Palatable hydrolyzed whey protein hydrolysate

Abstract

The present invention relates to novel whey protein hydrolysates having a high degree of hydrolysis, palatability and low turbidity without the need for ultrafiltration. The present invention also relates to a method for preparing the novel whey protein hydrolysates, the use of the novel whey protein hydrolysates and food products comprising these novel whey protein hydrolysates.

Description

Field of the Invention

[0001] The present invention relates to novel whey protein hydrolysates having a high degree of hydrolysis, a palatable taste and a low turbidity without the need for ultrafiltration. The present invention also relates to a method for preparing the novel whey protein hydrolysates, the use of the novel whey protein hydrolysates and food products comprising these novel whey protein hydrolysates. Background of the Invention

[0002] It is well known that whey protein hydrolysates can be used as ingredients in various food products. Whey protein hydrolysates are typically prepared by hydrolyzing whey protein materials, such as whey protein isolate or whey protein concentrate, to the desired degree of hydrolysis with a food-grade proteolytic and / or peptolytic preparation. In some cases, in order to prepare whey protein hydrolysates with low antigenicity or hydrolysates that are well absorbed in the intestine, it is necessary to prepare whey protein hydrolysates with a high degree of hydrolysis, such as a degree of hydrolysis of 15% or higher, such as 20 - 30%. However, the problem with existing whey protein hydrolysates is that when hydrolyzed excessively and a high degree of hydrolysis is obtained, the whey protein hydrolysates have a bitter and unpleasant taste and are therefore not suitable for use in large amounts in food products or beverages.

[0003] WO 02 / 19 837 A1 discloses a method for preparing whey protein hydrolysates from whey protein isolate (WPI) substrates with improved taste, functionality and ACE-I inhibitory properties. WO 02 / 19 837 A1 addresses the problem of bitterness of whey protein hydrolysates and solves the bitterness problem by controlling the enzymatic hydrolysis such that the hydrolysis is terminated when the degree of hydrolysis is at most 10%, such as 3 - 10%.

[0004] Therefore, it would be advantageous to have whey protein hydrolysates with a high degree of hydrolysis (degree of hydrolysis exceeding 15%) and thus with low antigenicity and improved absorption properties but without an unpleasant bitter taste at the same time. Summary of the Invention

[0005] The inventors of the present invention have surprisingly found that by using a specific combination of enzymes for the enzymatic hydrolysis of whey protein, whey protein hydrolysates are obtained with a high degree of hydrolysis, while the whey protein hydrolysates have an acceptable taste and no bitter taste or at least an acceptable level of bitter compounds.

[0006] Accordingly, one object of the present invention relates to a method for preparing a whey protein hydrolysate having a degree of hydrolysis of at least 15% and wherein the bitterness score of a 4% w / w protein solution corresponds to a solution of 0.08% w / v caffeine or less without any bitterness-reducing treatment of the whey protein hydrolysate.

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

[0008] In particular, an object of the present invention is to provide a whey protein hydrolysate that solves the above problems of the prior art having an unpleasant bitter taste at high degrees of hydrolysis.

[0009] Accordingly, one aspect of the present invention relates to a method for preparing a whey protein hydrolysate, comprising: a) providing a whey protein solution comprising whey protein in an amount of at least 50% by weight based on the total solids content;

[0010] b) subjecting the whey protein solution to enzymatic hydrolysis, wherein the enzymatic hydrolysis is carried out using any one of the following enzyme combinations:

[0011] i. comprising at least one serine endopeptidase from Bacillus, at least one serine endopeptidase from Aspergillus, and at least one trypsin-like protease

[0012] ii. comprising at least one serine endopeptidase from Bacillus, at least one serine endopeptidase from Aspergillus, and at least one leucine aminopeptidase from Aspergillus

[0013] iii. comprising at least one bacillolysin from Bacillus amyloliquefaciens, at least bromelain, and at least one leucine aminopeptidase from Aspergillus

[0014] c) stopping the enzymatic hydrolysis by inactivating the enzyme when the degree of hydrolysis (DH) is 15% or higher to obtain a whey protein hydrolysate;

[0015] Another aspect of the present invention relates to a whey protein hydrolysate comprising:

[0016] - free amino acids and peptides, and

[0017] - having a degree of hydrolysis of at least 15%, and

[0018] - peptides having a molecular weight of 2500 Da or higher, in an amount of 25% by weight or less of the total amount of peptides, and

[0019] - the amount of free amino acids is 15% by weight or less of the total amino acid content in the hydrolysis product, and the whey protein hydrolysate in a 4% w / w protein solution has a bitterness score corresponding to a caffeine solution of 0.08% w / v or less.

[0020] Another aspect of the present invention is to provide a food product comprising the whey protein hydrolysate according to the present invention.

[0021] Another aspect of the present invention is to provide a beverage comprising the whey protein hydrolysate according to the present invention, wherein the content of the whey protein hydrolysate in the beverage is equivalent to 2-25% by weight of the hydrolyzed whey protein.

[0022] Another aspect of the present invention is the use of the whey protein hydrolysate according to the present invention as a food ingredient. Description of the Drawings

[0023] Figure 1 A shows the nephelometric turbidity units (NTU) of Sample 16 (a whey protein hydrolysate according to the present invention prepared by enzymatic hydrolysis of WPI), Sample 14 (a whey protein hydrolysate according to the present invention prepared by enzymatic hydrolysis of whey protein concentrate (WPC)), and Sample 13 (a reference whey protein hydrolysate that was not prepared by using the enzyme combination of the present invention but was ultrafiltered from the whey protein hydrolysate) at different protein concentrations.

[0024] Figure 1 B shows the nephelometric turbidity units (NTU) of Sample 16 (a whey protein hydrolysate according to the present invention prepared by enzymatic hydrolysis of WPI) at different protein concentrations. Standard deviations are given.

[0025] Figure 1 C shows the nephelometric turbidity units (NTU) of Sample 14 (a whey protein hydrolysate according to the present invention prepared by enzymatic hydrolysis of WPC) at different protein concentrations. Standard deviations are given.

[0026] Figure 2 Samples of different protein concentrations of Samples 13, 14, and 16 are shown. The protein concentrations (w / w) from left to right are: 8%, 6.4%, 4.8%, 3.2%, and 1.8%. A) shows Sample 16, B) shows Sample 14, and C) shows Sample 13.

[0027] Figure 3 The bitterness scores of different concentrations of caffeine and the bitterness scores of Samples 13, 14, and 16 are shown.

[0028] Figure 4 A spider diagram showing the flavor and texture characteristics of Samples 13, 15, and 16 is shown. The significance level of the difference between the attributes with the highest and lowest scores is indicated by *** and is 99.9%, where P < 0.001 (ANOVA analysis).

[0029] Figure 5Shows the percentage of peptides of 7 - 19 amino acids that fall within the ranges of 7 - 10 amino acids and 11 - 19 amino acids when analyzed using size - exclusion chromatography (SEC) or LC - MS / MS (MS).

[0030] Figure 6 Shows peptides containing phenylalanine, presented as a percentage of all peptides from α - lactalbumin, β - lactoglobulin, and β - casein. The shortest peptide segments analyzed were 5 amino acids long.

[0031] Figure 7 Shows the percentage of the number of peptides of 5 - 19 amino acids from α - lactalbumin, β - lactoglobulin, and β - casein.

[0032] Figure 8 Shows beverage samples, from left to right: a beverage prepared without heat treatment, a beverage prepared by direct UHT treatment at 143 °C for 6 seconds, a beverage prepared by indirect UHT treatment for 6 seconds, and a beverage prepared by pasteurization at 90 °C for 6.5 minutes.

[0033] Figure 9 Shows an SDS - page gel picture used to determine the amount of undegraded BSA in different whey protein hydrolysate samples.

[0034] Figure 10 Shows the correlation between the carbonation and pH value of a beverage.

[0035] Figure 11 Shows the pH values of different samples that contain whey protein hydrolysate and are carbonated with 2.5 volumes of carbon dioxide per volume of composition

[0036] Figure 12 Shows the pH and turbidity during heating of a sample, which includes an 8% protein solution of sample 16 carbonated with 2.5 volumes of CO2 per volume of solution.

[0037] The present invention will now be described in more detail below. Detailed Description

[0038] Definitions

[0039] Before discussing the present invention in further detail, the following terms and conventions will first be defined:

[0040] Unless otherwise specified or clearly indicated to the contrary by the context of the reference, all references to singular features or limitations of the present invention shall include the corresponding plural features or limitations, and vice versa.

[0041] Unless otherwise specified, all percentages referred to herein are percentages by weight. Additionally, the terms "on a dry matter weight basis" and "on a dry matter basis" refer to the same concept and are used interchangeably.

[0042] For example, the term "w / w" in 1% w / w refers to a composition containing 1 weight % of a compound.

[0043] The term "palatable" means having a taste that is good enough to be eaten and / or drunk, i.e., having a taste acceptable or satisfactory to human consumers.

[0044] 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.

[0045] Whey protein solution:

[0046] In the context of the present invention, the term "solution" in "whey protein solution" includes compositions containing a combination of a liquid and solid compounds or semi-solid particles (such as protein particles). Thus, a "solution" can be a suspension or even 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 for the whey protein solution is usually water.

[0047] The whey protein solution generally contains protein in an amount of 2% by weight or more of the whey protein solution. In an embodiment of the present invention, the whey protein solution contains protein in the range of 2 - 20% by weight of the whey protein solution. Preferably, the whey protein solution contains protein in an amount of 5 - 15% by weight of the whey protein solution.

[0048] The whey protein solution for hydrolysis is obtained by dispersing a composition containing whey protein in a liquid (such as water). Preferably, the whey protein solution is prepared by mixing any one of whey protein concentrate, whey protein concentrate, whey protein isolate, and / or whey protein isolate with water. Thus, in an embodiment of the present invention, the whey protein solution contains whey protein concentrate, whey protein concentrate, whey protein isolate, and / or whey protein isolate.

[0049] The whey protein solution of the present invention contains whey protein in an amount of at least 50% by weight based on the total solids content. If the content of whey protein is less than 50% of the total solids content, the overall molecular composition (the ratio between protein, carbohydrate, lipid, and mineral) will be different, the behavior of the enzyme may be different, and thus the obtained product will also be different.

[0050] In addition to whey protein, the whey protein solution may also contain a small amount of other proteins, such as casein.

[0051] In addition to proteins, whey protein solutions typically also contain other components. The whey protein solution may contain other components that are normally present in whey or milk whey, such as minerals, carbohydrates, and / or lipids. Alternatively or additionally, the whey protein solution may contain components that are not natural to whey or milk whey. However, such non-natural milk components should be suitable and safe for use in food production.

[0052] Based on the total solids content, the lower the protein content in the whey protein solution, the higher the content of lipids, carbohydrates (mainly lactose), and other proteins compared to whey protein.

[0053] The whey protein solution may contain, for example, carbohydrates such as lactose, oligosaccharides, and / or hydrolysis products of lactose (i.e., glucose and galactose). The whey protein solution may contain, for example, carbohydrates in the range of 0 - 10% by weight based on the total solids content.

[0054] Whey protein isolate (WPI) and soy protein isolate (SPI) contain very small amounts of carbohydrates, such as lactose. Therefore, when preparing a whey protein solution using WPI or SPI, the carbohydrate content in the whey protein solution is in the range of 0 - 1% by weight based on the total solids content. If a whey protein concentrate (WPC) or soy protein concentrate (SPC) is used to prepare the whey protein solution, the amount of carbohydrates in the whey protein solution is preferably in the range of 2 - 8% by weight based on the total solids content.

[0055] The whey protein solution may also contain lipids, for example, in the form of triglycerides and / or other types of lipids (such as phospholipids).

[0056] In the context of the present invention, the terms "fat" and "lipid" have the same meaning and can be used interchangeably.

[0057] The whey protein solution according to the present invention contains whey protein in an amount of at least 50% based on the total solids content.

[0058] If the protein content in the whey protein solution is less than 50% of the total solids content, the whey protein hydrolysate obtained after hydrolysis may not have the characteristics of the whey protein hydrolysate of the present invention, i.e., no unpleasant bitterness in a 4% protein solution, a degree of hydrolysis above 15%, the amount of free amino acids being 15% by weight or less, and peptides with a molecular weight of 2500 Da or higher accounting for 25% by weight or less of the total amount of peptides.

[0059] Whey protein solutions with a whey protein content of less than 50% based on the total solids content will contain significant amounts of minerals, fats, and carbohydrates. It is not desirable to prepare whey protein hydrolysates with a whey protein content of less than 50% by weight of the solids content and with significant amounts of minerals, fats, 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 also applies to lipids and carbohydrates. Large amounts of lipids, minerals, and carbohydrates may also affect the taste and turbidity of the obtained whey protein hydrolysates.

[0060] Preferably, the whey protein solution contains at least 60% by weight of whey protein based on the total solids content, such as at least 70% by weight based on the total solids content, and even more preferably at least 80% by weight based on the total solids content. In a more preferred embodiment of the present invention, the whey protein solution contains at least 85% by weight of whey protein based on the total solids content, and most preferably, the whey protein solution contains at least 90% by weight of whey protein based on the total solids content.

[0061] The proteins present in the whey protein solution should be mainly whey proteins. However, small amounts of other proteins, such as casein, may be present. Thus, in an embodiment of the present invention, the whey protein solution contains 90% by weight or more of whey protein based on the total amount of proteins. Preferably, the whey protein solution contains 95% by weight or more of whey protein based on the total amount of proteins. Thus, in other embodiments of the present invention, the whey protein solution contains at most 10% by weight of casein or other non-whey proteins based on the total amount of proteins, preferably at most 5% by weight, and more preferably at most 3% by weight of casein or other non-whey proteins.

[0062] If the fat content in the whey protein solution is high, it will affect the clarity and taste of the resulting protein hydrolysate. Thus, in an embodiment of the present invention, the whey protein solution contains at most 10% by weight of lipids based on the total solids content, such as at most 8% by weight based on the total solids content, and even more preferably, the whey protein solution contains at most 6% by weight of lipids based on the total solids content.

[0063] If whey protein concentrate is used to prepare the whey protein solution, the lipid / fat content is approximately 6 - 8% by weight of the total solids content. In contrast, if whey protein isolate is used to prepare the whey protein solution, the whey protein solution is substantially free of fat.

[0064] In a preferred embodiment of the present invention, the whey protein solution is substantially free of fat. The term "substantially free of fat" means that the lipid content in the whey protein solution is less than 1% by weight based on the total solids content, preferably less than 0.5% by weight based on the total solids content, and even more preferably less than 0.1% by weight.

[0065] If the fat content in the whey protein solution is low, such as at most 0.5% based on the total solids content, the whey protein hydrolysate prepared according to the method of the present invention has a clear appearance. Preferably, the lipid content in the whey protein solution is less than 0.3% by weight of the total solids content, and more preferably less than 0.2% by weight of the lipid based on the total solids content.

[0066] In a preferred embodiment of the present invention, the whey protein hydrolysate is obtained from a whey protein solution using whey protein isolate and / or milk protein isolate. When using whey protein isolate and / or milk protein isolate for hydrolysis, the fat content will be very low (less than 0.5%). This makes the prepared whey protein hydrolysate not only have a high degree of hydrolysis (DH>15%) and good taste, but also have a clear appearance without any ultrafiltration step. Preferably, the whey protein solution is whey protein isolate or milk protein isolate mixed in water.

[0067] Whey protein:

[0068] In one aspect of the present invention, the whey protein hydrolysate is obtained by hydrolyzing a solution containing whey protein.

[0069] In the context of the present invention, the term "whey protein" refers to the proteins present in whey or milk serum. The whey protein in the whey protein solution can be a subset of the protein species found in whey or milk serum, or it can be the complete set of protein species found in whey and / or milk serum. Whey protein is a mixture of globular proteins isolated from whey, which is a liquid substance produced as a by-product of cheese production. Whey protein is the protein present in the whey phase of cow's milk or curdled milk. Proteins other than whey protein in the cow's milk whey phase are sometimes also referred to as cow's milk whey proteins.

[0070] The term "milk serum" refers to the liquid remaining after removing casein and milk fat globules from milk, for example, by microfiltration or macroporous ultrafiltration. Milk serum can also be referred to as "ideal whey".

[0071] The term "milk protein" or "serum protein" refers to the proteins present in milk serum.

[0072] The term "whey" refers to the liquid supernatant remaining after the precipitation and removal of casein in milk. The casein precipitation can be accomplished, for example, by acidification of milk and / or by using rennet.

[0073] There are various types of whey, such as sweet whey, acid whey, and casein whey.

[0074] The whey protein present in the whey protein solution of the present invention can be derived from whey of different sources, such as casein whey, acid whey, or sweet whey.

[0075] In a preferred embodiment of the present invention, the whey protein in the whey protein solution is derived from sweet whey. Sweet whey mainly 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 milk fat globule membrane proteins. CMP is not present in casein whey or acid whey. In an embodiment of the present invention, the whey protein in the whey protein solution is derived from sweet whey from which CMP has been completely or partially removed. This may be referred to as modified sweet whey. Removing CMP from sweet whey results in a protein material with threonine and tryptophan contents approaching those of human milk.

[0076] 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 refer to BLG from mammalian species. Additionally, the term "α-lactalbumin" may be referred to as "ALA" in the context of the present invention and refers to α-lactalbumin from mammalian species.

[0077] As used herein, the term "sweet whey" refers to the liquid remaining after milk is coagulated and filtered during the manufacture of curd-type cheese. "Sweet whey" is obtained during the production of curd-type hard cheeses such as cheddar or Swiss cheese. Sweet whey is obtained by adding rennet to a milk composition, which cleaves κ-casein into para-κ-casein and peptide caseinomacropeptide (CMP), thereby destabilizing the casein micelles and causing the casein to precipitate. The liquid surrounding the rennet-precipitated casein is called sweet whey. The pH value of sweet whey can be 5.2 - 6.7.

[0078] Sweet whey is a product from cheese production that contains approximately 10 - 15% by weight of protein and approximately 75 - 80% lactose. The proteins in sweet whey are mainly whey proteins, but small amounts of casein may also be present. Whey proteins include β-lactoglobulin (about 55 - 65%), α-lactalbumin (about 18 - 25%), bovine serum albumin, immunoglobulins, caseinomacropeptide (CMP), osteopontin, lactoferrin, and milk fat globule membrane proteins.

[0079] The term "casein whey" (sometimes also referred to as acid whey or acidic whey) refers to the whey obtained from the production of casein / caseinates. In the context of the present invention, casein whey is different from acid whey. Casein whey is the whey fraction obtained after separating casein / caseinates by microfiltration. Casein whey does not contain CMP.

[0080] The term acid whey is used for the whey obtained during the production of acid type cheeses such as cottage cheese and quark. In the preparation of acid type cheeses, casein is removed from milk by acid precipitation, i.e., the pH of the milk is lowered to below pH 4.6, which is the isoelectric point of casein and causes the decomposition and precipitation of casein micelles. The pH is usually lowered to the range of 3.8 - 4.6. The liquid surrounding the acid precipitated casein is usually called acid whey and does not contain CMP.

[0081] In an embodiment of the present invention, the whey protein used in the whey protein solution is not acid whey or casein whey.

[0082] The whey protein used in the whey protein solution of the present invention can be whey protein concentrate (WPC), serum protein concentrate (SPC), whey protein isolate (WPI) or serum protein isolate (SPI). The difference between whey protein concentrate and whey protein isolate is the composition of the product, especially the protein content. Whey protein isolate is purer than the concentrate and other non - protein components have been partially removed to "isolate" the whey protein. Therefore, whey protein isolate has a higher percentage of protein and can be pure enough to be almost lactose - free, carbohydrate - free, fat - free and cholesterol - free.

[0083] In this context, the terms "whey protein concentrate (WPC)" and "serum protein concentrate (SPC)" include dry and liquid compositions of whey protein. The protein content in the WPC and SPC used in the present invention is not less than 50% by weight based on the total solids content. However, whey protein concentrate can contain a higher content of whey protein, for example, 80% by weight of whey protein based on the dry matter content. The dry part of the liquid whey is obtained by removing sufficient non - protein components from the whey such that the dry product contains not less than 50% by weight of whey protein.

[0084] The WPC or SPC used in the present invention generally comprises:

[0085] 50 - 89% by weight of protein relative to the total solids content

[0086] 15 - 80% by weight of BLG relative to the total protein content

[0087] 8 - 50% by weight of ALA relative to the total protein content

[0088] 0 - 25% by weight of CMP relative to the total protein content.

[0089] Alternatively, but also preferably, the WPC or SPC comprises:

[0090] 50 - 89% by weight of protein relative to the total solids content

[0091] BLG at 15 - 80% by weight relative to the total protein content

[0092] ALA at 4 - 50% by weight relative to the total protein content

[0093] CMP at 0 - 25% by weight relative to the total protein content.

[0094] Preferably, WPC or SPC comprises:

[0095] Protein at 50 - 89% by weight relative to the total solids content

[0096] BLG at 15 - 80% by weight relative to the total protein content

[0097] ALA at 4 - 50% by weight relative to the total protein content

[0098] CMP at 0 - 25% by weight relative to the total protein content.

[0099] More preferably, WPC or SPC comprises:

[0100] Protein at 70 - 89% by weight relative to the total solids content

[0101] BLG at 30 - 80% by weight relative to the total protein content

[0102] ALA at 4 - 35% by weight relative to the total protein content

[0103] CMP at 0 - 25% by weight relative to the total protein content.

[0104] The terms "whey protein isolate" and "serum protein isolate" refer to dry or liquid compositions which are generally considered to be substantially free of lactose and cholesterol and have a whey protein content of at least 90% by weight based on the total solids content. A whey protein isolate may contain, for example, 92% by weight or more of whey protein based on the total solids content. Preferably, WPI and SPI contain 90 - 100% by weight of protein based on the total solids content, such as 92 - 99% by weight of protein based on the total solids content.

[0105] WPI or SPI may preferably comprise:

[0106] Protein at 90 - 100% by weight relative to the total solids content

[0107] BLG at 15 - 80% by weight relative to the total protein content

[0108] ALA at 8 - 50% by weight relative to the total protein content

[0109] CMP at 0 - 25 wt% relative to the total protein content.

[0110] Alternatively, and preferably, WPI or SPI may comprise:

[0111] Protein at 90 - 100 wt% relative to the total solids content

[0112] BLG at 30 - 80 wt% relative to the total protein content

[0113] ALA at 4 - 35 wt% relative to the total protein content

[0114] CMP at 0 - 25 wt% relative to the total protein content.

[0115] Preferably, WPI may preferably comprise:

[0116] Protein at 90 - 100 wt% relative to the total solids content

[0117] BLG at 60 - 80 wt% relative to the total protein content

[0118] ALA at 10 - 20 wt% relative to the total protein content

[0119] CMP at 10 - 20 wt% relative to the total protein content.

[0120] In an embodiment of the present invention, the whey protein solution for preparing the whey protein hydrolysate according to the present invention comprises a total amount of whey protein of 50 - 95 wt% of the dry matter, such as 70 - 97 wt%, preferably 72 - 95 wt%, and even more preferably 75 - 95 wt% of the dry matter.

[0121] 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 milk from cows, sheep, goats, buffalo, camels, llamas, mares, horses, and / or deer. In some preferred embodiments of the present invention, the whey protein is from bovine (cow) milk.

[0122] Preferably, the whey protein solution is a demineralized whey protein solution. Preferably, the whey protein solution has a low mineral content because, from a nutritional and health perspective, a low concentration of minerals such as sodium, calcium, potassium, magnesium, and phosphate is preferred in protein hydrolysates. In addition, minerals including sodium and calcium may interact with whey proteins and affect the product turbidity, aggregation behavior, and heat resistance of whey protein hydrolysates. At high concentrations, some minerals, including especially sodium, calcium, and zinc, may inhibit or promote the proteolytic activity of some proteases, and thus the presence of high concentrations of these ions may alter the cooperative cleavage pattern of proteases. Therefore, in an embodiment of the present invention, the mineral content in the whey protein solution is 10% or less, more preferably 8% or less, based on the total solids content. In the context of the present invention, the term "mineral" refers to the ash content. The terms "mineral" and "ash" can be used interchangeably and refer to the same thing. Therefore, with respect to the mineral content of the whey protein solution, it should be understood as the ash content of the whey protein solution.

[0123] In an embodiment of the present invention, the whey protein solution contains 30% by weight or more of BLG, such as 40% by weight or more of BLG, based on the total protein content. More preferably, the whey protein solution contains 50% by weight or more of BLG, based on the total protein content, and even more preferably, the whey protein solution contains 55% by weight or more of BLG, based on the total protein content. In another embodiment of the present invention, the whey protein solution contains 30 - 95% by weight of BLG, 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.

[0124] In the context of the present invention, the term "whey" refers to the liquid composition remaining when casein is removed from milk. Casein can be removed, for example, by microfiltration to provide a liquid permeate that is free or substantially free of micellar casein but contains native whey proteins. This liquid permeate is sometimes referred to as ideal whey, whey, or milk whey.

[0125] The proteins in the whey protein solution are preferably as close as possible to their native state and are preferably only subjected to mild heat treatment, if any.

[0126] Enzymatic hydrolysis:

[0127] Step b) in the method according to the present invention involves subjecting the whey protein solution to enzymatic hydrolysis, wherein the enzymatic hydrolysis is carried out using any one of the following enzyme combinations:

[0128] i. An enzyme combination comprising at least one serine endopeptidase from Bacillus, at least one serine endopeptidase from Aspergillus, and at least one trypsin-like protease

[0129] ii. An enzyme combination comprising at least one serine endopeptidase from Bacillus, at least one serine endopeptidase from Aspergillus, and at least one leucine aminopeptidase from Aspergillus

[0130] iii. An enzyme combination comprising at least one subtilisin from Bacillus amyloliquefaciens, at least one leucine aminopeptidase from Aspergillus, and at least bromelain.

[0131] The inventors of the present invention surprisingly found that by subjecting a whey protein solution to enzymatic hydrolysis by adding any one of the above three enzyme combinations, a whey protein hydrolysate with the following characteristics will be produced: a degree of hydrolysis higher than 15%, a free amino acid content of 15% or lower, and wherein the whey protein hydrolysate has an acceptable taste and a low content of bitter peptides. The inventors have found that the whey protein hydrolysates prepared by hydrolysis with the above enzyme combinations i to iii are not bitter in a 4% w / w protein solution.

[0132] In step c) of the present invention, when the degree of hydrolysis (DH) is 15% or higher, the enzymatic hydrolysis is stopped by inactivating the enzyme to obtain a whey protein hydrolysate. The degree of hydrolysis (DH) is defined as the percentage of peptide bonds in the original protein that have been cleaved by hydrolysis.

[0133] In an embodiment of the present invention, the serine endopeptidase from Aspergillus is a serine endopeptidase from Aspergillus oryzae and / or Aspergillus flavus. The serine endopeptidase from Aspergillus is preferably a subtilisin-like serine endopeptidase (EC 3.4.21).

[0134] In an embodiment of the present invention, enzyme combination i) further comprises a metalloendopeptidase from the genus Bacillus, such as subtilisin. Thus, in one embodiment, enzyme combination i) includes:

[0135] - At least one serine endopeptidase from Bacillus, at least one serine endopeptidase from Aspergillus, and at least one trypsin-like protease

[0136] In other embodiments of the present invention, enzyme combination ii) may comprise a metalloendopeptidase from the genus Bacillus, such as subtilisin. Thus, in one embodiment, enzyme combination ii) includes:

[0137] - At least one serine endopeptidase from Bacillus, at least one serine endopeptidase from Aspergillus, and at least one leucine aminopeptidase from Aspergillus

[0138] In an embodiment of the present invention, the leucine aminopeptidase from Aspergillus is from Aspergillus oryzae.

[0139] In one embodiment, the serine endopeptidase from Bacillus is subtilisin. Subtilisin is preferably from Bacillus licheniformis.

[0140] In another embodiment of the present invention, the metalloendopeptidase is bacillopeptidase. Bacillopeptidase is preferably from Bacillus, more preferably from Bacillus amyloliquefaciens. In a preferred embodiment of the present invention, bacillopeptidase is not from Bacillus subtilis.

[0141] In the context of the present invention, the term "trypsin-like protease" is a protease of microbial origin. Preferably, the trypsin-like protease of microbial origin is from a species of Fusarium, particularly Fusarium oxysporum. Thus, for example, the term "trypsin-like protease" does not include pancreatic enzymes of non-microbial origin. Instead, pancreatic enzymes are a mixture of enzymes derived from the pancreas, which include, for example, trypsin, chymotrypsin, amylase, and lipase. In addition, the term "trypsin-like protease" should not be confused with "trypsin".

[0142] In another embodiment, bromelain is from pineapple (Ananas comosus). Bromelain is a cysteine endopeptidase.

[0143] In an embodiment of the present invention, the enzymatic hydrolysis in step b) is carried out using any one of the following enzyme combinations:

[0144] i) An enzyme combination comprising at least one serine endopeptidase from Bacillus, at least one serine endopeptidase from Aspergillus, and at least one trypsin-like protease,

[0145] ii) An enzyme combination comprising at least one serine endopeptidase from Bacillus, at least one serine endopeptidase from Aspergillus, and at least one leucine aminopeptidase from Aspergillus,

[0146] iii) An enzyme combination comprising a combination of at least one serine endopeptidase from Bacillus and a metalloendopeptidase, at least one serine endopeptidase from Aspergillus, and at least one leucine aminopeptidase from Aspergillus,

[0147] iv) An enzyme combination comprising at least one bacillopeptidase from Bacillus amyloliquefaciens, at least bromelain, and at least one leucine aminopeptidase from Aspergillus.

[0148] In a preferred embodiment of the present invention, the preferred enzymatic hydrolysis in step b) is carried out using any one of the following enzyme combinations:

[0149] i) An enzyme combination comprising at least one serine endopeptidase from a Bacillus species, at least one serine endopeptidase from Aspergillus oryzae, and at least one trypsin-like protease of microbial origin,

[0150] ii) An enzyme combination comprising at least one subtilisin from a Bacillus species, at least one serine endopeptidase from Aspergillus oryzae, and at least one leucine aminopeptidase from Aspergillus oryzae,

[0151] iii) An enzyme combination comprising a combination of at least one bacillopeptidase and subtilisin from a Bacillus species, at least one serine endopeptidase from Aspergillus oryzae, and at least one leucine aminopeptidase from Aspergillus oryzae,

[0152] iv) An enzyme combination comprising at least one bacillopeptidase from Bacillus amyloliquefaciens, at least one bromelain from pineapple, and at least one leucine aminopeptidase from Aspergillus oryzae.

[0153] In a preferred embodiment of the present invention, the enzyme combination:

[0154] i) An enzyme combination comprising at least one enzyme from the EC 3.4.21.62 group, at least one other enzyme from the EC 3.4.21 group, and at least one other enzyme from the EC 3.4.21.4 group,

[0155] ii) An enzyme combination comprising at least one enzyme from the EC 3.4.21.62 group, at least one other enzyme from the EC 3.4.21 group, and at least one other enzyme from the EC 3.4.11 group,

[0156] iii) An enzyme combination comprising at least one enzyme from the EC 3.4.21.62 group, one other enzyme from the EC 3.4.24.28 group, at least one other enzyme from the EC 3.4.21 group, and at least one other enzyme from the EC 3.4.11 group,

[0157] iv) An enzyme combination comprising at least one enzyme from the EC 3.4.24.28 group, at least one other enzyme from the EC 3.4.22.32 group, and at least one other enzyme from the EC 3.4.11 group.

[0158] In another preferred embodiment of the present invention, the enzyme combination:

[0159] i) Comprises at least one serine endopeptidase from Bacillus licheniformis, at least one serine endopeptidase from Aspergillus oryzae, and at least one trypsin-like protease from Fusarium oxysporum, optionally also bacillopeptidase from Bacillus amyloliquefaciens,

[0160] ii) comprising at least one serine endopeptidase from Bacillus licheniformis, at least one serine endopeptidase from Aspergillus oryzae, and at least one leucine aminopeptidase from Aspergillus oryzae,

[0161] iii) comprising at least one serine endopeptidase from Bacillus licheniformis, subtilisin from Bacillus amyloliquefaciens, at least one serine endopeptidase from Aspergillus oryzae, and at least one leucine aminopeptidase from Aspergillus oryzae,

[0162] iv) comprising at least one subtilisin from Bacillus amyloliquefaciens, bromelain from pineapple, and at least one leucine aminopeptidase from Aspergillus oryzae.

[0163] The serine endopeptidase from Bacillus licheniformis is preferably subtilisin.

[0164] In another preferred embodiment of the present invention, the enzyme combination:

[0165] i) comprises at least one serine endopeptidase from Bacillus (EC 3.4.21.62), at least one serine endopeptidase from Aspergillus (EC 3.4.21), and at least one trypsin-like protease (EC 3.4.21.4),

[0166] ii) comprises at least one serine endopeptidase from Bacillus (EC 3.4.21.62), at least one serine endopeptidase from Aspergillus (EC 3.4.21), and at least one leucine aminopeptidase from Aspergillus (EC 3.4.11),

[0167] iii) comprises at least one serine endopeptidase from Bacillus (EC 3.4.21.62), subtilisin (EC 3.4.24.28), at least one serine endopeptidase from Aspergillus (EC 3.4.21.63), and at least one leucine aminopeptidase from Aspergillus (EC 3.4.11),

[0168] iv) comprises at least one subtilisin from Bacillus amyloliquefaciens (EC 3.4.24.28), at least bromelain [EC 3.4.22.32), and at least one leucine aminopeptidase from Aspergillus (EC 3.4.11).

[0169] The enzyme combinations i) to iv) used in the present method for preparing whey protein hydrolysates may contain other enzymes in addition to the mentioned main enzymes. The term "main enzyme" refers to the enzyme with the highest content in the preparation. In the following list, Uniprot accession numbers are given in parentheses to identify the proteases with specific nomenclature annotated. For example, the enzyme combination may contain one or more enzymes having a high sequence identity (95 - 100%) with enzymes selected from the group consisting of: peptidase (A0A364MDR7), subtilisin family protein (I8A6W5), fungalysin metallopeptidase M36 (A0A2P2H013), neutral protease 2 (A0A364MH70), aspergillopepsin-1 (B8NLY9), leucine aminopeptidase A (Q2U1F3), leucine aminopeptidase 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).

[0170] In one aspect of the present invention, the main enzymes of the enzyme combination i) are a serine endopeptidase from Bacillus, a serine endopeptidase from Aspergillus, and a trypsin-like protease. The serine endopeptidase from Bacillus is preferably subtilisin, and the serine endopeptidase from Aspergillus is preferably a subtilisin family protein. In an embodiment of the present invention, the enzyme combination i) may further contain one or more of the following: aminopeptidases (such as peptidase and leucine aminopeptidase), metalloendopeptidases (such as bacillopeptidase and fungalysin metallopeptidase, neutral protease), prolyl oligopeptidase family protein, and aspergillopepsin-1 (aspartic endopeptidase). It is expected that at least 80% of the enzymes present in the enzyme combination i) are a serine endopeptidase from Bacillus, a serine endopeptidase from Aspergillus, and a trypsin-like protease. An example of a preparation containing a serine endopeptidase from Bacillus is Protamex (Novozymes A / S). Protamex also contains bacillopeptidase. Examples of preparations containing a serine endopeptidase from Aspergillus are Promod 782 (Biocatalysts Ltd) and Protease A Amano 2SD (Amano Enzyme Ltd), where the main enzyme is a serine endopeptidase from Aspergillus oryzae. Promod 782 and Protease A Amano 2SD also include the enzymes peptidase, leucine aminopeptidase, fungalysin metallopeptidase M36, prolyl oligopeptidase family protein, neutral protease 2, and aspergillopepsin-1, and the main enzyme is a serine endopeptidase. The trypsin-like protease can be provided, for example, by Formea TL 1200BG (Novozymes A / S).

[0171] In one aspect of the present invention, the enzyme combination ii) comprises a serine endopeptidase from Bacillus, a serine endopeptidase from Aspergillus, and a leucine aminopeptidase from Aspergillus as the main enzymes. The serine endopeptidase from Bacillus is preferably subtilisin, and the serine endopeptidase from Aspergillus is preferably a subtilisin family protein and an alkaline protease. The leucine aminopeptidase from Aspergillus can be, for example, one or more of peptidase, leucine aminopeptidase A, and leucine aminopeptidase 2. In an embodiment of the present invention, the enzyme combination ii) may further comprise a metalloendopeptidase; one or more of fungalysin metallopeptidase M36, neutral protease 1, and neutral protease 2. The enzyme combination ii) may further comprise aspergillopepsin-1 (aspartic endopeptidase), dipeptidyl peptidase 4, and dipeptidyl peptidase 5. It is expected that at least 80% of the enzymes present in the enzyme combination i) are a serine endopeptidase from Bacillus, a serine endopeptidase from Aspergillus, and a leucine aminopeptidase from Aspergillus. Examples of preparations containing a serine endopeptidase from Bacillus are Alcalase (Novozymes A / S), which contains subtilisin. Examples of preparations containing a serine endopeptidase from Aspergillus are Protease A Amano 2SD and Promod 782, where the main enzyme is a serine endopeptidase from Aspergillus oryzae. Promod 782 and Protease A Amano 2SD also include the enzymes peptidase, leucine aminopeptidase, fungalysin metallopeptidase M36, prolyl oligopeptidase family protein, neutral protease 2, aspergillopepsin-1, and the main enzyme is a serine endopeptidase. Examples of preparations containing a leucine aminopeptidase from Aspergillus are Flavourzyme Conc BG (Novozymes A / S), where the main enzyme is leucine aminopeptidase. Flavourzyme Conc BG also contains leucine aminopeptidase A, leucine aminopeptidase 2, dipeptidyl peptidase 4, dipeptidyl peptidase 5, neutral protease 1, neutral protease 2, alkaline protease 1, and the main enzyme is leucine aminopeptidase.

[0172] In one aspect of the present invention, the enzyme combination (iii) comprises a serine endopeptidase from Bacillus, a metalloendopeptidase from Bacillus, a serine endopeptidase from Aspergillus, and a leucine aminopeptidase from Aspergillus as the main enzymes. The serine endopeptidase from Bacillus is preferably subtilisin, the metalloendopeptidase from Bacillus is preferably bacillopeptidase, and the serine endopeptidase from Aspergillus is preferably a subtilase family protein and an alkaline protease. The leucine aminopeptidase from Aspergillus can be, for example, one or more of peptidase, leucine aminopeptidase A, and leucine aminopeptidase 2. In an embodiment of the present invention, the enzyme combination (iii) may further comprise a metalloendopeptidase; one or more of fungalysin metallopeptidase M36, neutral protease 1, and neutral protease 2. The enzyme combination (ii) may further comprise aspergillopeptidase-1 (aspartic endopeptidase), dipeptidyl peptidase 4, and dipeptidyl peptidase 5. At least 80% of the enzymes present in the enzyme combination (iii) are a serine endopeptidase from Bacillus, bacillopeptidase, a serine endopeptidase from Aspergillus, and a leucine aminopeptidase from Aspergillus. Examples of preparations containing both a serine endopeptidase (subtilisin) from Bacillus and bacillopeptidase are Promod 950L (Biocatalysts Ltd) and Protamex. Examples of preparations containing a serine endopeptidase from Aspergillus are Protease A Amano 2SD and Promod 782. Promod 782 and Protease A Amano 2SD also include the enzymes peptidase, leucine aminopeptidase, fungalysin metallopeptidase M36, prolyl oligopeptidase family protein, neutral protease 2, and aspergillopeptidase-1, and the main enzyme is a serine endopeptidase. An example of a preparation containing a leucine aminopeptidase from Aspergillus is Flavourzyme conc BG, where the main enzyme is leucine aminopeptidase. Flavourzyme Conc BG also contains the following enzymes: leucine aminopeptidase A, leucine aminopeptidase 2, dipeptidyl peptidase 4, dipeptidyl peptidase 5, neutral protease 1, neutral protease 2, and alkaline protease 1, and where the main enzyme is leucine aminopeptidase.

[0173] In one aspect of the present invention, the enzyme combination iv) comprises subtilisin from Bacillus amyloliquefaciens, bromelain and leucine aminopeptidase from Aspergillus as the main enzymes. The leucine aminopeptidase from Aspergillus can be, for example, one or more of peptidase, leucine aminopeptidase A and leucine aminopeptidase 2. In an embodiment of the present invention, the enzyme combination iv) may further comprise a metalloendopeptidase; one or more of fungalysin metallopeptidase M36, neutral protease 1 and neutral protease 2. The enzyme combination ii) may further comprise dipeptidyl peptidase 4, dipeptidyl peptidase 5 and an alkaline protease (a serine protease from Aspergillus). It is expected that at least 80% of the enzymes present in the enzyme combination iv) are subtilisin from Bacillus amyloliquefaciens, bromelain and leucine aminopeptidase from Aspergillus. An example of a preparation containing subtilisin from Bacillus amyloliquefaciens is Neutrase. An example of bromelain is Promod 523MDP, while an example of leucine aminopeptidase from Aspergillus is Flavourzyme conc BG.

[0174] The method for preparing a whey protein hydrolysate of the present invention should not be limited to the amount of enzyme added in the hydrolysis step, as the amount of enzyme added depends on the type of enzyme and the activity of the enzyme. However, as a guide, the enzymatic hydrolysis is carried out with a combination of enzymes, wherein the total amount of enzyme is 0.05 - 10 g enzyme / 100 g protein, for example 0.1 - 7.5 g enzyme / 100 g protein. Preferably, the amount of enzyme is in the range of 0.2 - 5.0 g / 100 g protein.

[0175] The ratio between the three different enzymes in combinations i to iii can be, for example, in the range of 1 - 10:1 - 10:1 - 10, for example in the range of 1 - 8:1 - 8:1 - 8. However, the present invention should not be limited to the amount of enzyme added, as this will depend on the activity of the enzyme used.

[0176] 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 - 75°C, for example 40°C - 70°C. The enzymatic hydrolysis should be carried out at the 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 - 65°C.

[0177] The period of enzyme hydrolysis before the hydrolysis is stopped in step c) depends on the amount and activity of the enzyme used. The hydrolysis continues until the degree of hydrolysis is 15% or higher. The enzymatic hydrolysis in step b) is preferably carried out for a period in the range of 3 hours - 20 hours, for example 3.5 hours - 15 hours, preferably 4 hours - 10 hours, and even more preferably 4 hours - 7 hours.

[0178] The whey protein solution should preferably have a pH in the range of 6 - 9 during the enzymatic hydrolysis. In a preferred embodiment, the pH during the enzymatic hydrolysis in step b) is 6.5 - 8.0.

[0179] Within this pH range, the enzyme has the highest activity, and thus can most effectively cleave proteins into peptides and free amino acids. In addition, aggregation is avoided during hydrolysis and during the heat treatment for inactivating the enzyme within this pH range.

[0180] In step c) of the method for preparing a whey protein hydrolyzate according to the present 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 does not become active under other conditions.

[0181] The hydrolysis is stopped when the degree of hydrolysis is at least 15%, such as at least 18%, preferably at least 20%. In an embodiment of the present invention, the hydrolysis is stopped in step c) when the degree of hydrolysis is in the range of 15% - 35%, preferably 17% - 30%, even more preferably 18% - 28%.

[0182] The inactivation of the enzyme and thus the stopped hydrolysis in step c) can be carried out by any method known in the art. For example, the enzyme is inactivated by adjusting the temperature to the temperature at which the enzyme is inactivated and denatured. The inactivation and denaturation of the enzyme can also be achieved by adjusting the pH value of the solution to the pH value at which the enzyme is inactivated.

[0183] Therefore, in an embodiment of the present invention, the inactivation of the enzyme in step c) is carried out by heating the whey protein solution added with the enzyme to a temperature of at least 80°C. The inactivation of the enzyme is preferably carried out by heating to a temperature of 80°C - 130°C, such as 85°C - 125°C, even more preferably 90°C - 120°C. The inactivation of the enzyme by heating in step c) can be carried out, for example, by heating to a high temperature for a short period of time, such as heating to a temperature of 110°C - 130°C for 10 - 30 seconds. Alternatively, the inactivation of the enzyme in step c) can be carried out by heating to a relatively low temperature but for a longer period of time. This may involve heating to 80°C - 90°C for 5 - 10 minutes.

[0184] In another embodiment of the present invention, the irreversible inactivation of the enzyme in step c) includes increasing or decreasing the pH value of the whey protein solution added with the enzyme (i.e., the whey protein hydrolyzate) to the pH value at which the enzyme is inactivated. In an embodiment of the present invention, the pH value is increased to a pH value of 10 or higher. In another embodiment, the pH value is decreased to a pH value of 4 or lower.

[0185] In a preferred embodiment of the present invention, the method does not include any step of ultrafiltration of the whey protein hydrolysate obtained in step c). To the surprise of the inventors of the present invention, enzymatic hydrolysis of a whey protein solution having a small amount of lipid (i.e., WPI or SPI) using the specified enzyme combination results in a whey protein hydrolysate having a palatable taste and a clear appearance without involving any ultrafiltration step.

[0186] Whey protein hydrolysates known in the art can be classified into ultrafiltered and non-ultrafiltered hydrolysates. Known non-ultrafiltered protein hydrolysates will have an unclear or turbid appearance, while ultrafiltered protein hydrolysates are generally clear in appearance.

[0187] In the context of the present invention, the term "ultrafiltration" refers to membrane filtration using a membrane having a cut-off value in the range of 1500 Da - 50000 Da, preferably 2000 Da - 20000 Da.

[0188] During the ultrafiltration of protein hydrolysates, fats, intact proteins, and some larger peptides are retained by the ultrafiltration membrane and remain in the retentate, while free amino acids, smaller peptides, and minerals are in the ultrafiltration permeate.

[0189] In an embodiment of the present invention, the whey protein solution is prepared as a WPI and SPI solution having a low lipid content. To the surprise of the inventors of the present invention, preparing a whey protein hydrolysate with the enzyme combination of the present invention results in a whey protein hydrolysate having a high degree of hydrolysis, a good taste with low bitterness, and a clear appearance.

[0190] However, a low lipid content is not the only explanation for 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 using any one of the above enzyme combinations for enzymatic hydrolysis of a whey protein solution with a low lipid content, a whey protein hydrolysate with a degree of hydrolysis above 15%, no bitterness in a 4% protein solution, and a clear appearance can be prepared. For a clear appearance, a whey protein solution with a low fat content must be used. However, a low lipid content is not the only reason for obtaining a clear hydrolysate. The inventors of the present invention surprisingly found that hydrolysis using a specific enzyme combination produces a clear hydrolysis product. In a comparative experiment, it was observed that other whey protein hydrolysates with a low lipid content but hydrolyzed with enzymes other than those used in the method of the present invention did not produce whey protein hydrolysates with a clear appearance and low bitterness.

[0191] The whey protein hydrolysate obtained by the method of the present invention can preferably be concentrated and / or dried. Thus, in an embodiment of the present invention, the method includes step d) of concentrating and / or drying the whey protein hydrolysate obtained in step c). Concentration can be carried out, for example, by one or more of the unit operations of nanofiltration, reverse osmosis filtration, and evaporation.

[0192] In another embodiment of the present invention, the drying step comprises one or more of the unit operations spray drying, freeze drying and rotary flash drying, and rotary drying and / or fluidized bed drying may also be used.

[0193] Whey protein hydrolysate

[0194] In one aspect, the present invention relates to a whey protein hydrolysate comprising:

[0195] - free amino acids and peptides, and

[0196] - having a degree of hydrolysis of at least 15%, and

[0197] - peptides having a molecular weight of 2500 Da or higher, in an amount of 25% by weight or less of the total amount of peptides, and

[0198] - the amount of free amino acids is 15% by weight or less of the total amino acid content in the hydrolysate, and the whey protein hydrolysate in a 4% protein solution has a bitterness score corresponding to a solution of 0.08% w / v or less of caffeine.

[0199] In one aspect of the present invention, the degree of hydrolysis of the whey protein hydrolysate of the present invention is at least 15%. An object of the present invention is to prepare a highly hydrolyzed whey protein hydrolysate that has no unpleasant bitterness without any ultrafiltration step. It is well known that peptides are the cause of the bitterness of many hydrolysates. Generally, deep hydrolysis to provide a hydrolysate with a high degree of hydrolysis is expected to result in a bitter hydrolysate. To reduce the bitterness of a deeply hydrolyzed protein hydrolysate, the hydrolysate can be treated with activated carbon, which can be removed again together with the bitter peptides. Deeply hydrolyzed proteins may be desirable because these peptides have other functions in addition to intact proteins. For example, the peptides may be more heat-tolerant than intact whey protein.

[0200] However, the inventors of the present invention have surprisingly found that a method for preparing a whey protein hydrolysate with a high degree of hydrolysis has no unpleasant bitterness even if the whey protein hydrolysate has not been subjected to any additional treatment to remove bitter peptides.

[0201] In a preferred embodiment of the present invention, the degree of hydrolysis of the whey protein hydrolysate is at least 18%, and even more preferably the degree of hydrolysis of the whey protein hydrolysate is at least 20%.

[0202] In another embodiment of the present invention, the whey protein hydrolysate according to the present invention has a degree of hydrolysis of 15 - 35%, such as 18 - 30%, preferably 18 - 28%, and even more preferably 20 - 25%.

[0203] The whey protein hydrolysate may contain free amino acids, and if present, they are present in an amount of 15% by weight or less of the total amino acid content in the hydrolysate. Preferably, the free amino acid content is 12% by weight or less of the total amino acid content. 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 in peptides and proteins.

[0204] In some embodiments of the present invention, the content of free amino acids in the whey protein hydrolysate does not exceed 15% by weight of the total amino acid content, such as does not exceed 13% by weight of the total amino acid content, preferably does not exceed 10% by weight of the total amino acid content, and even more preferably, the content of free amino acids does not exceed 8% by weight of the total amino acid content.

[0205] In other embodiments of the present invention, the amount of free amino acids contained in the whey protein hydrolysate is 2 - 105% by weight of the total amino acid content in the hydrolysate, preferably, the amount of free amino acids is 4 - 13% by weight of the total amino acid content in the hydrolysate.

[0206] The inventors of the present invention have found that the peptides in the whey protein hydrolysate of the present invention have a molecular weight of 2500 Da or higher, and the amount thereof is 25% by weight or less of the total amount of peptides. Preferably, the whey protein hydrolysate contains peptides with a molecular weight of 2500 Da or higher, and the amount thereof is in the range of 8 - 25% by weight, and even more preferably 10 - 20% by weight.

[0207] In an embodiment of the present invention, the whey protein hydrolysate of the present invention contains peptides with a molecular weight of 375 Da or less, and the amount thereof is at least 10% by weight. Peptides with a molecular weight of 375 Da or less may be present, for example, in an amount in the range of 10 - 25% by weight in the whey protein hydrolysate.

[0208] The inventors of the present invention have found that, compared with known whey protein hydrolysates with a high degree of hydrolysis, the whey protein hydrolysate of the present invention has a reduced bitterness, and this is also the case if the known whey protein hydrolysate with a high degree of hydrolysis has been subjected to membrane filtration through an ultrafiltration membrane and / or treated with activated carbon.

[0209] The bitterness of the whey protein hydrolyzate was compared with the bitterness of caffeine, and the bitterness of the whey protein hydrolyzate in a 4% w / w protein solution was lower than that of a 0.08% w / v caffeine solution. Therefore, the bitterness score of the whey protein hydrolyzate in a 4% w / w protein solution corresponded to a bitterness score of 0.08% w / v caffeine or lower. Preferably, the bitterness score of the whey protein hydrolyzate in the 4% w / w protein solution of the present invention corresponded to a bitterness score of 0.07% w / v caffeine or lower, and even more preferably corresponded to a bitterness score of 0.065% w / v caffeine or lower. Most preferably, the bitterness score of the whey protein hydrolyzate in the 4% w / w protein solution of the present invention corresponded to a bitterness score of 0.060% w / v.

[0210] In other embodiments of the present invention, the scattered turbidity (NTU) of the whey protein hydrolyzate in a 4% w / w protein solution is 100 or lower. Other embodiments of the present invention are to obtain a whey protein hydrolyzate which, in addition to having a high degree of hydrolysis and an acceptable taste, is also clear in appearance. A whey protein hydrolyzate that needs to be clear and have a good taste because it can be used, for example, in beverages, gels, and shakes and enhances consumer appeal.

[0211] If the scattered turbidity measured in a 4% w / w protein solution is less than 100 NTU, the sample is considered transparent. If the scattered turbidity in a 4% protein solution is higher than 100 NTU, the measured whey protein hydrolyzate is considered opaque. If the scattered turbidity is less than 40 NTU, the solution is considered clear. However, a whey protein hydrolyzate with a turbidity between 40 and 100 NTU may be transparent (but not clear 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 an object behind the solution can be seen, that is, it is visible through the solution. The term "clear" refers to a colorless solution, so that the solution can be seen through without anything restricting the view. Therefore, the solution may be transparent but not clear.

[0212] In other embodiments of the present invention, the scattered turbidity (NTU) of the whey protein hydrolyzate in a 4% w / w protein solution is 80 or lower, for example, the scattered turbidity (NTU) in a 4% w / w protein solution is 60 or lower, preferably the scattered turbidity (NTU) in a 4% w / w protein solution is 50 or lower, and even more preferably the scattered turbidity (NTU) in a 4% w / w protein solution is 40 or lower.

[0213] In another embodiment of the present invention, the whey protein hydrolyzate has antioxidant activity. Preferably, the antioxidant activity of the whey protein hydrolyzate is measured to have a scavenging percentage of 54 - 60 in a 1.5% by weight protein solution.

[0214] Whey protein hydrolysates can contain other components in addition to proteins, such as carbohydrates, lipids, and minerals.

[0215] In embodiments of the present invention, the whey protein hydrolysate contains lipids in an amount of 8 wt% or less, such as 6 wt% or less, based on the total solids content. In another embodiment, the whey protein hydrolysate contains lipids in an amount of 1 wt% or less, such as 0.5 wt% or less, based on the total solids content.

[0216] The whey protein hydrolysate may also contain minerals, such as potassium, sodium, and calcium.

[0217] In embodiments of the present invention, the whey protein hydrolysate contains 3.0 wt% or less of potassium.

[0218] In another embodiment of the present invention, the whey protein hydrolysate contains 2 wt% or less of sodium.

[0219] In another embodiment of the present invention, the amount of citric acid contained in the whey protein hydrolysate is 4 - 10 g / kg of the whey protein hydrolysate solids content.

[0220] In embodiments of the present invention, the whey protein hydrolysate contains intact or undegraded bovine serum albumin (BSA) in an amount of 0.5 - 2 wt% based on the total protein content.

[0221] 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.

[0222] In another embodiment, the whey protein hydrolysate is a liquid composition.

[0223] Food product:

[0224] In one aspect, the present invention relates to providing a food product comprising a whey protein hydrolysate according to the present invention.

[0225] The food product can be, for example, any one selected from the group of dairy products, including beverages, milkshakes, gels, food bars, concentrates, or liquid shots.

[0226] In a preferred embodiment, the food product is selected from the group of protein beverages, protein concentrates, protein milkshakes, protein gels, or protein bars. The food product can also be an infant formula food or other infant nutrition products.

[0227] If the food product is in liquid form, such as a beverage, a milkshake, a gel or a concentrate, the food product may comprise a whey protein hydrolysate according to the invention. The whey protein hydrolysate is preferably present in the beverage in an amount corresponding to 2-25% by weight of hydrolyzed whey protein, preferably 3-20% by weight of hydrolyzed whey protein, such as 3-15% by weight of hydrolyzed whey protein, and even more preferably 3-10% by weight of hydrolyzed whey protein.

[0228] If the food product is a bar, such as a protein bar, the food product comprises a whey protein hydrolysate according to the invention in an amount equivalent to 2-30% by weight of hydrolyzed whey protein. Preferably, the amount of the whey protein hydrolysate according to the invention is present in the bar in an amount corresponding to 3-20% by weight of hydrolyzed whey protein, such as 4-15% by weight. If the whey protein hydrolysate according to the invention is used in a food bar, such as a protein bar, the whey protein hydrolysate can be used as a softening agent. It is well known that increasing the concentration of protein hydrolysate in a protein bar has a softening effect and prevents the bar from hardening during long-term storage.

[0229] In another aspect, the invention relates to providing a beverage comprising a whey protein hydrolysate according to the invention in an amount equivalent to 2-20% by weight of hydrolyzed whey protein. The beverage can be, for example, a protein beverage which further comprises carbohydrates, vitamins and minerals in addition to protein.

[0230] In a preferred embodiment of the invention, the beverage has a neutral pH, i.e., the pH in a 4% protein solution at 22 °C is in the range of 6.5-8.0.

[0231] In an embodiment of the invention, the whey protein hydrolysate according to the invention can be used as a component for preparing a carbonated beverage. Thus, the food product of the invention comprising the whey protein hydrolysate according to the invention is a carbonated beverage.

[0232] Other embodiments of the invention relate to carbonated beverages comprising the whey protein hydrolysate of the invention.

[0233] In one embodiment, the carbonated beverage comprises a whey protein hydrolysate in an amount corresponding to 2-10% by weight. The carbonated beverage can further comprise carbohydrates, and if present, the content thereof is 5% by weight or less. The carbonated beverage preferably does not contain fat.

[0234] In one embodiment, the amount of carbonation in a carbonated beverage comprising the whey protein hydrolysate of the present invention is from 0.1 volume carbonation (liquid present per volume of beverage) to 4 volume carbonation. More typically, the amount of carbonation ranges from about 1.6 volumes to about 3.5 volumes, most typically in the range from about 1.7 volumes to about 3.0 volumes, and most preferably the amount of carbonation is in the range from 2.0 to 3.0 volumes. In the context of the present invention, carbonation refers to the addition of carbon dioxide to the composition mixture of the beverage in an amount sufficient to obtain a carbonated protein beverage, wherein the amount of carbonation present in the beverage is from 0.1 volume to 4 volumes per volume of liquid mixture. In some embodiments of the method, carbon dioxide is added in the form of sterile carbonated water. In other embodiments, sterile carbon dioxide is bubbled through the liquid mixture until the desired amount of carbon dioxide is present.

[0235] Carbonation increases the acidity of the beverage. The more carbon dioxide added to the beverage, the lower the pH of the beverage. However, the inventors of the present invention have found that at a temperature of 5 °C, the addition of carbon dioxide reduces the pH of the beverage to a minimum pH of 5.5 - 6.0. Adding about 2.5 - 3.0 volumes of carbon dioxide per volume of beverage results in the pH of the beverage being reduced to about pH 6.0 at a temperature of 5 °C, while adding about 4 volumes of carbon dioxide per volume of beverage results in the pH of the beverage being reduced to about pH 5.5 at a temperature of 5 °C.

[0236] Thus, in an embodiment of the present invention, a carbonated beverage comprising the whey protein hydrolysate of the present invention has a pH of at least 5.5 at a temperature of 5 °C. The pH generally ranges from 5.5 - 8.25, for example in the range from 5.5 - 7.0, preferably in the range from 5.8 - 6.5. Carbonated beverages with a pH value higher than 5.5 are preferred.

[0237] A carbonated beverage comprising the whey protein hydrolysate of the present invention can be heat treated, for example pasteurized or sterilized. The inventors of the present invention have found that the turbidity of a carbonated beverage comprising the whey protein hydrolysate of the present invention does not change after heat treatment at a temperature of up to 120 °C for a long time (e.g., 20 minutes).

[0238] In other embodiments of the present invention, the carbonated beverage may comprise a combination of the whey protein hydrolysate according to the present invention and an unhydrolyzed whey protein isolate.

[0239] The whey protein hydrolysate of the present invention can also be used to prepare protein concentrates, protein shakes or protein gels. The protein concentrate, shake or gel may further comprise carbohydrates, vitamins and minerals in addition to the hydrolyzed whey protein in an amount of 2 - 20% by weight. The pH of the protein concentrate, protein shake or protein gel is preferably neutral, i.e., in the range from 6.5 - 8.0.

[0240] On the other hand, the present invention relates to the use of the whey protein hydrolyzate according to the present invention as a food ingredient. The whey protein hydrolyzate can be added as a food ingredient to any type of food. Preferably, the whey protein hydrolyzate of the present invention is used as a food ingredient for preparing cold or hot beverages.

[0241] In an embodiment of the present invention, the whey protein hydrolyzate is used as a food ingredient for preparing a UHT-stable beverage having a pH value in the range of 6.5 - 8.5.

[0242] In another embodiment of the present invention, the whey protein hydrolyzate is used as a food ingredient for preparing a beverage for sports nutrition. In the context of the present invention, the term "sports nutrition" refers to nutrition suitable for exercise or training-related, i.e., for enhancing muscle mass.

[0243] In another embodiment of the present invention, the whey protein hydrolyzate is used as a food ingredient for preparing a clinical beverage. In the context of the present invention, the term "clinical beverage" refers to a beverage having a clinical or medical indication. For example, a clinical beverage may have a health-related effect. Clinical beverages are typically used by inpatients or the elderly with nutritional difficulties or individuals who require pre-digested protein to recover from a medical condition. For example. A clinical beverage can be a beverage for individuals suffering from malnutrition or malabsorption. Clinical beverages can also be used by individuals suffering from gastrointestinal diseases. In the context of this article, the terms "clinical beverage" and "medical beverage" have the same meaning.

[0244] A clinical beverage can, for example, contain the whey protein hydrolyzate of the present invention in an amount corresponding to a beverage containing 2 - 20% by weight of hydrolyzed whey protein. A clinical beverage can contain carbohydrates in an amount of 5 - 50% by weight of the beverage. The amount of carbohydrates can, for example, be in the range of 10 - 40% by weight, such as 15 - 35% by weight. A clinical beverage can also contain fat. For example, the fat content in a clinical beverage can be in the range of 2 - 30% by weight, such as 3 - 20% by weight, more preferably 3 - 18% by weight.

[0245] In one example, a clinical beverage contains the hydrolyzed whey protein according to the present invention in an amount corresponding to 4 - 10% by weight, 3 - 15% by weight of fat, and 10 - 35% by weight of carbohydrates.

[0246] A clinical beverage preferably has a neutral pH value, i.e., a pH in the range of 6.5 - 8.0.

[0247] A clinical beverage can be in the form of a clear beverage, a milky beverage, a tube-fed form, or a powder form to be reconstituted in a liquid.

[0248] In one embodiment, the whey protein hydrolysate of the present invention can also be used to prepare fruit juice-based beverages. The fruit juice-based beverage preferably contains 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.

[0249] If the clinical beverage is in the form of tube feeding, it may contain 4-15% by weight of the hydrolyzed whey protein according to the present invention, about 5-35% by weight of carbohydrates, and about 3-15% by weight of fat.

[0250] The whey protein hydrolysate of the present invention can also be used in infant nutrition products, such as infant formula. In the context of the present invention, the term "infant formula" refers to any type of infant formula, including follow-up formula, growing formula, and premature formula.

[0251] If the whey protein hydrolysate according to the present invention is used in infant formula, the protein content in the infant formula is in the range of 1.6-5.0 g / 100 kcal. In addition to the whey protein hydrolysate, the infant formula may also contain carbohydrates, such as lactose, oligosaccharides, lipids, vitamins, and minerals.

[0252] The whey protein hydrolysate according to the present invention can also be used to prepare other infant nutrition products besides infant formula, such as for milkshakes, porridges, etc.

[0253] The whey protein hydrolysate of the present invention can also be used to prepare emulsions. Emulsions are usually prepared by reconstituting the powder of the whey protein hydrolysate in a liquid (such as water or milk) and fat. The whey protein hydrolysate will emulsify the water and fat. The powder usually contains the whey protein hydrolysate in an amount equivalent to 5-15% by weight of protein. After reconstitution, the emulsion contains 2-4% by weight of protein.

[0254] Protein hydrolysis causes changes in proteins, such as an increase in the number of charged groups, a decrease in the average molecular weight, and the exposure of reactive groups, etc. These are factors affecting the ability of protein hydrolysates to form and stabilize emulsions. The whey protein hydrolysate of the present invention can be used as an emulsifier, stabilizer, etc., by combining them with other ingredients.

[0255] The whey protein hydrolysate of the present invention can also be used in baked products, such as for cookies, crackers, and wafers.

[0256] In other aspects, the present invention relates to the use of the whey protein hydrolysate according to the present invention as an antioxidant. The inventors of the present invention surprisingly found that the whey protein hydrolysate of the present invention has an antioxidant effect. Therefore, the whey protein hydrolysate can be used in nutritional compositions as a source of antioxidant peptides.

[0257] Accordingly, the present invention relates to the whey protein hydrolysate of the present invention having antioxidant effects. More specifically, the present invention relates to a whey protein hydrolysate having antioxidant effects, which is defined as having a scavenging percentage of 54 - 60 in a solution containing 1.5 wt% protein. The scavenging percentage is measured by the DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) assay. The scavenging percentage is calculated as 100x(A0 - A S ) / A0, where A0 is the absorbance without the sample, and A S is the absorbance with the sample.

[0258] It should be noted that the embodiments and features described in the context of one aspect of the present invention are also applicable to other aspects of the present invention.

[0259] All patents and non-patent references cited in this application are hereby incorporated by reference in their entirety.

[0260] The present invention will now be described in more detail in the following non-limiting examples.

[0261] Examples

[0262] Example 1: Analytical Method

[0263] Example 1.1: Determination of degree of hydrolysis (DH)

[0264] The degree of hydrolysis (DH) is defined as the percentage of peptide bonds cleaved by hydrolysis, see Equation (1) below. The DH value provides information about the number of peptides formed, which is related to the number of available peptide bonds.

[0265] The DH of the whey protein hydrolysate was measured as described in the following references: 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, P.M., Petersen, D. & Dambmann, C. Improved method for determining food protein degree of hydrolysis. J. Food Sci. 66, 642 - 646 (2001). In Equation (1), h represents the number of cleaved peptide bonds, and h total represents the total number of available peptide bonds. Thus, DH gives the percentage of cleaved peptide bonds.

[0266] Equation (1): DH = (number of free amino termini) / (total number of available peptide bonds)·100% = h / h total ·100%

[0267] The free α-amino groups formed after hydrolysis react with o-phthalaldehyde (OPA) to form a yellow complex, which absorbs light at 340 nm. Therefore, it can be measured by spectrophotometry. Based on the formation of the color, DH can be calculated.

[0268] The hydrolyzate was resuspended in water at an appropriate concentration (0.03 - 0.08% protein) and reacted with 2 volumes of 15 volumes of OPA reagent (100 mM Na2B4O7, 0.1% sodium dodecyl sulfate, 6 mM DL-dithiothreitol, 6 mM o-phthalaldehyde, and 2% ethanol) at 25 °C for 2 minutes. Similar reactions were carried out to generate a concentration series of L-serine. Next, the absorbance at 340 nm (A340) was measured, and the A340 signal from the reaction of OPA with water was subtracted. To find the true DH, the serine equivalents measured in the supernatant were corrected as suggested by Adler-Nissen for the trinitrobenzenesulfonic acid method [Adler-Nissen J; Journal of Agricultural and Food Chemistry, 1979 27(6)1256], which gives the same response as the described OPA method. The factors for whey protein hydrolyzate are a = 1, b = 0.4, h total = 8.8.

[0269] Example 1.2: Determination of turbidity

[0270] The scattering turbidity of the whey protein hydrolyzate was used as a measure of clarity and transparency. If the scattering turbidity measured in a 4% w / w protein solution is less than 100 NTU, the sample is considered transparent. Additionally, if the scattering turbidity measured in a 4% w / w protein solution is less than 40 NTU, the sample is considered clear.

[0271] When measuring the scattering turbidity, the sample was diluted to 5 different protein concentrations, namely 1.8%, 3.2%, 4.8%, 6.4%, and 8%, and the scattering turbidity was measured using Merck's Turbiquant 3000IR.

[0272] Example 1.3: Determination of total protein

[0273] The total protein content (protein equivalent) of the sample was determined by the following method:

[0274] 1) Determine the total nitrogen of the sample in accordance with ISO 8968-1 / 2 IDF 020-1 / 2 - Milk - Determination of nitrogen content - Part 172: Use the Kjeldahl method to determine the nitrogen content.

[0275] 2) Calculate the total amount of protein as: N x 6.38

[0276] Example 1.4: Determination of total amino acid content

[0277] Amino acid composition analysis provides detailed information on protein hydrolysates as a source of amino acid supplementation.

[0278] Measure the total amino acid content by the method of ISO 13903:2005, EU 152 / 2009. Hydrolyze the sample in an aqueous hydrochloric acid solution to break the peptide bonds in the sample. After hydrolysis, adjust the pH value of the sample, make up the volume and filter. Separate the amino acids in an amino acid analyzer, detect using post-column derivatization with ninhydrin reagent, and measure at 440 and 570 nm. For quantification, use 1-point calibration. To ensure quality, analyze the internal standard for each run.

[0279] Before analysis on an amino acid analyzer, cysteine and methionine must be oxidized. Oxidize the sample with hydrogen peroxide and formic acid at low temperature, and then perform acid hydrolysis with an aqueous hydrochloric acid solution. The oxidation process oxidizes methionine and cysteine, thus preventing loss during hydrolysis. After hydrolysis, analyze the sample as described above.

[0280] Quantification of total tryptophan: Tryptophan is analyzed using another method because it cannot be quantified in the same way as other amino acids as it is destroyed during the acid hydrolysis of proteins. As an alternative, hydrolyze the sample by alkaline treatment and analyze and quantify by HPLC.

[0281] The results of each amino acid are normalized to the total amount of amino acids present: [g / 100 g amino acids].

[0282] Example 1.5: Determination of free amino acid content

[0283] Free amino acids in whey protein hydrolysate were determined by the methods of R. Schuster, "Determination of Amino Acids in Biological, Pharmaceutical, Plant and Food Samples by Automated Precolumn Derivatization and HPLC", Journal of Chromatography, 431:271-284(1988) and Henderson, J.W., Ricker, R.D., Bidlingmeyer, B.A., Woodward, C., "Rapid, Accurate, Sensitive, and Reproducible HPLC Analysis of Amino Acids, Amino Acid Analysis Us-ing Zorbax Eclipse-AAA columns and the Agilent 1100 HPLC," Agilent Publication, 2000.

[0284] Free amino acids were determined by extracting the amino acids into an aqueous or acidic solution. The sample could be deproteinized by molecular weight filtration. The sample was analyzed by HPLC after pre-injection derivatisation. Primary amino acids were derivatized with o-phthalaldehyde and secondary amino acids were derivatized with 9-fluorenylmethyl chloroformate before injection. The results were as follows:

[0285] [mg free amino acids / 100 g whey protein hydrolysate powder]

[0286] Example 1.6: Method for determining peptide distribution in whey protein hydrolysate

[0287] The molecular weight distribution of peptides in whey protein hydrolysate was analyzed using size exclusion chromatography (SEC). SEC separates molecules of the polymer type by size. A mixture of components of different sizes, which are peptides in this article, can be separated by SEC. The elution time depends on the size. The smaller the molecule, the longer the elution time.

[0288] The sample was dissolved in the mobile phase to a concentration of 0.5% w / v. Before injection, the sample was filtered through a 0.45 μm filter. In three TSK G2000 SWXL columns in series ( Chromatographic separation was carried out on a chromatographic column (5 μm, 7.5 mm x 300 mm). A buffer composed 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, and the flow rate was 0.7 mL / min. A UV detector measuring at 214 nm was used for the detection of peptides.

[0289] Based on the retention time, the peptide segment distribution was divided according to size, and the relative amounts were given according to the molecular weight.

[0290] Example 2: Screening of enzyme combinations

[0291] A total of 55 different enzyme combinations were tested for the enzymatic hydrolysis of whey protein. The clarity, bitterness, degree of hydrolysis, and peptide composition of the obtained whey protein hydrolysates were analyzed. The hydrolysis test was carried out on a 0.5 L scale.

[0292] The enzymes used to test different enzyme combinations were serine endopeptidase from Bacillus (EC 3.4.21.62), serine endopeptidase from Aspergillus (EC 3.4.21), trypsin-like protease from microbial origin (EC 3.4.21.4), aminopeptidase from Aspergillus (EC 3.4.11), metalloendopeptidase from Bacillus amyloliquefaciens (EC 3.4.24.28), endoprotease from pineapple (bromelain) (EC 3.4.22.32), proline-specific endopeptidase from Aspergillus niger (EC:3.4.21.26), aminopeptidase preparation from Aspergillus oryzae (EC 3.4.11), metalloendopeptidase preparation from Geobacillus stearothermophilus (EC 3.4.24), and endopeptidase preparation from Bacillus amyloliquefaciens (EC 3.4).

[0293] The enzyme preparations used for the experiment were:

[0294] Protamex (Novozymes A / S), which contains serine endopeptidase (subtilisin) and bacillopeptidase from Bacillus species.

[0295] Protease A Amano 2SD (Amano Enzymes Ltd.), which contains serine endopeptidase from Aspergillus oryzae.

[0296] Promod 782MDP (Biocatalysts Ltd.), which contains serine endopeptidase from Aspergillus species.

[0297] Formea TL 1200BG (Novozymes A / S), which contains trypsin-like protease from microbial origin.

[0298] Promod 950L (Biocatalysts Ltd.), which contains serine endopeptidases (subtilisins) and bacillopeptidases from Bacillus species.

[0299] Flavourzyme conc BG (Novozymes A / S), which contains leucine aminopeptidase from Aspergillus oryzae.

[0300] Alcalase AF 2.4L (Novozymes A / S), which contains serine endopeptidases (subtilisins) from Bacillus licheniformis.

[0301] Neutrase conc BG (Novozymes A / S), which contains metalloendopeptidases (bacillopeptidases) from Bacillus amyloliquefaciens.

[0302] Promod 523MDP (Bromelain) (Biocatalysts Ltd.), which contains cysteine endopeptidases from pineapple.

[0303] Maxipro PSP (DSM), a proline-specific endopeptidase from Aspergillus niger.

[0304] Flavorpro 766 (Biocatalyst Ltd.), which contains aminopeptidases from Aspergillus oryzae.

[0305] Thermoase PC10F (Amano Enzymes Ltd.), which contains metalloendopeptidases from Geobacillus stearothermophilus.

[0306] Protin NY100 (Amano Enzymes Ltd.), which contains endopeptidases from Bacillus amyloliquefaciens.

[0307] A combination of 55 enzymes was used for the hydrolysis of whey protein. As the hydrolysis substrate, a solution of whey protein isolate (WPI) (Lacprodan DI-9224, from Arla Food Ingredients) was used. The protein concentration of the WPI solution used for all experiments was 8 wt%. The hydrolysis reaction was carried out at 50 °C, where the pH-stat was pH = 7, and the reaction time since the addition of the first enzyme was 6 hours. After 6 hours of hydrolysis, the hydrolysis was stopped by heating to 99 °C and holding for 90 seconds to inactivate the enzymes. The amount of enzyme used is mentioned in Table 1 and is the amount of enzyme (grams) per 100 grams of protein. The products from each hydrolysis trial were freeze-dried and used for further analysis, including clarity (in the form of turbidity measurement), degree of hydrolysis, and flavor evaluation and scoring. The results are summarized in Table 1 below:

[0308] NEU: refers to Neutrase

[0309] FZ: refers to Flavourzyme conc BG

[0310] Alca: refers to Alcalase AF 2.4L

[0311] PA: refers to Protease AAmano 2SD

[0312] FTL: refers to Formea TL 1200BG

[0313] PM782: refers to Promod 782MDP

[0314] FP766: refers to Flavourpro 766

[0315] MP PSP: refers to Maxipro PSP

[0316] THER: refers to Thermoase PC10F

[0317] PRO: refers to Protin NY100

[0318] PM950: refers to Promod 950L

[0319] PTM: refers to Protamex

[0320] The enzymes used for the tests shown in Table 1 were selected from a large number of enzymes according to tests on a 96-well scale. These tests included hydrolysis with different enzyme combinations and visual scoring of apparent clarity, thermal stability, and hydrolysis (SDS-PAGE). To allow pH-stable hydrolysis and a larger amount of material for analysis, the combinations given in Table 1 were carried out on a 500 mL scale as described above. Thus, the enzymes listed in Table 1 were not randomly selected. Some of the enzyme combinations in Table 1 were repeated at different doses (e.g., combinations 1-3).

[0321] The term "Visual a inact" in Table 1 refers to "visible after inactivation".

[0322] Table 1

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330] Data on each whey protein hydrolyzate were evaluated. For a positive evaluation, the following conditions should be met:

[0331] - Clear appearance after enzyme inactivation

[0332] - A high degree of hydrolysis of more than 15%, preferably more than 20%

[0333] - No bitterness in a 4% w / w protein solution

[0334] - 25% by weight or less of the peptides should have a molecular weight higher than 2500 Da

[0335] 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).

[0336] Thus, as can be seen from the above table, 4 out of 55 enzyme combinations, namely the samples labeled as samples 4, 10, 11, and 30, met the conditions.

[0337] Sample 4 was obtained by hydrolysis using a combination of a serine endopeptidase from Bacillus, a serine endopeptidase from Aspergillus, and a trypsin-like protease.

[0338] Sample 10 was obtained by hydrolysis using a combination of a serine endopeptidase from Bacillus, a metalloendopeptidase from Bacillus, a serine endopeptidase from Aspergillus, and a leucine aminopeptidase from Aspergillus.

[0339] Sample 11 was obtained by hydrolysis using a combination of a serine endopeptidase from Bacillus, a serine endopeptidase from Aspergillus, and a leucine aminopeptidase from Aspergillus.

[0340] Sample 30 was obtained by hydrolysis with subtilisin from Bacillus amyloliquefaciens, bromelain, and leucine aminopeptidase from Aspergillus.

[0341] Example 3: According to the present invention The Further analysis of the whey protein hydrolysate.

[0342] Four whey protein hydrolysates of Example 2 that met the conditions of clarity, taste, degree of hydrolysis, and peptide distribution were further analyzed and are referred to as Samples 1 - 4 (S1 - S4) in the table below.

[0343] Samples 5 - 12 (S5 - S12) are whey protein hydrolysates from hydrolysis using other enzyme combinations.

[0344] Sample 13 (S13) is a spray - dried WPI hydrolysate obtained by the following method: hydrolysis using a combination of subtilisin (Alcalase) from Bacillus licheniformis and subtilisin (Neutrase) from Bacillus amyloliquefaciens, followed by ultrafiltration, activated carbon treatment, and microfiltration to remove the activated carbon (as described in WO 1993 / 024020A1). The degree of hydrolysis is approximately 25%.

[0345] Sample 14 (S14) was prepared by using the same enzyme combination as Sample 4 (the enzyme combination of the present invention), but using WPC as the substrate for hydrolysis.

[0346] Samples 15 and 16 (S15 - S16) are replicates of Sample 4. Samples 15 and 16 were prepared based on the WPI hydrolysate, using the same conditions as Sample 4 but without UF filtration.

[0347] The clarity, degree of hydrolysis, content of peptides with a molecular weight greater than 2500 Da, and content of peptides with a molecular weight less than 375 Da of whey protein hydrolysates obtained by enzymatic hydrolysis using different enzyme combinations were analyzed again.

[0348] The clarity (turbidity) of the samples was determined by the measurement according to Example 1.2, and for samples considered to be clear, the scattered turbidity should be below 40 NTU, while for samples considered to be transparent, it should be below 100 NTU.

[0349] If bitterness is perceived in a protein of 4% or less, the sample is scored as bitter.

[0350] The degree of hydrolysis was measured by the method described in Example 1.1, while bitterness was measured by tasting samples at different concentrations (2%, 4%, 8% protein concentration). The peptide distribution was measured as described in Example 1.5. The results are shown in Table 2:

[0351] Table 2:

[0352]

[0353]

[0354]

[0355] Thus, as can be seen from Table 2, using the specific enzyme combination according to the present invention can produce whey protein hydrolysates having the following characteristics: 1) a degree of hydrolysis greater than 20%, 2) less than 25% of the peptides having a molecular weight of 2500 Da or higher, and 3) no bitterness at a protein concentration of 4% or less.

[0356] In addition, Table 2 shows that if WPI is used for protein hydrolysis, using the specific enzyme combination of the present invention produces a whey protein hydrolysate with a clear appearance. Table 2 also shows that in the preparation of whey protein hydrolysates using WPC as a substrate, the whey protein hydrolysates produced using the specific enzyme combination have a degree of hydrolysis higher than 20%, less than 25% of the peptides having a molecular weight of 2500 Da or above, and no bitterness at a protein concentration of 4% or less. However, the whey protein hydrolysates prepared using WPC as a substrate are not clear in appearance (due to the presence of lipids in WPC).

[0357] In a preferred embodiment of the present invention, WPI is used as a substrate for protein hydrolysis to obtain a clear hydrolysate.

[0358] Example 4: Turbidity analysis

[0359] The scattering turbidity of Samples 1-16 from Example 3 at different protein concentrations was further analyzed. Samples with a turbidity less than 100 NTU were considered transparent, and samples with a turbidity below 40 were considered clear. Table 3 below shows the turbidity of the hydrolysates called Samples 1-12 in Example 3 measured at different protein concentrations. The protein concentrations were 1.8% protein, 3.2% protein, 4.8% protein, 6.4% protein, and 8% protein. The protein concentrations are weight percentages.

[0360] Before measuring the turbidity, the whey protein hydrolysate powder was hydrated at the specified concentration for at least 30 minutes (n = 3).

[0361] Table 3: Turbidity (NTU) of test samples of 1.8, 3.2, 4.8, 6.4 and 8% protein

[0362]

[0363]

[0364] Thus, Table 3 shows that the specific enzyme combination according to the present invention has a turbidity of less than 100 NTU at 8% protein concentration.

[0365] In Figure 1 A - C, the turbidity of samples 13, 14 and 16 measured is shown. Figure 1 A shows the turbidity of the whey protein hydrolysates of samples 13, 14 and 16 and shows the difference in turbidity. As can be seen from Figure 1 A, the ultrafiltered whey protein hydrolysate (not of the present invention, sample 13) has a very low turbidity (below 1 NTU) and is the clearest hydrolysate. The hydrolysate prepared using one of the specific enzyme combinations according to the method of the present invention (sample 16) has a turbidity of less than 100 NTU at 8% protein concentration and is thus transparent. In addition, the hydrolysate of the present invention has a turbidity of less than 40 NTU at 4% protein concentration and is thus considered clear at 4% protein concentration. In contrast, the whey protein hydrolysate prepared by using WPC as a substrate according to the present invention (sample 14) has a turbidity of more than 1000 NTU and is thus considered not clear.

[0366] Figure 1 B shows more clearly that at a protein concentration of 5% or less, the turbidity of sample 16 is less than 40 NTU.

[0367] Figure 1 C shows the turbidity of sample 14. The results show that even at a very low concentration (about 2%), the turbidity is higher than 2000 NTU. Sample 14 is considered very unclear and opaque.

[0368] Figure 2 A - C includes pictures of samples 13, 14 and 16 to visually show the contrast between the clear sample and the unclear sample. The samples are prepared at 8%, 6.4%, 4.8%, 3.2% and 1.8% protein from left to right respectively.

[0369] Figure 2 A shows that sample 16 is visually clear and transparent at 8%, 6.4%, 4.8%, 3.2% and 1.8% protein.

[0370] Figure 2Panel B shows Sample 14 and shows that Sample 14 is not clear and is opaque even at the lowest protein concentration of 1.8%.

[0371] Figure 2 Panel C shows Sample 13 and shows that Sample 13 is visually clear and transparent at all concentrations.

[0372] Example 5: Bitter taste evaluation relative to caffeine

[0373] In Example 5, the bitterness of the whey protein hydrolysate according to the present invention was compared with UF-filtered and activated-carbon-treated whey protein hydrolysates prepared by enzymatic hydrolysis of WPI with other enzymes outside the present invention (hydrolysis with Alcalase from Bacillus licheniformis and Neutrase from Bacillus amyloliquefaciens).

[0374] Sensory evaluations were conducted to compare the tastes of Samples 13, 15, and 16 of Example 3.

[0375] A caffeine solution was used as a reference to train a sensory panel to detect and quantify bitterness. The training consisted of first providing the panelists with reference samples, which included solutions composed of increasing concentrations of caffeine. The panelists were trained to assign bitterness scores to unknown solutions based on a 15-cm scale. The reference samples consisted of 3 caffeine solutions containing 0.025%, 0.05%, and 0.1% caffeine, respectively. After evaluating the reference samples, the panelists tasted the hydrolysate samples 3 times in 4 wt% protein solutions in a random order and ranked the bitterness intensity of the test solutions according to the bitterness of the reference solutions. The reference solution containing 0.025% caffeine was considered not bitter, while the reference solution containing 0.1% caffeine was considered bitter (scored 13 on a 15-cm bitterness scale). The sensory panel consisted of 7 panelists who participated in the evaluation.

[0376] In addition, the sensory analysis was conducted using a panel according to the international standard Quantitative Descriptive Profile ISO 13299:2016, 2nd Edition, 5.5, Annex F1-F6 & H3. 7 trained assessors participated in the evaluation. The evaluation was conducted in 3 replicates. The response scale used was a continuous line scale (15 cm). Approximately 2 ml of the sample was provided at ambient temperature. Red light was used during the evaluation process.

[0377] The bitterness scores given by the sensory panel with reference to the bitterness of caffeine are as Figure 3 shown, where the bitterness scores of the test samples are plotted relative to the caffeine concentration. The bitterness of 3 different concentrations of caffeine and the bitterness scores of Samples 13, 15, and 16 from Example 3 are as Figure 3 shown.

[0378] Figure 3 It is shown that sample 13 (the hydrolyzate of WPI, but without using the enzyme combination of the present invention, and the hydrolyzate is ultrafiltered and treated with activated carbon) has the highest relative bitterness of 0.095%. Sample 15 (the hydrolyzate of WPI, using the enzyme combination of the present invention, without ultrafiltration and activated carbon treatment) has the lowest relative bitterness of 0.054% among the 3 product samples, but is very close to the hydrolyzate in sample 16 (similar to sample 15), which has a relative bitterness of 0.061% relative to caffeine.

[0379] Therefore, the taste of the palatable whey protein hydrolyzates (sample 15 and sample 16) prepared according to the present invention is considered to have a bitterness lower than that of caffeine with a bitterness lower than 0.08% and lower than that of sample 13.

[0380] Example 6: Taste analysis

[0381] An example was conducted to evaluate the taste characteristics of the whey protein hydrolyzates in samples 13, 15, and 16. The taste analysis was carried out by a trained panel and 5 attributes were used to identify the differences between the samples, with an emphasis on odor, texture, and taste.

[0382] The analysis data was used to identify the significant differences in each attribute between the samples. The statistical evaluation of the data is shown in Table 4. In addition, a multiple range test has been used to identify the differences between the samples. In Table 4, samples with the same letter have no significant differences.

[0383] Table 4: Sensory scores

[0384]

[0385] ***p < 0.001 Duncan test: Samples marked with different letters are significantly different at the 95% level (p < 0.05).

[0386] Therefore, the bitterness scores of samples 15 and 16 are less than the bitterness score of sample 13.

[0387] The data mentioned in Table 4 can be represented by a spider web, see Figure 4 . The spider web shows the attributes of odor (O), texture (MF), and taste (T). The "high" intensity of each sensory attribute is shown on the periphery of the figure, and the "low" intensity is located at the center of the figure. Each product has a different label as shown below. For example, see the data of "bitterness_T", where *** indicates that the data of sample 13 is significantly different from the data of samples 15 and 16 (p < 0.001).

[0388] As can be seen from the spider chart of the flavor characteristics, sample 13 is significantly different from samples 15 and 16 in terms of flavor characteristics. Most importantly, samples 15 and 16 have low bitterness.

[0389] Example 7: Analysis by LC-MS / MS and peptide cleavage pattern

[0390] Peptides in liquid samples can be identified by mass spectrometry peptide analysis and database search. Samples 1, 2, 3, 13, 15, and 16 were analyzed by LC-MS / MS to identify peptide sequences and the proteins from which they originated. The 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 obtained MS / MS spectra were searched against a custom database containing bovine protein sequences. The overall results of the analysis are shown in Table 5.

[0391] Table 5: Proteins Identified by LC-MS / MS

[0392]

[0393]

[0394] * Glycosylation-dependent cell adhesion molecule 1

[0395] ** Bovine serum albumin

[0396] The data in Table 5 indicate that the analysis provided high sequence coverage for the most prevalent proteins in the samples, including β-lactoglobulin, α-lactalbumin, and β-casein. In addition, the number of peptides identified from each sample was also indicated. Since the data sets had the expected quality of LC-MS / MS analysis, they could be used for more detailed analysis (Example 9).

[0397] Example 8: LS-MS / MS Analysis of Bitter Peptides

[0398] First, it was confirmed by comparing peptide distributions that the LC-MS / MS data from Example 7 corresponded to the data obtained by SEC analysis (size exclusion chromatography). In this article, the SEC data were converted to percentages (the number of peptides of a given molecular weight) and plotted together with the same data calculated based on LC-MS / MS for each hydrolysate (see Figure 5 ). The LC-MS / MS data only included peptides from β-lactoglobulin, while the SEC analysis considered all proteins in the sample.

[0399] From Figure 5It can be seen that when using LC-MS / MS and SEC, the percentages of 7-10 amino acid and 11-19 amino acid β-lactoglobulin-derived peptides among β-lactoglobulin-derived peptides of 7-19 amino acids are similar.

[0400] Therefore, Figure 5 the data shown indicate that the number of peptides within different amino acid length ranges in the LC-MS / MS dataset can be used to extract quantitative information about the relative distribution of peptides, because the data are comparable to SEC data and the SEC method is quantitative. Additionally, Figure 5 it is shown that sample 13 (not belonging to the present invention) has more peptides with smaller peptide sizes than the hydrolysates according to the present invention.

[0401] The percentage of the amount of detectable peptides containing phenylalanine in samples 1, 2, 3, 13, 15, and 16 out of the total number of peptides was identified and analyzed by MS-LC / MS. The results are as Figure 6 shown.

[0402] Without being bound by any theory, the inventors of the present invention believe that the presence of phenylalanine in peptides is related to bitterness, and the bitterness of phenylalanine may be enhanced when the amino or carboxyl terminus of phenylalanine is blocked by a peptide bond with another amino acid residue. For example, the phenylalanine residue in the bitter peptide YPFPGPIPN identified in bitter whey protein hydrolysates is considered to be the main bitterness determinant (Liu. X., Jiang. D., and Peterson. D. G. Identification of Bitter Peptides in whey protein hydrolysate. J. Agric. Food Chem. 2014, 62: 5719-5725).

[0403] Figure 6 It shows the percentage of phenylalanine content in peptides out of the total number of peptides derived from β-lactoglobulin, α-lactalbumin, and β-casein. For the less bitter hydrolysates with peptide sizes less than 9 amino acid residues, the percentage of peptides containing phenylalanine is lower. Therefore, for the whey protein hydrolysates according to the present invention in samples 1 and 3, phenylalanine is present in peptides with larger peptide sizes. The whey protein hydrolysates according to the present invention represented by samples 2, 15, and 16 also have a low percentage of peptides containing phenylalanine, but in addition, samples 2, 15, and 16 also show an overall lower percentage of phenylalanine incorporated in peptides. This indicates that there may be more phenylalanine present in the form of free amino acids in samples 2, 15, and 16 compared to samples 1, 3, and 13. Therefore, an indication of the non-bitter whey protein hydrolysates according to the present invention is that a large percentage of phenylalanine is present in larger peptides or as free amino acids.

[0404] In Figure 7 it shows the percentage of peptides of 5 - 19 amino acids from β - lactoglobulin, α - lactalbumin, and β - casein.

[0405] From Figure 7 it can be seen 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 - containing peptides with smaller peptide sizes in the hydrolysate of sample 13 may be the reason for the higher bitterness in sample 13 compared to samples 1, 2, 3, 15, and 16 which are hydrolysates according to the present invention.

[0406] Example 9: SEC Size Distribution Data

[0407] The sizes of peptides in samples 1, 2, 3, 13, 15, and 16 were analyzed by size - exclusion chromatography (SEC).

[0408] The results are shown in Table 6 below:

[0409] Table 6: SEC Size Distribution Data, DH, and Free Amino Acid (FAA) Contents of Whey Protein Hydrolysates in Samples 1, 2, 3, 13, 15, and 16

[0410]

[0411]

[0412] The size - exclusion chromatography data cannot accurately account for free amino acids because the data was obtained by measuring at 214 nm which is the main expected absorption of peptide bonds.

[0413] Therefore, the content of free amino acids was measured by a different method (see Example 10). Compared with the reference whey protein hydrolysate in sample 13, the whey protein hydrolysates according to the present invention have a higher content of free amino acids. However, the content of small peptides in the reference whey protein hydrolysate (sample 13) is higher than that of the whey protein hydrolysates of the present invention. For example, the content of peptides below 750 Da in sample 13 exceeds 50%. In contrast, the content of peptides 750 Da or smaller in the whey protein hydrolysates of the present invention is less than 40%. In addition, sample 13 contains fewer peptides of 2500 Da or above than the whey protein hydrolysates of the present invention.

[0414] When comparing these data with those of Example 8, it is evident that the total content of phenylalanine in the β-lactoglobulin, α-lactalbumin, and β-casein-derived peptides of Samples 2, 15, and 16 is lower because these phenylalanine residues are released from the peptides into the free amino acid fraction. Thus, one way to reduce the bitterness of the hydrolysate could be to identify enzyme combinations that specifically concentrate phenylalanine in the free amino acid fraction of the total hydrolysate.

[0415] Example 10: Determination of the content of free amino acids

[0416] The content of free amino acids in the whey protein hydrolysate of the present invention was measured and compared with a reference whey protein hydrolysate (outside the present invention). The results are shown in Table 7 below.

[0417] Table 7: Content of free amino acids (mg / 100 g protein (Nx6.38))

[0418] Sample 1 Sample 2 Sample 3 Sample 13 Sample 15 Sample 16 Aspartic acid 113.51 <10 93.24 <10 32.56 17.70 Threonine 898.44 178.05 850.42 22.72 282.30 249.70 Serine 397.09 57.51 374.84 17.34 173.36 117.33 Glutamine 222.56 31.86 181.56 <10 194.45 117.21 Glutamic acid 87.62 <10 101.68 <10 42.29 29.33 Proline <10 <10 <10 <10 16.52 24.61 Glycine 42.05 26.59 24.48 <10 21.20 21.70 Alanine 663.0 189.76 445.12 32.68 343.21 253.33 Cystine <10 <10 <10 <10 <10 <10 Valine 1171.37 480.26 830.50 <10 730.94 627.88 Methionine 1055.41 473.23 1024.95 <10 500.18 386.67 Isoleucine 1311.94 524.77 981.61 44.75 763.73 665.45 Leucine 3736.68 1229.94 3127.56 152.28 2389.60 1903.03 Tyrosine 317.44 42.76 274.10 30.81 102.85 78.30 Phenylalanine <10 183.91 <10 <10 329.16 250.91 Lysine 1862.48 722.74 2237.32 81.76 742.65 723.64 Histidine 440.44 88.79 358.44 17.45 88.44 115.52 Arginine 957.01 138.22 715.71 13.47 384.21 266.67 Tryptophan 339.70 16.52 361.95 <10 128.85 87.64 Asparagine 208.50 19.91 257.70 <10 121.82 59.15 Total 13825 4404 12241 413.26 7266 5936

[0419] As can be seen from Table 7, the content of free amino acids in the whey protein hydrolysate of the present invention is 4% to 14% by weight of the total protein content. In contrast, the content of free amino acids in the reference hydrolysate (Sample 13) is approximately 0.4% by weight of the total protein content.

[0420] Furthermore, Table 7 shows that the content of free leucine in the whey protein hydrolysate of the present invention is much higher than that in Sample 13. The content of free leucine in the whey protein hydrolysate of the present invention is 1 - 4% by weight of the total protein content. In contrast, the content of free leucine in Sample 13 is approximately 0.15% by weight of the total protein content.

[0421] More importantly, as shown in Examples 8 and 9, the concentration of free phenylalanine in Samples 2, 15, and 16 is higher than that in Samples 1, 3, and 13. The percentage of free amino acids based on the total amino acid content is shown in Table 8.

[0422] Table 8: Free amino acids of total amino acids (%)

[0423]

[0424]

[0425] Of particular note is that the percentage of free leucine in the total leucine content in the whey protein hydrolysate of the present invention is much higher than that in the reference hydrolysate (Sample 13).

[0426] Importantly, Table 8 shows that for the whey protein hydrolysates according to the invention in Samples 2, 15, and 16, 6 - 12% of the phenylalanine is in the free phenylalanine form. For the whey protein hydrolysates according to the invention in Samples 1 and 3, no free phenylalanine was found.

[0427] Example 11: Mineral Content in the Whey Protein Hydrolysate of the Invention

[0428] Measure the amount of minerals in Samples 15 and 16. The results are shown in Table 9 below.

[0429] Table 9: Mineral Content

[0430]

[0431] Table 9 shows the mineral content and turbidity of Samples 15 and 16, as an example of the mineral content of the whey protein hydrolysate. The pH of the products in Table 9 in a 4% protein solution at 22 °C is 7.8.

[0432] Example 12: Undegraded BSA in the Hydrolysate

[0433] Measure the amount of undegraded bovine serum albumin (BSA) in the whey protein hydrolysates of the invention (Samples 1, 2, 3, 4, 15, and 16). The content of BSA was estimated by using SDS - PAGE and a BSA standard from Sigma Aldrich (product code A2153) ( Figure 9 A). The amount of BSA loaded into the 20% well corresponds to 10 μg of pure BSA. The total amount of protein added to each well of the SDS - PAGE gel shown in Figure 9 B corresponds to 50 μg of protein. The protein samples were mixed with Laemmli sample buffer and 2 - mercaptoethanol at 10 mg / ml to a final concentration of 3% protein, then incubated at 95 °C for 5 minutes, and then loaded onto the gel.

[0434] Figure 9 The SDS - PAGE gel in A is a titration series showing the expected intensity of BSA if it represents Figure 9 20%, 10%, 5%, 2.5%, 1.25%, or 0.63% of the total whey protein in B. The SDS - PAGE gels of different concentration standards were compared with Figure 9 the SDS - PAGE gels of different whey protein hydrolysate samples shown in B, from left to right: molecular weight standards, Sample 2, Sample 1, Sample 3, Sample 4, Sample 15, and Sample 16.

[0435] From Figure 9A and 9B can conclude that the undegraded BSA ( Figure 9 B) in the whey protein hydrolyzate of the present invention is in the range of 0.5 - 2% by weight because Figure 9 the intensity of the band in B corresponds to Figure 9 the sample intensities of 0.63% and 1.25% in A. BSA is resistant to proteolysis by the enzyme used according to the present invention.

[0436] Example 13: UHT - Treated Beverage for Sports Nutrition

[0437] Example 13 shows an example of the use of the whey protein hydrolyzate according to the present invention in the preparation of a beverage suitable for sports nutrition. The beverage is intended for use by athletes or for other sports or exercise - related applications.

[0438] The powder of Sample 16 was reconstituted in water and sugar and flavoring agents were added to prepare the beverage. The amounts of the ingredients are shown in Table 10 below.

[0439] Table 10 shows a beverage with a neutral taste, without an unpleasant bitter taste, which can be heat - treated by direct and indirect UHT treatment and pasteurization without further turbidity.

[0440] Table 10: Sports Beverage

[0441] Ingredients g / 100g Powder of Sample 16 4.9 Sucrose 2 Sucralose 0.006 Pineapple flavor 0.08 Lime flavor 0.17 Water 92.9

[0442] The sports beverage shown in Table 10 was subjected to 1) direct UHT, 2) indirect UHT, and 3) pasteurization. The direct UHT treatment was carried out by injection for 6 seconds at 143°C. The indirect UHT treatment was carried out on a tubular heat exchanger at 143°C for 6 seconds. The pasteurization was carried out at 90°C for 6.5 minutes. The beverage was poured into flasks at 5°C. The pH of all solutions was approximately 7.7 at 22°C. A content of 4.9 g / 100 g of the whey protein hydrolyzate of the present invention corresponds to 4.0% by weight of protein.

[0443] Figure 8 is a picture showing, from left to right, the untreated beverage, the directly UHT - treated beverage, the indirectly UHT - treated beverage, and the pasteurized beverage. The beverages are at room temperature. Figure 8 It shows that all samples are clear and transparent, and the background behind the bottle can be seen.

[0444] The scattering turbidity of the 4 beverages was measured, and the results are shown in Table 11.

[0445] Table 11: Measured Beverage Turbidity in NTU

[0446] Untreated Direct UHT Indirect UHT Pasteurization Sample 1 58.60 52.22 73.79 39.93 Sample 2 59.22 54.41 71.76 39.88

[0447] Therefore, as can be seen from Table 11, the turbidity of the heat-treated samples is all lower than 100 NTU. Therefore, heat treatment does not affect the turbidity of the beverage, and its appearance remains clear or transparent.

[0448] These beverages are not considered to be bitter.

[0449] Example 14: Clinical / Medical Beverage

[0450] Example 14 is an example of the use of the whey protein hydrolysate according to the present invention in the preparation of a beverage suitable for medical or clinical nutrition. In addition to the whey protein hydrolysate of the present invention, the clinical beverage also contains a large amount of carbohydrates.

[0451] The powder of Sample 16 was reconstituted in water and carbohydrates and flavorings were added to prepare a beverage. The amounts of the ingredients are shown in Table 12 below.

[0452] Table 12 shows a medicinal beverage with a neutral taste without an unpleasant bitter taste.

[0453] Table 12: Medical Beverage

[0454]

[0455]

[0456] Example 15: Carbonated Beverage

[0457] Example 15 shows an example of the use of the whey protein hydrolysate according to the present invention in the preparation of a carbonated beverage.

[0458] The powder of Sample 16 was reconstituted in water and sugar and flavorings 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 the beverage contained 2.5 volumes of carbon dioxide per volume of beverage.

[0459] Table 13: Carbonated Beverage

[0460] Ingredients g / 100g Powder of Sample 16 4.9 Sucrose 2 Sucralose 0.006 Pineapple flavor 0.08 Lime flavor 0.17 Water 92.9 Carbon dioxide (volume per volume of beverage) 2.5

[0461] Example 16: Carbonation - How the Amount of Carbonation Affects the pH Value

[0462] Sample 16 was resuspended in water to prepare an 8% protein solution. The solution was forced to carbonate at 5°C.

[0463] As more CO2 was introduced, the mass increase and pH value of the solution were measured over time. When the equilibrium pressure in the container reached approximately 1 bar, no more CO2 was absorbed (the pressure used under forced carbonation was 3 bar at 5°C).

[0464] Figure 10Shows the measured pH value, which depends on the amount of CO2 added to the carbonated solution. Figure 10 Shows that when a solution containing the whey protein hydrolysate of Sample 16 is carbonated to a CO2 content of 0 - 4 volumes per volume of solution, it results in a pH between 5.5 and 8.25. The pH of the non - carbonated solution is 8.25, and the pH decreases with an increase in the amount of carbonation.

[0465] Figure 10 Shows that adding CO2 to approximately 2.5 volumes (4.9 g / L) / volume of solution causes the pH to drop from 8.25 to approximately 6.0 at 5°C.

[0466] Figure 10 Also shows that the pH reaches a minimum value, approximately 5.5 - 6.0. Therefore, it can be concluded that adding more than 2.5 volumes of CO2 per volume of solution does not lower the pH below approximately 5.5 - 6.0.

[0467] The effect of carbonation on the pH of the solution or beverage was also analyzed by measuring the pH of the following solutions / beverages, which were carbonated to 2.5 volumes of CO2 per volume of solution / beverage:

[0468] - A 4% protein solution prepared by resuspending Sample 16 in water

[0469] - An 8% protein solution prepared by resuspending Sample 16 in water

[0470] - A beverage containing 8% of Sample 16

[0471] The results are as Figure 11 shown.

[0472] The beverage containing 8% of Sample 16 contains the following ingredients:

[0473] Ingredients g / 100g Powder of Sample 16 9.8 Sucrose 2 Sucralose 0.006 Pineapple flavor 0.08 Lime flavor 0.17 Water 88 Carbon dioxide (volume per volume of beverage) 2.5

[0474] Figure 11 Shows that when carbonated to a CO2 content of 2.5 volumes per volume of solution, both the 4% and 8% solutions of Sample 16 and the beverage prepared from Sample 16 have a pH of approximately 6.0.

[0475] Example 17: Analysis of heat treatment of carbonated solution

[0476] As disclosed in Example 16, an 8% protein solution of Sample 16 was carbonated with 2.5 volumes of CO2 per volume of solution.

[0477] The solution was heated to 95°C in a closed container with data recording. The temperature, pH, and turbidity were measured at different heating times, and the results are as Figure 12 shown.

[0478] As Figure 12 shown, the carbonated product remained clear during heating and after 5 minutes at 95 °C, as shown by the measured turbidity and visual inspection. By heating, the pH value remained at 6.2. These data indicate that the carbonated product may be suitable for treatments such as pasteurization and autoclaving, mainly for long - term treatments at temperatures up to 120 °C, for example 20 minutes.

[0479] Example 18: Protein bar

[0480] Example 17 shows an example of the use of the whey protein hydrolysate according to the present invention in the preparation of a protein bar.

[0481] Table 14 is an example of a protein bar with a content of the whey protein hydrolysate (powder) of the present invention of 5 g / 100 g.

[0482] Table 14: Protein bar

[0483] g / 100g Hydrolysate 5 Milk protein 37 Glycerol 4,9 Carbohydrates 39,5 Lipids 6 Flavoring agents 1,75

[0484] Another example of a protein bar is shown in Table 15, but in this example the content of the whey protein hydrolysate (powder) is 13 g / 100 g.

[0485] Table 15: Protein bar

[0486] g / 100g Hydrolysate 13 Milk protein 24 Glycerol 4,9 Carbohydrates 39,5 Lipids 6 Flavoring agents 1,75

[0487] Example 19: Analysis of antioxidant activity using DPPH assay

[0488] The antioxidant effect of the whey protein hydrolysate of the present invention was analyzed by using the DPPH assay, i.e., the free radical scavenging test. This assay measures the antioxidant effect by using DPPH (2,2 - diphenyl - 1 - picrylhydrazyl hydrate) because the color of DPPH changes from purple to yellow after reacting with antioxidant peptides.

[0489] 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 MilliQ water to different protein contents (0, 0.8, 1.5, 2.5, 3.0, 4.0, 5.0 and 6.0% protein) and mixed with an equal volume of DPPH (ratio 1:1). The mixture was incubated at 22 °C for 2 hours to allow the development of yellow due to antioxidant activity. The absorbance at 525 nm was measured and the scavenging percentage was calculated as 100x(A0 - A S ) / A0, where A0 is the absorbance without the sample and A S is the absorbance with the sample.

[0490] Table 16 shows the percentage clearance of the whey protein hydrolysate of the present invention (Sample 16) at different concentrations.

[0491] Table 16:

[0492]

[0493]

[0494] As can be seen from Table 16, the whey protein hydrolysate of Sample 16 has antioxidant activity when included at protein concentrations of 0.8 - 6% in the DPPH assay. As shown in Table 16, the antioxidant activity increases with increasing protein concentration.

Claims

1. A method for preparing a whey protein hydrolyzate, comprising: a) providing a whey protein solution comprising whey protein in an amount of at least 50% by weight based on the total solids content; b) subjecting the whey protein solution to enzymatic hydrolysis, wherein the enzymatic hydrolysis is carried out using any one of the following enzyme combinations; i) comprising at least one serine endopeptidase from Bacillus, at least one serine endopeptidase from Aspergillus, and at least one trypsin-like protein enzyme; ii) comprising at least one serine endopeptidase from Bacillus, at least one serine endopeptidase from Aspergillus, and at least one leucine aminopeptidase from Aspergillus; iii) comprising at least one subtilisin from Bacillus amyloliquefaciens, at least bromelain and at least one leucine aminopeptidase from Aspergillus; c) stopping the enzymatic hydrolysis by inactivating the enzyme when the degree of hydrolysis (DH) is in the range of 20% - 35% to obtain a whey protein hydrolyzate, the whey protein hydrolyzate comprising peptides having a molecular weight of 2500 Da or higher in an amount of 8 - 25% by weight of the total amount of peptides; wherein the method does not include any step of ultrafiltration of the whey protein hydrolyzate obtained in step c), and does not include any other additional treatment for removing bitter peptides from the obtained whey protein hydrolyzate.

2. The method according to claim 1, wherein the method further comprises step d) of concentrating and / or drying the whey protein hydrolyzate obtained in step c).

3. The method according to claim 1 or 2, wherein the whey protein solution in step a) comprises lipids in an amount of at most 10% by weight based on the total solids content.

4. The method according to claim 1, wherein the enzymatic hydrolysis in step b) is carried out at a temperature in the range of 40°C - 75°C.

5. The method according to any one of claims 1 - 2, wherein the inactivation of the enzyme in step c) is by heating to a temperature of at least 80°C.

6. The method according to claim 5, wherein the inactivation of the enzyme in step c) is by heating to a temperature of 80°C - 130°C.

7. The method according to any one of claims 1 - 2, wherein the whey protein solution comprises whey protein concentrate, whey protein isolate, whey protein concentrate, and / or whey protein isolate.

8. The method according to any one of claims 1 - 2, wherein the whey protein solution comprises 2% or more protein of the whey protein solution.

9. The method according to any one of claims 1 - 2, wherein the enzymatic hydrolysis in step b) is carried out using any one of the following enzyme combinations: i. comprising at least one serine endopeptidase from a Bacillus species, at least one serine endopeptidase from Aspergillus oryzae, and at least one microorganism source trypsin-like protease; ii. comprising at least one subtilisin from Bacillus licheniformis, at least one serine endopeptidase from Aspergillus oryzae, and at least one leucine aminopeptidase from Aspergillus oryzae; iii. comprising at least one subtilisin from Bacillus amyloliquefaciens, at least bromelain from pineapple, and at least one leucine aminopeptidase from Aspergillus oryzae.

10. A whey protein hydrolysate prepared by the method according to claim 1, comprising: - free amino acids and peptides, and - having a degree of hydrolysis in the range of 20%-35%, and - peptides with a molecular weight of 2500 Da or higher, in an amount of 8-25% by weight of the total amount of peptides, and - the amount of free amino acids is 15% by weight or less of the total amino acid content in the hydrolysate, and wherein the whey protein hydrolysate in a 4% w / w protein solution has a bitterness score corresponding to a caffeine solution of 0.08% w / v or less.

11. The whey protein hydrolysate according to claim 10, wherein the scattered turbidity (NTU) of the whey protein hydrolysate in a 4% (w / w) protein solution is 100 or less.

12. The whey protein hydrolysate according to any one of claims 10-11, wherein the amount of free amino acids contained in the whey protein hydrolysate is 2-15% by weight of the total protein content in the hydrolysate.

13. The whey protein hydrolysate according to any one of claims 10-11, wherein the whey protein hydrolysate has antioxidant activity.

14. The whey protein hydrolysate according to any one of claims 10-11, wherein the antioxidant activity of the whey protein hydrolysate is measured to have a scavenging percentage of 54-60 in a 1.5% by weight protein solution.

15. Use of the whey protein hydrolysate according to any one of claims 10-14 in a food product.

16. The use according to claim 15, wherein the amount of the whey protein hydrolysate used in the food product corresponds to the food product containing 2-25% by weight of hydrolyzed protein.

17. The use according to any one of claims 15 or 16, wherein the food product is selected from the group consisting of dairy products, beverages, milkshakes, gels, concentrates, and food bars.

18. Use of the whey protein hydrolysate according to any one of claims 10-14 as a food ingredient.

19. Use of the whey protein hydrolysate according to any one of claims 10-14 as a food ingredient in the preparation of a UHT-stable beverage with a pH value of 6.5-8.

0.

20. Use of the whey protein hydrolysate according to any one of claims 10-14 as a food ingredient in the preparation of a beverage used as a sports nutrition product.

21. Use of the whey protein hydrolysate according to any one of claims 10-14 as a food ingredient in the preparation of a clinical beverage.

22. Use of the whey protein hydrolysate according to any one of claims 10-14 as an ingredient in the preparation of a carbonated beverage.

23. Use of the whey protein hydrolysate according to any one of claims 10-14 as an antioxidant.

24. A carbonated beverage comprising the whey protein hydrolysate according to any one of claims 10-14.

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