Peptide manufacturing method

Abstract

The present invention addresses the problem of efficiently producing a specific peptide from a whey protein-containing ingredient. In particular, the present invention addresses the problem of providing: a flavorsome ingredient containing a high level of GTWY and having a low WY content; and a food in which the ingredient is used.　It was found that when hydrolyzing whey protein with protease derived from Aspergillus meleus, maintaining the pH of the reaction solution on the alkali side and performing an enzymatic reaction lasting a relatively short time not only accelerates the cutting out of GTWY from the whey protein but also inhibits degradation of GTWY by the protease, and makes it possible to produce a flavorsome material in which the GTWY content is increased.

Description

Technical Field

[0001] The present invention relates to a method for producing a peptide composition by decomposing a whey protein-containing material with a protein hydrolase. In particular, it relates to a method for efficiently producing a specific peptide composition with good flavor, the specific peptide composition, and foods using the same.

Background Art

[0002] The decline in brain function due to aging and stress causes memory loss, depression, insomnia, dementia, etc., and significantly reduces the quality of life (Quality of life: QOL). In Japan, where high stress and super-aging are progressing, the increase in the number of patients with mental diseases such as depression and dementia has become a social problem. Not only the development of therapeutic drugs for diseases, but also the search for substances that maintain and improve brain function from foods consumed daily from the perspective of preventing diseases is actively carried out.

[0003] So far, as food components expected to have an effect of maintaining and improving brain function, there are reports such as DHA, EPA, L-theanine, tea catechins, and flavonoid glycosides derived from ginkgo leaves. In recent years, reports have also been made on peptides derived from milk proteins. Among milk-derived peptides, glycine-threonine-tryptophan-tyrosine (hereinafter sometimes referred to as GTWY peptide or simply GTWY) has been reported to have an effect of enhancing memory learning ability and cognitive function, and it has been confirmed in a human clinical trial targeting healthy middle-aged and elderly people that memory is maintained and improved (Non-Patent Document 1). Therefore, developing materials and foods with a high content of such peptides is expected to greatly contribute to improving the QOL of middle-aged and elderly people.

[0004] The generation of GTWY from milk proteins has been reported in Non-Patent Document 2 and Patent Document 1. The GTWY sequence is contained in β-lactoglobulin, one of the whey proteins, and is also generated in fermented dairy products such as cheese and yogurt through protein degradation, but the amount is very small, and no effect can be expected from normal intake. There are also reports of obtaining GTWY by hydrolyzing whey protein material using commercially available general-purpose proteases, but generally, commercially available general-purpose proteases do not have strict cleavage specificity and also have side activity, making it difficult to generate and accumulate large amounts of specific peptides. In fact, while a certain amount of GTWY peptide is generated by hydrolysis of whey protein, further degradation into tryptophan-tyrosine (hereinafter sometimes referred to as WY peptide or simply WY) is expected thereafter. Furthermore, methods for concentrating specific peptides include obtaining them using reverse osmosis membranes or ultrafiltration membranes (Patent Documents 2 and 3), and eluting them by adsorption onto column resin (Patent Document 4). However, both of these methods require significant capital investment for industrial production. Thus, there is still no known method for effectively producing materials with a high GTWY content at low cost without special film treatment or fractionation processes.

[0005] Furthermore, it is generally known that peptide materials obtained by hydrolyzing proteins tend to have a stronger bitter or astringent taste compared to the original protein raw material, and therefore, measures are needed to suppress off-flavors when adding them to food. In fact, it has been reported that beverages to which a certain amount of GTWY-containing material has been added exhibit a bitter or astringent taste (Patent Document 1). Thus, when using this material in food, it is desirable to improve its flavor. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] WO2017 / 086303 Brochure [Patent Document 2] Japanese Patent Publication No. 2003-92996 [Patent Document 3] Japanese Patent Application Publication No. 6-7188 [Patent Document 4] Patent No. 3108518 [Non-Patent Document 1] Front. Neurosci., 2019, 13, 399 [Non-Patent Document 2] Neurobiology of Aging,2018,72,23-31 [Disclosure of the Invention] [Problems that the invention aims to solve]

[0007] The present invention aims to provide a method for efficiently producing specific peptides derived from whey protein. In particular, the present invention aims to provide a material and food product using the same that has a high GTWY content, a low WY content, and good flavor. [Means for solving the problem]

[0008] The present inventors have conducted extensive research to solve the above problems and have found that, when hydrolyzing a whey protein-containing material with a protease, maintaining the pH of the reaction solution on the alkaline side and performing the enzymatic reaction for a relatively short time not only promotes the cleavage of specific peptides such as GTWY from whey protein, but also suppresses the degradation of GTWY by the protease, making it possible to produce a flavorful material with a high GTWY content. In other words, the present invention relates to a method for producing a specific peptide composition that can efficiently generate specific peptides such as GTWY. More specifically, the present invention relates to providing a flavorful material with a high GTWY content and a low WY content, and food products using the same. Specifically, the present invention has the following configuration. <1> A method for producing a peptide composition by reacting a whey protein-containing material with a protease, characterized in that the pH of the reaction solution at the start of the reaction in which the protease is reacted is greater than 7.0. <2> The reaction time should be between 5 minutes and less than 5 hours. <1> The manufacturing method described above. <3> Whey protein is β-lactoglobulin. <1> or <2> The manufacturing method described above. <4> The protease is derived from Aspergillus meleus. <1> ~ <3> A manufacturing method described in any of the following. <5> The peptide composition is a GTWY-containing composition. <1> ~ <4> A manufacturing method described in any of the following. <6> The peptide composition is a GTWY-containing composition, having an average molecular weight of 500 or more, and a molar ratio of GTWY to WY of 2.0 or more. <1> ~ <5> A manufacturing method described in any of the following. <7> A method for producing a peptide composition by reacting a whey protein-containing material with a protease, characterized in that the pH of the reaction solution in which the protease is reacted is maintained at 6.6 or higher. <8> The reaction time should be between 5 minutes and less than 5 hours. <7> The manufacturing method described above. <9> Whey protein is β-lactoglobulin. <7> or <8> The manufacturing method described above. <10> The protease is derived from Aspergillus meleus. <7> ~ <9> A manufacturing method described in any of the following. <11> The peptide composition is a GTWY-containing composition. <7> ~ <10> A manufacturing method described in any of the following. <12> The peptide composition is a GTWY-containing composition, having an average molecular weight of 500 or more, and a molar ratio of GTWY to WY of 2.0 or more. <7> ~ <11> A manufacturing method described in any of the following. <13> A method for suppressing the degradation of GTWY in a method for producing a GTWY-containing composition by allowing a protease derived from Aspergillus melleus to act on a whey protein-containing material, characterized in that the degradation of GTWY is suppressed by making the pH in the reaction solution at the start of the reaction where the protease acts greater than 7.0. <14> A peptide composition derived from whey protein, wherein the peptide composition has an average molecular weight of 500 or more. <15> The peptide composition according to <14>, wherein the peptide composition is a GTWY-containing composition and the molar ratio of GTWY to WY is 2.0 or more. <16> A food or drink containing the peptide composition according to <15>, wherein the molar ratio of GTWY to WY in the food or drink is 2.0 or more.

Effects of the Invention

[0009] The production method of the present invention can increase the content of a specific peptide only by adjusting the pH when hydrolyzing a whey protein-containing material with a protease. Therefore, for example, GTWY peptides can be produced selectively and efficiently.

Brief Description of the Drawings

[0010] [Figure 1] It is a diagram showing the change over time of the amount of GTWY at each level when protease treatment is performed using β-lactoglobulin as a substrate. (Example 1) [Figure 2] It is a diagram showing the change over time of the pH at each level when protease treatment is performed using β-lactoglobulin as a substrate. (Example 1)

Modes for Carrying Out the Invention

[0011] The present invention provides a manufacturing method that controls the pH conditions during the enzymatic reaction when hydrolyzing a whey protein-containing material with a protease, thereby increasing the amount of specific peptides produced from whey protein while suppressing the degradation of those specific peptides. Therefore, this manufacturing method makes it possible to provide a material with good flavor and a high content of specific peptides. In particular, the manufacturing method of the present invention is a method that can increase the amount of GTWY produced from whey protein and suppress the degradation of GTWY by controlling the pH conditions during the enzymatic reaction when hydrolyzing a protein material containing the GTWY sequence with a protease derived from Aspergillus meleus. Therefore, this manufacturing method makes it possible to provide a material with good flavor and a high GTWY content. The present invention will be described in detail below.

[0012] (Whey protein-containing material) The raw materials used in the present invention are whey protein-containing materials, and among these, protein materials containing a GTWY sequence are preferably used. Protein materials containing a GTWY sequence are proteins that contain a peptide (GTWY) with an amino acid sequence consisting of glycine-threonine-tryptophan-tyrosine as part of their composition, and typically include materials containing β-lactoglobulin. Protein materials containing a GTWY sequence also include protein degradation products. Examples of materials containing β-lactoglobulin include milk, dairy products, whey, whey powder, whey protein concentrate (WPC), and whey protein isolate (WPI). The concentration of the raw materials when carrying out the protein hydrolysis reaction of the present invention is not particularly limited, but considering the normal manufacturing process, a protein content of about 0.01% to 50% (w / w) is preferred, and more preferably about 0.1% to 20%.

[0013] (Protease) In the present invention, a protease derived from Aspergillus melleus is preferred as the protease used for hydrolysis of the whey protein-containing material. Examples of commercially available enzymes derived from Aspergillus melleus include Protease P "Amano" 3SD (Amano Enzyme), Protease (Protease from Aspergillus melleus (Sigma-A)), and Sumizyme MP (Shin Nippon Chemical Industries). The amount of enzyme added is not particularly limited, but considering appropriate reaction time and manufacturing costs, the enzyme concentration in the reaction solution is preferably about 0.001% to 10%, more preferably 0.01% to 5%, and even more preferably 0.01% to 2%.

[0014] (Enzyme reaction) Our research has revealed that under neutral to alkaline pH conditions during the enzymatic reaction, specific peptides such as GTWY peptides are efficiently produced, and their degradation is suppressed. Generally, it is known that when proteins are hydrolyzed by proteases, the pH drops sharply due to the effect of the produced peptides. Therefore, when hydrolyzing protein solutions of several percent or more for material production, it is difficult to maintain the pH with buffer solutions of about 0.01 to 0.2 M used in typical enzymatic reaction tests. Thus, it is necessary to carry out the enzymatic reaction in a way that maintains the pH at a neutral to alkaline level. The pH at the start of the reaction is preferably greater than pH 7.0, and more preferably pH 7.5 or higher. A preferred range is greater than pH 7.0 and less than or equal to 10.0, and more preferably between pH 7.5 and 9.5. Furthermore, the pH during the reaction is preferably maintained at 6.6 or higher, more preferably at 6.7 or higher, and even more preferably at 7.0 or higher. A preferred range is pH 6.6 to 10.0, more preferably at pH 6.7 to 9.5, and even more preferably at 7.0 to 9.0. Methods for adjusting the pH during the reaction include adjusting the pH to neutral to alkaline with NaOH, KOH, (NH4)CO3, etc., before starting the enzyme reaction; intermittently adding an alkaline solution during the reaction to return the pH to alkaline; or using an alkaline solution as a neutralizing agent to maintain a constant pH while the reaction is carried out.

[0015] The enzyme reaction time should be set according to the amount of the target peptide, and is generally between 5 minutes and 5 hours. In particular, if the target peptide is GTWY, a time is desirable in which a large amount of GTWY is produced and GTWY degradation does not occur, for example, 10 minutes to 4 hours is preferable, and 15 minutes to 3 hours is even more preferable. Furthermore, as long as significant degradation of the specific peptide does not occur after its formation, the enzyme reaction time may be extended, the reaction conditions such as temperature may be changed, or other enzymes may be added to the reaction.

[0016] The reaction temperature can be anywhere from 10°C to 75°C, with 30°C to 65°C being more preferable. Stirring is not required during the reaction, or stirring at 10 to 500 rpm is permitted, or the reaction may be carried out while the liquid is being supplied in the line. After the reaction, the enzyme may be deactivated by heating, removed by ultrafiltration, or deactivated during the process of powdering the reaction solution or processing it into food.

[0017] (Peptide composition) By using the manufacturing method of the present invention, it is possible to efficiently recover specific peptides from raw materials containing whey proteins such as β-lactoglobulin. For example, if the specific peptide is GTWY, it is possible to recover 15-90% of the GTWY from the peptides containing the GTWY sequence in the raw material. Furthermore, since the degradation of GTWY is suppressed according to the manufacturing method of the present invention, the generation of its degradation product, WY, is suppressed, and the molar ratio of GTWY to WY (hereinafter sometimes simply referred to as the GTWY / WY molar ratio, GTWY / WY molar ratio, etc.) can be increased. The GTWY / WY molar ratio of the GTWY-containing composition of the present invention is approximately 2 or more, preferably 3 or more, and more preferably 4 or more. If the GTWY / WY molar ratio is less than 2, it means that more than one-third of the generated GTWY has been degraded, and this is not an efficient method for obtaining GTWY. Furthermore, the average molecular weight of the peptide composition of the present invention is preferably 500 or more, and more preferably 700 or more. Generally, proteins are known to produce peptides and amino acids that exhibit tastes such as bitterness and umami when broken down by proteases, and the more amino acids, dipeptides, and tripeptides with a molecular weight of less than 500 there are, the stronger the taste is perceived to be. By using the manufacturing method of the present invention, it is possible to produce specific peptides such as GTWY while suppressing excessive degradation of whey protein, thereby obtaining a flavorful peptide composition with an average molecular weight of 500 or more. When the peptide composition is a GTWY-containing composition, preferably the GTWY / WY molar ratio is 2 or more and the average molecular weight is 500 or more, more preferably the GTWY / WY molar ratio is 3 or more and the average molecular weight is 500 or more, and most preferably the GTWY / WY molar ratio is 4 or more and the average molecular weight is 700 or more. In materials, food and beverages, pharmaceuticals, etc., using the GTWY-containing composition of the present invention, detection is possible by measuring the amount of GTWY and WY contained and determining their ratio.

[0018] The peptide composition of the present invention can be used as is. Furthermore, the composition can also be used after desalting using a dialysis membrane or ion exchange resin, or after drying by freeze-drying or spray-drying to produce a powder. Furthermore, the peptide composition of the present invention can be used by incorporating it into food and beverages. When incorporating it into food and beverages, the peptide composition of the present invention can be incorporated as is. In addition, the peptide composition can be desalted using a dialysis membrane or ion exchange resin, or it can be dried by freeze-drying or spray-drying to produce a powder, and these can also be incorporated. Foods and beverages containing the peptide composition of the present invention include soft drinks, milk beverages, fermented milk, cheese, butter, skim milk, juice, jelly, bread, ice cream, noodles, sausages, infant formula, and baby food. Furthermore, the peptide composition of the present invention can also be used as a pharmaceutical or functional food. In this case, the peptide composition of the present invention can be processed into oral formulations such as tablets, capsules, granules, powders, pills, lozenges, sublingual preparations, or liquids. [Examples]

[0019] The present invention will be described in more detail below with reference to examples and manufacturing examples, but the present invention is not limited thereto.

[0020] [Example 1] Effect of pH during the enzymatic reaction on the production of GTWY from β-lactoglobulin 1. Test Method The effect of the initial pH (pH at the start of the reaction) on GTWY production was investigated using β-lactoglobulin as a substrate. β-lactoglobulin (SIGMA) was dissolved in 20 mM buffer to a concentration of 8% (w / w), and the pH was adjusted to 6.6, 6.9, 7.1, 7.5, and 7.8. After heating to 50°C, protease P "Amano" 3SD was added to a concentration of 0.05% (w / v) to start the reaction. Samples were taken 30 minutes and 1 hour after the reaction, and the pH and GTWY content of the reaction solution were measured. GTWY was quantified using a liquid chromatography-tandem mass spectrometer (LC-MS / MS) according to the method of Ano et al. (Non-Patent Literature 2).

[0021] 2. Test Results Figure 1 shows the change in the amount of GTWY in the reaction solution over time, and Figure 2 shows the change in pH over time. At an initial pH level of 6.6-7.1, GTWY was generated at a peak 30 minutes after the start of the reaction, and then decreased thereafter, confirming that decomposition occurred along with GTWY generation. In addition, the pH dropped sharply 30 minutes after the start of the reaction to approximately 6.0-6.5. On the other hand, at an initial pH level of 7.5-7.8, the amount of GTWY was high 30 minutes after the start of the reaction, and the amount of GTWY was maintained even 1 hour after the start of the reaction. During the reaction, the pH decreased 30 minutes after the start of the reaction, but remained above 6.6. From these results, it was confirmed that maintaining the pH of the enzyme reaction solution with protease at 6.6 or higher increases the production of GTWY and suppresses its degradation.

[0022] [Example 2] Effect of initial pH of enzyme reaction on GTWY production from WPC 1. Test Method The effect of the initial pH of an enzymatic reaction on GTWY production was investigated using WPC as a substrate. Commercial WPC was dissolved in deionized water heated to 50°C to a concentration of 13% (w / w), and the pH was adjusted to 6.5-9.0 with NaOH. Protease P "Amano" 3SD was added to a concentration of 0.1% (w / v) to start the reaction, and samples were taken 1 hour after the start of the reaction to measure the pH and GTWY content of the reaction solution.

[0023] 2. Test Results Table 1 shows the GTWY levels and pH after the reaction. At initial pH levels of 6.5 and 7.0, the GTWY levels after the reaction were low at 79.8 and 148.8 μg / ml, respectively, and the pH after the reaction decreased to 6.26-6.53. On the other hand, at an initial pH level of 7.7 to 9.0, the GTWY amount was high at 236.8 to 471.7 μg / ml, and the pH after the reaction remained above 6.6, at 6.73 to 7.02. From these results, it was confirmed that by setting the pH at the start of the enzymatic reaction by protease to an alkaline level greater than 7.0, it is possible to maintain a high pH during the enzymatic reaction and increase the amount of GTWY produced.

[0024] [Table 1]

[0025] [Example 3] Effect of pH during enzymatic reaction on GTWY degradation 1. Test Method The effect of pH conditions during the enzymatic reaction on the degradation of GTWY by protease was investigated using GTWY as a substrate. Chemically synthesized GTWY was added to 20 mM phosphate buffer prepared to a pH of 6.4–8.9 at a concentration of 0.2 mg / ml, and 0.01% protease P "Amano" 3SD was added. The reaction was carried out at 50°C for 2 hours. When GTWY was used as the substrate, no decrease in pH due to protein degradation occurred, and no change in pH was observed before and after the reaction. The amounts of GTWY and its degradation product, WY, remaining after the reaction were measured by high-performance liquid chromatography (HPLC).

[0026] 2. Test Results Table 2 shows the measurement results of the amount of GTWY at the start of the reaction, the amount of GTWY and WY after the reaction, and the decomposition rate of GTWY ((amount of GTWY at the start of the reaction - amount of GTWY after the reaction) / amount of GTWY at the start of the reaction). Under reaction conditions with a pH of 6.4, approximately 50% of GTWY was decomposed after 2 hours, producing 81.3 μM of its decomposition product, WY. Furthermore, under conditions where the pH during the reaction was 7.4, approximately 40% of GTWY was decomposed after 2 hours of reaction, and 57.3 μM of its decomposition product, WY, was produced. On the other hand, under conditions of pH 8.1-8.9, the decomposition of GTWY was suppressed to about 9-23%, and the amount of WY produced was also low (8.3-37.5 μM). From these results, it was found that the degradation of GTWY can be suppressed by maintaining the pH of the enzyme reaction solution with protease on the alkaline side.

[0027] [Table 2]

[0028] [Example 4] Prototype of GTWY-rich material by enzyme treatment at a constant pH 1. Test Method Using WPC as the protein material containing the GTWY sequence, a GTWY-containing composition was prepared under conditions where the pH during the enzymatic reaction was kept constant on the alkaline side. Commercially available WPC was dissolved in deionized water to a concentration of 16% (w / w), heated to 50°C, and then the pH was adjusted to 7.5 with NaOH (WPC solution). Protease P "Amano" 3SD was added to the WPC solution to a concentration of 0.1% (w / v) to initiate the reaction. Using a jar fermenter (2L total volume microbial culture system BMJ-02NC; ABLE Corporation), the reaction was carried out at 50°C for 5 hours while adding a neutralizing agent (NaOH) to maintain the pH at 7.5. Samples were taken 1 hour and 5 hours after the start of the reaction, and the amount of GTWY in the reaction solution was measured.

[0029] 2. Test Results The amount of GTWY in the reaction solution was 701.6 μg / ml after 1 hour and 851.6 μg / ml after 5 hours. When these were freeze-dried, the GTWY content was 4.4 mg / g and 5.3 mg / g, respectively. Therefore, a composition with a high GTWY content was obtained.

[0030] [Example 5] Prototype of GTWY-high content material 1. Test Method (1) Test method of the present invention Using WPC as a protein material containing the GTWY sequence, we prototyped a high-GTWY content material under conditions where the pH at the start of the enzymatic reaction was elevated to the alkaline side. Two levels of prototype were performed (Table 3). Commercial WPC was dissolved in deionized water to a concentration of 16% (w / w), heated to 50°C, and then the pH was adjusted to 8.5 with NaOH. Protease P "Amano" 3SD was added at a concentration of 0.06% (w / v) for level 1 and 0.1% (w / v) for level 2. The enzymatic reaction was carried out at 48°C for 2 hours for level 1 and at 50°C for 30 minutes for level 2. After inactivating the enzyme by heating at 90°C for 20 minutes, the samples were freeze-dried to obtain high-GTWY content materials 1 and 2.

[0031] (2) Comparative Example For comparison, a GTWY material was produced using an existing manufacturing method, referencing the method described in Patent Document 1. A commercially available WPC material was dissolved in deionized water to a concentration of 10% (w / w), heated to 50°C, and then the pH was adjusted to 7.0 with NaOH (WPC solution). Protease P "Amano" 3SD was added to the WPC solution to a concentration of 0.2% (w / v), and an enzymatic reaction was carried out at 50°C for 5 hours. After that, the reaction solution was heated at 90°C for 20 minutes to inactivate the enzyme. This reaction solution was freeze-dried to obtain a GTWY-containing material produced by an existing method (GTWY-containing material of the comparative example).

[0032] 2. Test Results Table 3 shows the GTWY content, WY content, GTWY / WY molar ratio, and average molecular weight of each material. The GTWY / WY molar ratio was calculated using a molecular weight of 525.54 for GTWY and 367.41 for WY. The GTWY content in the GTWY-high-content material of the present invention was very high, at 5.1 mg / g for level 1 and 3.4 mg / g for level 2, while the WY content in the material was low, at 0.2 mg / g for level 1 and 0.1 mg / g for level 2. Therefore, the GTWY / WY molar ratio was 17.0 for level 1 and 23.5 for level 2, both showing high values. In addition, the average molecular weight of the GTWY-high-content material was 786 Da for level 1 and 1024 Da for level 2. The comparative example's GTWY material contained 0.8 mg / g of GTWY and 0.3 mg / g of WY, resulting in a GTWY / WY molar ratio of 1.9. The average molecular weight of the material was 309 Da. The pH was 6.2 30 minutes after the start of the enzymatic reaction and 6.0 after 5 hours, suggesting that GTWY degradation occurred during the enzymatic reaction. The results above demonstrate that by using the technology of the present invention, it is possible to provide a material with a high GTWY content, a GTWY / WY molar ratio, and an average molecular weight in a shorter time.

[0033] [Table 3]

[0034] [Example 6] Sensory evaluation test of GTWY-high content material 1. Test Method A comparison of the bitterness of GTWY-rich material 2 prepared in Example 5 and GTWY material produced by an existing manufacturing method (comparative example) was conducted. Nine panelists were asked to taste 20 mg of the material powder placed on their tongues, and their bitterness was evaluated using a four-point scale as shown below. <Bitterness Evaluation Criteria> 3 points: I strongly felt the bitterness. I can taste two points of bitterness. One point: I can detect a slight bitterness. 0 points. I don't taste any bitterness at all.

[0035] 2. Test Results A comparison of the average scores of 10 participants revealed that the comparative GTWY material had an average score of 2.4, indicating a strong bitter taste, while the GTWY-rich material of the present invention had an average score of 1.4, confirming a reduction in bitterness.

[0036] [Table 4]

[0037] [Example 7] Sensory evaluation test of milk to which GTWY high-content material has been added. 1. Test Method A sensory evaluation was conducted on milk to which the GTWY-high content material prepared in Example 5 had been added. A sample was prepared by adding 0.67% of GTWY-high content material 2 to commercially available milk (GTWY content of 2.3 mg / 100g). For comparison, a milk sample to which 1.45% of the GTWY material prepared in the comparative example of Example 5 had been added (GTWY content of 1.1 mg / 100g) and milk without the GTWY material were also prepared. Ten panelists tasted the three samples and evaluated the bitterness and deliciousness using the four-point scale shown below. <Bitterness Evaluation Criteria> 3 points: I strongly felt the bitterness. I can taste two points of bitterness. One point: I can detect a slight bitterness. 0 points. I don't taste any bitterness at all. <Criteria for evaluating deliciousness> 3 out of 5 stars - Delicious 2 points - Slightly not tasty. 1 point - Not very tasty 0 points - Not tasty

[0038] 2. Test Results A comparison of the average scores of 10 participants revealed that the sample containing the GTWY material prepared in the comparative example tasted bitter and received a lower rating for deliciousness. On the other hand, the sample containing GTWY-high-content material 2 showed almost no bitterness, and the difference in deliciousness rating between it and the sample without GTWY material was minimal. This confirms that using GTWY-high-content material makes it possible to consume a high concentration of GTWY without compromising the flavor of the product.

[0039] [Table 5]

[0040] [Example 8] Effect of initial pH of enzymatic reaction on GTWY production from WPC when using different enzymes derived from Aspergillus meleus. 1. Test Method To investigate the enzymes usable in the patented technology, we used a protease derived from Aspergillus melleus (Sigma-A) that was different from the protease P "Amano" 3SD used in Example 1, and examined the effect of the initial pH of the enzymatic reaction on GTWY production. Commercially available WPC was dissolved in deionized water heated to 50°C to a concentration of 13% (w / w), and the pH was adjusted to 6.5-9.0 with NaOH. Protease from Aspergillus melleus (Sigma-A) was added to a concentration of 0.1% (w / v) to initiate the reaction. Two hours after the start of the reaction (after the reaction), a sample was taken, and the pH and GTWY content of the reaction solution were measured.

[0041] 2. Test Results Table 6 shows the initial pH and the amount of GTWY and pH after the reaction. Similar to the case using protease P "Amano" 3SD, it was confirmed that the amount of GTWY produced increased as the initial pH became more alkaline. Therefore, it was confirmed that in this invention, proteases derived from Aspergillus meleus can be widely used without any restrictions on type.

[0042] [Table 6]

[0043] [Example 9] Preparation of GTWY-containing capsules In Example 5, 10 g of the high-GTWY-content material 2 was prepared, and 33.5 g of lactose, 56.0 g of soluble starch, and 0.5 g of magnesium stearate were mixed together. The mixture was then granulated by a conventional method and filled into capsules to produce the GTWY-containing capsule of the present invention.

[0044] [Example 10] Preparation of a GTWY-containing beverage 10 g of the GTWY-high-content material 2 prepared in Example 5 was dissolved in 690 g of deionized water, heated to 40°C, and then stirred and mixed for 20 minutes at 9,500 rpm using an ultra-disperser (ULTRA-TURRAX T-25; manufactured by IKA Japan). After adding 100 g of maltitol, 2 g of acidulant, 20 g of reduced starch syrup, 2 g of flavoring, and 176 g of deionized water, the mixture was filled into 100 ml glass bottles, sterilized at 95°C for 15 seconds, and then sealed to prepare 10 bottles (100 ml each) of the GTWY-containing beverage of the present invention.

[0045] [Example 11] Preparation of a GTWY-containing milk beverage 100g of the GTWY-high-content material 2 prepared in Example 5 was mixed with 9.9kg of milk, heated to 40°C, and then stirred and mixed for 10 minutes at 6,000rpm using a TK homomixer (TK ROBO MICS; manufactured by Tokushu Kika Kogyo Co., Ltd.). After heat sterilization at 130°C for 2 seconds, it was cooled to below 10°C to produce 10kg of the GTWY-containing milk beverage of the present invention.

[0046] [Example 12] Preparation of GTWY-containing fermented milk 100g of the GTWY-rich material 2 prepared in Example 5, 1600g of skim milk powder, 300g of glucose, and 7700g of deionized water were mixed and heat-sterilized by holding at 95°C for 2 hours. This was cooled to 37°C, 300g of lactic acid bacteria starter (Lb. casei) was inoculated, and after stirring and mixing, fermentation was carried out in an incubator maintained at 37°C until the pH reached 4.0. After reaching pH 4.0, it was cooled to below 10°C to produce 10kg of the GTWY-containing fermented milk of the present invention.

[0047] [Example 13] Preparation of GTWY-containing cheese 9.5 kg of Gouda cheese, 9.5 kg of Cheddar cheese, 100 g of the GTWY-high-content material 2 prepared in Example 5, 200 g of sodium citrate, and 700 g of deionized water were mixed and emulsified at 85°C. After emulsification, the cheese was filled into cartons and cooled at 5°C for two days and nights to produce 20 kg of the GTWY-containing cheese of the present invention.

[0048] [Example 14] Preparation of GTWY-containing cream An oil phase was prepared by mixing 4.5 kg of hydrogenated rapeseed oil, 40 g of lecithin, 10 g of monoglycerin fatty acid ester, and 10 g of sorbitan fatty acid ester. Next, an aqueous phase was prepared by mixing 100 g of the GTWY-high-content material 2 prepared in Example 5, 308 g of skim milk powder, 10 g of sodium caseinate, 20 g of sugar ester, 10 g of phosphate, 5 g of xanthan gum, and 4.987 kg of deionized water. The aqueous phase was heated to 65°C, and the oil phase, heated to 70°C, was added little by little while stirring. The mixture was then stirred and mixed at 6,000 rpm for 10 minutes using a TK homomixer (TK ROBO MICS; manufactured by Tokushu Kika Kogyo Co., Ltd.). This mixture was homogenized using a homogenizer to produce 10 kg of the GTWY-containing cream of the present invention.

[0049] [Example 15] Preparation of GTWY-containing purine 2000g of honey, 100g of the GTWY-high-content material 2 prepared in Example 5, 770g of skim milk powder, 300g of mascarpone cheese, 700g of liquid starch syrup, 500g of granulated sugar, 250g of fresh cream, 200g of butter, 400g of sweetened egg yolk, 40g of gelatin, 15g of agar, 120g of locust bean gum, and 4605g of deionized water were mixed to make a pudding mix. This pudding mix was stirred and mixed at 6,000 rpm for 10 minutes using a TK Homomixer (TK ROBO MICS; manufactured by Tokushu Kika Kogyo Co., Ltd.), heated to 60°C to dissolve, and then filled into containers in 100g portions and cooled to produce 100 GTWY-containing puddings of the present invention.

[0050] [Example 16] Preparation of GTWY-containing milk powder In Example 5, 1 kg of the high-GTWY content material 2 was prepared, and 8.3 kg of skim milk powder and 90 kg of deionized water were mixed. After heating to 40°C, the mixture was stirred and mixed for 10 minutes at 6,000 rpm using a TK homomixer (TK ROBO MICS; manufactured by Tokushu Kika Kogyo Co., Ltd.). This solution was spray-dried to produce approximately 10 kg of the GTWY-containing milk powder of the present invention.

[0051] [Example 17] Prototype of a soft drink containing GTWY A GTWY-containing soft drink was prototyped by enzymatic treatment during the manufacturing process of the soft drink. 16g of commercially available WPC was dissolved in 84g of distilled water, heated to 50°C, and the pH was adjusted to 8.5 with NaOH. Protease P "Amano" 3SD was added to a concentration of 0.1% (w / v), and the enzymatic reaction was carried out at 50°C for 30 minutes. In a separate container, 10g of skim milk powder, 100g of sugar, 5g of citric acid, 2.5g of trisodium citrate, and 5g of pectin were dissolved in 778ml of distilled water. This was mixed with the enzymatically treated WPC solution, sterilized by heating at 90°C for 20 minutes, and then filled into 100ml plastic bottles. [Industrial applicability]

[0052] According to the manufacturing method of the present invention, the content of specific peptides can be increased simply by adjusting the pH when hydrolyzing whey protein-containing material with protease, thereby selectively concentrating peptides. Furthermore, since it does not require expensive equipment, it can be easily introduced into factories. Moreover, because the enzymatic reaction time is short, production efficiency is high, and continuous production on the manufacturing line is possible. Furthermore, the GTWY-containing composition of the present invention efficiently generates GTWY while suppressing excessive protein degradation, resulting in an average molecular weight of 500 or more and a good flavor. Therefore, it can be added to food and beverages to increase the peptide content without adversely affecting the original flavor of the food or beverage, making it highly valuable for various uses.

Claims

1. A GTWY-containing composition derived from whey protein, wherein the average molecular weight is 500 or more, and the molar ratio of GTWY to WY is 17.0 or more.

2. Food and beverages comprising the GTWY-containing composition described in claim 1, wherein the molar ratio of GTWY to WY in the food and beverage is 17.0 or more.

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