 Glutamine 222.56 31.86 181.56 <10 194.45 117.21 Glutamic acid 87.62 <10 101.68 <10 42.29 29.33 Proline <10 <10 <10 <10 16.52 24.61 Glycine 42.05 26.59 24.48 <10 21.20 21.70 Alanine 663.0 189.76 445.12 32.68 343.21 253.33 Cystine <10 <10 <10 <10 <10 <10 Valine 1171.37 480.26 830.50 <10 730.94 627.88 Methionine 1055.41 473.23 1024.95 <10 500.18 386.67 Isoleucine 1311.94 524.77 981.61 44.75 763.73 665.45 Leucine 3736.68 1229.94 3127.56 152.28 2389.60 1903.03 Tyrosine 317.44 42.76 274.10 30.81 102.85 78.30 Phenylalanine <10 183.91 <10 <10 329.16 250.91 Lysine 1862.48 722.74 2237.32 81.76 742.65 723.64 Histidine 440.44 88.79 358.44 17.45 88.44 115.52 Petition 870230043920, dated 05 / 25 / 2023, pages 84 / 101 76 / 86 Arginine 957.01 138.22 715.71 13.47 384.21 266.67 Tryptophan 339.70 16.52 361.95 <10 128.85 87.64 Asparagine 208.50 19.91 257.70 <10 121.82 59.15 Sum 13825 4404 12241 413.26 7266 5936

[255] From Table 7, it is shown that the free amino acid content in the whey protein hydrolysates of the invention is from 4% to 14% by weight of the total protein content. Conversely, the free amino acid content in the reference hydrolysate (sample 13) is about 0.4% by weight of the total protein content.

[256] Furthermore, Table 7 shows that the free leucine content in the whey protein hydrolysates of the invention is much higher than the free leucine in sample 13. The free leucine content in the whey protein hydrolysates of the invention is 1 to 4% by weight of the total protein content. Conversely, the free leucine content in sample 13 is about 0.15% by weight of the total protein content.

[257] More importantly, the concentration of free phenylalanine, as suggested in examples 8 and 9, is higher in samples 2, 15 and 16 than in samples 1, 3 and 13. Table 8 shows the percentage of free amino acids based on the total amino acid content. Table 8: Free amino acid (%) of total amino acids Sample 1 Sample 2 Sample 3 Sample 13 Sample 15 Sample 16 Aspartic Acid / Asparagine 2.00 0.19 2.47 0.00 1.48 0.76 Threonine 12.53 2.48 11.86 0.30 3.94 3.55 Serine 8.57 1.24 8.09 0.36 3.74 2.59 Glutamine 1.26 0.18 1.03 0.00 1.34 0.81 Petition 870230043920, dated 05 / 25 / 2023, pages 85 / 101 77 / 86 Glutamic acid Proline 0.00 0.00 0.00 0.00 0.27 0.41 Glycine 2.88 1.82 1.68 0.00 1.45 1.52 Alanine 12.30 3.52 8.26 0.59 6.37 4.72 Cystine 0.00 0.00 0.00 0.00 0.00 0.00 Valine 20.18 8.27 14.31 0.00 12.59 10.93 Methionine 46.79 20.98 45.44 0.00 22.17 15.96 Isoleucine 20.16 8.06 15.08 0.70 11.73 Leucine 10.25 35.75 11.77 29.93 1.55 22.86 18.07 Tyrosine 11.84 1.60 10.23 1.31 3.84 2.97 Phenylalanine 0.00 6.61 0.00 1.36 11.83 9.04 Lysine 19.80 7.68 23.79 0.82 7.90 7.77 Histidine 29.56 5.96 24.06 1.12 5.94 7.66 Arginine 47.67 6.89 35.65 0.75 19.14 14.59 Tryptophan 20.39 0.99 21.73 0.00 7.73 4.76

[258] It is particularly noteworthy that the percentage of free leucine in the total leucine content in the whey protein hydrolysates of the invention is much higher than in the reference hydrolysate (sample 13).

[259] It is important to note that Table 8 shows that for the whey protein hydrolysates according to the invention in samples 2, 15 and 16, between 6 and 12% of the phenylalanine was in the form of free phenylalanine. For the whey protein hydrolysates according to the invention in samples 1 and 3, phenylalanine was not found in its free form. Example 11: Mineral content in whey protein hydrolysates of the invention

[260] The amount of minerals in samples 15 and 16 was measured. The result is shown in Table 9 below. Petition 870230043920, dated 05 / 25 / 2023, pages 86 / 101 78 / 86 Table 9: Mineral content Sample K (%) Na (%) Ca (%) Citrate (%) Ash (%) Turbidity at 4% protein (NTU) 15 2.13 1.75 0.07 0.66 7.5 31.18 16 2.45 1.6 0.09 0.65 7.2 58.68

[261] Table 9 shows the mineral content and turbidity of samples 15 and 16 as an example of the mineral content of whey protein hydrolysates. All products in Table 9 have a pH of 7.8 in a 4% protein solution at 22 °C. Example 12: BSA not degraded into hydrolysates

[262] The amount of undegraded bovine serum albumin (BSA) was measured in the whey protein hydrolysates of the invention (samples 1, 2, 3, 4, 15 and 16). The BSA content was estimated using SDS-PAGE and a Sigma Aldrich BSA standard with product code A2153 (Figure 9A). The amount of BSA loaded into the 20% well corresponded to 10 pg of pure BSA. The total amount of protein added to each well of the SDS-PAGE gel shown in Figure 9B corresponded to 50 pg of protein. The protein samples were mixed at 10 mg / ml with Laemmli sample buffer and 2-mercaptoethanol to a final concentration of 3% protein followed by incubation at 95 °C for 5 minutes before loading onto the gels.

[263] The SDS-PAGE gel in Figure 9A is a titration series showing the expected intensity of BSA if it represents 20%, 10%, 5%, 2.5%, 1.25%, or 0.63% of the total amount of whey protein in Figure 9B. The standard SDS-PAGE gel at different concentrations was compared to the SDS-PAGE gel shown in Figure 9B with different whey protein hydrolysate samples, from left to right: a molecular weight standard, sample 2, sample 1, sample 3, sample 4, sample 15, and sample 16. Petition 870230043920, dated 05 / 25 / 2023, pages 87 / 101 79 / 86

[264] It can be concluded from Figures 9A and 9B that the undegraded BSA in the whey protein hydrolysates of the invention (Figure 9B) is in the range of 0.5 to 2% by weight, since the intensity of the bands in Figure 9B corresponded to the intensity of the samples at 0.63% and 1.25% in Figure 9A. The BSA was resistant to proteolysis by the enzymes used according to the invention. Example 13: UHT-treated beverage for sports nutrition

[265] Example 13 shows an example of the use of whey protein hydrolysate according to the invention in the preparation of a beverage suitable for use in sports nutrition. The beverage is intended for use by athletes or in other sports or exercise-related applications.

[266] A powder from sample 16 was reconstituted in water and sugars and flavorings were added to prepare a beverage. The quantities of the ingredients are shown in Table 10 below.

[267] Table 10 shows a neutral-tasting beverage with no unpleasant bitter taste that can be heat-treated by direct and indirect UHT treatment, as well as pasteurization, without developing additional turbidity. Table 10: Sports drink Ingredient g / 100 g Sample powder 16 4.9 Sucrose 2 Sucralose 0.006 Pineapple flavor 0.08 Lemon flavor 0.17 Water 92.9

[268] The sports drink shown in Table 10 was subjected to 1) direct UHT, 2) indirect UHT, and 3) pasteurization. The direct UHT treatment was performed by injection at 143 °C for 6 seconds. The indirect UHT treatment was performed in a Petition 870230043920, dated 05 / 25 / 2023, pages 88 / 101 80 / 86 tubular heat exchanger at 143 °C for 6 seconds. Pasteurization was carried out at 90 °C for 6.5 minutes. The beverages were placed in flasks at 5 °C. All solutions had a pH of approximately 7.7 at 22 °C. The 4.9 g / 100 g content of whey protein hydrolysate of the invention corresponds to 4.0% by weight of protein.

[269] Figure 8 shows images from left to right of the untreated beverage, the directly UHT-treated beverage, the indirectly UHT-treated beverage, and the pasteurized beverage. The beverages are at room temperature. Figure 8 shows that all samples were clear and transparent and the bottom behind the bottles can be seen.

[270] The nephelometric turbidity of the 4 drinks was measured and the result is shown in table 11. Table 11: Turbidity of beverages measured in NTU Untreated Direct UHT Indirect UHT Pasteurized Sample 1: 58.60 52.22 73.79 39.93 Sample 2: 59.22 54.41 71.76 39.88

[271] Thus, from table 11, it is shown that the turbidity of the thermally treated samples was all below 100 NTU. Therefore, the thermal treatment did not affect the turbidity of the beverages, which were still clear or transparent in appearance. These beverages were not perceived as bitter. Example 14: Beverage for clinical / medical use

[272] Example 14 is an example of the use of whey protein hydrolysate according to the invention in the preparation of a beverage suitable for use in medical or clinical nutrition. The clinical beverage contains a large amount of carbohydrates in addition to the whey protein hydrolysate of the invention.

[273] A powder from sample 16 was reconstituted in water and added Petition 870230043920, dated 05 / 25 / 2023, pages 89 / 101 81 / 86 carbohydrates and flavorings to prepare a drink. The quantities of the ingredients are shown in Table 12 below.

[274] Table 12 shows the neutral-tasting beverage for medical use, without any unpleasant bitter taste. Table 12: Medical Beverage Ingredients g / 100g Sample powder 16 4.9 Carbohydrates 33.0 Fat 5.0 Sucrose 2 Sucralose 0.006 Pineapple flavor 0.08 Lemon flavor 0.17 Water 54.8 Example 15: Carbonated beverage

[275] Example 15 shows an example of the use of whey protein hydrolysate according to the invention in the preparation of a carbonated beverage.

[276] A powder from sample 16 was reconstituted in water and added sugars and flavorings to prepare a beverage. The quantities of the ingredients are shown in Table 13 below. The beverage was carbonated by adding carbon dioxide to the beverage until the beverage comprised carbon dioxide in an amount of 2.5 volumes per volume of beverage. Table 13: Carbonated beverage Ingredient g / 100 g Sample powder 16 4.9 Sucrose 2 Petition 870230043920, dated 05 / 25 / 2023, pp. 90 / 101 82 / 86 Sucralose 0.006g Pineapple flavor 0.08g Lemon flavor 0.17g Water 92.9g Carbon dioxide (volume per volume of beverage) 2.5g Example 16: Carbonation - how the amount of carbonation influences pH

[277] Sample 16 was resuspended in water to make an 8% protein solution. The solution was forcibly carbonated at 5 °C.

[278] The mass increase and pH value of the solution were measured over time as more CO2 was introduced. When the equilibrium pressure in the container reached about 1 bar, no more CO2 could be absorbed (the pressure used under forced carbonation was 3 bar ((300 kPa)) at 5 °C).

[279] Figure 10 shows the measured pH dependent on the amount of CO2 added to the carbonated solution. Figure 10 shows that when a solution comprising whey protein hydrolysate from sample 16 is carbonated at a CO2 content of 0 to 4 volumes per volume of solution, it results in a pH between 5.5 and 8.25. The pH of the non-carbonated solution has a pH value of 8.25, and the pH value decreases as the amount of carbonation increases.

[280] Figure 10 shows that adding CO2 at approximately 2.5 volumes (4.9 g / L) per volume of solution resulted in a drop in pH from 8.25 to approximately 6.0 at 5 °C. Figure 10 also shows that the pH reaches a minimum at approximately 5.5 to 6.0. Therefore, it can be concluded that adding CO2 at a rate greater than 2.5 volumes per volume of solution would not decrease the pH to less than approximately Petition 870230043920, dated 05 / 25 / 2023, pp. 91 / 101 83 / 86 from 5.5 to 6.0.

[281] The effect of carbonation on the pH value of a solution or beverage was also analyzed by measuring the pH of the following carbonated solutions / beverages at a quantity of 2.5 volumes of CO2 per volume of solution / beverage: - A 4% protein solution prepared by resuspending sample 16 in water; - An 8% protein solution prepared by resuspending sample 16 in water; - A beverage comprising 8% of sample 16; The result is shown in Figure 11.

[282] The beverage comprising 8% of sample 16 comprises the following ingredients: Ingredient g / 100 g Sample powder 16 9.8 Sucrose 2 Sucralose 0.006 Pineapple flavor 0.08 Lemon flavor 0.17 Water 88 Carbon dioxide (volume per volume of beverage) 2.5

[283] Figure 11 shows that the 4% and 8% solution of sample 16 and the beverage prepared from sample 16 have a pH of about 6.0 when carbonated to a CO2 content of 2.5 volumes CO2 per volume solution. Example 17: Analysis of the heat treatment of a carbonated solution

[284] An 8% protein solution from sample 16 was carbonated with Petition 870230043920, dated 05 / 25 / 2023, pages 92 / 101 84 / 86 2.5 volumes of CO2 per volume of solution, as shown in example 16.

[285] The solution was heated to a temperature of 95 °C in a closed container with data logging. Temperature, pH and turbidity were measured at different heating times and the result is shown in figure 12.

[286] As shown in Figure 12, the carbonated product remained clear during heating and after 5 minutes at 95 °C, as indicated by the measured turbidity and visual inspection. The pH remained at 6.2 during heating. These data indicated that the carbonated product may be suitable for treatments such as pasteurization and autoclaving, essentially treatments at temperatures up to 120 °C for an extended period, for example, 20 minutes. Example 18: Protein bar

[287] Example 17 shows examples of the use of whey protein hydrolysate according to the invention in the preparation of a protein bar.

[288] Table 14 shows an example of a protein bar with a content of 5 g / 100 g of whey protein hydrolysate (powder) of the present invention. Table 14: Protein bar g / 100 g Hydrolyzed 5 Milk protein 37 Glycerol 4.9 Sugars 39.5 Lipids 6 Flavors 1.75

[289] Table 15 shows another example of a protein bar, but this example has a whey protein hydrolysate (powder) content. Petition 870230043920, dated 05 / 25 / 2023, pages 93 / 101 85 / 86 in a quantity of 13 g / 100 g. Table 15: Protein bar g / 100 g Hydrolyzed 13 Milk protein 24 Glycerol 4.9 Sugars 39.5 Lipids 6 Flavors 1.75 Example 19: Analysis of antioxidant activity using the DPPH assay.

[290] The antioxidant effect of the whey protein hydrolysate of the invention was analyzed using the DPPH assay, which is a radical scavenging assay. The assay measures the antioxidant effect using DPPH (2,2-diphenyl-1-picrylhydrazyl hydrate), since the color of DPPH changes from purple to yellow after reaction with antioxidant peptides.

[291] DPPH was dissolved in methanol to a concentration of 0.2 mM. The whey protein hydrolysate of the invention (sample 16) was dispersed in MilliQ water to different protein contents (0, 0.8, 1.5, 2.5, 3.0, 4.0, 5.0 and 6.0% protein) and mixed with DPPH in equal volumes (1:1 ratio). The mixtures were incubated for 2 hours at 22 °C to allow the yellow color to develop as a result of antioxidant activity. The absorbance at 525 nm was measured and the percentage of elimination calculated as 100x (Ao-As) / Ao, where Ao is the absorbance in the absence of sample, and As is the absorbance in the presence of sample.

[292] Table 16 shows the percentage of elimination for various concentrations of the whey protein hydrolysate of the invention (sample 16). Petition 870230043920, dated 05 / 25 / 2023, pages 94 / 101 86 / 86 Table 16: Sample 16 (% protein) Percentage of elimination (%) 0 0 0.8 48±2 1.5 57±3 2.5 67±1 3 72±1 4 75±2 5 77±2 6 78±0

[293] Table 16 shows that whey protein hydrolysate from sample 16 has antioxidant activity when included in the DPPH assay at protein concentrations of 0.8 to 6%. As shown in Table 16, antioxidant activity increased with increasing protein concentration. Petition 870230043920, dated 05 / 25 / 2023, pages 95 / 101

Claims

1 / 5 CLAIMS 1. A method for preparing a whey protein hydrolysate, characterized in that it comprises: a) providing a whey protein solution comprising whey protein in an amount of at least 50% by weight based on the total solids content; b) subjecting the whey protein solution to enzymatic hydrolysis, wherein the enzymatic hydrolysis is carried out using any one of the following enzyme combinations: i) comprising at least one Bacillus serine endopeptidase, at least one Aspergillus serine endopeptidase, and at least one trypsin-like protease; ii) comprising at least one Bacillus serine endopeptidase, at least one Aspergillus serine endopeptidase, and at least one Aspergillus leucilaminopeptidase; iii) comprising at least one bacillolysin from Bacillus amyloliquefaciens, at least one bromelain, and at least one leucilaminopeptidase from Aspergillus;c) to interrupt enzymatic hydrolysis by inactivating the enzymes when the degree of hydrolysis (DH) is in the range of 15% to 35% to obtain a whey protein hydrolysate; and wherein the method does not include any ultrafiltration step of the whey protein hydrolysate obtained in step c).

2. Method according to claim 1, characterized in that it further comprises a step d) of concentration and / or drying of the whey protein hydrolysate obtained in step c).

3. Method according to claim 1 or 2, characterized by the fact that the whey protein solution of step a) comprises lipids in an amount of no more than 10% by weight based on the total solids content.

4. Method according to claim 1, characterized in that the enzymatic hydrolysis in step b) is carried out at a temperature in the range of 40 °C to 75 °C.

5. Method according to any one of claims 1 to 4, characterized in that the enzyme inactivation in step c) is by heating to a temperature of at least 80 °C, preferably 80 °C to 130 °C.

6. A method according to any one of claims 1 to 5, characterized in that the whey protein solution comprises a whey protein concentrate, a whey protein concentrate, whey protein isolate and / or a whey protein isolate.

7. A method according to any one of claims 1 to 6, characterized in that the whey protein solution comprises protein in an amount of 2% by weight or more of the whey protein solution.

8. Method according to any one of claims 1 to 7, characterized in that the enzymatic hydrolysis in step b) is carried out using any one of the following enzyme combinations: i. comprising at least one serine endopeptidase from a Bacillus species, at least one serine endopeptidase from Aspergillus oryzae and at least one trypsin-like protease of microbial origin; ii. comprising at least one subtilisin from Bacillus licheniformis, at least one serine endopeptidase from Aspergillus oryzae and at least one leucyl aminopeptidase from Aspergillus oryzae; iii. comprising at least one bacillolysin from Bacillus amyloliquefaciens, at least one bromelain from Ananas comosus, and at least one leucil-aminopeptidase from Aspergillus oryzae.

9. Whey protein hydrolysate, characterized in that it comprises: - free amino acids and peptides, and - having a degree of hydrolysis of 15% to 35%; - peptides with a molecular weight of 2500 Da or more in an amount of 8% to 25% by weight of the total amount of peptides, and - having free amino acids in an amount of 8% by weight or less of the total amino acid content in the hydrolysate, and wherein the whey protein hydrolysate in a 4% w / w protein solution has a bitterness score corresponding to a 0.08% w / v or less caffeine solution.

10. Whey protein hydrolysate according to claim 9, characterized in that it has a nephelometric turbidity (NTU) of 100 or below in a 4% (w / w) protein solution.

11. Whey protein hydrolysate according to claim 9 or 10, characterized in that it comprises free amino acids in an amount of 2 to 15% by weight of the total protein content in the hydrolysate.

12. Whey protein hydrolysate according to any one of claims 9 to 11, characterized in that it has antioxidant activity.

13. Whey protein hydrolysate according to any one of claims 9 to 12, characterized in that the antioxidant activity of the whey protein hydrolysate is measured as having a percentage elimination of 54 to 60 in a 1.5% by weight protein solution.

14. Use of whey protein hydrolysate as defined in any one of claims 9 to 13, characterized by being in a food product.

15. Use according to claim 14, characterized in that the whey protein hydrolysate is used in the food product in an amount corresponding to the food product comprising from 2 to 25% by weight of hydrolyzed protein.

16. Use according to claim 14 or 15, characterized in that the food product is selected from the group consisting of a dairy product, a beverage, a shake, a gel, a shot, and a food bar.

17. Use of whey protein hydrolysate as defined in any one of claims 9 to 13, characterized by the fact that it is used as a food ingredient.

18. Use of whey protein hydrolysate defined in any of claims 9 to 13, characterized by being used as a food ingredient in the preparation of a stable UHT beverage with a pH of 6.5 to 8.

0.

19. Use of whey protein hydrolysate as defined in any one of claims 9 to 13, characterized by its use as a food ingredient in the preparation of beverages for use as sports nutrition.

20. Use of whey protein hydrolysate as defined in any one of claims 9 to 13, characterized by its use as a food ingredient in the preparation of a clinical beverage.

21. Use of whey protein hydrolysate defined in any of claims 9 to 13, characterized by being an ingredient in the preparation of a carbonated beverage.

22. Use of whey protein hydrolysate defined in any of claims 9 to 13, characterized by its antioxidant properties.

23. Carbonated beverage, characterized in that it comprises whey protein hydrolysate as defined in any one of claims 9 to 13. Petition 870250076698, dated 08 / 28 / 2025, page 18 / 18

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