Selectively hydrolysed WHEY protein compositions

Abstract

The present disclosure relates to treatment of whey proteins for improved properties in food use, particularly in infant formulas. More particularly, the present disclosure relates to selective hydrolysis of whey proteins to provide whey protein compositions with altered protein composition. A selectively hydrolysed whey protein composition, a method of producing a selectively hydrolysed whey protein composition and a food product comprising the selectively hydrolysed whey protein composition are provided.

Description

[0001] SELECTIVELY HYDROLYSED WHEY PROTEIN COMPOSITIONS

[0002] FIELD OF THE DISCLOSURE

[0003] The present disclosure relates to treatment of whey proteins for improved properties in food use, particularly in infant formulas. More particularly, the present disclosure relates to selective hydrolysis of whey proteins to provide whey protein compositions with altered protein composition.

[0004] BACKGROUND OF THE DISCLOSURE

[0005] Infant formulas are needed in various social and / or physiological situations where breast feeding is not possible. The properties of being immunologically tolerated and not inducing digestive discomfort are of great importance to infant formulas.

[0006] Infant formulas have conventionally been prepared by using a mixture of whey and milk. Therefore, these formulas contain whey proteins and caseins. Beta-lactoglobulin (BLG, p- LG) is the primary protein in bovine whey, forming 50-70% of whey protein. BLG, however, is not present in human milk. Alpha-lactalbumin (ALA, a-LA) is another major bovine whey protein. ALA forms 15-20% of bovine whey proteins and shows 72% sequence homology with human ALA which is abundantly present in human milk. Thus, bovine ALA is often easily tolerated by humans, making it a suitable protein for infant formulas.

[0007] In many conventional infant formulas, BLG is the main protein constituent and also a major allergen which may potentially induce discomfort and / or intolerance in infants due to its non-human origin. To improve digestive tolerance, enhance digestibility, and to reduce allergenicity, milk proteins in infant formulas can be hydrolysed with food grade proteases. For production of reduced allergen and allergen-free formulas, extensive enzymatic hydrolysis of proteins is required. Hydrolysis must often be combined with an appropriate filtration step to produce a protein ingredient without large intact protein fractions.

[0008] Thus, there is a constant need to develop products that more closely resemble human milk with respect to compositional, nutritional, functional, and physiological factors.

[0009] BRIEF DESCRIPTION OF THE DISCLOSURE

[0010] An object of the present disclosure is to provide a selectively hydrolysed whey protein composition, a method of producing a selectively hydrolysed whey protein composition and a food product comprising the selectively hydrolysed whey protein composition so as to alleviate the above disadvantages.

[0011] The object of the disclosure is achieved by the selectively hydrolysed whey protein composition, the method of producing a selectively hydrolysed whey protein composition and the food product comprising the selectively hydrolysed whey protein composition which are characterised by what is stated in the independent claims. The preferred embodiments of the disclosure are disclosed in the dependent claims.

[0012] The disclosure is based on the idea of selectively hydrolysing a whey protein derivative such that BLG is targeted for degradation while ALA is retained. Advantages of the present disclosure relate for example to the expected enhanced digestibility and digestive tolerance of the BLG-reduced whey protein composition which makes it suitable for use in e.g. “comfort-type” infant formulas. Comfort-type infant formulas are special formulas designed to be easier on a baby's digestive system, particularly for those who experience mild digestive discomforts like gas, colic, or fussiness. Selective hydrolysis may also maintain and even improve other functional properties such as taste compared to conventional hydrolysed whey protein.

[0013] The selectively hydrolysed whey protein composition according to the disclosure has a high degree of hydrolysis which also predicts better digestibility and lower allergenicity. Moreover, the preserved intactness of ALA may bring additional value due to its similarity to human milk ALA. The similar biological activity may have a positive influence on infant growth and development. The selectively hydrolysed whey protein composition possesses good solubility and will likely confer enhanced heat stability since the heat sensitive BLG fraction has been degraded into smaller peptides.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In the following the disclosure will be described in greater detail by means of preferred embodiments with reference to the accompanying drawings, in which

[0016] Figure 1 shows gel-electrophoresis (SDS-PAGE) images illustrating how selectivity of enzymatic hydrolysis of whey protein i.e. intactness of BLG and ALA is either maintained or lost upon terminating hydrolysis by heat-treatment, compared to a sample where hydrolysis has been terminated by lowering pH to a value < 3. A) Hydrolysate samples terminated by indirect heating at 97°C for 1 min). B) Hydrolysate samples terminated by direct heating at 125°C for 9 s);

[0017] Figure 2 shows a gel-electrophoresis image of protein bands illustrating the selective degradation of BLG and maintaining of intact ALA after selective hydrolysis of whey protein. 1 . Whey protein concentrate (WPC) before hydrolysis; 2. After hydrolysis reaction terminated by lowering the pH to < 3; 3. After hydrolysis reaction terminated by heating to 95°C for 20 s; 4. After hydrolysis reaction terminated by heating 90°C for 2 min. Abbreviation: BSA = bovine serum albumin; Figure 3 shows a gel-electrophoresis image related to table 5. in the patent, showing how intact ALA is maintained in the hydrolysates. In A), lane 1 . shows unhydrolysed whey protein concentrate, and lanes 2.-5. selective hydrolysates from Formea TL treatment inactivated by pH < 3 (2.), heat treatment at pH 8.5 (3.), heat treatment at pH 7.0 (4.), and heat treatment at pH 6.0 (5.). In B), lane 6. shows unhydrolysed whey protein concentrate, and lanes 7.-12. show selective hydrolysates from Alcalase treatment inactivated by pH < 3 (7.), heat treatment at pH 8.5 (8.), heat treatment at 7.0 (9.), heat treatment at 6.7 (10.), heat treatment at 6.4 (11 .), and heat treatment at 6.0 (12.); and

[0018] Figure 4 shows a gel-electrophoresis image of samples taken from different steps in a pilot run of whey protein selective hydrolysis. 1. WPC before hydrolysis; 2. After hydrolysis reaction terminated by lowering the pH to < 3; 3. After hydrolysis reaction terminated by pasteurisation at 90°C for 2 min; 4. Dried selective hydrolysate powder.

[0019] The Figures are available on PATENTSCOPE in colour or grayscale format.

[0020] DETAILED DESCRIPTION OF THE DISCLOSURE

[0021] Selective hydrolysis of whey protein refers to a process in which specific target proteins among whey proteins are preferably hydrolysed i.e. cleaved into smaller fragments by enzymes, leading to a decrease in relative content of said target proteins in the whey protein. Total protein content in the whey protein remains the same, but the proportion of smaller-size peptides and amino acids in the total protein increases. Particularly, digestion of BLG is preferred over digestion of other proteins such as ALA, leading to a decrease in content of intact BLG. The relative content of intact ALA in comparison to intact BLG, i.e. the intact ALA to intact BLG ratio thus increases in selective hydrolysis.

[0022] Thus, a key to obtaining selectively hydrolysed whey protein is to find suitable conditions in which enzymatic digestion of BLG is preferred over digestion of ALA. Additionally or alternatively, suitable conditions should be found for terminating the proteolytic enzyme activity while avoiding degradation of particularly ALA and also other proteins than BLG by the conditions used during selective hydrolysis and / or termination.

[0023] Previously, separation of ALA and BLG fractions in bovine whey has been studied using for instance chromatographic methods, salting out / precipitation processes, and selective heat denaturation. All of these methods are laborious, require a specific skillset and instrumentation, and may be difficult to perform in an industrial scale.

[0024] Previous attempts to selectively hydrolyse whey protein by enzymes have been hampered by lack of enzymes with sufficiently selective behaviour as well as the difficulty of finding hydrolysis conditions that provide satisfactory selectiveness. For instance, selective enzymatic hydrolysis of BLG in whey protein isolate has been carried out under highly specific, narrow-range hydrolysis conditions using as hydrolytic enzyme animal-based trypsin and chymotrypsin or experimental Bacillus licheniformis protease. Previous attempts at selective hydrolysis have also often been associated with a generally low degree of protein hydrolysis. It is often beneficial that BLG is not only selectively hydrolysed but is further degraded into smaller fragments which is reflected by an increase in overall degree of hydrolysis (DH) %.

[0025] As used herein, the term “degree of hydrolysis” or “DH” is defined as the percentage of peptide bonds in the original proteins that have been cleaved by hydrolysis. With “original proteins” is meant the total protein contained in the whey protein derivative that is subjected to protein hydrolysis. Suitable methods for determining degree of hydrolysis include the o-phthaldialdehyde (OPA; also known as benzene-1 ,2-dicarbaldehyde and o- phthalaldehyde) method, based on the method of Nielsen et al. (2001) to determine free amino nitrogen in the product. The free alpha-amino groups formed in hydrolysis react with OPA and form a yellow complex which absorbs light at 340 nm and therefore can be measured spectrophotometrically. Based on the colour formation, DH can be calculated. The degree of hydrolysis (DH) % is calculated according to equation 1 :

[0026] DH % = (a / atotai) *100 [1]

[0027] Where: atotai is the total amount of amino nitrogen in the protein substrate (determined through acid hydrolysis), and a is the amount of free amino nitrogen liberated upon hydrolysis (free amino nitrogen in the hydrolysate subtracted by the free amino nitrogen in the substrate before hydrolysis).

[0028] In addition to choosing the suitable enzymes, selective hydrolysis of whey protein typically requires defined hydrolysis conditions, such as a suitable temperature, pH, enzyme to substrate ratio, and hydrolysis time. It has now been discovered that enzymatic hydrolysis with endoproteases produces a selectively hydrolysed whey protein composition having an increased amount of intact ALA in relation to intact BLG. Notably, the present disclosure is not based on fractionation of whey protein, but the whey protein composition of the present disclosure comprises essentially the entire milk protein content of the starting material, whey protein derivative. In other words, total protein content in the selectively hydrolysed whey protein composition remains same although whey proteins of the starting material are partially degraded. Thus, in an aspect, the disclosure relates to a method of producing a selectively hydrolysed bovine whey protein composition. The method comprises providing a whey protein derivative comprising beta-lactoglobulin (BLG) and alpha-lactalbumin (ALA) and subjecting the whey protein derivative to an enzymatic treatment with at least one endoprotease. Optionally, the serine endoprotease is a member of the class EC 3.4.21. Enzymatic hydrolysis of the whey protein composition provides a selectively hydrolysed whey protein composition.

[0029] Whey is the liquid remaining after a part of milk protein has been removed from milk. As used herein, the term “whey” may refer to cheese whey or sweet whey. “Whey” may also refer to types of whey prepared from milk by filtration steps such as ideal whey, whey protein concentrate, whey protein isolate, nanofiltered whey and demineralised whey.

[0030] Herein, “milk” and “whey” refer particularly to bovine milk and bovine whey. Bovine milk is the milk produced by cows. Cow's milk is dominated by caseins, which make up about 80% of the milk proteins. Milk of other mammals is also suitable such as milk of sheep, goats, buffaloes, horses, camels, llamas and deer.

[0031] Cheese whey is a liquid by-product that is left after milk has been curdled and strained during cheese production. It contains water, lactose, vitamins, minerals, and various proteins that do not coagulate into curd during the cheese-making process. Infant formulas are often produced from cheese whey as a source of lactose and protein. Sweet whey is a common form of cheese whey and results when enzymes like rennet are used to curdle the milk in the production of cheeses such as Cheddar, Swiss, and mozzarella. Sweet whey has a pH of about 5.6 or above and contains a moderate amount of lactose, which gives it a slightly sweet taste. Other types of cheese whey include acid whey which is produced when milk is coagulated with acid (e.g., vinegar or lactic acid bacteria) instead of rennet, typically in the making of acidic cheeses like ricotta or soft cheeses like cottage cheese, as well as strained yogurts like Greek yogurt. Acid whey has a lower pH (below 5) due to its higher acid content, making it tangier. Salty whey can be a by-product of cheese-making process where salt is heavily used.

[0032] The protein content of whey is a collection of proteins. About 80% of the protein in cow's milk is casein, the remaining 20% being whey protein. Casein forms a curd when milk is acidified or coagulated, leaving whey proteins in the liquid fraction. The three principal whey proteins are p-lactoglobulin (BLG), a-lactalbumin (ALA), and caseinomacropeptide (CMP) which is released from casein by rennet. When CMP is glycosylated, it is often referred to as glycomacropeptide (GMP). Casein and whey protein in milk can be separated into casein rich and whey protein rich fractions by microfiltration. When milk is filtered using 0.1 to 0.5 pm membranes, whey proteins penetrate through the membrane into permeate whereas casein is retained in retentate. This whey protein fraction separated from milk by filtration is called ideal whey. In the production of ideal whey, casein is not precipitated before the separation of whey. The protein composition of ideal whey differs from the composition of conventional cheese whey for example in that ideal whey does not contain metabolism products of starters, such as lactic acid, that are released to the cheese whey in cheese-making. Similarly, the presence of CMPs released by rennet enzymes from kappa casein to the whey is avoided. In human milk, the most important types of protein are a-lactalbumin and p-casein. When release of caseinomacropeptides to whey is avoided, a-lactalbumin contained in the whey protein forms a larger portion of total protein. Thus, by using microfiltration it is possible to achieve in ideal whey a protein composition which is closer to that of human milk, compared to the use of cheese whey.

[0033] Whey protein concentrate (WPC) is a high-protein product produced by removing lactose and minerals from whey by ultrafiltration and diafiltration. Ultrafiltration membranes typically have pore sizes of around 10 nanometres, and they retain larger molecules like whey proteins and fats while allowing smaller molecules like water, lactose, and minerals to pass through. Diafiltration is a variation of ultrafiltration, where additional water is added to the retentate to further reduce the content of smaller molecules like lactose and salts in the retentate. WPC typically has set protein levels, e.g. WPC34 with 34% protein or WPC80 with 80% protein.

[0034] Whey protein isolate (WPI) is a highly purified form of whey protein, which has undergone additional processing to remove most of the non-protein components such as fats and lactose. WPI typically contains 90 wt-% or more of protein and less lactose, fat and other milk solids than WPC. Compared to WPC, WPI is further processed through microfiltration or ion exchange to concentrate protein and to remove fats and lactose.

[0035] Nanofiltration is a membrane filtration process that utilises membranes having a pore size of 1 -2 nm. Nanofiltration can be used to remove small molecules, minerals and ions from whey while retaining larger molecules, like lactose and proteins, in the retentate.

[0036] Demineralised whey has undergone a demineralisation process to remove a significant portion of its mineral content, such as calcium, phosphorus, magnesium, sodium, and potassium. Demineralised whey may be produced from cheese whey or ideal whey by filtration, electrodialysis and / or ion exchange. Protein and lactose content of demineralised whey do not necessarily differ significantly from those of the cheese whey or ideal whey used as starting material. Due to the lower mineral content, demineralised whey is often easier on the digestive system of infants. Demineralised whey is usually categorised by the percentage of minerals removed: in D40 40% of the mineral content is removed, in D70 70% of the mineral content is removed, and in D90 90% of the mineral content is removed. D90 is commonly used in sensitive applications like infant formulas.

[0037] As used herein, the term “whey protein derivative” refers to a whey protein comprising composition that is produced from bovine milk. The whey protein derivative may be a cheese whey or derived from cheese whey, or the whey protein derivative may be an ideal whey or derived from ideal whey. Also, the whey protein derivative may be a whey protein concentrate, a whey protein isolate, a nanofiltered whey or a demineralised whey processed from cheese whey, ideal whey or any mixture of cheese whey and ideal whey.

[0038] As used herein, the terms “native”, “native protein” and the like refer to a protein that has the specific three-dimensional structure that the protein assumes under physiological conditions in order to perform its biological function. A protein maintaining it native conformation can be said to be in its native form or native state. Native conformation is typically the most energetically stable and biologically active form of the protein.

[0039] As used herein, the terms “intact, “intact protein” and the like refer to a protein that does not necessarily maintain its native 3-D structure, but the amino acid chain(s) that form(s) the protein is(are) whole and no parts of it(them) have been cleaved off. An intact protein can be said to be in its intact form or intact state. In intact protein, intermolecular covalent bonds such as disulfide bridges often persist, but are sometimes cleaved by e.g. chemical treatment. In case all intermolecular covalent bonds are cleaved, subunits of a multimeric protein may no longer be bonded to each other and the protein may degrade into its subunits. Native protein is always intact protein, but not all intact protein necessarily has native conformation.

[0040] Herein, the term “ALA” may refer to intact ALA or native ALA or ALA that is both intact and native. Similarly, the term “BLG” may refer to intact BLG or native BLG or BLG that is both intact and native.

[0041] As used herein, the terms “denatured” and “denatured protein” and the like refer to a protein that has lost its native, three-dimensional structure without breaking its primary amino acid sequence. This structural change typically disrupts the protein's functional and biological properties, as the specific folding pattern of a protein is crucial to its activity. Denaturation can be caused by various physical and chemical factors, including heat, pH changes, denaturing chemical agents and mechanical stress. Once denatured, a protein's secondary, tertiary, or quaternary structures are altered, often resulting in loss of solubility and function. Unlike hydrolysis, denaturation does not break the protein into smaller peptides or amino acids - it only unfolds the structure. Thus, intact protein may be denatured, but native protein may not.

[0042] Bovine ALA is a monomeric protein of 123 amino acid residues and a molecular weight of 14 kDa. It’s native, active structure is stabilised by the presence of four intramolecular disulfide bridges and the binding of calcium.

[0043] Bovine BLG is a globular protein comprising 162 amino acid residues and has a molecular weight of 18.3 kDa. It normally occurs as a non-covalently linked dimer or octamer at neutral pH but separates into monomers at low or high pH or under reducing conditions. A significant feature of BLG is the sulfur chemistry of this protein: in addition to two intramolecular disulfide bridges that stabilise the protein, there is a single sulfhydryl group at Cys 121 that is buried in the protein, protected by an alpha helix region. If exposed as a result of heating or other disruption of the secondary and tertiary structure of the protein, this sulfhydryl can react with other sulfhydryl groups, leading to disulfide exchange and cross-linking reactions with other beta-lactoglobulin molecules or other whey or food proteins, depending on the environment.

[0044] As used herein, the terms “digestion”, “enzymatic digestion” and the like refer to breaking down of proteins into smaller peptides and amino acids by enzymes. Enzymatic digestion is typically specific and selective, as different proteases recognize and cleave specific amino acid sequences or types of bonds, allowing controlled breakdown of proteins. For enzymatic digestion, terms such as “enzymatic hydrolysis”, “enzymatic degradation”, proteolysis and the like may also be used.

[0045] As used herein, the term “enzymatic treatment” refers to subjecting proteins to enzymatic activity whereby at least a part of the proteins are digested by enzymes into smaller molecules. In some instances, the terms “enzymatic treatment”, “enzymatic hydrolysis”, “enzymatic degradation” and the like may be used interchangeably.

[0046] As used herein, the term “unhydrolysed” refers to protein that has not undergone enzymatic hydrolysis. "Unhydrolysed protein" can imply that it is the original, native and intact form of the protein before any hydrolysis process took place, whereas “nonhydrolyzed protein” can imply that the protein has undergone an enzymatic treatment but has remained in intact form and possibly also in native form and was not cleaved by the hydrolytic enzyme. An endoprotease, also known as endopeptidase, is a type of enzyme that cleaves peptide bonds within the interior of a protein or polypeptide chain, as opposed to cutting at the terminal ends. By targeting internal peptide bonds, endoproteases break down proteins into smaller peptide fragments.

[0047] In the method according to the present disclosure, the at least one endoprotease is preferably a serine endoprotease.

[0048] Serine endoproteases (also known as serine endopeptidases or serine proteases) are a class (EC 3.4.21) of enzymes that cleave internal peptide bonds in proteins and polypeptides using a serine residue in their active site. Particularly, the mechanism of action of serine endoproteases involves a so-called catalytic triad, typically composed of three key amino acids: serine, histidine, and aspartate. These residues work together to cleave the peptide bond.

[0049] As shown herein, serine endoproteases including trypsin-like endoproteases, chymotrypsin-like endoproteases and subtilisins are suitable enzymes for providing the selectively hydrolysed whey protein composition. Examples of suitable serine endoproteases include trypsin-like endoproteases such as trypsin-like endoprotease from Fusarium venenatum. It is sold under commercial names including Formea TL and Formea Prime which are both manufactured by Novonesis. Also, chymotrypsin-like endoproteases such as chymotrypsin-like endoprotease from Bacillus licheniformis which is sold e.g. under the commercial name Formea CTL (Novonesis) express selective hydrolysis behaviour towards BLG. In addition, subtilisins such as subtilisin from B. licheniformis, which is sold e.g. under the commercial name Alcalase (Novonesis), show selectivity towards BLG in whey protein hydrolysis. In addition to enzymes derived from microbes, animal-origin serine endoprotease enzymes such as trypsin and chymotrypsin may in some instances be used for selective hydrolysis of whey protein.

[0050] Thus, the at least one serine endoprotease employed in the method of the disclosure may be selected from trypsin-like endoproteases, chymotrypsin-like endoproteases, subtilisins and any mixtures thereof. Particularly, the serine endoprotease may be of microbial origin. The use of commercial microbial enzymes allows broad and rapid utilisation of the technology across different consumer groups.

[0051] Trypsin-like endoproteases cleave peptide bonds following a positively charged amino acid residue (lysine or arginine), whereas chrymotrypsin-like endoproteases cleave peptide bonds at aromatic residues like phenylalanine, tyrosine, and tryptophan. Subtilisins tend to favour large, hydrophobic amino acids such as phenylalanine, leucine, and tyrosine as the amino acid immediately adjacent to the cleavage site on the N-terminal side.

[0052] Particularly, the serine endoprotease may be an alkaline serine endoprotease. Alkaline serine endoproteases are a group of enzymes that belong to the serine protease family, characterized by their ability to break down proteins into smaller peptides or amino acids. They operate optimally under alkaline conditions (typically pH 8-1 1 ) and rely on a catalytic triad including serine, histidine, and aspartate in their active site for catalytic activity.

[0053] In addition to choosing the correct enzymes, selective hydrolysis typically requires defined hydrolysis conditions, such as temperature, pH, and time.

[0054] Selective hydrolysis may be performed at cold temperatures such as temperatures between above freezing point (>0°C) and 10°C. A benefit of using cold temperatures for hydrolysis is that less energy for heating is required for performing the hydrolysis. Also, a cold temperature may protect the proteins from nonspecific degradation and denaturation, thus contributing to specificity of selective hydrolysis. Hydrolysis may also be performed at higher temperatures of up to e.g. 45°C, depending for example on the temperature optimum of the endoprotease enzyme(s) used. At temperatures below the optimum range of enzymes the reaction speed slows down and a longer reaction time is required for completing the hydrolysis reaction. Employing a higher temperature increases the enzymatic reaction speed but requires a larger energy input to heating the hydrolysis mix and increases the risk of nonspecific degradation and denaturation of proteins. Generally, hydrolysis temperatures that are both technically feasible and economically viable in manufacturing processes range from 1 °C to 30°C.

[0055] In the method of the disclosure, enzymatic treatment of a whey protein derivative is typically performed at a temperature in the range of 1 °C to 30°C, preferably 2°C to 25°C, more preferably 3°C to 12°C. Alternatively, the enzymatic treatment is performed at a temperature of 1 °C to 30°C, or 2°C to 25°C, or 1 °C to 12°C, or 20°C to 30°C. Alternatively, the enzymatic treatment is performed at a temperature ranging between any of said values.

[0056] During hydrolysis, pH of the enzymatic treatment has an effect on both the endoprotease enzyme(s) used and the whey protein to be hydrolysed. The hydrolysis mix contains components of the hydrolysis reaction, including the protease enzyme and whey protein substrate which are typically dissolved in an aqueous solution. Additionally, the hydrolysis mix may contain components such as a pH adjusting agent, buffer, salt, and detergent. Endoproteases function most efficiently in their respective pH optimum ranges. A pH within the optimum pH range facilitates functioning of the chemical reaction occurring at the catalytic site and / or maintaining functional conformation of the enzyme molecule. On the other hand, pH has an effect on the protein to be hydrolysed. A too low or too high pH may cause aggregation and / or denaturation of substrate proteins, rendering them unavailable for efficient hydrolysis. In selective hydrolysis of whey protein, pH is a particularly important factor in promoting selectivity. A suitable pH promotes BLG degradation over that of other proteins such as ALA.

[0057] It is believed that a combination of low temperature and high pH creates an environment where BLG-molecules appear as monomers with a “loosened” tertiary structure, thus rendering them more susceptible to enzymatic proteolysis. At the same conditions, ALA in turn remains as a compact globular molecule that can more efficiently resist enzymatic degradation.

[0058] In the method disclosed herein, enzymatic hydrolysis of a whey protein derivative may be performed at a pH ranging from about 7.0 to about 9.5, preferably about 7.5 to about 9.5, more preferably about 7.5 to about 9.0, yet more preferably about 8.0 to about 8.5. Additionally or alternatively, the enzymatic treatment may be performed at a pH ranging from more than 7.0 to 9.5, preferably from more than 7.5 to 9.5, more preferably from more than 7.5 to 9.0, yet more preferably from more than 8.0 to 8.5. Additionally or alternatively, enzymatic hydrolysis of a whey protein derivative may be performed at a pH of about 7.0, about 7.1 , about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1 , about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1 , about 9.2, about 9.3, about 9.4, about 9.5, or at a pH ranging between any two of said values.

[0059] In the enzymatic hydrolysis in the method disclosed herein, a pH that is neutral or slightly above neutral is suitable for efficient activity of the endoprotease(s) while maintaining the substrate whey protein, particularly BLG, in a conformation that facilitates efficient selective hydrolysis. In the hydrolysis mix, pH may be adjusted by adding a base. Suitable bases include hydroxide compounds such as alkali metal hydroxides.

[0060] Hydrolysis time is also an important factor in selective hydrolysis. When conditions that favour selective hydrolysis such as the type of enzyme, pH and temperature are applied, it is also important to control the hydrolysis time. A short hydrolysis time may not suffice to cleave the target protein in amounts required for selective hydrolysis, while a too long hydrolysis time may impair selectivity by allowing degradation of proteins other than the target protein. Additionally or alternatively to the pH values of enzymatic treatment disclosed above, in the method disclosed herein, enzymatic hydrolysis of a whey protein derivative may be performed for a period of time ranging from about 2 hours to about 72 hours, preferably about 5 hours to about 48 hours. In general, the higher the temperature used for hydrolysis, the shorter the time required for optimally selective hydrolysis. Alternatively or additionally, the enzymatic treatment may be performed for a period of time ranging from about 2 hours to about 72 hours, or about 5 hours to about 48 hours, or about 3 hours to about 12 hours, or about 15 hours to about 72 hours. Alternatively or additionally, the enzymatic treatment may be performed for a period of time ranging between any two of the values 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h, 27 h, 28 h, 29 h, 30 h, 31 h, 32 h, 33 h, 34 h, 35 h, 36 h, 37 h, 38 h, 39 h, 40 h, 41 h, 42 h, 43 h, 44 h, 45 h, 46 h, 47 h, 48 h, 49 h, 50 h, 51 h, 52 h, 53 h, 54 h, 55 h, 56 h, 57 h, 58 h, 59 h, 60 h, 61 h, 62 h, 63 h, 64 h, 65 h, 66 h, 67 h, 68 h, 69 h, 70 h, 71 h, and 72 h.

[0061] Additionally or alternatively to the pH values of enzymatic treatment disclosed above, the enzymatic treatment may be performed at a temperature of about 1 °C to about 15°C, preferably about 3°C to about 12°C, more preferably about 6°C to about 10°C, and / or for a period of time ranging from 12 to 72 hours, preferably 15 to 72 hours, more preferably 15 to 48 hours. Additionally or alternatively, the enzymatic treatment may be performed at a temperature ranging between any two of the values 0.5°C, 1 °C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 1 1°C, 12°C, 13°C, 14°C, and 15°C. Additionally or alternatively, the enzymatic treatment may be performed for a period of time ranging between any two of the values 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 25 h,

[0062] 26 h, 27 h, 28 h, 29 h, 30 h, 31 h, 32 h, 33 h, 34 h, 35 h, 36 h, 37 h, 38 h, 39 h, 40 h, 41 h,

[0063] 42 h, 43 h, 44 h, 45 h, 46 h, 47 h, 48 h, 49 h, 50 h, 51 h, 52 h, 53 h, 54 h, 55 h, 56 h, 57 h,

[0064] 58 h, 59 h, 60 h, 61 h, 62 h, 63 h, 64 h, 65 h, 66 h, 67 h, 68 h, 69 h, 70 h, 71 h, and 72 h.

[0065] In an exemplary embodiment, the enzymatic treatment is performed at a temperature of about 1 °C to about 15°C, preferably about 3°C to about 12°C, more preferably about 6°C to about 10°C, and for a period of time ranging from about 12 hours to about 72 hours, preferably about 15 hours to about 72 hours, more preferably about 15 hours to about 48 hours. In this exemplary embodiment, the enzymatic treatment is performed at a pH ranging from about 7.0 to about 9.5, preferably about 7.5 to about 9.5, more preferably about 7.5 to about 9.0, yet more preferably about 8.0 to about 8.5. Additionally or alternatively, the enzymatic treatment is performed at a pH ranging from more than 7.0 to 9.5, preferably from more than 7.5 to 9.5, more preferably from more than 7.5 to 9.0, yet more preferably from more than 8.0 to 8.5. Additionally or alternatively, the enzymatic treatment is performed at a pH of about 7.0, about 7.1 , about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1 , about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1 , about 9.2, about 9.3, about 9.4, about 9.5, or at a pH ranging between any two of said values.

[0066] Additionally or alternatively to the pH values of enzymatic treatment disclosed above, the enzymatic treatment may be performed at a temperature of about 15°C to about 45°C, preferably about 15°C to about 35°C, more preferably about 20°C to about 30°C, and / or for a period of time ranging from about 2 hours to 12 hours, preferably about 3 hours to about 10 hours, more preferably about 5 hours to about 10 hours. Additionally or alternatively, the enzymatic treatment may be performed at a temperature ranging between any two of the values 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41 °C, 42°C, 43°C, 44°C, and 45°C. Additionally or alternatively, the enzymatic treatment may be performed for a period of time ranging between any two of the values 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, and 12 h.

[0067] In an exemplary embodiment, the enzymatic treatment is performed at a temperature of about 15°C to about 45°C, preferably about 15°C to about 35°C, more preferably about 20°C to about 30°C, and for a period of time ranging from about 2 hours to 12 hours, preferably about 3 hours to about 10 hours, more preferably about 5 hours to about 10 hours. In this exemplary embodiment, the enzymatic treatment is performed at a pH ranging from about 7.0 to about 9.5, preferably about 7.5 to about 9.5, more preferably about 7.5 to about 9.0, yet more preferably about 8.0 to about 8.5. Additionally or alternatively, the enzymatic treatment is performed at a pH ranging from more than 7.0 to 9.5, preferably from more than 7.5 to 9.5, more preferably from more than 7.5 to 9.0, yet more preferably from more than 8.0 to 8.5. Additionally or alternatively, the enzymatic treatment is performed at a pH of about 7.0, about 7.1 , about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1 , about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1 , about 9.2, about 9.3, about 9.4, about 9.5, or at a pH ranging between any two of said values.

[0068] In order to stabilise the selectively hydrolysed whey protein composition achieved by the enzymatic treatment, the enzymatic degradation reaction is preferably terminated. Termination facilitates maintaining the characteristics of the produced whey protein composition, such as the intact ALA to intact BLG ratio, degree of hydrolysis and size distribution of proteins and / or peptides. Termination is performed by inactivating the hydrolytic enzyme(s) at least in part. Inactivation may be carried out by heat treatment or pH adjustment or both. Some conventionally employed enzyme inactivation methods may have poor industrial applicability: for example, heat treatment as the enzyme inactivation method may have better industrial applicability compared to employing enzyme inhibitors and / or lowering the pH to a strongly acidic value such as to a value below pH 3.

[0069] Termination is carried out to completely or effectively abolish enzymatic activity. Its success can be verified by monitoring the reaction over time, for example by measuring the degree of hydrolysis (DH). In a properly terminated reaction, DH remains stable, showing no or only negligible increase during the observation period (e.g., 1-7 days).

[0070] The method used for enzyme inactivation is important in maintaining the selectivity achieved during selective hydrolysis. In some cases, termination of the enzymatic reaction by heat treatment can lead to a major loss of hydrolysis selectivity. This is because during heat treating, the temperature applied must surpass optimum temperature of the enzyme, which may also lead to degradation of ALA. In addition, heat treating may cause protein aggregation, especially when heat treating is combined with a very low pH value such as pH < 3. As used herein, the term “loss of selectivity” refers to a change in the whey protein composition achieved by selective hydrolysis, particularly by protein or peptide degradation, more particularly by degradation leading to a decrease in content of intact ALA or a decrease in intact ALA to intact BLG (weight) ratio.

[0071] Selective degradation of BLG may also improve heat stability of the resulting whey protein ingredient. Improved heat stability is beneficial because powdery ingredients may be used, for example, in the production of UHT (ultra-high temperature) infant formulas. Increased heat stability reduces the risk of the powdery ingredient coagulating or burning onto surfaces during heat treating. However, when heat inactivation is applied, a high temperature and a short heat treating time are sometimes preferred to ensure rapid inactivation of the enzyme and a low denaturation rate of ALA.

[0072] It has now been discovered that heat treating at a temperature exceeding the optimum temperature of the at least one endoprotease used in enzymatic hydrolysis, such as a temperature of at least about 60°C, may be used for termination of the enzymatic treatment while maintaining selectivity of hydrolysis. BLG is a relatively heat sensitive protein and its degradation during heat inactivation may enhance the relative decrease of BLG content achieved in selective hydrolysis. Terminating the enzymatic treatment may be achieved by heat treating at a temperature exceeding the optimum temperature of the at least one endoprotease used in the enzymatic treatment. Additionally or alternatively, the temperature of heat treating may be at least about 60°C, optionally in the range of about 60°C to about 160°C, or about 60°C to about 95°C, or about 65°C to about 95°C, or about 70°C to about 90°C, or about 95°C to about 160°C, or about 95°C to about 155°C, or about 100°C to about 150°C. Additionally or alternatively, the heat treating may be performed at a temperature ranging between any two of the values 50°C, 51 °C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61 °C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71 °C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81 °C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91 °C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, 100°C, 105°C, 110°C, 115°C, 120°C, 125°C, 130°C, 135°C, 140°C, 145°C, 150°C, 155°C, 160°C, 165°C, and 170°C.

[0073] Additionally or alternatively, the heat treating at a temperature or temperature range disclosed above may be performed for at least about 0.05 s. Additionally or alternatively, the heat treating may be performed for a period of time ranging from about 0.05 s to about 60 min, or about 0.05 s to about 1 min, or about 0.1 s to about 30 s, or about 0.5 s to about 15 s or about 30 s to about 60 min, or about 30 s to about 30 min, or about 1 min to about 10 min. Additionally or alternatively, the heat treating may be performed for a period of time ranging between any two of 0.05 s. 0.1 s, 0.5 s, 1 s, 2 s, 3 s, 4 s, 5 s, 6 s, 7 s, 8 s, 9 s, 10 s, 15 s, 20 s, 25 s, 30 s, 35 s, 40 s, 45 s, 50 s, 55 s, 1 min, 1 .5 min, 2 min, 2.5 min, 3 min, 3.5 min, 4 min, 4.5 min, 5 min, 5.5 min, 6 min, 7.5 min, 8 min, 8.5 min, 9 min, 9.5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, and 60 min.

[0074] In an exemplary embodiment, terminating the enzymatic treatment is performed by heat treating at a temperature in the range of about 70°C to about 95°C, preferably about 75°C to about 95°C, more preferably about 80°C to about 90°C, for a period of time ranging from about 30 s to about 5 min, preferably about 30 s to about 3 min, more preferably about 1 min to about 2 min.

[0075] In an exemplary embodiment, terminating the enzymatic treatment is performed by heat treating at a temperature in the range of about 95°C to about 160°C, preferably about 95°C to about 155°C, more preferably about 100°C to about 150°C, for a period of time ranging from about 0.05 s to about 1 min, preferably about 0.1 s to about 30 s, more preferably about 0.5 s to about 15 s. Alternatively or additionally to the temperatures and / or durations of heat treating disclosed above, employing for termination of the enzymatic reaction a pH lower than that applied during enzymatic hydrolysis is a suitable method for maintaining selectivity of hydrolysis during enzyme inactivation. The pH may be adjusted to a pH value of above pH 3, or in the range of about 3 to about 8. Optionally, the pH may be adjusted to a pH value of about 4.0 to about 7.5, preferably about 4.5 to about 7.0, more preferably about 5.0 to about 7.0, even more preferably about 6.0 to about 7.0. Additionally or alternatively, the pH may be adjusted to a value in the range between any two of the values 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5,

[0076] 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5,

[0077] 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5,

[0078] 7.6, 7.7, 7.8, 7.9, and 8.0.

[0079] When protein undergoes enzymatic hydrolysis, peptide bonds are cleaved, producing smaller peptides and free amino acids. This introduces new carboxyl (-COOH) groups into the hydrolysis mixture, which can act as weak acids and lower the pH. Buffer capacity exhaustion may contribute to the decrease in pH. To counteract the accumulation of acidity, pH is often controlled during hydrolysis e.g. by introducing a base to maintain the pH at or above a certain pH value. This also ensures that the hydrolysis mixture remains at a pH that is favourable or optimal for enzyme activity.

[0080] In cases where the pH is maintained at or above a certain pH value during enzymatic hydrolysis, the pH may be adjusted for termination to a value that is at least 0.1 pH units lower than the pH employed during hydrolysis, preferably 0.1 to 5 pH units, more preferably 0.5 to 5.0 pH units, even more preferably 0.5 to 4.0 pH units, still more preferably 0.8 to 4.0 pH units, yet more preferably 1 .0 to 3.5 pH units.

[0081] Alternatively or additionally, the pH may be adjusted to a value that is at least 0.1 pH units lower than that employed during hydrolysis, or to a value that is 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6,

[0082] 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6,

[0083] 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0 pH units lower than that employed during hydrolysis, or to a value that is in the range of any two pH units selected from 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1 .1 , 1.2, 1.3, 1.4, 1 .5, 1.6, 1.7, 1.8, 1 .9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, and 6.0 lower than that employed during hydrolysis, such as to a value in the range of 0.1 to 5.0 pH units lower than that employed during hydrolysis. Adjusting pH to a value lower than that applied during enzymatic treatment and optionally to a value of for example above 3.0 before or simultaneously with termination by heat treatment may result in enhanced selectivity as intact BLG remaining after enzymatic treatment is further degraded during termination while intact ALA is retained.

[0084] Accordingly, the method according to the present disclosure may comprise terminating the enzymatic treatment by at least one of i) to ii): i) adjusting pH to a value lower than the pH applied during hydrolysis, optionally to a pH in the range of about 3.0 to about 8.0; and / or ii) heat treating at a temperature exceeding the optimum temperature of the at least one endoprotease, optionally at a temperature of at least about 60°C. Alternatively, pH in i) may adjusted to a pH in the range of about 4.0 to about 7.5, or about 5.0 to about 7.0, or about 5.5 to about 7.0, or about 6.0 to about 7.0. and / or heat treating in ii) may be performed at a temperature in the range of about 60°C to about 160°C, or about 60°C to about 95°C, or about 65°C to about 95°C, or about 70°C to about 90°C, or about 95°C to about 160°C, or about 95°C to about 155°C, or about 100°C to about 150°C. Additionally or alternatively, in ii), the heat treating may be performed for a period of time ranging from about 0.05 s to about 60 min, or about 0.05 s to about 1 min, or about 0.1 s to about 30 s, or about 0.5 s to about 15 s or about 30 s to about 60 min, or about 30 s to about 30 min, or about 1 min to about 10 min. Additionally or alternatively, in i), the pH may be adjusted to a value in the range between any two of the values 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,

[0085] 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,

[0086] 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,

[0087] 7.8, 7.9, and 8.0

[0088] In termination the enzymatic treatment, i) i.e. lowering the pH may be performed before ii) i.e. heat treating, or i) may be performed simultaneously with ii). Preferably, i) is performed before ii).

[0089] In an exemplary embodiment, terminating the enzymatic treatment is performed by adjusting pH to a value lower than that applied during hydrolysis, preferably to a pH in the range of about 3.0 to about 8.0, more preferably about 4.0 to about 7.5, even more preferably about 4.5 to about 7.0, yet more preferably about 5.0 to about 7.0, still more preferably about 6.0 to about 7.0, and heat treating at a temperature exceeding the optimum temperature of the at least one endoprotease applied for the enzyme treatment. Optionally, the temperature may be at least about 60°C, preferably in the range of about 60°C to about 160°C, or about 60°C to about 95°C, or about 65°C to about 95°C, or about 70°C to about 90°C, or about 95°C to about 160°C, or about 95°C to about 155°C, or about 100°C to about 150°C. Optionally, heat treating is performed for a period of time ranging from about 0.05 s to about 60 min, or about 0.05 s to about 1 min, or about 0.1 s to about 30 s, or about 0.5 s to about 15 s or about 30 s to about 60 min, or about 30 s to about 30 min, or about 1 min to about 10 min.

[0090] In an exemplary embodiment, terminating the enzymatic treatment is performed by adjusting pH to a value lower than that applied during hydrolysis, preferably to a pH in the range of about 3.0 to about 8.0, more preferably about 4.0 to about 7.5, even more preferably about 4.5 to about 7.0, yet more preferably about 5.0 to about 7.0, still more preferably about 6.0 to about 7.0, and heat treating at a temperature in the range of about 60°C to about 95°C, preferably about 65°C to about 95°C, more preferably about 70°C to about 90°C. Optionally, heat treating is performed for a period of time ranging from about 30 s to about 60 min, preferably about 30 s to about 30 min, more preferably about 1 min to about 10 min, even more preferably about 1 min to about 5 min.

[0091] In an exemplary embodiment, terminating the enzymatic treatment is performed by adjusting pH to a value lower than that applied during hydrolysis, preferably to a pH in the range of about 3.0 to about 8.0, more preferably about 4.0 to about 7.5, even more preferably about 4.5 to about 7.0, yet more preferably about 5.0 to about 7.0, still more preferably about 6.0 to about 7.0, and heat treating at a temperature in the range of about 95°C to about 160°C, preferably about 95°C to about 155°C, more preferably about 100°C to about 150°C. Optionally, heat treating is performed for a period of time ranging from about 0.05 s to about 1 min, preferably about 0.1 s to about 30 s, more preferably about 0.5 s to about 15 s.

[0092] In an exemplary embodiment, in the method according to the present disclosure, the enzymatic treatment is performed at a temperature of 1 °C to 15°C, preferably 3°C to 12°C, more preferably 6°C to 10°C, and for a period of time ranging from 12 to 72 hours, preferably 15 to 72 hours, more preferably 15 to 48 hours. The enzymatic treatment is performed with at least one endoprotease, preferably a serine endoprotease, more preferably an alkaline serine endoprotease. In this exemplary embodiment, the enzymatic treatment is performed at a pH ranging from 7.0 to 9.5, preferably 7.5 to 9.5, more preferably about 7.5 to about 9.0, yet more preferably 8.0 to 8.5. The enzymatic treatment is terminated by adjusting pH to a value lower than the applied during hydrolysis, preferably to a pH in the range of 3.0 to 8.0, more preferably 4.0 to 7.5, even more preferably 4.5 to 7.0, yet more preferably 5.0 to 7.0, still more preferably 6.0 to 7.0, and heat treating at a temperature above the temperature optimum of the at least one endoprotease, particularly in the range of 60°C to 95°C, preferably 65°C to 95°C, more preferably 70°C to 90°C. Optionally, heat treating is performed for a period of time ranging from 30 s to 60 min, preferably 30 s to 30 min, more preferably 1 min to 10 min, even more preferably 1 min to 2 min.

[0093] In an exemplary embodiment, in the method according to the present disclosure, the enzymatic treatment is performed at a temperature of 1 °C to 15°C, preferably 3°C to 12°C, more preferably 6°C to 10°C, and for a period of time ranging from 12 to 72 hours, preferably 15 to 72 hours, more preferably 15 to 48 hours. The enzymatic treatment is performed with at least one endoprotease, preferably a serine endoprotease, more preferably an alkaline serine endoprotease. In this exemplary embodiment, the enzymatic treatment is performed at a pH ranging from 7.0 to 9.5, preferably 7.5 to 9.5, more preferably about 7.5 to about 9.0, yet more preferably 8.0 to 8.5. The enzymatic treatment is terminated by adjusting pH to a value lower than that applied during hydrolysis, preferably to a pH in the range of 3.0 to 8.0, more preferably 4.0 to 7.5, even more preferably 4.5 to 7.0, yet more preferably 5.0 to 7.0, still more preferably 6.0 to 7.0, and heat treating at a temperature above the temperature optimum of the at least one endoprotease, particularly in the range of 95°C to 160°C, preferably 95°C to 155°C, more preferably 100°C to 150°C. Optionally, heat treating is performed for a period of time ranging from 0.05 s to 1 min, preferably 0.1 s to 30 s, more preferably 0.5 s to 15 s.

[0094] In an exemplary embodiment, the enzymatic treatment is performed at a temperature of 15°C to 45°C, preferably 15°C to 35°C, more preferably 20°C to 30°C, and for a period of time ranging from 2 to 12 hours, preferably 3 to 10 hours, more preferably 5 to 10 hours. The enzymatic treatment is performed with at least one endoprotease, preferably a serine endoprotease, more preferably an alkaline serine endoprotease. In this exemplary embodiment, the enzymatic treatment is performed at a pH ranging from 7.0 to 9.5, preferably 7.5 to 9.5, more preferably about 7.5 to about 9.0, yet more preferably 8.0 to 8.5. The enzymatic treatment is terminated by adjusting pH to a value lower than that applied during hydrolysis, preferably to a pH in the range of 3.0 to 8.0, more preferably 4.0 to 7.5, even more preferably 4.5 to 7.0, yet more preferably 5.0 to 7.0, still more preferably 6.0 to 7.0, and heat treating at a temperature above the temperature optimum of the at least one endoprotease, particularly in the range of 60°C to 95°C, preferably 65°C to 95°C, more preferably 70°C to 90°C. Optionally, heat treating is performed for a period of time ranging from 30 s to 60 min, preferably 30 s to 30 min, more preferably 1 min to 10 min, even more preferably 1 min to 2 min.

[0095] In an exemplary embodiment, the enzymatic treatment is performed at a temperature of 15°C to 45°C, preferably 15°C to 35°C, more preferably 20°C to 30°C, and for a period of time ranging from 2 to 12 hours, preferably 3 to 10 hours, more preferably 5 to 10 hours. The enzymatic treatment is performed with at least one endoprotease, preferably a serine endoprotease, more preferably an alkaline serine endoprotease. In this exemplary embodiment, the enzymatic treatment is performed at a pH ranging from 7.0 to 9.5, preferably 7.5 to 9.5, more preferably about 7.5 to about 9.0, yet more preferably 8.0 to

[0096] 8.5. The enzymatic treatment is terminated by adjusting pH to a value lower than that applied during hydrolysis, preferably to a pH in the range of 3.0 to 8.0, more preferably 4.0 to 7.5, even more preferably 4.5 to 7.0, yet more preferably 5.0 to 7.0, still more preferably 6.0 to 7.0, and heat treating at a temperature above the temperature optimum of the at least one endoprotease, particularly in the range of 95°C to 160°C, preferably 95°C to 155°C, more preferably 100°C to 150°C. Optionally, heat treating is performed for a period of time ranging from 0.05 s to 1 min, preferably 0.1 s to 30 s, more preferably 0.5 s to 15 s.

[0097] It is also disclosed a method for terminating selective enzymatic hydrolysis of a whey protein derivative, comprising adjusting pH to a value lower than that applied during the enzymatic treatment, preferably to a pH in the range of 3.0 to 8.0, more preferably 4.0 to

[0098] 7.5, even more preferably 5.0 to 7.0, yet more preferably 6.0 to 7.0, and heat treating at a temperature above the optimum temperature of the enzyme(s) used in enzymatic treatment. Optionally, the temperature of heat treating may be at least 60°C, preferably in the range of 60°C to 160°C, or 60°C to 95°C, or 65°C to 95°C, or 70°C to 90°C, or 95°C to 160°C, or 95°C to 155°C, or 100°C to 150°C. Optionally, heat treating is performed for a period of time ranging from 0.05 s to 60 min, or 0.05 s to 1 min, or 0.1 s to 30 s, or 0.5 s to 15 s or 30 s to 60 min, or 30 s to 30 min, or 1 min to 10 min. Selective hydrolysis of the whey protein derivative is performed by enzymatically treating with at least one endoprotease, preferably at least one serine endoprotease, more preferably with at least one alkaline serine endoprotease. Optionally, the enzymatic treatment may further comprise treating the whey protein derivative with at least one exopeptidase.

[0099] In addition to hydrolysis with at least one endoprotease, it has been discovered that it is possible to introduce an exopeptidase enzyme to the enzymatic treatment. The idea in the exopeptidase treatment is that after selective hydrolysis of a whey protein derivative with the endopeptidase has proceeded to a desired extent, e.g. for a certain period of time, an exopeptidase is introduced to the hydrolysis mix. In some instances, the exopeptidase may be introduced to the whey protein derivative simultaneously with the endoprotease.

[0100] The exopeptidase hydrolysis may further increase the overall degree of hydrolysis and may also enhance flavour properties of the selectively hydrolysed whey protein composition. The exopeptidase cleaves peptides originating from degradation of proteins such as BLG into smaller peptides which may have reduced bitterness. It is hypothesised that the exopeptidase has a stronger affinity towards peptides as a substrate than towards intact or native proteins. Thus, as BLG is selectively hydrolysed by the endoprotease, producing smaller peptides, these peptides are preferred substrates for the exopeptidase over intact, nonhydrolysed proteins such as ALA. Preferably, the exopeptidase should possess little or no endoprotease activity. Suitable exopeptidases, also known as exoproteases, include aminopeptidases such as leucine aminopeptidase, aminopeptidase N and aminopeptidase B, and carboxypeptidases such as carboxypeptidase A, carboxypeptidase B and carboxypeptidase Y, and any mixtures thereof.

[0101] Thus, the method of the disclosure may further comprise subjecting the whey protein derivative to an enzymatic treatment with at least one exopeptidase. The at least one exopeptidase may be introduced to the whey protein derivative after or simultaneously with the at least one endoprotease. Preferably, the exopeptidase is introduced into the hydrolysis mix after the endoprotease treatment has proceeded for a period of time, such as at least 1 hour or at least 1 .5 hours, so it may exert its effect on the peptides produced by the endoprotease.

[0102] In the method according to the present disclosure, the selectively hydrolysed whey protein composition obtained by or obtainable by the method is characterised by comprising one or more of i) to ii): i) a decreased relative concentration of intact BLG based on the relative content of intact BLG in the whey protein derivative before the enzymatic treatment; and / or ii) an increased relative concentration of intact ALA based on the relative content of intact ALA in the whey protein derivative before the enzymatic treatment.

[0103] Additionally or alternatively, in the method according to the present disclosure, the selectively hydrolysed whey protein composition obtained by or obtainable by the method comprises up to 20% intact BLG, preferably up to 15% intact BLG, more preferably up to 10% intact BLG, yet more preferably up to 5% intact BLG, based on the amount of intact BLG present in the whey protein derivative before the enzymatic treatment.

[0104] Additionally or alternatively, in the method according to the present disclosure, the selectively hydrolysed whey protein composition obtained by or obtainable by the method comprises at least 60% intact ALA, preferably at least 65% intact ALA, more preferably at least 75% intact ALA, even more preferably at least 80% intact ALA, based on the amount of intact ALA present in the whey protein derivative before the enzymatic treatment.

[0105] The whey protein used as substrate for selective hydrolysis may be a concentrated and / or a demineralised whey protein derivative. Concentration of the whey protein derivative may have been performed by microfiltration, nanofiltration, ultrafiltration, diafiltration or any combination thereof. Additionally or alternatively, demineralisation of the whey protein derivative may have been performed by filtration, electrodialysis, ion exchange or any combination thereof. The whey protein derivative may be a whey protein concentrate (WPC), a whey protein isolate (WPI), an ideal whey, a nanofiltered whey or a demineralised whey.

[0106] In the whey protein derivative, the concentration of ALA may be in the range of 5 wt-% to 25 wt-% and the concentration of BLG in the range of 30 wt-% to 90 wt-% of the protein composition.

[0107] In an aspect, the disclosure relates to a selectively hydrolysed whey protein composition. In the selectively hydrolysed whey protein composition, a larger proportion of p- lactoglobulin (BLG) than a-lactalbumin (ALA) is degraded into smaller peptides. Thus, the intact ALA to intact BLG ratio increases.

[0108] Optionally, the selectively hydrolysed whey protein composition is obtainable by the method of the present disclosure or obtained by the method of the present disclosure.

[0109] The selectively hydrolysed whey protein composition according to the present disclosure comprises a weight ratio of intact ALA to intact BLG of at least 1 .0, preferably at least 1 .5, more preferably at least 2.0, even more preferably at least 5.0, yet more preferably at least 8.0, still more preferably at least 10.0, most preferably at least 12.0. In some instances, the weight ratio of intact ALA to intact BLG may be at least 3.0, at least 3.5, at least 4.0, at least 4.5, at least 5.0, at least 5.5, at least 6.0, at least 6.5, at least 7.0, at least 7.5, at least 8.0, at least 8.5, at least 9.0, at least 9.5, at least 10.0, at least 10.5, at least 11.0, at least 11.5, at least 12.0, at least 125., at least 13.0, at least 13.5, at least 14.5, or at least 15.0. In the starting material, whey protein derivative, the weight ratio of intact ALA to intact BLG is typically in the range of 0.3 to 0.5.

[0110] The overall degree of hydrolysis is relatively high in the selectively hydrolysed whey protein composition according to the disclosure. A high degree of hydrolysis in connection with a high content of intact (or native) ALA reflects the high specificity of selective hydrolysis. BLG is preferably degraded, and also peptides derived from BLG are effectively degraded before intactness of ALA is severely compromised.

[0111] Additionally or alternatively to the weight ratios of intact ALA to intact BLG disclosed above, the degree of protein hydrolysis in the selectively hydrolysed whey protein composition according to the present disclosure is at least 5%, preferably in the range of 5% to 25%, more preferably in the range of 5% to 20%, even more preferably in the range of 5% to 18%, still more preferably in the range of 5% to 10%. The degree of protein hydrolysis may be determined by any suitable method such as the o-phthaldialdehyde (OPA) method. OPA may be performed according to Nielsen et al. (2001 ).

[0112] In an exemplary embodiment, the selectively hydrolysed whey protein composition comprises a weight ratio of intact ALA to intact BLG of at least 1 .0, preferably at least 1 .5, more preferably at least 2.0, even more preferably at least 5.0, yet more preferably at least 8.0, still more preferably at least 10.0, most preferably at least 12.0, and a degree of protein hydrolysis of at least 5%, preferably in the range of 5% to 25%, more preferably in the range of 5% to 20%, even more preferably in the range of 5% to 18%, still more preferably in the range of 5% to 10%.

[0113] Additionally or alternatively to the weight ratios of intact ALA to intact BLG and / or degrees of protein hydrolysis disclosed above, the selectively hydrolysed whey protein composition according to the disclosure may comprise up to 50 wt-% of peptides having a size of more than 10 kDa, preferably up to 40 wt-%, more preferably up to 30 wt-%, based on weight of total protein. Additionally or alternatively, the selectively hydrolysed whey protein composition according to the disclosure may comprise 10 wt-% to 50 wt-% of peptides having a size of more than 10 kDa, preferably 15 wt-% to 40 wt-%, more preferably 18 wt- % to 30 wt-%, based on weight of total protein. Additionally or alternatively, the selectively hydrolysed whey protein composition according to the disclosure may comprise 10 wt-%, 11 wt-%, 12 wt-%, 13 wt-%, 14 wt-%, 15 wt-%, 16 wt-%, 17 wt-%, 18 wt-%, 19 wt-%, 20 wt-%, 21 wt-%, 22 wt-%, 23 wt-%, 24 wt-%, 25 wt-%, 26 wt-%, 27 wt-%, 28 wt-%, 29 wt- %, 30 wt-%, 31 wt-%, 32 wt-%, 33 wt-%, 34 wt-%, 35 wt-%, 36 wt-%, 37 wt-%, 38 wt-%, 39 wt-%, 40 wt-%, 44 wt-%, 42 wt-%, 43 wt-%, 44 wt-%, 45 wt-%, 46 wt-%, 47 wt-%, 48 wt-%, 49 wt-%, or 50 wt-% of peptides having a size of more than 10 kDa, based on weight of total protein, or an amount of peptides having a size of more than 10 kDa that is in a range between any two of said values.

[0114] Additionally or alternatively to the weight ratios of intact ALA to intact BLG and / or degrees of protein hydrolysis and / or contents of peptides having a size of more than 10 kDa disclosed above, the selectively hydrolysed whey protein composition according to the present disclosure may comprise at least 5 wt-% of peptides having a size of less than 1 kDa, preferably at least 10 wt-%, more preferably at least 15 wt-%, based on weight of total protein. Additionally or alternatively, the selectively hydrolysed whey protein composition according to the present disclosure may comprise 5 wt-% to 50 wt-% of peptides having a size of less than 1 kDa, preferably 10 wt-% to 48 wt-%, more preferably 15 wt-% to 45 wt-%, based on weight of total protein. Additionally or alternatively, the selectively hydrolysed whey protein composition according to the disclosure may comprise 5 wt-%, 6 wt-%, 7 wt-%, 8 wt-%, 9 wt-%, 10 wt-%, 1 1 wt-%, 12 wt-%, 13 wt-%, 14 wt-%, 15 wt-%, 16 wt-%, 17 wt-%, 18 wt-%, 19 wt-%, 20 wt-%, 21 wt-%, 22 wt-%, 23 wt-%, 24 wt-%, 25 wt-%, 26 wt-%, 27 wt-%, 28 wt-%, 29 wt-%, 30 wt-%, 31 wt-%, 32 wt-%, 33 wt- %, 34 wt-%, 35 wt-%, 36 wt-%, 37 wt-%, 38 wt-%, 39 wt-%, 40 wt-%, 44 wt-%, 42 wt-%, 43 wt-%, 44 wt-%, 45 wt-%, 46 wt-%, 47 wt-%, 48 wt-%, 49 wt-%, or 50 wt-% of peptides having a size of less than 1 kDa, based on weight of total protein, or an amount of peptides having a size of less than 1 kDa that is in a range between any two of said values.

[0115] As is clear to the skilled person, in the selectively hydrolysed whey protein composition according to the present disclosure, the total amount of peptides having different molecular weights is at maximum 100 wt-%.

[0116] In an exemplary embodiment, the selectively hydrolysed whey protein composition comprises a weight ratio of intact ALA to intact BLG of at least 1 .0, preferably at least 1 .5, more preferably at least 2.0, even more preferably at least 5.0, yet more preferably at least 8.0, still more preferably at least 10.0, most preferably at least 12.0, and up to 50 wt-% of peptides having a size of more than 10 kDa, preferably up to 40 wt-%, more preferably up to 30 wt-%, based on weight of total protein.

[0117] In an exemplary embodiment, the selectively hydrolysed whey protein composition comprises a weight ratio of intact ALA to intact BLG of at least 0.7, preferably at least 0.8, more preferably at least 1 .0, even more preferably at least 1 .5, yet more preferably at least 2.0, and at least 5 wt-% of peptides having a size of less than 1 kDa, preferably at least 10 wt-%, more preferably at least 15 wt-%, based on weight of total protein.

[0118] In an exemplary embodiment, the selectively hydrolysed whey protein composition comprises a weight ratio of intact ALA to intact BLG of at least 1 .0, preferably at least 1 .5, more preferably at least 2.0, even more preferably at least 5.0, yet more preferably at least 8.0, still more preferably at least 10.0, most preferably at least 12.0, and up to 50 wt-% of peptides having a size of more than 10 kDa, preferably up to 40 wt-%, more preferably up to 30 wt-%, and at least 5 wt-% of peptides having a size of less than 1 kDa, preferably at least 10 wt-%, more preferably at least 15 wt-%, based on weight of total protein.

[0119] In an exemplary embodiment, the selectively hydrolysed whey protein composition comprises a weight ratio of intact ALA to intact BLG of at least 1 .0, preferably at least 1 .5, more preferably at least 2.0, even more preferably at least 5.0, yet more preferably at least 8.0, still more preferably at least 10.0, most preferably at least 12.0, and a degree of protein hydrolysis of at least 5%, preferably in the range of 5% to 25%, more preferably in the range of 5% to 20%, even more preferably in the range of 5% to 18%, still more preferably in the range of 5% to 10%, and up to 50 wt-% of peptides having a size of more than 10 kDa, preferably up to 40 wt-%, more preferably up to 30 wt-%, based on weight of total protein.

[0120] In an exemplary embodiment, the selectively hydrolysed whey protein composition comprises a weight ratio of intact ALA to intact BLG of at least 1 .0, preferably at least 1 .5, more preferably at least 2.0, even more preferably at least 5.0, yet more preferably at least 8.0, still more preferably at least 10.0, most preferably at least 12.0, and a degree of protein hydrolysis of at least 5%, preferably in the range of 5% to 25%, more preferably in the range of 5% to 20%, even more preferably in the range of 5% to 18%, still more preferably in the range of 5% to 10%, and at least 5 wt-% of peptides having a size of less than 1 kDa, preferably at least 10 wt-%, more preferably at least 15 wt-%, based on weight of total protein.

[0121] In an exemplary embodiment, the selectively hydrolysed whey protein composition comprises a weight ratio of intact ALA to intact BLG of at least 1 .0, preferably at least 1 .5, more preferably at least 2.0, even more preferably at least 5.0, yet more preferably at least 8.0, still more preferably at least 10.0, most preferably at least 12.0, and a degree of protein hydrolysis of at least 5%, preferably in the range of 5% to 25%, more preferably in the range of 5% to 20%, even more preferably in the range of 5% to 18%, still more preferably in the range of 5% to 10%, and up to 50% of peptides having a size of more than 10 kDa, preferably up to 40%, more preferably up to 30%, and at least 5 wt-% of peptides having a size of less than 1 kDa, preferably at least 10 wt-%, more preferably at least 15 wt-%, based on weight of total protein.

[0122] It has now been discovered that the selective hydrolysis method according to the disclosure is effective in maintaining native conformation of whey derivative proteins such as ALA. Particularly, the hydrolysis termination conditions applied in the method allow for maintaining the native conformation. Performing a reduction of pH to a level lower than that applied in the enzymatic treatment, particularly lowering to a pH value in the range of about 3.0 to about 8.0, preferably about 4.0 to about 7.5, more preferably about 4.5 to about 7.0, even more preferably about 5.0 to about 7.0, yet more preferably about 6.0 to about 7.0 simultaneously with or before performing heat treating promotes maintaining native conformation of particularly ALA while the residual BLG remaining after the enzymatic treatment may be further degraded to enhance selectivity. Additionally or alternatively, for terminating hydrolysis, the pH may be adjusted to a value in the range between any two of the values 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2,

[0123] 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0. Enzymatic hydrolysis of the whey protein derivative may be performed at a pH ranging from about 7.0 to about 9.5, preferably about 7.5 to about 9.5, more preferably about 7.5 to about 9.0, yet more preferably about 8.0 to about 8.5. Additionally or alternatively, the enzymatic treatment may be performed at a pH ranging from more than 7.0 to 9.5, preferably from more than 7.5 to 9.5, more preferably from more than 7.5 to 9.0, yet more preferably from more than 8.0 to 8.5. Additionally or alternatively, enzymatic hydrolysis may be performed at a pH of about 7.0, about 7.1 , about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1 , about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1 , about 9.2, about 9.3, about 9.4, about 9.5, or at a pH ranging between any two of said values.

[0124] A heat treating performed simultaneously with or after the pH reduction may be performed at a temperature above the optimum temperature of the at least one endoprotease used in enzyme treatment. Additionally or alternatively, the temperature may be at least about 60°C, preferably in the range of about 60°C to about 160°C, or about 60°C to about 95°C, or about 65°C to about 95°C, or about 70°C to about 90°C, or about 95°C to about 160°C, or about 95°C to about 155°C, or about 100°C to about 150°C. Additionally or alternatively, the heat treating may be performed at a temperature ranging between any two of the values 50°C, 51 °C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61 °C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71 °C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81 °C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91 °C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, 100°C, 105°C, 1 10°C, 115°C, 120°C, 125°C, 130°C, 135°C, 140°C, 145°C, 150°C, 155°C, 160°C, 165°C, and 170°C. Optionally, the heat treatment may be performed for a period of time ranging from 0.05 s to 60 min, or 0.05 s to 1 min, or 0.1 s to 30 s, or 0.5 s to 15 s or 30 s to 60 min, or 30 s to 30 min, or 1 min to 10 min. Additionally or alternatively, the heat treatment is performed for a period of time ranging between any two of 0.05 s. 0.1 s, 0.5 s, 1 s, 2 s, 3 s, 4 s, 5 s, 6 s, 7 s, 8 s, 9 s, 10 s, 15 s, 20 s, 25 s, 30 s, 35 s, 40 s, 45 s, 50 s, 55 s, 1 min, 1 .5 min, 2 min, 2.5 min, 3 min, 3.5 min, 4 min, 4.5 min, 5 min, 5.5 min, 6 min, 7.5 min, 8 min, 8.5 min, 9 min, 9.5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, and 60 min.

[0125] Native proteins maintain their biological activity and are readily quantifiable. In the Examples herein, native ALA and BLG were quantified with UPLC, but if it was detected in the UPLC quantification that ALA and BLG had lost their native state, intact ALA and BLG were analysed by PAGE.

[0126] Thus, in an exemplary embodiment, the intact ALA or the intact BLG or both is (are) present in the selectively hydrolysed whey protein composition essentially in native form. As used herein, the term “essentially in native form” means that as determined by routinely used laboratory methods capable of detecting native protein conformation such as ultraperformance liquid chromatography (UPLC) employed in the Examples herein, the protein is detected to maintain its native form. This means for example that the protein has not aggregated or changed in mass or hydrodynamic size. Other suitable methods for routine monitoring of native state of proteins include spectroscopy such as circular dichroism (CD) spectroscopy and fluorescence spectroscopy; size-exclusion chromatography; native polyacrylamide gel electrophoresis (PAGE); differential scanning fluorimetry; and determining biological functionality of active proteins.

[0127] In an aspect, the present disclosure relates to use of the selectively hydrolysed whey protein composition according to the disclosure in manufacturing a food product. The food product may be an infant formula.

[0128] In an aspect, the present disclosure relates to a food product comprising the selectively hydrolysed whey protein composition according to the disclosure.

[0129] The food product may comprise at least 0.1 wt-% of the selectively hydrolysed whey protein composition, based on total weight of the food product. Alternatively, the content of the selectively hydrolysed whey protein composition in the food product may be at least 0.5 wt-%, or at least 1 wt-%, or in the range of 0.1 wt-% to 99 wt-% or 0.1 wt-% to 50 wt- % or 0.1 wt-% to 25 wt-% or 0.5 wt-% to 15 wt-% or 0.5 wt-% to 10 wt-% or 1 wt-% to 5 wt- %, based on total weight of the food product. Additionally or alternatively, the selectively hydrolysed whey protein composition may represent the entirety of intact ALA and intact BLG content in the food product.

[0130] Additionally or alternatively, the food product may comprise the selectively hydrolysed whey protein composition at an amount in the range of 0.38 to 1 .7 grams per 100 kJ. With respect to the protein content of infant formulae, it is generally required in most markets that the product contains from about 0.38 g to about 1 .7 g of protein per 100 kJ of total energy. Furthermore, it is contemplated that the selectively hydrolyzed whey protein composition according to the present disclosure may fully satisfy this protein requirement in an infant formula. The food product may be an infant formula. Human breast milk and breast feeding represent the uncontested gold standard in terms of infant nutrition. Infant formulae that serve as a substitute for or complement to human breast milk should satisfy the nutritional requirements of infants, have an acceptable taste and be hypoallergenic when targeted to infants at risk of allergy and / or food intolerances.

[0131] An advantage of the present disclosure relates to the expected enhanced digestibility and digestive tolerance of the BLG-reduced whey protein composition which makes it suitable for use in e.g. “comfort-type” infant formulas. Comfort-type infant formulas are special formulas designed to be easier on a baby's digestive system, particularly for those who experience mild digestive discomforts like gas, colic, or fussiness.

[0132] The selectively hydrolysed whey protein composition according to the disclosure also has a high degree of protein hydrolysis which predicts better digestibility and lower allergenicity. Moreover, the preserved intactness of ALA may bring additional value due to its similarity to human milk ALA. The similar biological activity may have a positive influence on infant growth and development. The selectively hydrolysed whey protein composition possesses good solubility and will likely confer enhanced heat stability since the heat sensitive BLG fraction has been degraded into smaller peptides.

[0133] Terms such as "about", "generally", "substantially", “typically” and suchlike shall be understood with their function of modifying a term or value that is not absolute, but is not reported in the state of the art. Such terms shall be defined by the specific circumstances and by the terms that they are intended to modify according to the common acceptance of such terms in the specific field. They shall take into account at least the degree of experimental error expected, the technical error and the instrumental error for a given technique adopted to measure a value. For example, the term “about” may refer to a value that is within ±10% of the mentioned value. That is, for example “about 7.0” may refer to any value between 6.3 and 7.7.

[0134] As used herein, the term "or" has the meaning of both "and"' and "or" (i.e. "and / or"). Furthermore, the meaning of a singular noun includes that of a plural noun and thus a singular term, unless otherwise specified, may also carry the meaning of its plural form. In other words, the term "a" or "an" may mean one or more.

[0135] As used herein, the term “comprising” includes the broader meanings "including”, "containing”, and "comprehending", as well as the narrower expressions “consisting of’ and “consisting only of’. It will be obvious to a person skilled in the art that, as technology advances, the inventive concept can be implemented in various ways. The disclosure and its embodiments are not limited to the examples described below but may vary within the scope of the claims.

[0136] EXAMPLES

[0137] Example 1

[0138] Analytical methods

[0139] Quantification of native ALA and BLG as well as molecular weight distribution of proteins / peptides was performed as described in Hong et al. (2012). Samples were analysed with UPLC Premier system coupled with UV-detector and the system was controlled with Empower software (Waters, Milford, MA, USA). Analytes were resolved by ACQUITY UPLC® BEH125 SEC, 1 .7 pm column (Waters, Milford, MA, USA) with isocratic run consisting of solvent A (20% of 0.1% TFA in H2O) and B (80% of 0.1 % TFA in acetonitrile). Flow rate was 0.2 ml / minutes, column temperature 30°C and injection volume 2 pl. Proteins and peptides for molecular weight calibration were purchased as individual standards from Sigma-Aldrich and Cytiva.

[0140] Degree of hydrolysis was measured with the o-phthaldialdehyde (OPA) method, based on the method of Nielsen et al. (2001 ) to determine free amino nitrogen in the product. The degree of hydrolysis (DH) % was then calculated according to equation 1 :

[0141] DH % = (a / atotai) *100 [1]

[0142] Where: atotai is the total amount of amino nitrogen in the protein substrate (determined through acid hydrolysis), and a is the amount of free amino nitrogen liberated upon hydrolysis (free amino nitrogen in the hydrolysate subtracted by the free amino nitrogen in the substrate before hydrolysis).

[0143] Gel electrophoresis to determine intact ALA and BLG was performed according to Bio-Rad peptide-PAGE protocol (Criterion Precast Gels, Instruction Manual and Application Guide, 2011 ) using 16.5 % Criterion Tris-Tricine Gel (Bio-Rad), tricine sample buffer with dithiothreitol (DTT) (reducing PAGE) and Coomassie-staining.

[0144] Example 2

[0145] Selective hydrolysis of whey protein by endoproteases Selective hydrolysis by endoproteases was studied using as substrate bovine whey protein concentrate (WPC) solution adjusted to 5% (w / v) protein concentration. Of the initial protein, 55% (w / w) was BLG and 15% (w / w) ALA. The concentration of ALA in the 5% (w / v) WPC was 0.7-0.8 g / 100 ml and the concentration of BLG 2.0-2.6 g / 100 ml. The remaining protein in WPC comprised a caseinomacropeptide / glycomacropeptide (CMP / GMP) fraction, serum albumin, denatured whey proteins and other minor whey protein fractions such as immunoglobulins and lactoferrin.

[0146] For selective hydrolysis, pH of the WPC solution was adjusted to 8.5 with 3 M NaOH or KOH. The enzyme preparates used in the experiments were Formea TL, comprising a trypsin-like endoprotease from Fusarium venenatum, Formea CTL, comprising a chymotrypsin-like endoprotease from Bacillus licheniformis, and Alcalase, comprising a subtilisin endoprotease from B. licheniformis. All enzymes were obtained from Novozymes. Enzymes were added to the whey protein solution according to the following enzyme to substrate (E / S) ratios given as volume of enzyme used per volume of substrate (in this case 5% (w / v) WPC):

[0147] • Formea TL: 1 / 100 (12 KMTU / g of protein),

[0148] • Formea CTL: 1 / 200 (1 .5 KPROT / g of protein), and

[0149] • Alcalase: 1 / 100 (0.024 AU-A / g of protein).

[0150] Hydrolysis was carried out by incubating the hydrolysis mix at a temperature of 25°C under static conditions or with gentle mixing. During hydrolysis, pH of the hydrolysis mix solution was maintained between 8.2 and 8.7 using 3 M NaOH or KOH for pH adjustment. Hydrolysis was terminated by lowering pH to a value below 3 with 1 M HCI to inactivate the hydrolytic enzyme. Amounts of native ALA and BLG after hydrolysis (Table 1) and molecular weight distribution (Table 2) were determined by UPLC as described in Example 1 . Results are given in Table 1 as percentage of native ALA or BLG present after hydrolysis as calculated from ALA or BLG content before hydrolysis, respectively.

[0151] All of the tested enzymes produced a hydrolysate containing 70-90% of ALA remaining intact and in native conformation while a significantly smaller amount, such as only up to 10%, typically less than 3%, of the BLG remained intact and in native conformation. Total degree of hydrolysis (DH) in the hydrolysates after 7 h hydrolysis time ranged between 8 and 12%. Minimum and maximum weight ratios of ALA to BLG were calculated based on the known amounts of ALA (0.7-0.8 g / 100 ml) and BLG (2.0-2.6 g / 100 ml) in the 5 % (w / v) WPC substrate. The minimum ALA to BLG weight ratio in the WPC substrate is 0.3 and maximum is 0.4. Selective hydrolysis increased the ratio with all of the tested enzymes. After a 7-hour hydrolysis, the BLG content of WPC was degraded into smaller peptides having a molecular weight ranging from 0.2 to 10 kDa (Table 2). The remaining > 10 kDa fraction in the selectively hydrolysed whey protein contained mostly native ALA, serum albumin, and other minor whey proteins as well as the remnants of BLG.

[0152] Table 1. Selective hydrolysis of whey protein with endoproteases at 25°C. The relative amounts (%) of native BLG and native ALA remaining in samples taken at different time points during hydrolysis are given. Min and max values refer to minimum and maximum ALA to BLG weight ratios in the selectively hydrolysed whey protein composition. Abbreviations: HT = hydrolysis time; DH = degree of hydrolysis; n.d. = not determined.

[0153] Table 2. Molecular weight distribution (peptide profile) of unhydrolysed whey protein and whey protein selectively hydrolysed for 7 hours with different endoproteases. The molecular weight distribution was determined based on peak areas of fractions from total peak area in the UPLC chromatogram. Abbreviation: WPC = whey protein concentrate.

[0154] Example 3

[0155] Selective hydrolysis in cold temperatures

[0156] In Example 2, hydrolysis was performed at 25°C. To improve the process in terms of microbiological safety, and thus, industrial applicability, we performed selective hydrolysis of WPC otherwise under same conditions but at lower temperatures than in Example 2. In the cold hydrolysis experiments performed at 10°C, different Formea TL dosages (E / S 1 / 1000-1 / 100) were also tested. Hydrolysis was terminated by lowering the pH to < 3. Amounts of native ALA and native BLG after hydrolysis were determined as described in Example 1 . Results are given as percentage of native ALA or BLG present after hydrolysis as calculated from ALA or BLG content before hydrolysis, respectively.

[0157] The results of cold hydrolysis at either 6°C (Table 3) or 10°C (Table 4) showed good selectivity with all of the enzymes tested, Formea TL, Formea CTL and Alcalase. However, hydrolysis times needed to be extended from those used in Example 2 to reach sufficient BLG degradation. Minimum and maximum weight ratios of ALA to BLG were calculated based on the known amounts of ALA (0.7-0.8 g / 100 ml) and BLG (2.0-2.6 g / 100 ml) in the 5 % (w / v) WPC substrate. The minimum ALA to BLG weight ratio in the WPC substrate is 0.3 and maximum is 0.4. Selective hydrolysis increased the ratio with all of the tested enzymes and hydrolysis times.

[0158] To reach a satisfactory selectivity with Formea TL enzyme, the dependency of temperature on hydrolysis time and enzyme dosage was studied. 5 % (w / v) WPC was used as substrate and hydrolysis was terminated by lowering the pH to < 3. The amounts of native ALA and native BLG after hydrolysis were determined as described in Example 1 (Table 4). For instance, at a temperature of 10°C, an enzyme dose of 0.05% (v / v) (E / S approximately 1 / 100) produced a similar result after 24h hydrolysis than an enzyme dose of 0.015% (v / v) (E / S approximately 1 / 330) after 48h of hydrolysis.

[0159] Table 3. The remaining relative amount (%) of native BLG and native ALA in WPC hydrolysates after enzymatic hydrolysis at 6°C. Min and max values refer to minimum and maximum ALA to BLG weight ratios in the selectively hydrolysed whey protein composition. Abbreviations: EZ = enzyme; HT = hydrolysis time.

[0160] Table 4. The remaining relative amount (%) of native BLG and native ALA in WPC hydrolysates after Formea TL (FTL) hydrolysis at 10°C using different enzyme doses. Min and max values refer to minimum and maximum ALA to BLG weight ratios in the selectively hydrolysed whey protein composition. Abbreviation: HT = hydrolysis time.

[0161] Example 4

[0162] Inactivation of enzyme after selective hydrolysis Inactivation of hydrolytic enzyme after selective hydrolysis of WPC was studied by terminating hydrolysis either by lowering pH of the hydrolysis mix solution to below 3 with 1 M HCI or heating the hydrolysis mix solution to a temperature in the range of 85°C to 125°C and for a period of time ranging from 9 seconds to 2 minutes.

[0163] 4. 1 Heat treatment inactivation at different temperatures

[0164] Except for the different enzyme inactivation options tested, hydrolysis of 5% (w / v) WPC with Formea TL, Formea CTL and Alcalase was performed as described in Example 2 with the exception that the hydrolysis time was 5 hours. When selective hydrolysis was terminated by heat-treatment instead of lowering the pH as in Example 2, proteins including ALA were mostly denatured and partially disulfide-aggregated, thus making quantification of native ALA and native BLG by the UPLC method described in Example 1 challenging. Therefore, analysis of intact ALA and intact BLG was performed with polyacrylamide gel electrophoresis (PAGE) under reducing conditions as described in Example 1 .

[0165] It was detected that Formea TL enzyme could be inactivated by indirect heating at 95- 97°C for 1 min (Figure 1A) while maintaining hydrolysis selectivity. Direct heating to 125°C for 9s was also a suitable inactivation method for Formea TL (Figure 1 B) as hydrolysis selectivity was not compromised. Loss of selectivity is seen in Figure 1 as degradation of the protein-peptide composition achieved by selective hydrolysis, particularly as degradation of intact ALA.

[0166] Inactivation of Formea CTL and Alcalase enzymes while maintaining hydrolysis selectivity required a higher temperature than inactivation of Formea TL, but a long inactivation time was not beneficial. A rapid heat treatment at 125°C for 9s (Figure 1 B) by direct heating provided suitable conditions for inactivation of Formea CTL and Alcalase, while a longer inactivation time at a lower temperature (95-97°C for 1 min) resulted in loss of selectivity i.e. degradation of intact ALA (Figure 1 A).

[0167] 4.2 Pilot-scale inactivation of WPC cold hydrolysed with Formea TL

[0168] Since in manufacturing processes indirect heating under moderate conditions is favored in terms of practicality, Formea TL was selected for a larger, pilot-scale run where hydrolysis was performed on WPC at 5% (w / v) protein concentration using Formea TL at an E / S ratio of 1 / 100 i.e. 12 KMTU / g of protein at 6°C for 26 h and at a pH maintained between 8.2 and 8.7. When hydrolysis was terminated by lowering pH to a value of < 3, the amounts of remaining native BLG and ALA in the hydrolysate were 3.2% and 71.0%, respectively. Degree of hydrolysis was 6.5%. When selective hydrolysis was terminated by heat-treatment (heating to 95°C for 20s or 90°C for 2 min) instead of lowering the pH, ALA was mostly denatured and partially disulfide-aggregated, thus losing its native conformation and making quantification by the UPLC method described in Example 1 challenging. However, an illustration by PAGE under reducing conditions (Figure 2) showed that ALA remained intact during heat treatment inactivation at a level that was similar to the level of intact ALA in a hydrolysate inactivated by lowering pH.

[0169] 4.3 Heat-inactivation at lowered pH

[0170] The influence of pH adjustment before heat inactivation on native ALA content was studied. 5% (w / v) protein concentration WPC was hydrolysed with Formea TL (24 h hydrolysis) or Alcalase (28 h hydrolysis) at 10°C. Hydrolysis was performed at a pH maintained between 8.0 and 8.5 using Formea TL at an amount of E / S 1 / 100 i.e. 12 KMTU / g of protein or Alcalase at an amount of E / S 1 / 100 i.e. 0.024 AU-A / g of protein. Heat inactivation was performed at 90 °C for 2 minutes. Amounts of native ALA after hydrolysis were determined as described in Example 1. Results are given as percentage of native ALA present after hydrolysis as calculated from ALA content before hydrolysis. Intact ALA was determined by gel electrophoresis.

[0171] As seen from the results in Table 5, lowering the pH of the hydrolysate to 6.0-7.0 prior to heat inactivation retained a larger proportion of native ALA compared to heat inactivation at the pH of 8.0-8.5 used during hydrolysis (Table 5). Also, a gel electrophoresis analysis of the hydrolysates (Figure 3) shows that intact ALA was partially degraded upon heat treatment if pH is not lowered from hydrolysis pH before heating. However, decreasing the pH led to an increase in the amount of intact ALA.

[0172] Table 5. The influence of inactivation method and pH adjustment on native ALA and native BLG content after 24 h hydrolysis of WPC with Formea TL (FTL) or 28 h with Alcalase (AL) at 10°C. Results are shown in percentage of native ALA or native BLG present after hydrolysis as calculated from ALA content before hydrolysis. Min and max values refer to minimum and maximum ALA to BLG weight ratios in the selectively hydrolysed whey protein composition. Abbreviations: HT = heat treatment; n.d. = not determined.

[0173] Example 5

[0174] Nano filtered whey concentrate and demineralised whey as substrates for hydrolysis

[0175] To demonstrate production of selectively hydrolysed demineralised whey protein composition, protein hydrolysis was performed on nanofiltered (concentrated) cheese whey, demineralised whey and ideal whey.

[0176] Hydrolysis of demineralised whey was performed at 5% (w / v) protein concentration for up to 7 hours at 25°C and a pH maintained between 8.2 and 8.7. Formea TL was used at a concentration of E / S 1 / 100 i.e. 12 KMTU / g of protein, Formea CTL at a concentration of E / S 1 / 200 i.e. 1.5 KPROT / g of protein, and Alcalase at a concentration of E / S 1 / 100 i.e. 0.024 AU-A / g of protein. Hydrolysis reactions were terminated by inactivating the enzyme by lowering the pH to < 3.

[0177] Table 6 shows the remaining level (%) of native BLG and native ALA, and degree of hydrolysis (DH) % in demineralised whey hydrolysed at 25°C for 5h or 7h with Formea TL, Formea CTL or Alcalase. According to the results, demineralised whey can be selectively hydrolysed. However, the remaining content of native ALA is typically lower than in Examples 2-4 that use WPC as substrate. It is believed that this reduction in selectivity occurs due to molecular changes in ALA structure during the demineralisation process. Despite the reduced selectivity, demineralised whey may act as a substrate for selective hydrolysis in cases where selectively hydrolysed demineralised whey powders are needed. Weight ratios of ALA to BLG were calculated based on the known amounts of ALA (0.4 g / 100 ml) and BLG (1.1 g / 100 ml) in the 5 % (w / v) demineralised whey substrate. The ALA to BLG weight ratio in the demineralised whey substrate is 0.4. Selective hydrolysis increased the ratio with all of the tested enzymes and hydrolysis times. Table 6. The remaining level (%) of native BLG and native ALA, and DH % in hydrolysates using demineralised whey as a substrate at 25°C and 5-7h hydrolysis conditions. ALA / BLG values refer to ALA to BLG weight ratios in the selectively hydrolysed whey protein composition. Abbreviations: DH = degree of hydrolysis; HT = hydrolysis time.

[0178] Nanofiltered whey was also tested as a substrate of selective hydrolysis at a protein concentration of 2.5-3.0% (w / v). The hydrolysis was performed using different dosages of Formea TL enzyme, at 10°C, a pH maintained at 8.2-8.7 and for 24-48 h. Formea TL doses were as follows: 0.05 % = E / S 1 / 60 i.e. 20 KMTU / g; 0.03 % = E / S 1 / 100 i.e. 12 KMTU / g; and 0.015 % = E / S 1 / 200 i.e. 6 KMTU / g. Hydrolysis reactions were terminated by inactivating the enzyme by lowering the pH to < 3.

[0179] Results are given as percentage of native ALA or BLG present after hydrolysis from ALA or BLG content before hydrolysis, respectively. The results on the remaining level (%) of native BLG and ALA (Table 7) show that BLG-selective hydrolysis occurs also in nanofiltered whey. However, the degradation of BLG was significantly slower compared to hydrolysis on WPC-substrate in Example 3. The change in hydrolysis kinetics is believed to be related to the high lactose and mineral content in nanofiltered whey. Minimum and maximum weight ratios of ALA to BLG were calculated based on the known amounts of ALA (0.3-0.4 g / 100 ml) and BLG (0.8-1 .0 g / 100 ml) in the 2.5-3.0% (w / v) nanofiltered whey substrate. The minimum ALA to BLG weight ratio in the nanofiltered whey substrate is 0.3 and maximum is 0.5. Selective hydrolysis increased the ratio with all of the tested enzyme concentrations.

[0180] Table 7. The remaining level (%) of native BLG and native ALA in samples from selective hydrolysis of nanofiltered whey with Formea TL at 10°C for 24-48 h. Min and max values refer to minimum and maximum ALA to BLG weight ratios in the selectively hydrolysed whey protein composition. Abbreviation: HT = hydrolysis time.

[0181] An ideal whey produced from milk by membrane filtration and containing beta-casein was tested as a substrate for selective hydrolysis. The ideal whey was adjusted to 5% (w / v) protein concentration and hydrolysed with Alcalase (1 / 70 i.e. 0.034 AU-A / g of protein) for 28 h at 10°C at a pH of 8.5. Hydrolysis was terminated by indirect heating at 90°C for 2 min. Results are given as percentage of native ALA or BLG present after hydrolysis from ALA or BLG content before hydrolysis, respectively.

[0182] Selective hydrolysis of ideal whey was slightly slower compared to hydrolysis of WPC in Example 3, because beta-casein was hydrolysed in addition to BLG. Also, heatinactivation of hydrolysed ideal whey caused beta-casein fragments to precipitate (results not shown). However, as shown in Table 8, ALA remained intact in native form similarly to after selective hydrolysis of WPC in Example 3. Weight ratios of ALA to BLG were calculated based on the known amounts of ALA (0.8 g / 100 ml) and BLG (2.4 g / 100 ml) in the 5 % (w / v) ideal whey substrate. The ALA to BLG weight ratio in the ideal whey substrate is 0.3. Inactivation by lowering the pH to 6-7 before heat treatment increased the ALA to BLG ratio compared to inactivation by lowering the pH to < 3 only.

[0183] Table 8. Selective hydrolysis of ideal whey with Alcalase enzyme (28 h at 10°C), content of native BLG and native ALA, and degree of hydrolysis after different inactivation methods. ALA / BLG values refer to ALA to BLG weight ratios in the selectively hydrolysed whey protein composition. Abbreviations: DH = degree of hydrolysis; HT = heat treatment.

[0184] Example 6

[0185] Multiple stage hydrolysis

[0186] In addition to batch hydrolysis in one step as done in the above Examples, we determined if it is possible to further increase the degree of hydrolysis by introducing an additional hydrolysis step using an exopeptidase enzyme. In the exopeptidase treatment, an exopeptidase is introduced to the hydrolysis mix after selective hydrolysis with an endopeptidase has proceeded for a period of time. Total hydrolysis time includes the first step of hydrolysis with endoprotease and the second step with exopeptidase.

[0187] Multiple stage hydrolysis was tested by adding a small amount of Flavourzyme (Novozymes) to an ongoing selective hydrolysis. Flavourzyme, a blend of endo- and exopeptidases, has been determined (Merz et al., 2015) to comprise eight enzymes: two aminopeptidases, two dipeptidyl peptidases, three endopeptidases, and one a-amylase from the Aspergillus oryzae strain ATCC 42149 / RIB 40. Selective hydrolysis was performed on WPC at a 5% (w / v) protein concentration and adjusted to a pH of 8.5. Hydrolysis was performed with 1 / 70 E / S addition (0.07%) of Alcalase enzyme at a temperature of 10°C and at a pH of 8.5. Flavourzyme was added to the hydrolysis mix at 1 / 167 E / S level (0.03%) after 6 h, and hydrolysis was continued for 18 or 22 hours (total hydrolysis time 24 h or 28 h). Hydrolysis was terminated by either lowering pH to a value below 3 or by heat treatment as presented above. Results are given as percentage of native ALA or BLG present after hydrolysis from ALA or BLG content before hydrolysis, respectively. The results (Table 9) show that the exopeptidase treatment can be used to increase the degree of hydrolysis by up to 5 %-points without significantly influencing native ALA-content. Minimum and maximum weight ratios of ALA to BLG were calculated based on the known amounts of ALA (0.7-0.8 g / 100 ml) and BLG (2.0-2.6 g / 100 ml) in the 5 % (w / v) WPC substrate. The minimum ALA to BLG weight ratio in the WPC substrate is 0.3 and maximum is 0.4. Inactivation by lowering the pH to 6-7 before heat treatment increased the ALA to BLG ratio compared to inactivation by lowering the pH to < 3 only. Table 9. The remaining level (%) of native BLG and native ALA, and DH % in multiple stage hydrolysis of WPC with Alcalase (AL) and Flavourzyme (FZ), performed at 10°C for 24-28 h. Min and max values refer to minimum and maximum ALA to BLG weight ratios in the selectively hydrolysed whey protein composition. Abbreviations: HyT = hydrolysis time; HT = heat treatment; DH = degree of hydrolysis.

[0188] In a further experiment, WPC-substrate was hydrolysed at 5% (w / v) protein content with Formea Prime (8.4 KMTU / g of protein) or Alcalase (0.034AU-A / g of protein). Formea Prime contains the same active protease, trypsin-like endoprotease from F. venenatum, as Formea TL and hydrolysis results with these two enzyme preparations are essentially equal, so Formea Prime and Formea TL can in practice be used interchangeably.

[0189] The hydrolysis was performed at 10°C with pH 8.5. After 6 h hydrolysis with Formea Prime or Alcalase, Flavourzyme was added at 6.6 LAPU / g protein, and hydrolysis was continued for 18 h or 24 h to give a total hydrolysis time of 24 h or 30 h, respectively. Hydrolysis was terminated by lowering solution pH to < 3. Results are given as percentage of native ALA or BLG present after hydrolysis from ALA or BLG content before hydrolysis, respectively. As presented in Table 10, multiple stage hydrolysis with leucyl aminopeptidase can increase DH% of the hydrolysate. Surprisingly, the hydrolysate produced with Alcalase (subtilisin) and Flavourzyme (leucyl aminopeptidase) can reach even above 18 % DH without significantly influencing the native ALA content of the hydrolysate. Minimum and maximum weight ratios of ALA to BLG were calculated based on the known amounts of ALA (0.7-0.8 g / 100 ml) and BLG (2.0-2.6 g / 100 ml) in the 5 % (w / v) WPC substrate. The minimum ALA to BLG weight ratio in the WPC substrate is 0.3 and maximum is 0.4. The enzyme treatments increased the increased the ALA to BLG weight ratios. However, in the conditions tested, combining Formea Prime with Flavourzyme appeared to decrease selectivity of hydrolysis as seen from the decreased ALA to BLG ratios, whereas combining Alcalase with Flavourzyme increased the ALA to BLG ratios and selectivity.

[0190] Table 10. The remaining level (%) of native BLG and ALA, and DH % in multiple stage hydrolysis of WPC with Formea Prime (FP), Alcalase (AL) and Flavourzyme (FZ), performed at 10°C for 24-48 h. Min and max values refer to minimum and maximum ALA to BLG weight ratios in the selectively hydrolysed whey protein composition. Abbreviation: HyT = hydrolysis time; DH = degree of hydrolysis. Example 7

[0191] Pilot-scale selective hydrolysis

[0192] Selective hydrolysis was tested at a larger scale at Lapinlahti Eritehdas pilot factory (Lapinlahti, Finland) in October 2023. In the pilot run, approximately 5400 kg of nanofiltered whey was ultrafiltered and diafiltered to obtain a whey protein concentrate (WPC) solution containing 20% protein (w / w). The WPC was diluted to approximately 5 % (w / v) protein content, and selective hydrolysis was carried out using Formea TL enzyme (0.05% i.e. E / S 1 / 100; 12 KMTU / g) for 25 h at 6°C maintaining a pH of 8.3-8.7 with KOH. At this point, an aliquot was taken from the selectively hydrolysed WPC and the enzyme was inactivated by lowering pH to < 3. The remaining selectively hydrolysed WPC was subjected to enzyme inactivation by pasteurisation at 90°C for 2 min. Thereafter, the hydrolysate was heat-treated to reduce moisture content by evaporation until a concentration of approximately 17%-20% dry matter was achieved. The hydrolysate was then spray-dried to a powder and collected to 15 kg bags.

[0193] In the pilot run, good hydrolysis selectivity was achieved throughout the process. Intact ALA was maintained and BLG was degraded (Figure 4) in a manner similar to the laboratory tests of the preceding Examples. This confirms the industrial applicability of the hydrolysis process.

[0194] The characteristics of the obtained selectively hydrolysed WPC-powder showed similar flowing and solubilising behavior compared to intact (non-hydrolysed) WPC80 powder as well as a commercial partially hydrolysed whey protein. However, unlike intact WPC80 and other hydrolysates, the selectively hydrolysed WPC-powder does not foam at all, which is likely related to degradation of BLG. Selectively hydrolysed WPC had some bitter taste, but it was evaluated to be less bitter than two commercial partially hydrolysed WPCs.

[0195] References

[0196] Hong et al., 2012. Advances in Size-Exclusion Chromatography for the Analysis of Small Proteins and Peptides: Evaluation of Calibration Curves for Molecular Weight Estimation. Application Note, Waters Corporation, Milford, MA, USA. Available in: https: / / www.waters.com / content / dam / waters / en / app-notes / 2012 / 720004412 / 720004412- en.pdf (accessed on 13.9.2024).

[0197] Merz et al. 2015. Flavourzyme, an Enzyme Preparation with Industrial Relevance: Automated Nine-Step Purification and Partial Characterization of Eight Enzymes. J. Agric. Food Chem. 2015, 63, 23, 5682-5693

[0198] Nielsen et al., 2001. Improved Method for Determining Food Protein Degree of Hydrolysis. J Food Sci, Vol. 66, No. 5, 642-646.

Claims

CLAIMS1 . A method of producing a selectively hydrolysed whey protein composition, comprising: providing a whey protein derivative comprising beta-lactoglobulin (BLG) and alphalactalbumin (ALA), subjecting the whey protein derivative to an enzymatic treatment with at least one endoprotease.

2. The method according to claim 1 , wherein the at least one endoprotease is a serine endoprotease, optionally a serine endoprotease of class EC 3.4.21 , preferably an alkaline serine endoprotease.

3. The method according to claim 1 or 2, wherein the enzymatic treatment is performed at a pH ranging from about 7.0 to about 9.5, preferably about 7.5 to about 9.5, more preferably about 7.5 to about 9.0, yet more preferably about 8.0 to about 8.5.

4. The method according to any one of the preceding claims, wherein the enzymatic treatment is performed at a temperature of about 1 °C to about 30°C, or about 2°C to about 25°C, or about 3°C to about 12°C, or about 20°C to about 30°C.

5. The method according to any one of the preceding claims, wherein the enzymatic treatment is performed for a period of time ranging from about 2 to about 72 hours, or about 5 to about 48 hours, or about 3 to about 12 hours, or about 15 to about 72 hours.

6. The method according to any one of the preceding claims, comprising: subjecting the whey protein derivative to an enzymatic treatment with at least one exopeptidase, wherein preferably the at least one exopeptidase is introduced to the whey protein derivative after or simultaneously with the at least one endoprotease.

7. The method according to any one of the preceding claims, comprising: terminating the enzymatic treatment by at least one of i) to ii): i) adjusting pH to a value lower than the pH applied during hydrolysis, optionally to a pH value in the range of about 3.0 to about 8.0; ii) heat treating at a temperature exceeding the optimum temperature of the at least one endoprotease, optionally at a temperature of at least about 60°C.

8. The method according to claim 7, wherein i) is performed before or simultaneously with ii), preferably i) is performed before ii).

9. The method according to claim 7 or 8, wherein heat treating in ii) is performed at a temperature in the range of about 60°C to about 160°C, or about 60°C to about 95°C, or about 65°C to about 95°C, or about 70°C to about 90°C, or about 95°C to about 160°C, or about 95°C to about 155°C, or about 100°C to about 150°C.

10. The method according to any one of claims 7 to 9, wherein in i), pH is adjusted to a pH in the range of about 4.0 to about 7.5, or about 5.0 to about 7.0, or about 5.5 to about 7.0, or about 6.0 to about 7.0.

11. The method according to any one of claims 7 to 10, wherein in ii), the heat treating is performed for a period of time ranging from about 0.05 s to about 60 min, or about 0.05 s to about 1 min, or about 0.1 s to about 30 s, or about 0.5 s to about 15 s or about 30 s to about 60 min, or about 30 s to about 30 min, or about 1 min to about 10 min.

12. The method according to any one of the preceding claims, wherein the selectively hydrolysed whey protein composition is characterised by comprising one or more of i) to ii): i) a decreased relative content of intact BLG based on the relative content of intact BLG in the whey protein derivative before the enzymatic treatment; ii) an increased relative content of intact ALA based on the relative content of intact ALA in the whey protein derivative before the enzymatic treatment.

13. The method according to any one of the preceding claims, wherein the selectively hydrolysed whey protein composition comprises up to 20% intact BLG, preferably up to 15% intact BLG, more preferably up to 10% intact BLG, yet more preferably up to 5% intact BLG based on the amount of intact BLG present in the whey protein derivative before the enzymatic treatment.

14. The method according to any one of the preceding claims, wherein the selectively hydrolysed whey protein composition comprises at least 60% intact ALA preferably at least 65% intact ALA, more preferably at least 75% intact ALA, even more preferably at least 80% intact ALA, based on the amount of intact ALA present in the whey protein derivative before the enzymatic treatment.

15. The method according to any one of the preceding claims, wherein the whey protein derivative is a concentrated and / or a demineralised whey, optionally wherein the concentrating is performed by microfiltration, nanofiltration, ultrafiltration, diafiltrationor any combination thereof, and / or the demineralisation is performed by filtration, electrodialysis, ion exchange or any combination thereof.

16. A selectively hydrolysed whey protein composition obtainable by the method according to any one of claims 1 to 15.

17. A selectively hydrolysed whey protein composition comprising a weight ratio of intact ALA to intact BLG of at least 1 .0, preferably at least 1 .5, more preferably at least 2.0, even more preferably at least 5.0, yet more preferably at least 8.0, still more preferably at least 10.0, most preferably at least 12.0, optionally wherein the selectively hydrolysed whey protein composition is obtainable by the method according to any one of claims 1 to 15.

18. The selectively hydrolysed whey protein composition according to claim 17, wherein degree of protein hydrolysis is at least 5%, preferably in the range of 5% to 25%, more preferably in the range of 5% to 20%, even more preferably in the range of 5% to 18%, still more preferably in the range of 5% to 10%.

19. The selectively hydrolysed whey protein composition according to any one of claims 17 to 18, comprising up to 50 wt-% of peptides having a size of more than 10 kDa, preferably up to 40 wt-%, more preferably up to 30 wt-%, based on weight of total protein.

20. The selectively hydrolysed whey protein composition according to any one of claims 17 to 19, comprising at least 5 wt-% of peptides having a size of less than 1 kDa, preferably at least 10 wt-%, more preferably at least 15 wt-%, based on weight of total protein.21 . Use of the selectively hydrolysed whey protein composition according to any one of claims 16 to 20 in manufacturing a food product, optionally wherein the food product is an infant formula.

22. The use according to claim 21 , wherein the food product comprises at least 0,1 wt-% of the selectively hydrolysed whey protein composition, based on total weight of the food product, and / or wherein the selectively hydrolysed whey protein composition represents the entirety of intact ALA and intact BLG content in the food product.

23. The use according to claim 21 or 22, wherein the food product comprises the selectively hydrolysed whey protein composition at an amount in the range of 0.38 to 1 .7 grams per 100 kJ.

24. A food product comprising or manufactured from the selectively hydrolysed whey protein composition according to any one of claims 16 to 20, optionally wherein the food product is an infant formula.

25. The food product according to claim 24, wherein the food product comprises at least 0,1 wt-% of the selectively hydrolysed whey protein composition, based on total weight of the food product and / or wherein the selectively hydrolysed whey protein composition represents the entirety of intact ALA and intact BLG content in the food product.

26. The food product according to claim 24 or 25, wherein the food product comprises the selectively hydrolysed whey protein composition at an amount in the range of 0.38 to 1 .7 grams per 100 kJ.

27. The method according to any one of claims 1 to 15, the selectively hydrolysed whey protein composition according to any one of claims 16 to 20, the use according to any one of claims 21 to 23, or the food product according to any one of claims 24 to 26, wherein the whey protein is from one or more of cow milk, sheep milk, goat milk, buffalo milk, horse milk, camel milk, llama milk, or deer milk, preferably from cow milk.

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