Palatable extensively hydrolysed whey protein hydrolyzates

Abstract

To provide new whey protein hydrolyzates having a high degree of hydrolysis, good taste and low turbidity even without being subjected to ultrafiltration, a method for preparing the new whey protein hydrolyzates, use of the new whey protein hydrolyzates and a food containing the new whey protein hydrolyzates.SOLUTION: A whey protein hydrolysate: -contains free amino acids and peptides, -has a degree of hydrolysis of 15% to 35%, -has peptides with a molecular weight of 2500 Da or more in an amount of 8 to 25% by weight of the total amount of peptides, and -has free amino acids in an amount of 8% by weight or less of the total amino acid content of the hydrolysate. Further provided is a whey protein hydrolysate having a bitterness score equivalent to a solution of 0.08% (wt / vol) or less caffeine in a 4% (wt / wt) protein solution; or use thereof.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to novel whey protein hydrolysates that have a high degree of hydrolysis, good taste, and low turbidity even without ultrafiltration. The present invention also relates to methods for preparing the novel whey protein hydrolysates, uses of the novel whey protein hydrolysates, and foods containing the novel whey protein hydrolysates. [Background technology]

[0002] The use of whey protein hydrolysates as ingredients in various foods is well known. Typically, whey protein hydrolysates are prepared by hydrolyzing whey protein substances, such as whey protein isolates or whey protein concentrates, to a desired degree of hydrolysis using food-grade proteolytic and / or peptidolytic preparations. In some situations, it is desirable to prepare whey protein hydrolysates with a high degree of hydrolysis, for example, 15% or more, e.g., 20-30%, in order to prepare whey protein hydrolysates with low antigenicity or hydrolysates that are well absorbed from the intestine. However, existing whey protein hydrolysates have a problem in that when hydrolysis is too advanced and a high degree of hydrolysis is obtained, the whey protein hydrolysates have a bitter, unpleasant taste and are therefore unsuitable for use in large amounts in foods or beverages.

[0003] WO 02 / 19837 A1 discloses a method for preparing whey protein hydrolysates from whey protein isolate (WPI) substrates that have improved flavor, functionality, and ACE-I inhibitory properties. WO 02 / 19837 A1 discusses the problem of bitter flavors in whey protein hydrolysates and solves the bitter flavor problem by controlling the enzymatic hydrolysis so that hydrolysis terminates when the degree of hydrolysis is at most 10%, such as 3-10%.

[0004] Therefore, it would be advantageous for whey protein hydrolysates to have a high degree of hydrolysis (greater than 15% hydrolysis) that provides low antigenicity and improved absorption properties while not having an unpleasant bitter taste. Summary of the Invention

[0005] The inventors of the present invention have surprisingly found that the use of a particular combination of enzymes for the enzymatic hydrolysis of whey proteins results in whey protein hydrolysates with a high degree of hydrolysis, while the whey protein hydrolysates have an acceptable taste and are free of bitter flavors or at least have acceptable levels of bitter compounds.

[0006] An object of the present invention therefore relates to a method for preparing a whey protein hydrolysate having a degree of hydrolysis of at least 15%, wherein a 4% (wt / wt) protein solution has a bitterness score equivalent to a solution of 0.08% (wt / vol) or less caffeine, without subjecting the whey protein hydrolysate to a bitterness reduction treatment.

[0007] Preferably, the method of the present invention relates to a method for preparing whey protein hydrolysates with low turbidity and therefore clear appearance without any ultrafiltration step.

[0008] In particular, it is an object of the present invention to provide a whey protein hydrolysate that overcomes the above-mentioned problems of the prior art, which are accompanied by an unpleasant bitter taste at high degrees of hydrolysis.

[0009] Thus, one aspect of the present invention is a method for preparing a whey protein hydrolysate, comprising the steps of: a) providing a whey protein solution containing whey protein in an amount of at least 50% by weight based on total solids; b) treating said whey protein solution with a combination of the following enzymes: i. a combination comprising at least a serine endopeptidase derived from the genus Bacillus, at least a serine endopeptidase derived from the genus Aspergillus, and at least a trypsin-like protease; ii. A combination comprising at least a serine endopeptidase derived from the genus Bacillus, at least a serine endopeptidase derived from the genus Aspergillus, and at least a leucyl aminopeptidase derived from the genus Aspergillus; iii. A combination comprising at least bacillolysin from Bacillus amyloliquefaciens, at least bromelain, and at least leucyl aminopeptidase from Aspergillus sp.; subjecting the resulting product to enzymatic hydrolysis using any one of c) Stopping the enzymatic hydrolysis by inactivating the enzyme when the degree of hydrolysis (DH) is 15% or more to obtain whey protein hydrolysate. The present invention relates to a method comprising:

[0010] Another aspect of the present invention is a whey protein hydrolysate comprising: -containing free amino acids and peptides, - has a degree of hydrolysis of at least 15%; - containing peptides having a molecular weight of 2500 Da or more in an amount of 25% by weight or less of the total amount of peptides, - for whey protein hydrolysates containing free amino acids in an amount not exceeding 15% by weight of the total amino acid content of the hydrolysate, The whey protein hydrolysate, in a 4% (wt / wt) protein solution, has a bitterness score equivalent to a solution of 0.08% (wt / vol) or less caffeine.

[0011] A further aspect of the present invention is to provide a food product comprising a whey protein hydrolysate according to the present invention.

[0012] A further aspect of the present invention provides a beverage comprising a whey protein hydrolysate according to the present invention, wherein the whey protein hydrolysate is present in the beverage in an amount equivalent to 2 to 25% by weight of hydrolyzed whey protein.

[0013] Yet another aspect of the present invention is the use of a whey protein hydrolysate according to the present invention as a food ingredient. [Brief explanation of the drawings]

[0014] [Figure 1A] FIG. 1A shows the nephelometric turbidity (NTU) at various protein concentrations for Sample 16 (a whey protein hydrolysate according to the invention made by enzymatic hydrolysis of a WPI), Sample 14 (a whey protein hydrolysate according to the invention made by enzymatic hydrolysis of a whey protein concentrate (WPC)), and Sample 13 (a reference whey protein hydrolysate not prepared by using the enzyme combination of the invention, this whey protein hydrolysate having been subjected to ultrafiltration). [Figure 1B] Figure 1B shows the nephelometric turbidity (NTU) of Sample 16 (a whey protein hydrolysate according to the invention made by enzymatic hydrolysis of WPI) at various protein concentrations. Standard deviations are shown. [Figure 1C] 1C shows the nephelometric turbidity (NTU) of Sample 14 (a whey protein hydrolysate according to the invention made by enzymatic hydrolysis of WPC) at various protein concentrations. Standard deviations are shown. [Figure 2] Figure 2 shows samples with various protein concentrations, Samples 13, 14, and 16. From left to right, the protein concentrations (wt / wt) are 8%, 6.4%, 4.8%, 3.2%, and 1.8%. A) shows Sample 16, B) shows Sample 14, and C) shows Sample 13. [Figure 3] FIG. 3 shows the bitterness scores of various concentrations of caffeine as well as the bitterness scores of samples 13, 14 and 16. [Figure 4] Figure 4 shows a radar chart representation of the taste and mouthfeel profiles of samples 13, 15 and 16. The significance level of the difference between the attribute with the highest and the attribute with the lowest score is indicated by *** and is P<0.001 at 99.9% (ANOVA analysis). [Figure 5]Figure 5 shows the proportions of amino acids in the range of 7 to 10 and 11 to 19 for peptides with 7 to 19 amino acids when analyzed using size exclusion chromatography (SEC) or LC-MS / MS (MS). [Figure 6] Figure 6 shows the phenylalanine-containing peptides expressed as a percentage of all peptides of α-lactalbumin, β-lactoglobulin and β-casein. The shortest peptide analyzed was 5 amino acids long. [Figure 7] FIG. 7 shows the percentages of the number of peptides of 5 to 19 amino acids derived from α-lactalbumin, β-lactoglobulin, and β-casein. [Figure 8] Figure 8 shows, from left to right, samples of a beverage prepared without heat treatment, a beverage prepared with direct UHT treatment at 143°C for 6 seconds, a beverage prepared with indirect UHT treatment for 6 seconds, and a beverage prepared with pasteurization at 90°C for 6.5 minutes. [Figure 9] FIG. 9 shows a photograph of an SDS-PAGE gel used to measure the amount of undegraded BSA in various samples of whey protein hydrolysate. [Figure 10] Figure 10 shows the correlation between beverage carbonation and pH. [Figure 11] FIG. 11 shows the pH of various samples containing whey protein hydrolysate carbonated with 2.5 volumes of carbon dioxide per volume of composition. [Figure 12] FIG. 12 shows the pH and turbidity during heating of samples including an 8% protein solution of Sample 16 carbonated with 2.5 volumes of CO 2 per volume of solution.

[0015] The present invention will now be described in more detail. DETAILED DESCRIPTION OF THE INVENTION

[0016] definition Before describing the present invention in further detail, the following terms and conventions will first be defined.

[0017] All references to one feature or limitation of the invention include the corresponding multiple features or limitations, and vice versa, unless otherwise specified or clearly suggested to the contrary by the context in which the reference is made.

[0018] All percentages referred to herein are percent by weight unless otherwise stated, and the terms "by weight of dry matter" and "on a dry matter basis" refer to the same concept and are used interchangeably.

[0019] The term "wt / wt," such as 1% (wt / wt), refers to a composition containing 1% of the compound by weight.

[0020] "Pleasant palatable" refers to having a taste sufficient to be eaten or drunk, i.e., having a taste that is acceptable or pleasing to the human consumer.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0022] whey protein solution In the context of the present invention, the term "solution," as in "whey protein solution," encompasses compositions containing a combination of liquid and solid compounds or semi-solid particles, such as protein particles. Thus, the "solution" may be a suspension or a slurry. However, the "whey protein solution" is preferably pumpable, and the amount of liquid in the whey protein solution is preferably 70-98%, more preferably 80-96%. The liquid used in the whey protein solution is typically water.

[0023] The whey protein solution will typically contain 2% or more of protein by weight of the whey protein solution. In one embodiment of the present invention, the whey protein solution contains 2-20% of protein by weight of the whey protein solution. Preferably, the whey protein solution contains 5-15% of protein by weight of the whey protein solution.

[0024] The whey protein solution used for hydrolysis is obtained by dispersing a composition containing whey protein in a liquid such as water. Preferably, the whey protein solution is made by mixing any of a whey protein concentrate, a whey protein concentrate, a whey protein isolate, and / or a whey protein isolate with water. Thus, in one embodiment of the present invention, the whey protein solution comprises a whey protein concentrate, a whey protein concentrate, a whey protein isolate, and / or a whey protein isolate.

[0025] The whey protein solution of the present invention contains whey protein in an amount of at least 50% of the total solids. If the whey protein content is less than 50% of the total solids, the overall molecular composition (ratio between protein, carbohydrates, lipids, and minerals) will be different, and the enzymes may behave differently, resulting in a different product.

[0026] In addition to whey proteins, the whey protein solution may contain small amounts of other proteins, such as casein.

[0027] Whey protein solutions typically contain other components in addition to protein. Whey protein solutions may contain other components typically found in whey or milk serum, such as minerals, carbohydrates, and / or lipids. Alternatively, or in addition, whey protein solutions may contain components not naturally found in whey or milk serum. However, such non-naturally occurring milk components should be suitable and safe for use in food manufacturing.

[0028] The lower the protein content of a whey protein solution relative to the total solids, the higher the amount of lipids, carbohydrates (mainly lactose), and other proteins other than whey protein.

[0029] The whey protein solution may contain carbohydrates such as lactose, oligosaccharides, and / or lactose hydrolysis products (i.e., glucose and galactose), for example, in an amount of 0 to 10% by weight based on the total solid content.

[0030] Whey protein isolate (WPI) and whey protein isolate (SPI) contain very small amounts of carbohydrates such as lactose. Therefore, when WPI or SPI is used to prepare a whey protein solution, the carbohydrate content in the whey protein solution is in the range of 0 to 1 wt.% based on the total solids. When whey protein concentrate (WPC) or whey protein concentrate (SPC) is used to prepare a whey protein solution, the carbohydrate content in the whey protein solution is preferably in the range of 2 to 8 wt.% based on the total solids.

[0031] The whey protein solution may also contain lipids, for example in the form of other types of lipids such as triglycerides and / or phospholipids.

[0032] In the context of the present invention, the terms "fats" and "lipids" have the same meaning and can be used interchangeably.

[0033] The whey protein solution according to the present invention should contain whey protein in an amount of at least 50% based on the total solids. If the protein content of the whey protein solution is less than 50% of the total solids, the whey protein hydrolysate obtained after hydrolysis may not have the characteristics defining the whey protein hydrolysate according to the present invention, i.e., the absence of an unpleasant bitter taste in a 4% protein solution, a degree of hydrolysis of more than 15%, a content of free amino acids of 15% by weight or less, and a content of peptides having a molecular weight of 2500 Da or more of 25% by weight or less of the total amount of peptides.

[0034] A whey protein solution containing less than 50% whey protein based on the total solids will contain large amounts of minerals, fat, and carbohydrates. It is undesirable to produce a whey protein hydrolysate containing less than 50% whey protein based on the total solids and containing large amounts of minerals, fat, and carbohydrates. Without being bound by any theory, the inventors of the present invention believe that minerals may affect the activity of some enzymes. This may also apply to lipids and carbohydrates. Furthermore, large amounts of lipids, minerals, and carbohydrates may affect the taste and turbidity of the resulting whey protein hydrolysate.

[0035] Preferably, the whey protein solution comprises whey protein in an amount of at least 60% by weight based on total solids, such as at least 70% by weight based on total solids, even more preferably at least 80% by weight based on total solids. In a more preferred embodiment of the invention, the whey protein solution comprises at least 85% by weight based on total solids, and most preferably the whey protein solution comprises at least 90% by weight based on total solids.

[0036] The proteins present in the whey protein solution should be mainly whey proteins. However, trace amounts of other proteins, such as casein, may also be present. In one embodiment of the present invention, the whey protein solution therefore comprises whey proteins in an amount of 90% by weight or more, based on the total amount of proteins. Preferably, the whey protein solution comprises whey proteins in an amount of 95% by weight or more, based on the total amount of proteins. Thus, in a further embodiment of the present invention, the whey protein solution comprises casein or other non-whey proteins in an amount of up to 10% by weight, based on the total amount of proteins, preferably up to 5% by weight, more preferably up to 3% by weight, based on the total amount of proteins.

[0037] A high fat content in the whey protein solution will affect the clarity and taste of the resulting protein hydrolysate. Thus, in one embodiment of the present invention, the whey protein solution comprises lipids in an amount of up to 10% by weight based on total solids, such as up to 8% by weight based on total solids, and even more preferably, the whey protein solution comprises lipids in an amount of up to 6% by weight based on total solids.

[0038] When whey protein concentrate is used to prepare the whey protein solution, the lipid / fat content is about 6-8% by weight of the total solids, whereas when whey protein isolate is used to prepare the whey protein solution, the whey protein solution is essentially fat-free.

[0039] In a preferred embodiment of the present invention, the whey protein solution is essentially fat-free. The term "essentially fat-free" means that the lipid content of the whey protein solution is less than 1% by weight based on total solids, preferably less than 0.5% by weight, and more preferably less than 0.1% by weight based on total solids.

[0040] When the fat content of the whey protein solution is low, such as up to 0.5% based on total solids, the whey protein hydrolysate prepared according to the method of the present invention will be clear in appearance. Preferably, the fat content in the whey protein solution is less than 0.3% by weight of total solids, more preferably less than 0.2% by weight of total solids.

[0041] In a preferred embodiment of the present invention, the whey protein hydrolysate is obtained by using a whey protein solution of whey protein isolate and / or whey protein isolate. When whey protein isolate and / or whey protein isolate are used for hydrolysis, the fat content is low (less than 0.5%). This allows the whey protein hydrolysate to have a high degree of hydrolysis (DH>15%), good taste, and a clear appearance without any ultrafiltration step. Preferably, the whey protein solution is obtained by mixing whey protein isolate or whey protein isolate with water.

[0042] whey protein In one aspect of the invention, the whey protein hydrolysate is obtained by hydrolyzing a solution containing whey protein.

[0043] In the context of the present invention, the term "whey protein" relates to proteins found in whey or whey. The whey proteins of a whey protein solution may be a subset of the protein species found in whey or whey, or may be the complete set of protein species found in whey and / or whey. Whey proteins are a mixture of globular proteins isolated from whey, a liquid material produced as a by-product of cheese production. Whey proteins are proteins present in the serum phase of either milk or coagulated milk. Proteins in the serum phase of milk are sometimes referred to as whey proteins as well as whey proteins.

[0044] The term "whey" refers to the liquid remaining when casein and milk fat globules have been removed from milk, for example by microfiltration or large pore ultrafiltration. Whey is also sometimes called "ideal whey".

[0045] The term "whey protein" or "serous protein" relates to proteins present in whey.

[0046] The term "whey" refers to the liquid supernatant remaining after the casein of milk has been precipitated and removed. Precipitation of casein can be achieved, for example, by acidifying the milk and / or by using rennet enzymes.

[0047] There are several types of whey, including sweet whey, acid whey, and casein whey.

[0048] The whey protein present in the whey protein solution of the present invention can be derived from different sources of whey, such as casein whey, acid whey or sweet whey.

[0049] In a preferred embodiment of the present invention, the whey protein in the whey protein solution is from sweet whey. Sweet whey primarily contains the proteins β-lactoglobulin (BLG), α-lactalbumin (ALA), and caseinomacropeptide (CMP). However, sweet whey may contain other proteins, such as immunoglobulins, osteopontin, lactoferrin, and fat globule membrane proteins. CMP is not present in sweet whey or acid whey. In one embodiment of the present invention, the whey protein in the whey protein solution is from sweet whey from which the CMP has been completely or partially removed. This is sometimes referred to as denatured sweet whey. Removing CMP from sweet whey results in a protein material with a threonine and tryptophan content closer to that of human milk.

[0050] In the context of the present invention, the term "β-lactoglobulin" may also be referred to as "BLG". These terms can be used interchangeably and relate to BLG derived from mammalian species. Furthermore, the term "α-lactalbumin" may also be referred to as "ALA" in the context of the present invention and relate to α-lactalbumin derived from mammalian species.

[0051] As used herein, the term "sweet whey" refers to the liquid remaining after coagulating and straining milk during the production of rennet-type cheeses. Sweet whey is obtained during the manufacture of rennet-type hard cheeses, such as Cheddar and Swiss cheese. Sweet whey is obtained by adding rennet enzymes to a milk composition, which cleave κ-casein into para-κ-casein and the peptide caseinomacropeptide (CMP), thereby destabilizing casein micelles and causing casein precipitation. The liquid surrounding the rennet-precipitated casein is called sweet whey. Sweet whey has a pH value ranging from 5.2 to 6.7.

[0052] Sweet whey is a product of cheese manufacturing and contains approximately 10-15% protein by weight and approximately 75-80% lactose. The proteins in sweet whey are primarily whey proteins, although small amounts of casein may also be present. Whey proteins include β-lactoglobulin (approximately 55-65%), α-lactalbumin (approximately 18-25%), bovine serum albumin, immunoglobulins, caseinomacropeptide (CMP), osteopontin, lactoferrin, and milk fat globule membrane proteins.

[0053] The term "sweet whey" (sometimes called sour whey or acid whey) relates to whey obtained from the production of casein / caseinates. In the context of the present invention, sweet whey is not the same as acid whey. Sweet whey is the whey fraction obtained after separating casein / caseinates by microfiltration. Sweet whey does not contain CMP.

[0054] The term acid whey is used to describe whey obtained during the production of acid-type cheeses, such as cottage cheese and quark. In acid-type cheese production, casein is removed from milk by acid precipitation, i.e., by lowering the pH of the milk to a pH below 4.6, the isoelectric point of casein, thereby disrupting and precipitating the casein micelles. The pH is often lowered to the range of 3.8 to 4.6. The liquid surrounding the acid-precipitated casein is often called acid whey and does not contain CMP.

[0055] In one embodiment of the present invention, the whey protein used in the whey protein solution is not acid whey or casein whey.

[0056] The whey protein used in the whey protein solution of the present invention can be a whey protein concentrate (WPC), a whey protein concentrate (SPC), a whey protein isolate (WPI), or a whey protein isolate (SPI). The difference between a whey protein concentrate and a whey protein isolate is the product's composition, particularly its protein content. Whey protein isolates are purer than concentrates, with other non-protein components partially removed to "isolate" the whey protein. Thus, whey protein isolates have a higher protein content and are sufficiently pure that they can be virtually lactose-free, carbohydrate-free, fat-free, and cholesterol-free.

[0057] In the present context, the terms "whey protein concentrate (WPC)" and "serum protein concentrate (SPC)" encompass both dry and liquid compositions of whey protein. The protein content of the WPC and SPC used in the present invention is 50% by weight or more based on the total solids content. However, whey protein concentrates may contain higher amounts of whey protein, for example, 80% by weight of whey protein based on the dry matter content. The dry portion of liquid whey is obtained by removing sufficient non-protein components from whey so that the dry product contains 50% by weight or more of whey protein.

[0058] Typically, the WPC or SPC used in the invention comprises: 50-89% protein by weight based on total solids 15-70% by weight of BLG based on total protein content 8-50% ALA by weight of total protein content 0-40% CMP by weight based on total protein content

[0059] Alternatively, but also preferred, is a WPC or SPC comprising: 50-89% protein by weight based on total solids 15-80% by weight of BLG based on total protein content 4-50% ALA by weight of total protein content 0-40% CMP by weight based on total protein content

[0060] Preferably, the WPC or SPC comprises: 50-89% protein by weight based on total solids 15-80% by weight of BLG based on total protein content 4-50% ALA by weight of total protein content 0-40% CMP by weight based on total protein content

[0061] More preferably, the WPC or SPC comprises: 70-89% protein by weight based on total solids BLG: 30-80% by weight of total protein content 4-35% ALA by weight of total protein content 0-25% CMP by weight based on total protein content

[0062] The terms "whey protein isolate" and "serous protein isolate" refer to dry or liquid compositions generally considered to be substantially lactose- and cholesterol-free and to have a whey protein content of at least 90% by weight based on total solids. Whey protein isolates may, for example, contain 92% or more whey protein by weight based on total solids. Preferably, WPIs and SPIs contain 90-100% protein by weight based on total solids, such as 92-99% protein by weight based on total solids.

[0063] The WPI or SPI may preferably include: 90-100% protein by weight based on total solids 15-70% by weight of BLG based on total protein content 8-50% ALA by weight of total protein content 0-40% CMP by weight based on total protein content

[0064] Alternatively, but also preferably, the WPI or SPI may include: 90-100% protein by weight based on total solids BLG: 30-80% by weight of total protein content 4-35% ALA by weight of total protein content 0-25% CMP by weight based on total protein content

[0065] Preferably, the WPI may preferably include: 90-100% protein by weight based on total solids 60-70% by weight of BLG based on total protein content 10-20% ALA by weight of total protein content 10-20% CMP by weight based on total protein content

[0066] In one embodiment of the invention, the whey protein solution used for preparing the whey protein hydrolysate according to the invention comprises a total amount of whey protein in the range of 50-98% by weight, such as 70-97% by weight of dry substance, preferably 72-95% by weight, even more preferably 75-95% by weight of dry substance.

[0067] Any suitable whey protein source can be used to prepare the whey protein solution according to the present invention. The whey protein used in the whey protein solution according to the present invention is preferably whey protein from mammalian milk, such as cow, sheep, goat, buffalo, camel, llama, mare, horse and / or deer milk. In some preferred embodiments of the present invention, the whey protein is derived from bovine (dairy cow) milk.

[0068] The whey protein solution is preferably a demineralized whey protein solution. From a nutritional and health standpoint, it is preferable that minerals such as sodium, calcium, potassium, magnesium, and phosphate are present at low concentrations in protein hydrolysates, so the whey protein solution preferably has a low mineral content. Furthermore, minerals, including sodium and calcium, for example, can interact with whey proteins and affect the turbidity, aggregation behavior, and heat resistance of whey protein hydrolysate products. At high concentrations, some minerals, particularly sodium, calcium, and zinc, can inhibit or promote the proteolytic activity of some proteases; therefore, the presence of high concentrations of these ions can alter the concerted cleavage pattern of proteases. Therefore, in an embodiment of the present invention, the mineral content of the whey protein solution is 10% or less, more preferably 8% or less, based on the total solids. In the context of the present invention, the term "mineral" refers to ash content. The terms "mineral" and "ash" are used interchangeably and can refer to the same thing. Therefore, a reference to the mineral content of a whey protein solution should be understood as the ash content of the whey protein solution.

[0069] In one embodiment of the present invention, the whey protein solution comprises 30% or more by weight of BLG, such as 40% or more by weight of BLG based on the total protein content. Most preferably, the whey protein solution comprises 50% or more by weight of BLG based on the total protein content, and even more preferably, the whey protein solution comprises 55% or more by weight of BLG based on the total protein content. In another embodiment of the present invention, the whey protein solution comprises 30-95% by weight of BLG based on the total protein content, such as 40-90% by weight of BLG based on the total protein content, and even more preferably, 45-80% by weight of BLG based on the total protein content.

[0070] In the context of the present invention, the term "whey" refers to the liquid composition remaining when casein has been removed from milk. The casein can be removed, for example, by microfiltration, resulting in a liquid permeate that is free or essentially free of micellar casein but contains the whey proteins that are naturally present. This liquid permeate is sometimes referred to as ideal whey, serum, or whey.

[0071] The proteins of the whey protein solution are preferably as close to their native state as possible and have preferably been subjected to only mild heat treatment, if any.

[0072] Enzymatic hydrolysis Step b) of the method according to the invention involves subjecting the whey protein solution to enzymatic hydrolysis, which is carried out using any one of the following enzyme combinations: i. A combination of enzymes including at least a serine endopeptidase derived from the genus Bacillus, at least a serine endopeptidase derived from the genus Aspergillus, and at least a trypsin-like protease. ii. A combination of enzymes including at least a serine endopeptidase derived from the genus Bacillus, at least a serine endopeptidase derived from the genus Aspergillus, and at least a leucyl aminopeptidase derived from the genus Aspergillus. iii. A combination of enzymes including at least bacillolysin from Bacillus amyloliquefaciens, at least leucyl aminopeptidase from Aspergillus sp., and at least bromelain.

[0073] The inventors of the present invention have surprisingly found that enzymatic hydrolysis of whey protein solutions by adding any of the above three enzyme combinations results in whey protein hydrolysates having a degree of hydrolysis greater than 15% and a content of free amino acids of 15% or less, which whey protein hydrolysates have an acceptable taste and a low content of bitter peptides.The inventors have found that whey protein hydrolysates prepared by hydrolysis with the above enzyme combinations i. to iii. are not bitter in a 4% (wt / wt) protein solution.

[0074] In step c) of the present invention, the enzymatic hydrolysis is stopped by inactivating the enzyme when the degree of hydrolysis (DH) is 15% or higher to obtain a whey protein hydrolysate, where the degree of hydrolysis (DH) is defined as the percentage of peptide bonds of the original protein that are cleaved by hydrolysis.

[0075] In one embodiment of the present invention, the serine endopeptidase derived from the genus Aspergillus is a serine endopeptidase derived from Aspergillus oryzae and / or Aspergillus flavus. The serine endopeptidase derived from the genus Aspergillus is preferably a subtilisin-like serine endopeptidase (EC 3.4.21).

[0076] In one embodiment of the invention, the enzyme combination i) further comprises a metalloendopeptidase derived from the genus Bacillus, such as bacillolysin. - serine endopeptidase derived from at least the genus Bacillus, metalloendopeptidase derived from at least the genus Bacillus, serine endopeptidase derived from at least the genus Aspergillus, and at least trypsin-like protease

[0077] In a further embodiment of the invention, the enzyme combination ii) may further comprise a metalloendopeptidase from the genus Bacillus, such as bacillolysin. - serine endopeptidase derived from at least the genus Bacillus, metalloendopeptidase derived from at least the genus Bacillus, serine endopeptidase derived from at least the genus Aspergillus, and leucyl aminopeptidase derived from at least the genus Aspergillus

[0078] In one embodiment of the present invention, the leucyl aminopeptidase from an Aspergillus species is from Aspergillus oryzae.

[0079] In one embodiment, the serine endopeptidase from the Bacillus genus is a subtilisin, preferably from Bacillus licheniformis.

[0080] In another embodiment of the present invention, the metalloendopeptidase is bacillolysin. The bacillolysin is preferably derived from the genus Bacillus, more preferably from Bacillus amyloliquefaciens. In a preferred embodiment of the present invention, the bacillolysin is not derived from Bacillus subtilis.

[0081] In the context of the present invention, the term "trypsin-like protease" refers to a protease of microbial origin. Preferably, the microbial trypsin-like protease is derived from a Fusarium species, particularly Fusarium oxysporum. Therefore, the term "trypsin-like protease" does not include, for example, pancreatin, which is not derived from a microorganism. In contrast, pancreatin is a mixture of enzymes derived from the pancreas, including, for example, trypsin, chymotrypsin, amylase, and lipase. Furthermore, the term "trypsin-like protease" should not be confused with "trypsin."

[0082] In yet another embodiment, the bromelain is from Ananas comosus.Bromelain is a cysteine ​​endopeptidase.

[0083] In one embodiment of the present invention, the enzymatic hydrolysis of step b) is carried out using any one of the following enzyme combinations: i) a combination of enzymes comprising at least a serine endopeptidase derived from the genus Bacillus, at least a serine endopeptidase derived from the genus Aspergillus, and at least a trypsin-like protease ii) A combination of enzymes including at least a serine endopeptidase derived from the genus Bacillus, at least a serine endopeptidase derived from the genus Aspergillus, and at least a leucyl aminopeptidase derived from the genus Aspergillus. iii) A combination of enzymes including a combination of a serine endopeptidase and a metalloendopeptidase derived from at least the genus Bacillus, a serine endoprotease derived from at least the genus Aspergillus, and a leucyl aminopeptidase derived from at least the genus Aspergillus. iv) a combination of enzymes including at least bacillolysin from Bacillus amyloliquefaciens, at least bromelain, and at least leucyl aminopeptidase from Aspergillus sp.

[0084] In a preferred embodiment of the invention, the enzymatic hydrolysis of step b) is carried out using any one of the following enzyme combinations: i) a combination of enzymes including at least a serine endopeptidase from a Bacillus species, at least a serine endopeptidase from Aspergillus oryzae, and at least a trypsin-like protease from a microorganism; ii) a combination of enzymes including a subtilisin from at least a Bacillus species, a serine endopeptidase from at least an Aspergillus oryzae strain, and a leucyl aminopeptidase from at least an Aspergillus oryzae strain. iii) a combination of enzymes including a combination of bacillolysin and subtilisin from at least a Bacillus species, a serine endoprotease from at least Aspergillus oryzae, and a leucyl aminopeptidase from at least Aspergillus oryzae; iv) a combination of enzymes including bacillolysin from at least Bacillus amyloliquefaciens, bromelain from at least Ananas comosus, and leucyl aminopeptidase from at least Aspergillus oryzae;

[0085] In a preferred embodiment of the invention, the enzyme combination is: i) Enzyme combinations comprising at least an enzyme from group EC 3.4.21.62, at least a further enzyme from group EC 3.4.21, and at least a further enzyme from group EC 3.4.21.4 ii) a combination of enzymes comprising at least an enzyme from group EC 3.4.21.62, at least a further enzyme from group EC 3.4.21, and at least a further enzyme from group EC 3.4.11 iii) A combination of enzymes comprising at least an enzyme from group EC 3.4.21.62, a further enzyme from group EC 3.4.24.28, at least a further enzyme from group EC 3.4.21 and at least a further enzyme from group EC 3.4.11. iv) A combination of enzymes comprising at least an enzyme from group EC 3.4.24.28, at least a further enzyme from group EC 3.4.22.32 and at least a further enzyme from group EC 3.4.11.

[0086] In another preferred embodiment of the invention, the enzyme combination is: i) a combination comprising at least a serine endopeptidase derived from Bacillus licheniformis, at least a serine endopeptidase derived from Aspergillus oryzae, and at least a trypsin-like protease derived from Fusarium oxysporum, optionally further comprising bacillolysin derived from Bacillus amyloliquefaciens; ii) A combination comprising at least a serine endopeptidase derived from Bacillus licheniformis, at least a serine endopeptidase derived from Aspergillus oryzae, and at least a leucyl aminopeptidase derived from Aspergillus oryzae. iii) a combination comprising at least a serine endopeptidase derived from Bacillus licheniformis, a bacillolysin derived from Bacillus amyloliquefaciens, a serine endopeptidase derived from at least Aspergillus oryzae, and a leucyl aminopeptidase derived from at least Aspergillus oryzae; iv) A combination comprising at least bacillolysin from Bacillus amyloliquefaciens, bromelain from at least Ananas comosus, and leucyl aminopeptidase from at least Aspergillus oryzae. The serine endopeptidase from Bacillus licheniformis is preferably a subtilisin.

[0087] In another preferred embodiment of the invention, the enzyme combination is: i) A combination comprising at least a serine endopeptidase (EC 3.4.21.62) derived from the genus Bacillus, at least a serine endopeptidase (EC 3.4.21) derived from the genus Aspergillus, and at least a trypsin-like protease (EC 3.4.21.4). ii) A combination comprising at least a serine endopeptidase (EC 3.4.21.62) derived from the genus Bacillus, at least a serine endopeptidase (EC 3.4.21) derived from the genus Aspergillus, and at least a leucyl aminopeptidase (EC 3.4.11) derived from the genus Aspergillus. iii) A combination comprising at least a serine endopeptidase (EC 3.4.21.62) derived from the genus Bacillus, a bacillolysin (EC 3.4.24.28), a serine endopeptidase (EC 3.4.21.63) derived from at least the genus Aspergillus, and a leucyl aminopeptidase (EC 3.4.11) derived from at least the genus Aspergillus. iv) A combination comprising at least bacillolysin (EC 3.4.24.28) from Bacillus amyloliquefaciens, at least bromelain (EC 3.4.22.32), and at least leucyl aminopeptidase (EC 3.4.11) from Aspergillus sp.

[0088] The enzyme combinations i) to iv) used in the method of the present invention for preparing whey protein hydrolysates may contain other enzymes in addition to the major enzymes listed above. The term "major enzyme" refers to the enzyme most abundant in the preparation. In the list below, Uniprot accession numbers are given in parentheses to identify the proteases annotated with a particular nomenclature. For example, the enzyme combination may include peptide hydrolase (A0A364MDR7), subtilase family protein (I8A6W5), fungalysin metallopeptidase M36 (A0A2P2H013), neutral protease 2 (A0A364MH70), aspergillopepsin-1 (B8NLY9), leucyl aminopeptidase A (Q2U1F3), leucyl aminopeptidase B (Q2U1F4), and leucyl aminopeptidase C (Q2U1F5). The present invention may include one or more enzymes having high identity (95 to 100%) to an enzyme selected from the group consisting of dipeptidyl peptidase 2 (Q2ULM2), dipeptidyl peptidase 4 (Q2UH35), dipeptidyl peptidase 5 (Q9Y8E3), neutral protease 1 (Q2U1G7), neutral protease 2 (P46076), alkaline protease 1 (P12547), and prolyl oligopeptidase family protein (B8NBM3).

[0089] In one aspect of the present invention, the main enzymes in the enzyme combination i) are a Bacillus-derived serine endopeptidase, an Aspergillus-derived serine endopeptidase, and a trypsin-like protease. The Bacillus-derived serine endopeptidase is preferably a subtilisin, and the Aspergillus-derived serine endopeptidase is preferably a subtilase family protein. In one embodiment of the present invention, the enzyme combination i) can further include one or more of an aminopeptidase (e.g., peptide hydrolase or leucyl aminopeptidase), a metalloendopeptidase (e.g., bacillolysin or fungalin metallopeptidase, or neutral protease), a prolyl oligopeptidase family protein, and aspergillopepsin-1 (aspartic endopeptidase). At least 80% of the enzymes present in enzyme combination i) are expected to be Bacillus-derived serine endopeptidase, Aspergillus-derived serine endopeptidase, and trypsin-like protease. An example of a preparation containing Bacillus-derived serine endopeptidase is Protamex (Novozymes A / S). Protamex also contains bacillolysin. Examples of preparations containing Aspergillus-derived serine endopeptidase are Promod 782 (Biocatalysts Ltd) and Protease A Amano 2 SD (Amano Enzyme Ltd), with the predominant enzyme being Aspergillus oryzae-derived serine endopeptidase. Promod 782 and Protease A Amano 2 SD contain the enzymes peptide hydrolase, leucyl aminopeptidase, fungalin metallopeptidase M36, prolyl oligopeptidase family protein, neutral protease 2, and aspergillopepsin-1, with the main enzyme being serine endopeptidase. Trypsin-like proteases can be supplied, for example, by Formea ​​TL 1200 BG (Novozymes).

[0090] In one aspect of the present invention, the enzyme combination ii) comprises, as major enzymes, a serine endopeptidase derived from Bacillus, a serine endopeptidase derived from Aspergillus, and a leucyl aminopeptidase derived from Aspergillus. The serine endopeptidase derived from Bacillus is preferably a subtilisin, and the serine endopeptidase derived from Aspergillus is preferably a subtilase family protein and an alkaline protease. The leucyl aminopeptidase derived from Aspergillus can be, for example, one or more of peptide hydrolase, leucyl aminopeptidase A, and leucyl aminopeptidase 2. In one embodiment of the present invention, the enzyme combination ii) can further comprise one or more of a metalloendopeptidase: fungalin metallopeptidase M36, neutral protease 1, and neutral protease 2. Enzyme combination ii) can also include aspergillopepsin-1 (aspartic endopeptidase), dipeptidyl peptidase 4, and dipeptidyl peptidase 5. It is expected that at least 80% of the enzymes present in enzyme combination ii) are Bacillus-derived serine endopeptidase, Aspergillus-derived serine endopeptidase, and Aspergillus-derived leucyl aminopeptidase. An example of a preparation containing Bacillus-derived serine endopeptidase is Alcalase (Novozymes), which contains subtilisin. Examples of preparations containing Aspergillus-derived serine endopeptidase are Protease A Amano 2 SD and Promod 782, in which the primary enzyme is Aspergillus oryzae-derived serine endopeptidase. Promod 782 and Protease A Amano 2 SD also contain the enzymes peptide hydrolase, leucyl aminopeptidase, fungalin metallopeptidase M36, prolyl oligopeptidase family proteins, neutral protease 2, and aspergillopepsin-1, the primary enzyme being a serine endopeptidase. An example of a preparation containing leucyl aminopeptidase from Aspergillus is Flavorzyme Conc BG (Novozymes), the primary enzyme being leucyl aminopeptidase.Flavorzyme Conc BG also contains leucyl aminopeptidase A, leucyl aminopeptidase 2, dipeptidyl peptidase 4, dipeptidyl peptidase 5, neutral protease 1, neutral protease 2, and alkaline protease 1, with the primary enzyme being leucyl aminopeptidase.

[0091] In one embodiment of the present invention, the enzyme combination iii) comprises a Bacillus-derived serine endopeptidase, a Bacillus-derived metalloendopeptidase, an Aspergillus-derived serine endopeptidase, and an Aspergillus-derived leucyl aminopeptidase as major enzymes. The Bacillus-derived serine endopeptidase is preferably subtilisin, the Bacillus-derived metalloendopeptidase is preferably bacillolysin, and the Aspergillus-derived serine endopeptidase is preferably a subtilase family protein and an alkaline protease. The Aspergillus-derived leucyl aminopeptidase can be, for example, one or more of peptide hydrolase, leucyl aminopeptidase A, and leucyl aminopeptidase 2. In one embodiment of the present invention, the enzyme combination iii) can further comprise one or more of the following metalloendopeptidases: fungalin metallopeptidase M36, neutral protease 1, and neutral protease 2. The enzyme combination iii) can also comprise aspergillopepsin-1 (aspartic endopeptidase), dipeptidyl peptidase 4, and dipeptidyl peptidase 5. At least 80% of the enzymes present in the enzyme combination iii) are Bacillus-derived serine endopeptidase, bacillolysin, Aspergillus-derived serine endopeptidase, and Aspergillus-derived leucyl aminopeptidase. Examples of preparations containing both Bacillus-derived serine endopeptidase (subtilisin) and bacillolysin are Promod 950L (Biocatalysts) and Protamex. Examples of preparations containing serine endopeptidase from Aspergillus are Protease A Amano 2 SD and Promod 782. Promod 782 and Protease A Amano 2 SD also contain the enzymes peptide hydrolase, leucyl aminopeptidase, fungalin metallopeptidase M36, prolyl oligopeptidase family proteins, neutral protease 2, and aspergillopepsin-1, with the major enzyme being serine endopeptidase.An example of a preparation containing leucyl aminopeptidase from Aspergillus is Flavourzyme conc BG, in which the primary enzyme is leucyl aminopeptidase. Flavourzyme conc BG also contains the enzymes leucyl aminopeptidase A, leucyl aminopeptidase 2, dipeptidyl peptidase 4, dipeptidyl peptidase 5, neutral protease 1, neutral protease 2 and alkaline protease 1, in which the primary enzyme is leucyl aminopeptidase.

[0092] In one aspect of the present invention, the enzyme combination iv) comprises bacillolysin from Bacillus amyloliquefaciens, bromelain, and leucyl aminopeptidase from Aspergillus sp. as the main enzymes. The leucyl aminopeptidase from Aspergillus sp. can be, for example, one or more of peptide hydrolase, leucyl aminopeptidase A, and leucyl aminopeptidase 2. In one embodiment of the present invention, the enzyme combination iv) can further comprise one or more of metalloendopeptidases: fungalin metallopeptidase M36, neutral protease 1, and neutral protease 2. The enzyme combination ii) can also comprise dipeptidyl peptidase 4, dipeptidyl peptidase 5, and alkaline protease (a serine protease from Aspergillus sp.). At least 80% of the enzymes present in enzyme combination iv) are expected to be bacillolysin from Bacillus amyloliquefaciens, bromelain, and leucyl aminopeptidase from Aspergillus sp. An example of a preparation containing bacillolysin from Bacillus amyloliquefaciens is Neutrase. An example of a bromelain preparation is Promod 523 MDP, while an example of leucyl aminopeptidase from Aspergillus sp. is Flavourzyme conc BG.

[0093] The amount of enzyme used in the hydrolysis step of the method for preparing whey protein hydrolysates of the present invention is not limited by the amount of enzyme added, since the amount depends on the type and activity of the enzyme. However, as a guideline, enzymatic hydrolysis is carried out using a combination of enzymes, and the total amount of enzymes ranges from 0.05 to 10 g per 100 g of protein, such as 0.1 to 7.5 g per 100 g of protein. Preferably, the amount of enzyme is 0.2 to 5.0 g per 100 g of protein.

[0094] The ratio between the three different enzymes in combinations i. to iii. can be in the range of 1-10:1-10:1-10, for example, in the range of 1-8:1-8:1-8. However, the amount of enzyme added depends on the activity of the enzyme used, so the present invention should not be limited to the amount of enzyme added.

[0095] The enzymatic hydrolysis carried out in step b) of the present invention is preferably carried out at a temperature in the range of 40°C to 75°C, such as 40°C to 70°C. The enzymatic hydrolysis should be carried out at a temperature at which the enzyme has optimal activity. In a preferred embodiment, the enzymatic hydrolysis is carried out at a temperature in the range of 45°C to 65°C.

[0096] The time for enzymatic hydrolysis before hydrolysis is stopped in step c) depends on the amount and activity of the enzyme used. Hydrolysis is continued until the degree of hydrolysis is 15% or more. The enzymatic hydrolysis in step b) is preferably carried out for a time in the range of 3 to 20 hours, such as 3.5 to 15 hours, preferably 4 to 10 hours, more preferably 4 to 7 hours.

[0097] The whey protein solution should preferably have a pH during the enzymatic hydrolysis in the range of 6 to 9. In a preferred embodiment, the pH during the enzymatic hydrolysis of step b) is 6.5 to 8.0.

[0098] In this pH range, the enzyme has maximum activity and therefore most efficiently cleaves proteins into peptides and free amino acids. In addition, this pH range avoids aggregation not only during the hydrolysis process but also during heat treatment to inactivate the enzyme.

[0099] In step c) of the method for preparing whey protein hydrolysates according to the invention, the enzymatic hydrolysis is stopped by inactivating the enzyme. In the context of the present invention, the term "inactivation" refers to the irreversible inactivation of the enzyme. The inactivation of the enzyme must be irreversible so that the enzyme is not active under other conditions.

[0100] The hydrolysis is stopped when the degree of hydrolysis is at least 15%, such as at least 18%, preferably at least 20%. In one embodiment of the present invention, in step c), the hydrolysis is stopped when the degree of hydrolysis is in the range of 15% to 35%, preferably 17% to 30%, even more preferably 18% to 28%.

[0101] Inactivation of the enzyme in step c), and thus terminating the hydrolysis, can be achieved by any method known in the art, such as by changing the temperature to a temperature at which the enzyme is inactivated. Alternatively, the enzyme can be inactivated and denatured by changing the pH of the solution to a pH at which the enzyme is inactivated.

[0102] Thus, in one embodiment of the present invention, the enzyme inactivation in step c) is achieved by heating the whey protein solution to which the enzyme has been added to a temperature of at least 80°C. The enzyme is preferably inactivated by heating to a temperature of 80°C to 130°C, such as 85°C to 125°C, even more preferably 90°C to 120°C. Inactivating the enzyme in step c) by heating can be achieved by short heating to a high temperature, for example, by heating to a temperature of 110°C to 130°C for 10 to 30 seconds. Alternatively, the enzyme inactivation in step c) can be achieved by heating at a lower temperature for a longer period of time, which may involve heating to 80°C to 90°C for 5 to 10 minutes.

[0103] In another embodiment of the invention, irreversible inactivation of the enzyme in step c) comprises increasing or decreasing the pH of the whey protein solution to which the enzyme has been added, i.e., the whey protein hydrolysate, to a pH at which the enzyme is inactivated. In one embodiment of the invention, the pH is increased to a pH of 10 or higher. In another embodiment, the pH is decreased to a pH of 4 or lower.

[0104] In a preferred embodiment of the present invention, the method of the present invention does not include any step of ultrafiltration of the whey protein hydrolysate obtained in step c).To the surprise of the present inventors, enzymatic hydrolysis of whey protein solutions with low amounts of lipids, i.e., WPI or SPI, using a specific enzyme combination resulted in whey protein hydrolysates that were good tasting and clear in appearance without any ultrafiltration step.

[0105] Whey protein hydrolysates known in the art can be divided into ultrafiltered and non-ultrafiltered hydrolysates, with known non-ultrafiltered protein hydrolysates being unclear or cloudy in appearance, and ultrafiltered protein hydrolysates generally being clear in appearance.

[0106] In the context of the present invention, the term "ultrafiltration" means membrane filtration using membranes with a cut-off in the range of 1500 Da to 50000 Da, preferably 2000 Da to 20000 Da.

[0107] During ultrafiltration of protein hydrolysates, fat, intact proteins, and some large peptides are retained by the ultrafiltration membrane and in the retentate, while free amino acids, small peptides, and minerals are contained in the ultrafiltration permeate.

[0108] In one embodiment of the present invention, a whey protein solution is prepared as a solution of WPI and SPI with a low lipid content. Surprisingly to the inventors of the present invention, the preparation of whey protein hydrolysate using the enzyme combination of the present invention resulted in a whey protein hydrolysate with a high degree of hydrolysis, a good taste with little bitterness, and a clear appearance.

[0109] However, the low lipid content is not the only reason why the inventors of the present invention were able to prepare a clear protein hydrolysate. The inventors of the present invention surprisingly found that when a whey protein solution with a low lipid content was subjected to enzymatic hydrolysis using any one of the above enzyme combinations, it was possible to prepare a whey protein hydrolysate with a degree of hydrolysis of more than 15%, a non-bitter taste, and a clear appearance in a 4% protein solution. To achieve a clear appearance, a whey protein solution with a low lipid content had to be used. However, the low lipid content was not the only reason for the clear hydrolysate. The inventors of the present invention surprisingly found that hydrolysis using a specific enzyme combination resulted in a clear hydrolysate. In comparative tests, it was observed that other whey protein hydrolysates with a low lipid content but hydrolyzed using enzymes other than those used in the method of the present invention did not produce a clear whey protein hydrolysate with a low bitter taste.

[0110] The whey protein hydrolysate obtained by the method of the present invention can preferably be concentrated and / or dried. Thus, in one embodiment of the present invention, the method of the present invention comprises a step d) of concentrating and / or drying the whey protein hydrolysate obtained in step c). Concentration can be, for example, by one or more of the unit operations nanofiltration, reverse osmosis filtration, and evaporation.

[0111] In another embodiment of the invention, the drying step comprises one or more of the unit operations spray drying, freeze drying and spin flash drying, although rotary drying and / or fluidized bed drying may also be used.

[0112] Whey Protein Hydrolysate In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: -containing free amino acids and peptides, - has a degree of hydrolysis of at least 15%; - containing peptides having a molecular weight of 2500 Da or more in an amount of 25% by weight or less of the total amount of peptides, - whey protein hydrolysates containing free amino acids in an amount of not more than 15% by weight of the total amino acid content of the hydrolysate, The whey protein hydrolysate, in a 4% protein solution, has a bitterness score equivalent to a solution of 0.08% (wt / vol) or less caffeine.

[0113] In one embodiment of the present invention, the degree of hydrolysis of the whey protein hydrolysate of the present invention is at least 15%. The objective of the present invention was to produce a whey protein hydrolysate with a high degree of hydrolysis that does not have an unpleasant bitter taste, even without an ultrafiltration step. It is well known that peptides are the cause of bitterness in many hydrolysates. Generally, extensive hydrolysis resulting in a highly hydrolyzed hydrolysate is expected to result in a bitter-tasting hydrolysate. To reduce the bitterness of highly hydrolyzed protein hydrolysates, the hydrolysate can be treated with activated carbon, but this treatment may also remove bitter-tasting peptides and other hydrolysates. Highly hydrolyzed proteins are desirable because peptides have additional functionalities compared to intact proteins. For example, peptides may be able to withstand heat treatment better than intact whey proteins.

[0114] However, the inventors of the present invention have surprisingly found a method for preparing whey protein hydrolysates with a high degree of hydrolysis that do not have an unpleasant bitter taste, without subjecting the whey protein hydrolysate to additional treatment to remove bitter-tasting peptides.

[0115] In a preferred embodiment of the present invention, the whey protein hydrolysate has a degree of hydrolysis of at least 18%, and even more preferably the whey protein hydrolysate has a degree of hydrolysis of at least 20%.

[0116] In another embodiment of the invention, the whey protein hydrolysate according to the invention has a degree of hydrolysis of 15-35%, such as 18-30%, preferably 18-28%, even more preferably 20-25%.

[0117] The whey protein hydrolysate may contain free amino acids, if present, in an amount of 15% by weight or less of the total amino acid content of the hydrolysate. Preferably, the free amino acid content is 12% by weight or less of the total amino acid content. The term "total amino acid content" in the context of the present invention refers to the total amount of amino acids present, including free amino acids and amino acids bound to peptides and proteins.

[0118] In some embodiments of the present invention, the whey protein hydrolysate has a free amino acid content of 15% by weight or less of the total amino acid content, such as 13% by weight or less of the total amino acid content, preferably 10% by weight or less of the total amino acid content, and even more preferably the free amino acid content is 8% by weight or less of the total amino acid content.

[0119] In another embodiment of the invention, the whey protein hydrolysate comprises free amino acids in an amount of 2 to 15% by weight of the total amino acid content of the hydrolysate, preferably free amino acids in an amount of 4 to 13% by weight of the total amino acid content of the hydrolysate.

[0120] The inventors of the present invention have found that the peptides in the whey protein hydrolysate of the present invention contain peptides having a molecular weight of 2500 Da or more in an amount of 25% by weight or less of the total amount of peptides. Preferably, the whey protein hydrolysate contains peptides having a molecular weight of 2500 Da or more in an amount ranging from 8 to 25% by weight, and even more preferably from 10 to 20% by weight.

[0121] In one embodiment of the present invention, the whey protein hydrolysate of the present invention comprises peptides having a molecular weight of 375 Da or less in an amount of at least 10% by weight. The peptides having a molecular weight of 375 Da or less may be present in the whey protein hydrolysate in an amount ranging from 10 to 25% by weight, for example.

[0122] The inventors of the present invention have found that the whey protein hydrolysate of the present invention, when subjected to membrane filtration using an ultrafiltration membrane and / or treatment with activated carbon, exhibits reduced bitterness compared to known whey protein hydrolysates with a high degree of hydrolysis.

[0123] The bitterness of whey protein hydrolysates was compared with that of caffeine. A whey protein hydrolysate in a 4% (wt / wt) protein solution has a less bitter taste than a solution containing 0.08% (wt / vol) caffeine. Thus, the bitterness score of a whey protein hydrolysate in a 4% (wt / wt) protein solution corresponds to the bitterness score of 0.08% (wt / vol) or less of caffeine. Preferably, the bitterness score of a whey protein hydrolysate in a 4% (wt / wt) protein solution of the present invention corresponds to the bitterness score of 0.07% (wt / vol) or less of caffeine, more preferably to the bitterness score of 0.065% or less of caffeine. Most preferably, the bitterness score of a whey protein hydrolysate in a 4% (wt / wt) protein solution of the present invention corresponds to the bitterness score of 0.060% (wt / vol).

[0124] In a further embodiment of the present invention, the whey protein hydrolysate has a nephelometric turbidity (NTU) of 100 or less in a 4% (wt / wt) protein solution. In a further embodiment, a whey protein hydrolysate is obtained that, in addition to having a high degree of hydrolysis and an acceptable taste, also has a clear appearance. A clear, good-tasting whey protein hydrolysate is desirable because it can be used, for example, in beverages, gels, and shakes, thereby improving consumer appeal.

[0125] In a 4% (wt / wt) protein solution, if the measured nephelometric turbidity is less than 100 NTU, the sample is recognized as clear. In a 4% (wt / wt) protein solution, if the measured nephelometric turbidity is more than 100 NTU, the measured whey protein hydrolysate is recognized as not clear. If the nephelometric turbidity is less than 40 NTU, the solution is recognized as clear. However, whey protein hydrolysates with a turbidity between 40 NTU and 100 NTU may be clear (but opaque or cloudy). In the context of the present invention, the term "transparent" refers to a solution that allows some light to pass through, so that objects behind the solution can be seen, i.e., it is possible to see through the solution. The term "clear" refers to a solution that is colorless, and therefore it is possible to see through the solution without any obstructions. Therefore, a solution may be clear but not transparent.

[0126] In a further embodiment of the invention, the whey protein hydrolysate has a nephelometric turbidity (NTU) of 80 or less in a 4% (wt / wt) protein solution, such as a nephelometric turbidity (NTU) of 60 or less in a 4% (wt / wt) protein solution, preferably a nephelometric turbidity (NTU) of 50 or less in a 4% (wt / wt) protein solution, even more preferably a nephelometric turbidity (NTU) of 40 or less in a 4% (wt / wt) protein solution.

[0127] In one embodiment of the present invention, the whey protein hydrolysate also has antioxidant activity, preferably as measured by a scavenging ratio of 54 to 60 in a 1.5 wt% protein solution.

[0128] The whey protein hydrolysate may contain other components in addition to protein, such as carbohydrates, lipids, and minerals.

[0129] In one embodiment of the invention, the whey protein hydrolysate comprises lipids in an amount of 8% or less by weight based on total solids, such as 6% or less by weight based on total solids, hi another embodiment, the whey protein hydrolysate comprises lipids in an amount of 1% or less by weight based on total solids, such as a lipid content of 0.5% or less by weight based on total solids.

[0130] The whey protein hydrolysate may also contain minerals such as potassium, sodium and calcium.

[0131] In one embodiment of the present invention, the whey protein hydrolysate comprises potassium in an amount of less than or equal to 3.0% by weight.

[0132] In another embodiment of the present invention, the whey protein hydrolysate contains sodium in an amount of less than or equal to 2% by weight.

[0133] In yet another embodiment of the present invention, the whey protein hydrolysate comprises citric acid in an amount ranging from 4 to 10 g per kg solids of whey protein hydrolysate.

[0134] In one embodiment of the invention, the whey protein hydrolysate comprises intact or undegraded bovine serum albumin (BSA) in an amount of 0.5 to 2% by weight relative to the total protein content.

[0135] In a preferred embodiment of the present invention, the whey protein hydrolysate is in the form of a dry composition, such as a powder or granules.

[0136] In another embodiment, the composition is a whey protein hydrolysate liquid composition.

[0137] food In one aspect, the present invention relates to providing a food product comprising a whey protein hydrolysate according to the present invention.

[0138] For example, the food product may be any one selected from the group of dairy products including drinks, shakes, gels, food bars, concentrates or liquid shots.

[0139] In a preferred embodiment, the food product is selected from the group of a protein drink, a protein shot, a protein shake, a protein gel or a protein bar. The food product may also be an infant formula or other infant nutritional product.

[0140] When the food product is in a liquid form such as a drink, shake, gel or shot, the food product may comprise a whey protein hydrolysate according to the present invention, which is preferably present in the drink in an amount corresponding to 2 to 25% by weight of hydrolyzed whey protein, preferably 3 to 20% by weight of hydrolyzed whey protein, such as 3 to 15% by weight of hydrolyzed whey protein, and even more preferably 3 to 10% by weight of hydrolyzed whey protein.

[0141] When the food product is a bar, such as a protein bar, the food product comprises the whey protein hydrolysate according to the present invention in an amount corresponding to 2 to 30% by weight of hydrolyzed whey protein. Preferably, the amount of whey protein hydrolysate according to the present invention is present in the bar in an amount corresponding to 3 to 20% by weight of hydrolyzed whey protein, such as 4 to 15% by weight. When the whey protein hydrolysate according to the present invention is used in a food bar, such as a protein bar, the whey protein hydrolysate can be used as a softener. It is well known that increasing the concentration of protein hydrolysate in a protein bar has a softening effect and prevents the bar from becoming hard during long-term storage.

[0142] In a further aspect, the present invention relates to a beverage comprising the whey protein hydrolysate according to the present invention in an amount corresponding to 2 to 20% by weight of hydrolyzed whey protein. The beverage may be a protein beverage containing carbohydrates, vitamins and minerals in addition to protein.

[0143] In a preferred embodiment of the invention, the beverage has a neutral pH, ie a pH in the range of 6.5 to 8.0 in a 4% protein solution at 22°C.

[0144] In one embodiment of the present invention, the whey protein hydrolysate according to the present invention can be used as an ingredient in the preparation of a carbonated beverage. Thus, the food product of the present invention comprising the whey protein hydrolysate according to the present invention is a carbonated beverage.

[0145] A further embodiment of the present invention relates to a carbonated beverage comprising the whey protein hydrolysate of the present invention.

[0146] In one embodiment, the carbonated beverage contains whey protein hydrolysate in an amount equivalent to 2-10% by weight. The carbonated beverage may also contain carbohydrates, if present, in an amount of 5% by weight or less. The carbonated beverage is preferably fat-free.

[0147] In one embodiment, the amount of carbonation in carbonated beverages containing the whey protein hydrolysates of the present invention ranges from 0.1 volumes to 4 volumes of carbonation (per volume of liquid present in the beverage). More typically, the amount of carbonation ranges from about 1.6 volumes to about 3.5 volumes, with the most typical concentrations being from about 1.7 volumes to about 3.0 volumes, and the amount of carbonation most preferably ranging from 2.0 to 3.0 volumes. Carbonation, in the context of the present invention, means the addition of carbon dioxide to a mixture of beverage ingredients in an amount sufficient to obtain a carbonated protein beverage in which the amount of carbonation present in the beverage ranges from 0.1 volumes to 4 volumes per volume of liquid mixture.

[0148] In some embodiments of the method of the present invention, the carbon dioxide is added in the form of sterile carbonated water, while in other embodiments, sterilized carbon dioxide is bubbled through the liquid mixture until the desired amount of carbon dioxide is present.

[0149] Carbonation increases the acidity of a beverage. The more carbon dioxide added to a beverage, the lower the beverage's pH. However, the inventors of the present invention have found that adding carbon dioxide lowers the beverage's pH to a lower limit of 5.5-6.0 at 5°C. Adding approximately 2.5-3.0 volumes of carbon dioxide per volume of beverage lowers the beverage's pH to approximately 6.0 at 5°C, while adding approximately 4 volumes of carbon dioxide per volume of beverage lowers the beverage's pH to approximately 5.5 at 5°C.

[0150] Thus, in one embodiment of the present invention, carbonated beverages comprising the whey protein hydrolysate of the present invention have a pH of at least 5.5 at a temperature of 5° C. The pH will typically be in the range of 5.5 to 8.25, such as in the range of 5.5 to 7.0, preferably in the range of 5.8 to 6.5. Carbonated beverages having a pH above 5.5 are preferred.

[0151] Carbonated beverages containing the whey protein hydrolysates of the present invention can be heat-treated, for example, by pasteurization or autoclaving, and the inventors have found that the turbidity of carbonated beverages containing the whey protein hydrolysates of the present invention remains unchanged after heat treatment at temperatures up to 120°C for extended periods of time, for example, 20 minutes.

[0152] In a further embodiment of the present invention, a carbonated beverage may comprise a whey protein hydrolysate according to the present invention in combination with a non-hydrolyzed whey protein isolate.

[0153] The whey protein hydrolysate of the present invention can also be used to prepare protein shots, protein shakes, or protein gels, which contain, in addition to hydrolyzed whey protein, carbohydrates, vitamins, and minerals in amounts of 2 to 20% by weight. The pH of the protein shots, protein shakes, or protein gels is preferably neutral, i.e., in the range of 6.5 to 8.0.

[0154] In a further aspect, the present invention relates to the use of the whey protein hydrolysate according to the present invention as a food ingredient. The whey protein hydrolysate can be added as a food ingredient to any type of food. Preferably, the whey protein hydrolysate according to the present invention is used as a food ingredient in the preparation of cold or hot beverages.

[0155] In one embodiment of the present invention, the whey protein hydrolysate is used as a food ingredient in the preparation of a UHT stable beverage having a pH in the range of 6.5 to 8.5.

[0156] In another embodiment of the present invention, the whey protein hydrolysate is used as a food ingredient in the preparation of a beverage for use in sports nutrition, in the context of the present invention the term "sports nutrition" refers to nutrition suitable in connection with exercise or training, i.e. for increasing muscle mass.

[0157] In yet another embodiment of the present invention, the whey protein hydrolysate is used as a food ingredient in the preparation of a clinical beverage. In the context of the present invention, the term "clinical beverage" refers to a beverage with clinical or medical efficacy. For example, a clinical beverage may have health-related effects. Clinical beverages are typically used by hospitalized patients or elderly people with nutritional problems, or by individuals who require pre-digested protein to recover from a medical condition. For example, a clinical beverage may be a beverage used by individuals suffering from malnutrition or malabsorption. A clinical beverage may also be for individuals suffering from gastrointestinal disorders. In the present context, the terms "clinical beverage" and "medical beverage" have the same meaning.

[0158] A clinical beverage may, for example, contain the whey protein hydrolysate of the present invention in an amount equivalent to a beverage containing 2-20% by weight of hydrolyzed whey protein. The clinical beverage may contain carbohydrates in an amount of 5-50% by weight of the beverage. The amount of carbohydrates may, for example, be in the range of 10-40% by weight, such as 15-35% by weight. The clinical beverage may also contain fat. For example, the fat content of the clinical beverage may be in the range of 2-30% by weight, such as 3-20% by weight, more preferably 3-18% by weight.

[0159] In one example, the clinical beverage comprises a hydrolyzed whey protein according to the invention corresponding to an amount of 4-10% by weight, 3-15% by weight fat and 10-35% by weight carbohydrates.

[0160] Clinical beverages preferably have a neutral pH value, ie a pH in the range of 6.5 to 8.0.

[0161] Clinical drinks may be in the form of clear drinks, milky drinks, tube fed or in the form of a powder that is redissolved in liquid.

[0162] In one embodiment, the whey protein hydrolysates of the present invention can also be used to prepare juice-style beverages, which preferably contain the hydrolyzed whey protein according to the present invention in an amount corresponding to 4-10% by weight of protein, 0-1% by weight of fat, and 15-35% by weight of carbohydrates.

[0163] When the clinical beverage is in tube-fed form, it may contain 4-15% by weight of hydrolyzed whey protein according to the present invention, about 5-35% by weight of carbohydrates and about 3-15% by weight of fat.

[0164] The whey protein hydrolysate according to the invention can also be used in infant nutrition, such as in infant formula. In the context of the present invention, the term "infant formula" refers to any type of infant formula, including follow-on formula, growing-up formula and preterm formula.

[0165] When the whey protein hydrolysate according to the present invention is used in infant formula, the protein content of the infant formula is in the range of 1.6 to 5.0 g / 100 kcal. In addition to the whey protein hydrolysate, the infant formula may contain carbohydrates such as lactose, oligosaccharides, lipids, vitamins, and minerals.

[0166] The whey protein hydrolysate according to the invention can also be used in the preparation of other infant nutrition products than infant formula, such as smoothies, porridges, etc.

[0167] The whey protein hydrolysate of the present invention can also be used to prepare emulsions. Emulsions are typically prepared by redissolving whey protein hydrolysate powder in a liquid, such as water or milk, and fat. The whey protein hydrolysate has an emulsifying effect on water and fat. The powder typically contains whey protein hydrolysate in an amount equivalent to 5-15% by weight of protein. After redissolution, the emulsion contains 2-4% by weight of protein.

[0168] Protein hydrolysis causes changes in the protein, such as an increase in the number of charged groups, a decrease in average molecular weight, and exposure of reactive groups. These changes affect the emulsion-forming and emulsion-stabilizing abilities of the protein hydrolysate. The whey protein hydrolysate of the present invention can be used as an emulsifier, stabilizer, etc. by combining it with other ingredients.

[0169] The whey protein hydrolysates of the present invention can also be used in bakery products such as biscuits, cookies and crackers.

[0170] In a further aspect, the present invention relates to the use of whey protein hydrolysate according to the present invention as an antioxidant.The inventors of the present invention have surprisingly found that whey protein hydrolysate according to the present invention has antioxidant effect.Therefore, whey protein hydrolysate can be used in nutritional compositions as a source of antioxidant peptides.

[0171] Thus, the present invention relates to whey protein hydrolysates of the present invention having antioxidant effect. More particularly, the present invention relates to whey protein hydrolysates having antioxidant effect as defined by a scavenging ratio of 54 to 60 in a solution containing 1.5% by weight of protein. The scavenging ratio is measured by the DPPH (2,2-diphenyl-1-picryl-hydrazyl hydrate) assay. The scavenging ratio is calculated as 100 x (A0 - A0). S) / A0, where A0 is the absorbance in the absence of sample and A S is the absorbance in the presence of sample.

[0172] It should be noted that embodiments and features described in the context of one aspect of the invention also apply to other aspects of the invention.

[0173] All patent and non-patent literature cited in this application is incorporated herein by reference in its entirety.

[0174] The invention is further illustrated in the following non-limiting examples. [Example]

[0175] Example 1: Method of analysis Example 1.1: Determination of the degree of hydrolysis (DH) The degree of hydrolysis (DH) is defined as the percentage of peptide bonds that are broken by hydrolysis, see equation (1) below: The DH value gives information about the number of peptides formed in relation to the number of available peptide bonds.

[0176] The DH of whey protein hydrolysates was measured as described in Adler-Nissen, J. Determination of the degree of hydrolysis of food protein hydrolysates by trinitrobenzenesulfonic acid. J. Agric. Food Chem. 27, 1256-1262 (1979) and Nielsen, PM, Petersen, D. & Dambmann, C. Improved method for determining food protein degree of hydrolysis. J. Food Sci. 66, 642-646 (2001). In formula (1), h represents the number of peptide bonds broken, and h total represents the total number of available peptide bonds. Thus, DH gives the percentage of broken peptide bonds. Equation (1): DH = (number of free amino termini) / (total number of available peptide bonds) 100% = h / h total 100%

[0177] The free α-amino group formed after hydrolysis reacts with o-phthalaldehyde (OPA) to form a yellow complex that absorbs light at 340 nm and can be measured spectrophotometrically. Based on this color formation, the DH can be calculated.

[0178] The hydrolysate was resuspended in water at an appropriate concentration (0.03–0.08% protein), and 2 volumes were reacted with 15 volumes of OPA reagent (100 mM Na2B4O7, 0.1% sodium dodecyl sulfate, 6 mM DL-dithiothreitol, 6 mM o-phthalaldehyde, and 2% ethyl alcohol) at 25°C for 2 min. A similar reaction was performed to generate a concentration series of L-serine. The absorbance at 340 nm (A340) was then measured, and the A340 signal from the OPA-water reaction was subtracted. To determine the true DH, the serine equivalents measured in the supernatant were corrected as proposed by Adler-Nissen (Adler-Nissen J; Agricultural and Food Chemistry, 1979 27 (6) 1256) for the trinitrobenzenesulfonic acid method, which gives the same response as the OPA method described above. The factors used for whey protein hydrolysate were a = 1, b = 0.4, h total =8.8.

[0179] Example 1.2: Turbidity Measurement The nephelometric turbidity of whey protein hydrolysates is used as a measure of clarity and transparency. A sample is considered clear if the measured nephelometric turbidity is less than 100 NTU in a 4% (wt / wt) protein solution. Furthermore, a sample is considered transparent if the measured nephelometric turbidity is less than 40 NTU in a 4% (wt / wt) protein solution.

[0180] For measuring nephelometric turbidity, the sample is diluted to five different concentrations of protein: 1.8%, 3.2%, 4.8%, 6.4% and 8% and the nephelometric turbidity is measured using a Merck Turbiquant 3000 IR.

[0181] Example 1.3: Determining total protein The total protein content of the sample (protein equivalent) is calculated as follows: 1) Measure the total nitrogen content of the sample in accordance with ISO 8968-1 / 2IIDF 020-1 / 2-Milk - Determination of nitrogen content - Part 172: Determination of nitrogen content using the Kjeldahl method. 2) Calculate the total amount of protein as N x 6.38.

[0182] Example 1.4: Determination of Amino Acid Content Analysis of amino acid composition provides detailed information about protein hydrolysates as a source of amino acid supplementation.

[0183] Total amino acid content was determined according to the methods of ISO13903:2005 and EU152 / 2009. Samples were hydrolyzed in aqueous hydrochloric acid to cleave peptide bonds in the samples. After hydrolysis, the samples were pH adjusted, made up to volume, and filtered. Amino acids were separated on an amino acid analyzer and detected using post-column derivatization with ninhydrin reagent at 440 nm and 570 nm. A single-point calibration curve was used for quantification. For quality assurance, in-house standards were analyzed with every analytical run.

[0184] Cysteine ​​and methionine must be oxidized before analysis on an amino acid analyzer. Samples were oxidized at low temperature using hydrogen peroxide and formic acid, followed by acid hydrolysis using aqueous hydrochloric acid. The oxidation process oxidizes methionine and cysteine, preventing their loss during hydrolysis. After hydrolysis, samples were analyzed as described above.

[0185] Total tryptophan quantification: Because tryptophan is destroyed during acid hydrolysis of proteins and therefore cannot be quantified in the same manner as other amino acids, it was analyzed using a different method: instead, samples were hydrolyzed by alkaline treatment and quantified by HPLC analysis.

[0186] The results for each amino acid were normalized to the total amount of amino acid detected [g / 100g amino acid].

[0187] Example 1.5: Determination of free amino acid content Free amino acids in whey protein hydrolysates are measured by the method of R. Schuster, "Determination of Amino Acids in Biological, Pharmaceutical, Plant and Food Samples by Automated Precolumn Derivatization and HPLC", Journal of Chromatography, 431:271-284 (1988) and Henderson, JW, Ricker, RD, Bidlingmeyer, BA, Woodward, C., "Rapid, Accurate, Sensitive and Reproducible HPLC Analysis of Amino Acids, Amino Acid Analysis Using Zorbax Eclipse-AAA columns and the Agilent 1100 HPLC", Agilent Publication, 2000.

[0188] Free amino acids are measured by extracting the amino acids into aqueous or acidic solutions. Samples may be deproteinized by molecular weight filtration. Samples are analyzed by HPLC after derivatization prior to injection. Prior to injection, primary amino acids are derivatized with o-phthalaldehyde and secondary amino acids are derivatized with fluorenylmethyl chloroformate. Results are presented as follows: [mg free amino acids / 100g whey protein hydrolysate powder]

[0189] Example 1.6: Method for determining peptide distribution in whey protein hydrolysates Size exclusion chromatography (SEC) was used to analyze the molecular weight distribution of peptides in whey protein hydrolysates. SEC is used to separate polymeric molecules by size. Mixtures of different size components, such as peptides, can be separated by SEC. The elution time depends on the size of the molecule; the smaller the molecule, the longer the elution time.

[0190] The sample was dissolved in the mobile phase to a concentration of 0.5% (wt / vol). Prior to injection, the sample was filtered through a 0.45 μm filter. Chromatographic separation was performed on three TSK G2000 SWXL (125 Å, 5 μm, 7.5 mm × 300 mm) columns connected in series. A buffer of 0.0375 M phosphate buffer, 0.375 M ammonium chloride, 0.1% trifluoroacetic acid (TFA), and 25% acetonitrile (CH3CN) was used as the mobile phase, with a flow rate of 0.7 mL per minute. Peptides were detected using a UV detector measuring at 214 nm.

[0191] Based on retention time, the peptide distribution was sized to give relative amounts according to molecular weight.

[0192] Example 2: Screening of enzyme combinations A total of 55 different enzyme combinations were tested for use in the enzymatic hydrolysis of whey proteins. The resulting whey protein hydrolysates were analyzed for clarity, bitterness, degree of hydrolysis, and peptide composition. Hydrolysis studies were performed at a 0.5 liter scale.

[0193] The enzymes used to test the various enzyme combinations were serine endopeptidase from Bacillus (EC 3.4.21.62), serine endopeptidase from Aspergillus (EC 3.4.21), trypsin-like protease from microorganisms (EC 3.4.21.4), aminopeptidase from Aspergillus (EC 3.4.11), metalloendopeptidase from Bacillus amyloliquefaciens (EC 3.4.24.28), endoprotease from Ananas comosus (bromelain) (EC 3.4.22.32), and proline-specific endopeptidase from Aspergillus niger (EC 3.4.21.62). 3.4.21.26), an aminopeptidase preparation derived from Aspergillus oryzae (EC 3.4.11), a metalloendopeptidase preparation derived from Geobacillus stearothermophilus (EC 3.4.24), and an endopeptidase preparation derived from Bacillus amyloliquefaciens (EC 3.4).

[0194] The enzyme preparations used in the experiments were as follows: Protamex (Novozymes), which contains serine endopeptidase (subtilisin) and bacillolysin from Bacillus species Protease A Amano 2 SD (Amano Enzyme Co., Ltd.), containing serine endopeptidase derived from Aspergillus oryzae Promod 782 MDP (Biocatalysts), containing serine endopeptidase from Aspergillus species Formea ​​TL 1200 BG (Novozymes), containing a trypsin-like protease derived from a microorganism Promod 950L (Biocatalysts), containing serine endopeptidase (subtilisin) and bacillolysin from Bacillus species Flavorzyme conc BG (Novozymes), containing leucyl aminopeptidase derived from Aspergillus oryzae Alcalase AF 2.4L (Novozymes), containing serine endopeptidase (subtilisin) derived from Bacillus licheniformis Neutrase conc BG (Novozymes), containing metalloendopeptidase (bacillolysin) derived from Bacillus amyloliquefaciens Promod 523 MDP (Bromelain) (Biocatalysts), containing cysteine ​​endopeptidase from Ananas comosus Maxipro PSP (DSM), a proline-specific endopeptidase from Aspergillus niger Flavorpro 766 (Biocatalyst), which contains aminopeptidase derived from Aspergillus oryzae Thermoase PC10F (Amano Enzyme Co., Ltd.), which contains a metalloendopeptidase derived from Geobacillus stearothermophilus Protin NY100 (Amano Enzyme Co., Ltd.), containing endopeptidase derived from Bacillus amyloliquefaciens

[0195] Fifty-five enzyme combinations were used for whey protein hydrolysis. A solution of whey protein isolate (WPI) (Lacprodan DI-9224, manufactured by Arla Food Ingredients) was used as the hydrolysis substrate. In all experiments, the WPI solution had a protein concentration of 8% by weight. The hydrolysis reaction was carried out at 50°C with a pH stat of pH = 7, with a reaction time of 6 hours from the first enzyme addition. After 6 hours of hydrolysis, the enzyme was stopped by inactivating it by heating to 99°C with a 90-second hold time. The amount of enzyme used is listed in Table 1 as grams of enzyme per 100 g of protein. The product of each hydrolysis test was lyophilized and used for further analysis, including evaluation and scoring of clarity (in the form of turbidity measurements), degree of hydrolysis, and taste. The results are shown in Table 1 below. NEU: Refers to Neutrase. FZ: Flavorzyme conc BG. Alca: Refers to Alcalase AF 2.4L. PA: Protease A Amano 2 SD. FTL: Refers to Formea ​​TL 1200 BG. PM782: Refers to Promod 782 MDP. FP766: Refers to Flavorpro 766. MP PSP: Refers to Maxipro PSP. THER: Refers to thermoase PC10F. PRO: Refers to Protin NY100. PM950: Refers to Promod 950L. PTM: refers to Protamex.

[0196] The enzymes used in the tests presented in Table 1 were selected from a larger pool of enzymes based on 96-well scale testing. These tests included hydrolysis with various enzyme combinations and visual scoring of apparent transparency, thermal stability, and hydrolysis (SDS-PAGE). To allow for large amounts of material for pH-stat hydrolysis and analysis, the combinations presented in Table 1 were performed at a 500 ml scale, as described above. Therefore, the enzymes presented in Table 1 were not randomly selected. Some of the enzyme combinations in Table 1 (e.g., combinations 1–3) were repeated at different volumes.

[0197] The term "Visual a inact." in Table 1 refers to the appearance after inactivation.

[0198] Table 1 [Table 1] TIFF2025157549000003.tif254151TIFF2025157549000004.tif254146TIFF2025157549000005.tif254144

[0199] The data for each whey protein hydrolysate was evaluated. For a positive evaluation, the following conditions had to be met: - Transparent appearance after enzyme inactivation - have a high degree of hydrolysis, greater than 15%, preferably greater than 20% - No bitterness in a 4% (w / w) protein solution - Not more than 25% by weight of the peptides should have a molecular weight greater than 2500 Da.

[0200] The sample should be clear after 90 seconds at 99°C, as this indicates UHT stability (stable and clear after treatment of a 4% solution at 143°C for 6 seconds).

[0201] Thus, from the table above, it can be seen that out of the 55 enzyme combinations, four samples, designated as samples 4, 10, 11 and 30, are satisfactory.

[0202] Sample 4 was obtained by hydrolysis using a combination of enzymes: Bacillus-derived serine endopeptidase, Aspergillus-derived serine endopeptidase, and trypsin-like protease.

[0203] Sample 10 was obtained by hydrolysis using a combination of the enzymes Bacillus-derived serine endopeptidase, Bacillus-derived metalloendopeptidase, Aspergillus-derived serine endopeptidase and Aspergillus-derived leucyl aminopeptidase.

[0204] Sample 11 was obtained by hydrolysis using serine endopeptidase derived from Bacillus, serine endopeptidase derived from Aspergillus, and leucyl aminopeptidase derived from Aspergillus.

[0205] Sample 30 was obtained by hydrolysis using bacillolysin from Bacillus amyloliquefaciens, bromelain, and leucyl aminopeptidase from Aspergillus sp.

[0206] Example 3: Further analysis of whey protein hydrolysates according to the invention The four whey protein hydrolysates of Example 2 that met the criteria of clarity, taste, degree of hydrolysis and peptide distribution were further analyzed and listed as Samples 1 to 4 (S1 to S4) in the table below.

[0207] Samples 5-12 (S5-S12) are whey protein hydrolysates from hydrolysis using other enzyme combinations.

[0208] Sample 13 (S13) is a spray-dried WPI hydrolysate obtained by hydrolysis of sweet whey with a combination of Bacillus licheniformis subtilisin (alcalase) and Bacillus amyloliquefaciens bacillolysin (neutrase), followed by ultrafiltration, treatment with activated carbon, and microfiltration to remove the activated carbon (as described in WO 1993 / 024020 A1). The degree of hydrolysis is approximately 25%.

[0209] Sample 14 (S14) was prepared by using the same enzyme combination (enzyme combination of the present invention) as Sample 4, with WPC used as the substrate for hydrolysis.

[0210] Samples 15 and 16 (S15-S16) are replicates of Sample 4. Samples 15 and 16 are WPI-based hydrolysates prepared using the same conditions as Sample 4 but without UF filtration.

[0211] Whey protein hydrolysates produced by enzymatic hydrolysis using different enzyme combinations were again analyzed for clarity, degree of hydrolysis and content of peptides with molecular weights greater than 2500 Da and less than 375 Da.

[0212] The clarity (turbidity) of the sample is determined by measurement according to Example 1.2; the nephelometric turbidity should be less than 40 NTU for the sample to be recognized as transparent, and less than 100 NTU for the sample to be recognized as clear.

[0213] Samples were rated as bitter if bitterness was detected at 4% protein or less.

[0214] The degree of hydrolysis was measured as described in Example 1.1, while bitterness was measured by tasting samples of different concentrations (protein concentrations of 2%, 4%, and 8%). The peptide distribution was measured as described in Example 1.5. The results are shown in Table 2.

[0215] Table 2: [Table 2] TIFF2025157549000007.tif241158TIFF2025157549000008.tif98158

[0216] Thus, Table 2 shows that using the particular enzyme combinations according to the present invention, whey protein hydrolysates 1) have a degree of hydrolysis greater than 20%, 2) have less than 25% of the peptides with a molecular weight of 2500 Da or greater, and 3) are free of bitterness at concentrations of 4% protein or less.

[0217] Furthermore, Table 2 shows that when WPI is used for protein hydrolysis, the use of a specific enzyme combination of the present invention results in a whey protein hydrolysate that is clear in appearance. Table 2 also shows that the use of a specific enzyme combination in the preparation of whey protein hydrolysates using WPC as a substrate results in whey protein hydrolysates with a degree of hydrolysis of more than 20%, in which less than 25% of the peptides have a molecular weight of 2500 Da or more, and in which the proteins do not have a bitter taste at concentrations of 4% or less. However, the whey protein hydrolysates prepared by using WPC as a substrate are not clear in appearance (due to the lipids present in WPC).

[0218] In a preferred embodiment of the present invention, WPI is used as the substrate for proteolysis to obtain a clear hydrolysate.

[0219] Example 4: Turbidity analysis Additionally, Samples 1-16 in Example 3 were analyzed for nephelometric turbidity at various protein concentrations. Samples with a turbidity of less than 100 NTU are considered clear, and samples with a turbidity of less than 40 are considered transparent. Table 3 below shows the turbidity of the hydrolysates listed as Samples 1-12 in Example 3 measured at various protein concentrations. The protein concentrations are 1.8% protein, 3.2% protein, 4.8% protein, 6.4% protein, and 8% protein. Protein concentrations are in wt%.

[0220] Whey protein hydrolysate powders were allowed to hydrate for at least 30 minutes at the indicated concentrations before measuring turbidity (n=3).

[0221] Table 3: Turbidity (NTU) of test samples at 1.8, 3.2, 4.8, 6.4 and 8% protein [Table 3]

[0222] Thus, Table 3 shows that certain enzyme combinations according to the present invention have turbidity of less than 100 NTU at a concentration of 8% protein.

[0223] Figures 1A-C show the measured turbidities of Samples 13, 14, and 16. Figure 1A shows the turbidity of the whey protein hydrolysates of Samples 13, 14, and 16, illustrating the differences in turbidity. Figure 1A shows that the whey protein hydrolysate subjected to ultrafiltration (not of the present invention, Sample 13) has very low turbidity (less than 1 NTU), making it the clearest hydrolysate. The hydrolysate prepared according to the method of the present invention using one of the specific enzyme combinations (Sample 16) has a turbidity of less than 100 NTU at a protein concentration of 8% and is therefore clear. Furthermore, the hydrolysate of the present invention has a turbidity of less than 40 NTU at a protein concentration of 4%, and is therefore recognized as clear at a protein concentration of 4%. In contrast, the whey protein hydrolysate prepared according to the present invention by using WPC as a substrate (Sample 14) has a turbidity of more than 1000 NTU and is therefore recognized as opaque.

[0224] Figure 1B more clearly shows that the turbidity of sample 16 is less than 40 NTU at protein concentrations of 5% or less.

[0225] Figure 1C shows the turbidity of Sample 14. It shows that even at very low concentrations (approximately 2%), the turbidity exceeds 2000 NTU. Sample 14 is perceived as very opaque and not transparent.

[0226] Figures 2A-C contain photographs of samples 13, 14, and 16 to visually illustrate the clear versus opaque samples. From left to right, the samples were prepared with 8%, 6.4%, 4.8%, 3.2%, and 1.8% protein.

[0227] FIG. 2A shows that sample 16 appeared clear and transparent at 8%, 6.4%, 4.8%, 3.2% and 1.8% protein.

[0228] FIG. 2B shows sample 14 and shows that even at the lowest protein level of 1.8%, sample 14 appears opaque and not clear.

[0229] FIG. 2C shows Sample 13, which appears clear and transparent at all concentrations.

[0230] Example 5: Evaluation of bitterness associated with caffeine In Example 5, the bitterness of whey protein hydrolysates according to the present invention is analyzed in comparison with UF-filtered and activated carbon-treated whey protein hydrolysates prepared by enzymatic hydrolysis of WPI using enzymes other than those of the present invention (hydrolysis with Bacillus licheniformis subtilisin (alcalase) and Bacillus amyloliquefaciens bacillolysin (neutrase)).

[0231] A sensory evaluation was carried out to compare the taste of Samples 13, 15 and 16 of Example 3.

[0232] The sensory panel was trained to detect and quantify bitterness using caffeine solutions as a reference. Panelists were first presented with a reference sample consisting of solutions with increasing concentrations of caffeine. They were then trained to assign bitterness scores to unknown solutions on a 15-cm scale. The reference samples consisted of three caffeine solutions containing 0.025%, 0.05%, and 0.1% caffeine, respectively. After evaluating the reference samples, panelists tasted the hydrolysate samples in a 4% protein solution by weight three times in random order and ranked the bitterness intensity of the test solutions based on the bitterness of the reference solutions. The reference solution containing 0.025% caffeine was perceived as not bitter, while the standard solution containing 0.1% caffeine was perceived as bitter (a score of 13 on the 15-cm bitterness scale). The sensory panel included seven panelists participating in the evaluation.

[0233] Additionally, a sensory profiling was performed using a panel according to the international standard, Quantitative Descriptive Profile ISO 13299:2016 2nd ed. 5.5, Annex F1-F6&H3. Seven trained evaluators participated in the evaluation. Evaluations were conducted in triplicate. The response scale used was a continuous line scale (15 cm). Approximately 2 ml of sample was provided at room temperature. Red light was used during the evaluation.

[0234] The bitterness scores from the sensory panel related to the bitterness of caffeine are shown in Figure 3. The bitterness scores of the test samples are plotted against the concentration of caffeine. The bitterness of three different concentrations of caffeine and the bitterness scores of Samples 13, 15, and 16 from Example 3 are shown in Figure 3.

[0235] 3 shows that Sample 13 (a WPI hydrolysate, but not with the enzyme combination of the present invention, in which the hydrolysate was ultrafiltered and treated with activated carbon) had the highest relative bitterness of 0.095%. Sample 15 (a WPI hydrolysate with the enzyme combination of the present invention, but without ultrafiltration and activated carbon treatment) had the lowest relative bitterness of the three product samples, at 0.054%, which was quite close to the hydrolysate of Sample 16 (similar to Sample 15), which had a relative bitterness to caffeine of 0.061%.

[0236] Thus, the taste of the good tasting whey protein hydrolysates prepared according to the present invention (Samples 15 and 16) was perceived as less bitter than 0.08% caffeine and less bitter than Sample 13.

[0237] Example 6: Taste profiling An example was conducted to evaluate the taste profiles of whey protein hydrolysates Samples 13, 15, and 16. Taste profiling was performed by a trained panel, who used five attributes to distinguish between the samples, focusing on odor, mouthfeel, and taste.

[0238] The data was analyzed to reveal significant differences between samples for each attribute. Statistical evaluation of the data is shown in Table 4. Furthermore, multiple comparison tests were used to reveal differences between samples. In Table 4, samples with the same letter are not significantly different.

[0239] Table 4: Sensory scores [Table 4]

[0240] ***p<0.001 Duncan test. Samples with different letters for attributes are significantly different at the 95% level (p<0.05).

[0241] Therefore, the bitterness scores of samples 15 and 16 are less than the bitterness score of sample 13.

[0242] The data in Table 4 can be shown as a radar chart representation. See Figure 4. The radar chart shows the attributes of odour (O), mouthfeel (MF) and taste (T). 'High' intensity of each sensory attribute is shown on the outside of the plot, while 'little' intensity is shown in the centre of the plot. Each product has a different label shown below the figure. See the 'Bitter_T' data as an example. *** indicates that the data for sample 13 and samples 15 and 16 are significantly different (p<0.001).

[0243] The taste profile radar chart shows that sample 13 has a significantly different taste profile than samples 15 and 16, and most importantly, samples 15 and 16 are less bitter.

[0244] Example 7: Analysis by LC-MS / MS and peptide cleavage patterns Peptides in liquid samples can be identified by mass spectrometry peptide analysis and database searching. Samples 1, 2, 3, 13, 15, and 16 were analyzed by LC-MS / MS to identify peptide sequences and their proteins of origin. Peptides present in each sample were dissolved in water and injected onto a Dionex nano-LC system for MS / MS analysis on a Bruker Maxis Impact QTOF mass spectrometer. The resulting MS / MS spectra were searched against a custom-made database containing bovine protein sequences. The overall results of the analysis are shown in Table 5.

[0245] Table 5: Proteins identified by LC-MS / MS [Table 5]

[0246] *Glycosylation-dependent cell adhesion molecule 1 **Bovine serum albumin

[0247] The data in Table 5 demonstrate that this analysis yielded high sequence coverage for the most abundant proteins in the samples, including β-lactoglobulin, α-lactalbumin, and β-casein. Additionally, the number of peptides identified from each sample is shown. The dataset was of the quality expected for LC-MS / MS analysis and therefore warranted further detailed analysis (Example 9).

[0248] Example 8: LS-MS / MS analysis of bitter peptides First, we confirmed that the LC-MS / MS data in Example 7 corresponded to the data obtained by SEC analysis (size exclusion chromatography) by comparing the peptide distributions. Here, the SEC data were converted to percentage numbers (number of peptides of a given molecular weight) and plotted for each hydrolysate alongside the same data calculated based on LC-MS / MS (see Figure 5). The LC-MS / MS data included only β-lactoglobulin peptides, whereas the SEC analysis included all proteins in the sample.

[0249] Figure 5 shows that the proportion of β-lactoglobulin-derived peptides in the range of 7–10 amino acids and 11–19 amino acids in the number of β-lactoglobulin-derived peptides is similar when using LC-MS / MS and SEC.

[0250] Therefore, since the data is comparable to SEC data and the SEC method is quantitative, the data shown in Figure 5 demonstrate that the number of peptides in different amino acid length ranges in the LC-MS / MS dataset can be used to extract quantitative information about the relative distribution of peptides. Furthermore, Figure 5 shows that sample 13 (not according to the invention) has more peptides with smaller peptide sizes than the hydrolysate according to the invention.

[0251] The amount of detectable peptides containing phenylalanine as a percentage of the total number of peptides was determined and analyzed by MS-LC / MS in samples 1, 2, 3, 13, 15, and 16. The results are shown in FIG.

[0252] Without being bound by any theory, the inventors of the present invention believe that the presence of phenylalanine in a peptide correlates with bitterness, and that the bitterness of phenylalanine is enhanced when its amino or carboxy terminus is blocked by a peptide bond with another amino acid residue. For example, the phenylalanine residue of the bitter peptide YPFPGPIPN identified in bitter whey protein hydrolysate has been suggested to be the major bitterness determinant (Liu, X., Jiang, D., and Peterson, DG Identification of Bitter Peptides in Whey Protein Hydrolysate. J. Agric. Food Chem. 2014. 62: 5719-5725).

[0253] FIG. 6 shows the phenylalanine content in peptides as a percentage of the total number of peptides derived from β-lactoglobulin, α-lactalbumin, and β-casein. The proportion of peptides containing phenylalanine was lower in the less bitter hydrolysates for peptides with a peptide size of less than nine amino acid residues. Thus, for the whey protein hydrolysates according to the present invention, Samples 1 and 3, phenylalanine was present in peptides with larger peptide sizes. Furthermore, the whey protein hydrolysates according to the present invention, represented by Samples 2, 15, and 16, also showed a lower proportion of phenylalanine-containing peptides. Furthermore, Samples 2, 15, and 16 also showed an overall lower proportion of phenylalanine bound to peptides. This suggests that Samples 2, 15, and 16 may contain more phenylalanine as a free amino acid than Samples 1, 3, and 13. Therefore, an indication of a non-bitter whey protein hydrolysate according to the present invention is that the majority of phenylalanine is present in larger peptides or as a free amino acid.

[0254] In Figure 7, the proportions of peptides of 5 to 19 amino acids from β-lactoglobulin, α-lactalbumin and β-casein are shown.

[0255] 7 shows that Sample 13 (outside the present invention) contains more small peptides (5-9 amino acids) than the other five whey protein hydrolysates. Without being bound by any theory, the inventors of the present invention believe that the higher content of phenylalanine in the hydrolysate of Sample 13, which contains peptides with smaller peptide sizes, may be the reason for the higher bitterness of Sample 13 compared to the hydrolysates of the present invention, Samples 1, 2, 3, 15, and 16.

[0256] Example 9: SEC Size Distribution Data The sizes of the peptides in samples 1, 2, 3, 13, 15 and 16 were analyzed by size exclusion chromatography (SEC), and the results are shown in Table 6 below.

[0257] Table 6: SEC size distribution data, DH and free amino acid (FAA) content for whey protein hydrolysates of samples 1, 2, 3, 13, 15 and 16 [Table 6]

[0258] The size exclusion chromatography data does not accurately take into account free amino acids because it was obtained by measuring at 214 nm, where peptide bonds are expected to absorb primarily.

[0259] Therefore, the content of free amino acids was measured by a different method (see Example 10). Compared with the reference whey protein hydrolysate of Sample 13, the whey protein hydrolysate according to the present invention has a higher content of free amino acids. However, the content of small peptides is higher in the reference whey protein hydrolysate (Sample 13) than in the whey protein hydrolysate according to the present invention. For example, the content of peptides of 750 Da or less is more than 50% in Sample 13. In contrast, the content of peptides of 750 Da or less in the whey protein hydrolysate according to the present invention is less than 40%. Furthermore, Sample 13 contains fewer peptides of 2500 Da or more than the whey protein hydrolysate according to the present invention.

[0260] Comparing these data with those of Example 8, it becomes clear that the overall low phenylalanine content of peptides derived from beta-lactoglobulin, alpha-lactalbumin and beta-casein in samples 2, 15 and 16 is a result of these phenylalanine residues being released from the peptides into the free amino acid fraction. Thus, a way to reduce the bitterness of hydrolysates may be to identify enzyme combinations that specifically enrich phenylalanine in the free amino acid fraction of the total hydrolysate.

[0261] Example 10: Measured free amino acid content The free amino acid content in the whey protein hydrolysates of the present invention was measured and compared with a reference whey protein hydrolysate (not of the present invention), and the results are shown in Table 7 below.

[0262] Table 7: Free amino acid content (mg / 100g protein (N x 6.38)) [Table 7]

[0263] Table 7 shows that the free amino acid content in the whey protein hydrolysates of the present invention is between 4% and 14% by weight of the total protein content, whereas the free amino acid content of the reference hydrolysate (sample 13) is about 0.4% by weight of the total protein content.

[0264] Furthermore, Table 7 shows that the free leucine content in the whey protein hydrolysates of the present invention is much higher than that in Sample 13. The free leucine content in the whey protein hydrolysates of the present invention is 1-4 wt% of the total protein content, whereas the free leucine content in Sample 13 is about 0.15 wt% of the total protein content.

[0265] More importantly, the concentrations of free phenylalanine indicated in Examples 8 and 9 are higher in Samples 2, 15 and 16 than in Samples 1, 3 and 13. Table 8 shows the percentage of free amino acids relative to the total amino acid content.

[0266] Table 8: Free amino acids out of total amino acids (%) [Table 8]

[0267] It is particularly noteworthy that the proportion of free leucine in the total leucine content of the whey protein hydrolysate according to the invention is much higher than that of the reference hydrolysate (sample 13).

[0268] Importantly, Table 8 shows that 6-12% of the phenylalanine was in the form of free phenylalanine for the whey protein hydrolysates according to the invention in samples 2, 15 and 16. In the whey protein hydrolysates according to the invention in samples 1 and 3, no phenylalanine was found in the free form.

[0269] Example 11: Mineral Content in Whey Protein Hydrolysates of the Present Invention The amount of minerals in samples 15 and 16 was measured and the results are shown in Table 9 below.

[0270] Table 9: Mineral Content [Table 9]

[0271] Table 9 shows the mineral content and turbidity of samples 15 and 16 as examples of the mineral content of whey protein hydrolysates. All of the products in Table 9 have a pH of 7.8 in a 4% protein solution at 22°C.

[0272] Example 12: Undegraded BSA in hydrolysates The amount of undegraded bovine serum albumin (BSA) was measured in the whey protein hydrolysates of the present invention (samples 1, 2, 3, 4, 15 and 16).

[0273] BSA content was estimated using SDS-PAGE and a BSA standard from Sigma-Aldrich, product code A2153 (Figure 9A). The amount of BSA loaded in 20% of the wells corresponded to 10 μg of pure BSA. The total amount of protein added to each well of the SDS-PAGE gel shown in Figure 9B corresponded to 50 μg of protein. Protein samples were mixed with 10 mg / ml Laemmli sample buffer and 2-mercaptoethanol to a final protein concentration of 3%, then incubated at 95°C for 5 minutes before loading onto the gel.

[0274] The SDS-PAGE gel in Figure 9A is a titration series showing the expected intensity when BSA in Figure 9B represents 20%, 10%, 5%, 2.5%, 1.25%, or 0.63% of the total whey protein. SDS-PAGE gels of standards at various concentrations were compared to SDS-PAGE gels shown in Figure 9B using various samples of whey protein hydrolysate. From left to right: molecular weight standard, sample 2, sample 1, sample 3, sample 4, sample 15, and sample 16.

[0275] Since the band intensities in Figure 9B correspond to those of the 0.63% and 1.25% samples in Figure 9A, it can be concluded from Figures 9A and 9B that the amount of undegraded BSA in the whey protein hydrolysate of the present invention (Figure 9B) is in the range of 0.5-2 wt. %. BSA was resistant to proteolysis by the enzymes used in accordance with the present invention.

[0276] Example 13: UHT processed beverage for sports nutrition Example 13 illustrates the use of whey protein hydrolysates according to the invention in the preparation of beverages suitable for use in sports nutrition, which beverages are intended for use by athletes or in other sports or exercise-related applications.

[0277] The powder of Sample 16 was redissolved in water and sugar and flavorings were added to prepare a beverage. The amounts of the ingredients are shown in Table 10 below.

[0278] Table 10 shows that the beverages were neutral tasting, without any unpleasant bitterness, and could be heat treated by direct and indirect UHT processes as well as pasteurized without developing any additional haze.

[0279] Table 10: Sports drinks [Table 10]

[0280] The sports drinks shown in Table 10 were subjected to 1) direct UHT, 2) indirect UHT, and 3) pasteurization. Direct UHT treatment was performed by spraying at 143°C for 6 seconds. Indirect UHT treatment was performed in a tubular heat exchanger at 143°C for 6 seconds. Pasteurization was at 90°C for 6.5 minutes. The drinks were poured into flasks at 5°C. All solutions had a pH of approximately 7.7 at 22°C. A whey protein hydrolysate of the present invention at a content of 4.9g / 100g corresponds to 4.0% protein by weight.

[0281] Figure 8 shows, from left to right, photographs of an untreated beverage, a direct UHT treated beverage, an indirect UHT treated beverage, and a pasteurized beverage. The beverages are at room temperature. Figure 8 shows that all samples are clear and transparent, allowing the background behind the bottle to be seen.

[0282] The nephelometric turbidity of the four beverages was measured and the results are shown in Table 11.

[0283] Table 11: Turbidity of beverages measured in NTU [Table 11]

[0284] Therefore, Table 11 shows that the turbidity of the heat treated samples was all less than 100 NTU. Therefore, the heat treatment did not affect the turbidity of the beverages, which appeared clear or transparent.

[0285] These drinks were not perceived as bitter.

[0286] Example 14: Beverages for clinical / medical use Example 14 illustrates the use of whey protein hydrolysates according to the invention in the preparation of beverages suitable for use in medical or clinical nutrition, which clinical beverages contain a high amount of carbohydrates in addition to those from the whey protein hydrolysates of the invention.

[0287] The powder from Sample 16 was redissolved in water and carbohydrates and flavorings were added to prepare a beverage. The amounts of the ingredients are shown in Table 12 below.

[0288] Table 12 shows a medical beverage that has a neutral taste without unpleasant bitterness.

[0289] Table 12: Medical drinks [Table 12]

[0290] Example 15: Carbonated Drink Example 15 illustrates the use of whey protein hydrolysates according to the invention in the preparation of carbonated beverages.

[0291] The powder from Sample 16 was redissolved in water and sugar and flavoring were added to prepare a beverage. The amounts of the ingredients are shown in Table 13 below. The beverage was carbonated by adding carbon dioxide to the beverage until it contained 2.5 volumes of carbon dioxide per volume of beverage.

[0292] Table 13: Carbonated drinks [Table 13]

[0293] Example 16: Carbonation - How does the amount of carbonation affect pH? Sample 16 was suspended in water to make an 8% protein solution, and the solution was force carbonated at 5°C.

[0294] The mass gain and pH of the solution were measured over time as more CO2 was introduced. Once the equilibrium pressure in the vessel reached approximately 1 bar, no more CO2 could be absorbed (the pressure used for forced carbonation was 3 bar at 5 °C).

[0295] Figure 10 shows that pH measurements depend on the amount of CO2 added to the carbonated solution. Figure 10 shows that solutions containing whey protein hydrolysate, Sample 16, when carbonated to a CO2 content of 0 to 4 volumes per volume of solution resulted in a pH range of 5.5 to 8.25. The pH of the uncarbonated solution had a p-value of 8.25, and the pH decreased as the amount of carbonation increased.

[0296] FIG. 10 shows that adding CO2 to about 2.5 volumes per volume of solution (4.9 g / L) reduced the pH from 8.25 to about 6.0 at 5°C.

[0297] Figure 10 also shows that the pH reaches a minimum of approximately 5.5-6.0. Therefore, it can be concluded that adding more than 2.5 volumes of CO2 per volume of solution will not decrease the pH below approximately 5.5-6.0.

[0298] Furthermore, the effect of carbonation on the pH value of solutions and beverages was analyzed by measuring the pH of the following solutions / beverages carbonated to an amount of 2.5 volumes of CO2 per volume of solution / beverage: - A 4% protein solution prepared by resuspending sample 16 in water. - 8% protein solution prepared by resuspending sample 16 in water - Beverage containing 8% of Sample 16 The results are shown in Figure 11.

[0299] A beverage containing 8% of Sample 16 contains the following ingredients:

[0300] [Table 14]

[0301] FIG. 11 shows that the 4% and 8% solutions of Sample 16 and the beverage prepared from Sample 16 both have a pH of approximately 6.0 when carbonated to a CO2 content of 2.5 volumes of CO2 per volume of solution.

[0302] Example 17: Analysis of the heat treatment of carbonated solutions An 8% protein solution of Sample 16 was carbonated with 2.5 volumes of CO2 per volume of solution as disclosed in Example 16.

[0303] The solution was heated in a sealed container with a data log to a temperature of 95° C. Temperature, pH and turbidity were measured at various times during heating and the results are shown in FIG.

[0304] As shown in Figure 12, the carbonated product remained clear throughout heating and after 5 minutes at 95°C, as indicated by measured turbidity and visual inspection. The pH remained at 6.2 throughout heating. These data indicated that the carbonated product may be suitable for processes such as pasteurization and autoclaving, which is essentially a process at temperatures up to 120°C for extended periods of time, e.g., 20 minutes.

[0305] Example 18: Protein Bar Example 18 illustrates the use of whey protein hydrolysates according to the invention in the preparation of protein bars.

[0306] Table 14 shows an example of a protein bar containing 5 g / 100 g of whey protein hydrolysate (powder) of the present invention.

[0307] Table 14: Protein bars [Table 15]

[0308] Table 15 shows another example of a protein bar, in which the whey protein hydrolysate (powder) content is 13g / 100g.

[0309] Table 15: Protein bars [Table 16]

[0310] Example 19: Analysis of antioxidant activity using the DPPH assay The antioxidant effect of the whey protein hydrolysate of the present invention was analyzed using the DPPH assay, a radical scavenging assay. This assay measures the antioxidant effect by using DPPH (2,2-diphenyl-1-picrylhydrazyl hydrate), whose color changes from purple to yellow when reacting with antioxidant peptides.

[0311] DPPH was dissolved in methanol to a concentration of 0.2 mM. The whey protein hydrolysate of the present invention (Sample 16) was dispersed in Milli-Q water to various protein concentrations (0, 0.8, 1.5, 2.5, 3.0, 4.0, 5.0, and 6.0% protein) and mixed with DPPH in equal volumes (1:1 ratio). The mixture was incubated at 22°C for 2 hours to allow a yellow color to develop as a result of antioxidant activity. The absorbance at 525 nm was measured and calculated as 100 x (A0 - A0). S The quenching rate was calculated as ) / A0, where A0 is the absorbance in the absence of sample, and A S is the absorbance in the presence of sample.

[0312] Table 16 shows the scavenging rate for various concentrations of whey protein hydrolysate of the present invention (Sample 16).

[0313] Table 16 [Table 17]

[0314] Table 16 shows that whey protein hydrolysate sample 16 has antioxidant activity when included in the DPPH assay at concentrations of 0.8-6% protein. As shown in Table 16, the antioxidant activity increased as the protein concentration increased.

Claims

1. Whey protein hydrolysate, - containing free amino acids and peptides, -having a degree of hydrolysis of 15% to 35%; - having a peptide having a molecular weight of 2500 Da or more in an amount of 8 to 25% by weight of the total amount of peptides, - having free amino acids in an amount of not more than 8% by weight of the total amino acid content of the hydrolysate, and 1. A whey protein hydrolysate having a bitterness score equivalent to a solution of 0.08% (wt / vol) or less caffeine in a 4% (wt / wt) protein solution.

2. 2. The whey protein hydrolysate of claim 1, wherein the whey protein hydrolysate has a nephelometric turbidity (NTU) of 100 or less in a 4% (wt / wt) protein solution.

3. 3. The whey protein hydrolysate according to claim 1, wherein the whey protein hydrolysate contains free amino acids in an amount of 2 to 15% by weight of the total protein content of the hydrolysate.

4. The whey protein hydrolysate according to any one of claims 1 to 3, wherein the whey protein hydrolysate has antioxidant activity.

5. 5. The whey protein hydrolysate according to claim 1, wherein the antioxidant activity of the whey protein hydrolysate is measured as having a scavenging ratio of 54 to 60 in a 1.5 wt.% protein solution.

6. Use of the whey protein hydrolysate according to any one of claims 1 to 5 in a food product.

7. 7. The use according to claim 6, wherein the whey protein hydrolysate is used in the food product in an amount corresponding to 2 to 25% by weight of hydrolyzed protein.

8. 8. The use according to claim 6 or 7, wherein the food product is selected from the group consisting of dairy products, drinks, shakes, gels, shots and food bars.

9. 6. Use of the whey protein hydrolysate according to any one of claims 1 to 5 as a food ingredient.

10. 6. Use of the whey protein hydrolysate according to any one of claims 1 to 5 as a food ingredient in the preparation of a UHT stable beverage having a pH of 6.5 to 8.

0.

11. 6. Use of the whey protein hydrolysate according to any one of claims 1 to 5 as a food ingredient in the preparation of a beverage for use in sports nutrition.

12. 10. Use of the whey protein hydrolysate according to any one of claims 1 to 5 as a food ingredient in the preparation of a clinical beverage.

13. 6. Use of the whey protein hydrolysate according to any one of claims 1 to 5 as an ingredient in the preparation of a carbonated drink.

14. 6. Use of the whey protein hydrolysate according to any one of claims 1 to 5 as an antioxidant.

15. A carbonated drink comprising the whey protein hydrolysate according to any one of claims 1 to 5.

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