AU2022353630A1

AU2022353630A1 - Method for producing peptide

Info

Publication number: AU2022353630A1
Application number: AU2022353630A
Authority: AU
Inventors: Kazuki Maeyama; Nana Suzuki; Tomohiro Tsuda; Hiroshi Uenishi; Norihiro Yokoyama
Original assignee: Megmilk Snow Brand Co Ltd
Current assignee: Megmilk Snow Brand Co Ltd
Priority date: 2021-09-30
Filing date: 2022-09-29
Publication date: 2024-04-11
Also published as: JPWO2023054594A1; KR20240070539A; CN118043335A; TW202323532A; WO2023054594A1

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

METHOD FOR PRODUCING PEPTIDE

Technical Field

[0001]

The present invention relates to a method for producing a peptide composition by

degrading a whey protein-containing material with a protein hydrolase. In particular, the

present invention relates to a specific peptide composition with good flavor, a method for

efficiently producing the same, and a food product using the same.

Background Art

[0002]

Deterioration of brain function due to aging and stress causes a decline in memory,

depression, insomnia, dementia, etc., and significantly reduces the quality of life (QOL).

In Japan, where stress levels are high and the population is rapidly aging, the increasing

number of people suffering from mental illnesses such as depression and dementia has

become a social issue. In addition to the development of therapeutic drugs for such

illnesses, the search for substances that can be ingested from the foods we consume daily

and can maintain and improve brain function is actively underway from the perspective of

preventing the illnesses.

[0003] So far, DHA, EPA, L-theanine, tea catechins, flavonoid glycosides from Ginkgo

biloba, and the like have been reported as food ingredients that may help maintain and

improve brain function. In recent years, peptides derived from milk protein have also been

reported. Among milk-derived peptides, glycine-threonine-tryptophan-tyrosine (GTWY)

peptide has been reported to enhance memory, learning ability, and cognitive function and

has been confirmed to maintain and improve memory in human clinical trials on healthy

middle-aged and elderly individuals (Non-Patent Literature 1). Thus, the development of

materials and food products with a high content of the peptide can be expected to greatly

contribute to improving the QOL of middle-aged and elderly people.

[0004]

The production of GTWY from milk protein has been reported in Non-Patent

Literature 2 and Patent Literature 1. GTWY sequences are found in P-lactoglobulin, a type of whey protein, and are also produced by protein degradation in fermented dairy products such as cheese and yogurt; however, the amount is so small that no effect is expected with normal dairy consumption. There has also been a report that GTWY is obtained by hydrolyzing a whey protein material with a commercially available general-purpose protease. However, commercially available general-purpose proteases generally lack strict cleavage specificity and have side activities, making it difficult to produce and accumulate a large amount of a specific peptide. Although GTWY peptides are indeed produced to some extent by hydrolysis of whey protein, they would then be degraded to a tryptophan tyrosine (WY) peptide or the like.

In addition, there have been proposed methods to obtain a specific peptide using a

reverse osmosis membrane or an ultrafiltration membrane (Patent Literatures 2 and 3) and

methods to adsorb specific peptides onto column resin and elute them (Patent Literature

4). These methods may be used to concentrate specific peptides; however, they require

expensive equipment investment for industrial production.

Thus, there is still no known method for effectively producing a high GTWY content

material at low cost without special membrane treatment or fractionation treatment.

[0005]

Furthermore, it is known that peptide materials produced by protein hydrolysis

generally have a more bitter and harsh taste compared to the original protein material, and

it is necessary to find a way to reduce the strange taste when adding such peptide material

to food. In fact, it has been reported that a beverage to which a certain amount of GTWY

containing material is added has a bitter and harsh taste (Patent Literature 1). Thus, for the

use of peptide materials in food products, it is also desired to improve their flavor.

Citation List

[0006] Patent Literature

Patent Literature 1: PCT International Publication No. W02017/086303

Patent Literature 2: Japanese Patent Application Publication No. 2003-92996

Patent Literature 3: Japanese Patent Application Publication No. H06-7188

Patent Literature 4: Japanese Patent No. 3108518

Non-Patent Literature

Non-Patent Literature 1: Front. Neurosci., 2019, 13, 399

Non-patent Literature 2: Neurobiology of Aging, 2018, 72, 23-31

Summary of Invention

Technical Problem

[0007]

An object of the present invention is to provide a method for efficiently producing a

specific peptide derived from whey protein. In particular, an object of the present invention

is to provide a material with a high content of GTWY, a low content of WY, and good

flavor as well as a food product using the material.

Solution to Problem

[0008] As a result of intensive studies to achieve the above objects, the present inventors

have found that when a whey protein-containing material is hydrolyzed with a protease,

maintaining the pH of a reaction solution on the alkaline side and carrying out an

enzymatic reaction in a relatively short time promote the cleavage of a specific peptide

such as GTWY from whey protein and suppress the degradation of GTWY by the protease,

thereby making it possible to produce a material with an increased GTWY content and

good flavor. That is, the present invention relates to a method for producing a specific

peptide composition, which allows a specific peptide such as GTWY to be efficiently

produced. Particularly, the present invention relates to the provision of a material with a

high content of GTWY, a low content of WY, and good flavor as well as a food product

using the material. Specifically, the present invention has the following configurations:

<1> A method for producing a peptide composition, wherein the peptide composition is

produced by reaction of a protease with a whey protein-containing material, and a reaction

solution has a pH of more than 7.0 at a start of the reaction of the protease.

<2> The method according to <1>, wherein the reaction is carried out for 5 minutes or

more and 5 hours or less.

<3> The method according to <1> or <2>, wherein the whey protein is P-lactoglobulin. <4> The method according to any one of<1> to <3>, wherein the protease is derived from

Aspergillus melleus.

<5> The method according to any one of <1> to <4>, wherein the peptide composition is

a GTWY-containing composition.

<6> The method according to any one of <1> to <5>, wherein 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> A method for producing a peptide composition, wherein the peptide composition is

produced by reaction of a protease with a whey protein-containing material, and a pH of

6.6 or more is maintained in a reaction solution in which the reaction of the protease takes

place.

<8> The method according to <7>, wherein the reaction is carried out for 5 minutes or

more and 5 hours or less.

<9> The method according to <7> or <8>, wherein the whey protein is P-lactoglobulin. <10> The method according to any one of <7> to <9>, wherein the protease is derived

from Aspergillus melleus.

<11> The method according to any one of <7> to <10>, wherein the peptide composition

is a GTWY-containing composition.

<12> The method according to any one of <7> to <11>, wherein 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.

<13> A method for suppressing degradation of GTWY in a method for producing a

GTWY-containing composition, wherein the GTWY-containing composition is produced

by reaction of a protease derived from Aspergillus melleus with a whey protein-containing

material, and the degradation of GTWY is suppressed by setting a pH in a reaction solution

at a start of the reaction of the protease to be more 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 having a molar ratio of GTWY to WY of 2.0 or more.

<16> A food or drink product, comprising the peptide composition according to <15>,

wherein the food or drink product has a molar ratio of GTWY to WY of 2.0 or more.

Advantageous Effects of Invention

[0009] The method of the present invention can increase the content of a specific peptide

simply by adjusting the pH when a whey protein-containing material is hydrolyzed with a

protease. Accordingly, it is possible to produce, for example, a GTWY peptide selectively

and efficiently.

Brief Description of Drawings

[0010]

[FIG. 1] FIG. 1 is a diagram illustrating changes over time in the amount of GTWY for

each level when protease treatment was performed using p-lactoglobulin as a substrate.

(Example 1)

[FIG. 2] FIG. 2 is a diagram illustrating changes over time in the pH for each level when

protease treatment was performed using p-lactoglobulin as a substrate. (Example 1)

Description of Embodiments

[0011]

A method of the present invention can increase the amount of a specific peptide

produced from whey protein and suppress degradation of the specific peptide by

controlling a pH condition during an enzymatic reaction when a whey protein-containing

material is hydrolyzed with a protease. Therefore, according to the present method, it is

possible to provide a material with good flavor and a high content of a specific peptide.

Particularly among these, the method of the present invention can increase the amount

of GTWY produced from whey protein and suppress degradation of GTWY by controlling

a pH condition during an enzymatic reaction when a protein material containing a GTWY

sequence is hydrolyzed with a protease derived from Aspergillus melleus. Therefore, the

present method is capable of providing a material with good flavor and a high GTWY

content.

[0012]

(Whey protein-containing material)

In an embodiment of the invention, whey protein-containing materials are used as

source materials. Among these, a protein material containing a GTWY sequence is

preferably used. The protein material containing a GTWY sequence refers to a protein that contains, as part of its structure, a peptide (GTWY) having an amino acid sequence consisting of glycine-threonine-tryptophan-tyrosine. A typical example thereof is a material containing P-lactoglobulin. The protein material containing a GTWY sequence also includes a protein degradation product. Examples of the material containing p lactoglobulin include milk, dairy products, whey, whey powder, whey protein concentrate

(WPC), and whey protein isolate (WPI). Although the concentration of the source material

used in the protein hydrolysis reaction of the embodiment is not particularly limited, the

protein amount is preferably about 0.01% to 50% (w/w), more preferably about 0.1% to

20%, in view of the normal production process.

[0013]

(Protease)

In an embodiment of the invention, a protease derived from Aspergillus melleus may

preferably be used to hydrolyze the whey protein-containing material. As the protease

derived from Aspergillus melleus, commercially available enzymes such as Protease P

"Amano" 3SD (Amano Enzyme Inc.), "Proteinase from Aspergillus melleus" (Sigma), and

Sumiteam MP (SHINNIHON CHEMICALS Corporation) are known. The amount of

enzyme added to a reaction solution is not particularly limited, but the reaction solution

preferably has an enzyme concentration of about 0.001% to 10%, more preferably 0.01%

to 5%, further preferably 0.01% to 2%, in view of an appropriate reaction time, production

cost, etc.

[0014]

(Enzymatic reaction)

Through the studies conducted by the present inventors, it has been found that specific

peptides such as GTWY peptides are efficiently produced and their degradation is

suppressed under the condition that the pH is neutral to alkaline during an enzymatic

reaction. Generally, it is known that when a protein is hydrolyzed with a protease, the pH

rapidly decreases due to the influence of peptides produced. Hence, it is difficult to

maintain the pH with a buffer solution of about 0.01 to 0.2 M used in normal enzymatic

reaction tests when a protein solution containing several percent or more of protein is

hydrolyzed to produce a material. Therefore, it is necessary to carry out an enzymatic

reaction in such a way that the pH is maintained at neutral to alkaline during the reaction.

The pH at the start of the reaction is preferably more than 7.0, more preferably 7.5 or more. The preferred range thereof is preferably more than 7.0 and 10.0 or less, more preferably 7.5 or more and 9.5 or less.

Further, the pH during the reaction is preferably maintained at 6.6 or more, more

preferably 6.7 or more, and even more preferably 7.0 or more. The preferred range thereof

is preferably 6.6 or more and 10.0 or less, more preferably 6.7 or more and 9.5 or less, and

further more preferably 7.0 or more and 9.0 or less.

The pH may be adjusted during the enzymatic reaction as follows: the pH at the start

of the reaction is adjusted to neutral or alkaline with NaOH, KOH, (NH 4 )CO 3 , or the like

before the reaction starts; an alkaline solution is added intermittently during the reaction

to bring the pH back to alkaline; or the pH is maintained constant using an alkaline solution

as a neutralizing agent while the reaction is carried out.

[0015]

The enzymatic reaction may be carried out in a period of time which is set depending

on the desired amount of a specific peptide and is preferably carried out for about 5 minutes

to 5 hours. When the specific peptide is GTWY, the enzymatic reaction time is preferably

set such that a large amount of GTWY is produced and the degradation of GTWY does

not occur. For example, the enzymatic reaction time is preferably 10 minutes to 4 hours,

more preferably 15 minutes to 3 hours.

As long as the specific peptide does not undergo significant degradation after the

production thereof, the enzymatic reaction time may be extended; the reaction may be

carried out under different conditions by changing, e.g., the temperature; or the reaction

may be carried out by adding another enzyme.

[0016]

The reaction temperature may be about 10°C to 75°C, more preferably 30°C to 65°C.

The reaction may be carried out without stirring or with stirring at about 10 to 500 rpm, or

the reaction may be carried out while the reaction solution is being fed through the line.

After the reaction, the enzyme may be inactivated by heating, removed by ultrafiltration

membrane treatment, or inactivated during the process of powdering the reaction solution

or processing it into food.

[0017]

(Peptide composition)

By using the method of the present invention, it becomes possible to efficiently obtain specific peptides from a source material containing whey protein such as P-lactoglobulin. For example, when the specific peptide is GTWY, it is possible to recover 15 to 90% of

GTWY of the peptides containing GTWY sequences in the source material. Furthermore,

according to the method of the present invention, since the degradation of GTWY is

suppressed, the production of WY, which is a degradation product of GTWY, is

suppressed, so that the molar ratio of GTWY to WY (hereinafter also simply referred to

as the molar ratio of GTWY/WY, the GTWY/WY molar ratio, and the like) can be

increased. In an embodiment of the invention, the GTWY/WY molar ratio of a GTWY

containing composition is about 2 or more, preferably 3 or more, and more preferably 4 or

more. When the molar ratio of GTWY/WY is less than 2, it means that one-third or more

of the produced GTWY has undergone the degradation, which is not an efficient way to

obtain GTWY.

Further, the peptide composition of the present invention preferably has an average

molecular weight of 500 or more, and more preferably 700 or more. Generally, it is known

that proteins produce peptides and amino acids that have tastes such as bitterness and

umami as they are degraded by proteases, and as the number of amino acids, dipeptides,

and tripeptides with a molecular weight of less than 500 increases, the tastes become

stronger.

Since using the method of the present invention makes it possible to produce a specific

peptide such as GTWY while suppressing excessive degradation of whey protein, it is

possible to obtain a flavorful peptide composition with an average molecular weight of

500 or more.

When the peptide composition is a GTWY-containing composition, it preferably has

a GTWY/WY molar ratio of 2 or more and an average molecular weight of 500 or more;

more preferably a GTWY/WY molar ratio of 3 or more and an average molecular weight

of 500 or more; and most preferably a GTWY/WY molar ratio of 4 or more and an average

molecular weight of 700 or more.

For materials, foods, beverages, pharmaceuticals, and the like using the GTWY

containing composition of the present invention, the GTWY/WY molar ratio can be

determined by measuring the amounts of GTWY and WY contained and calculating the

ratio.

[0018]

The peptide composition of the present invention can be used as is. In addition, the

peptide composition can also be utilized after being desalted using a dialysis membrane,

an ion exchange resin, or the like, or after being dried and powdered by freeze drying,

spray drying, or the like.

Further, the peptide composition of the present invention can be used by blending it

into a food or drink. When blended into a food or drink, the peptide composition of the

present invention can be blended as is. In addition, the peptide composition can also be

utilized after being desalted using a dialysis membrane, an ion exchange resin, or the like,

or after being dried and powdered by freeze drying, spray drying or the like.

Examples of 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 utilized as

a pharmaceutical or functional food. In this case, the peptide composition of the present

invention can be processed into a preparation for oral administration such as a tablet,

capsule, fine granule, powder, pill, troche, sublingual preparation, or liquid preparation

for use.

Example

[0019]

Hereinafter, the present invention will be explained in more detail by presenting

Examples and Production Examples of the present invention, but the present invention is

not limited thereto.

[0020]

[Example 1] Influence of pH during enzymatic reaction on the production of GTWY from

p-lactoglobulin 1. Test method

Using p-lactoglobulin as a substrate, the influence of the initial pH of an enzymatic

reaction (the pH at the start of the reaction) on the production of GTWY was examined.

p-lactoglobulin (Sigma) was dissolved in a 20mM buffer solution 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 the resultant

solution to 50°C, Protease P "Amano" 3SD was added to a concentration of 0.05% (w/v) to start the reaction. Samples were collected at 30 minutes and 1 hour after the start of the reaction to measure the pH and GTWY content of the reaction solution. GTWY was measured in accordance with the method of Ano et al. (Non-Patent Literature 2) and quantified using a liquid chromatograph-tandem mass spectrometer (LC-MS/MS).

[0021] 2. Test results FIG. 1 illustrates changes over time in the amount of GTWY in the reaction solution. FIG. 2 illustrates changes over time in the pH of the reaction solution. At the initial pH levels of 6.6 to 7.1, GTWY production peaked at 30 minutes after the start of the reaction and then decreased, confirming that the degradation occurred along with the GTWY production. Further, the pHs rapidly decreased and were about 6.0 to 6.5 at 30 minutes after the start of the reaction. On the other hand, at the initial pH levels of 7.5 to 7.8, a large amount of GTWY was produced at 30 minutes after the start of the reaction, and the production level was maintained even 1 hour after the start of the reaction. Although the pHs decreased in 30 minutes after the start of the reaction, they remained at 6.6 or more throughout the reaction. The above results have confirmed that maintaining the pH of the protease enzymatic reaction solution at 6.6 or more can increase the production of GTWY and suppress the degradation of GTWY.

[0022]

[Example 2] Influence of the initial pH of enzymatic reaction on the production of GTWY from WPC 1. Test method Using WPC as a substrate, the influence of the initial pH of an enzymatic reaction on the production of GTWY was examined. A commercially available WPC was dissolved in ion-exchanged water heated to 50°C to a concentration of 13% (w/w), and the pH was adjusted to 6.5 to 9.0 with NaOH. Protease P "Amano" 3SD was added thereto to a concentration of 0.1% (w/v) to start the reaction. Samples were collected at 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 amounts of GTWY and the pHs after the reaction. At the initial pH levels of 6.5 and 7.0, the amounts of GTWY after the end of the reaction were as low as

79.8 and 148.8 tg/mL, respectively. The pHs decreased to 6.26 to 6.53 after the end of the

reaction.

On the other hand, at the initial pH levels of 7.7 to 9.0, the amounts of GTWY were

as high as 236.8 to 471.7 tg/ml. The pHs after the reaction were 6.73 to 7.02, indicating

that the pHs were maintained at 6.6 or more.

The above results have confirmed that setting the pH at the start of the enzymatic

reaction with a protease to the alkaline side of 7.0 can maintain the pH high during the

enzymatic reaction, thereby increasing the amount of GTWY produced.

[0024]

[Table 1]

Level Initial pH pH after reaction GTWY amount after reaction (pg/mL) T 6.5 6.26 79.8 7.0 6.53 148.8 7.7 6.73 236.8 8.0 6.80 251.5 9.0 7.02 471.7

[0025]

[Example 3] Influence of pH during enzymatic reaction on the degradation of GTWY

1. Test method

Using GTWY as a substrate, the influence of the pH condition during an enzymatic

reaction on the degradation of GTWY by a protease was examined. Chemically

synthesized GTWY was added to a concentration of 0.2 mg/ml to a 20 mM phosphate

buffer prepared to a pH of 6.4 to 8.9, and Protease P "Amano" 3SD was added at 0.01%,

and the reaction was carried out at 50°C for 2 hours. When GTWY was used as a substrate,

no decrease in pH due to protein degradation occurred, and no change in pH was observed

before and after the reaction. The amount of GTWY remaining after the reaction and the

amount of its degradation product WY were measured using high performance liquid

chromatography (HPLC).

[0026]

2. Test results

Table 2 shows the measurement results of the amounts of GTWY at the start of the reaction, the amounts of GTWY after the reaction, and the amounts of WY after the reaction as well as the degradation rate of GTWY ((the amount of GTWY at the start of the reaction - the amount of GTWY after the reaction)/the amount of GTWY at the start of the reaction).

Under the condition that the pH during the reaction was 6.4, about 50% of GTWY

was degraded in 2 hours of reaction, and 81.3 M of WY, a degradation product thereof,

was produced.

Under the condition that the pH during the reaction was 7.4, about 40% of GTWY

was degraded in 2 hours of reaction, and 57.3 M of WY, a degradation product thereof,

was produced.

On the other hand, under the conditions that the pHs were 8.1 to 8.9, the degradation

of GTWY was suppressed to about 9 to 23%, and the amounts of WY produced were small

(8.3 to 37.5 [M).

From the results above, it has been found that maintaining the pH of the protease

enzymatic reaction solution on the alkaline side can suppress the degradation of GTWY.

[0027]

[Table 2]

W C)V C) Cf)

~4-1

CntU C cr) Lfl

U4 CD]C

07) q~ 0 ,1

r__ Cq1- C) C-1

0 0 ;0

[0028]

[Exaple]TralpoducionfahghGW~cotenmatriabyenymereamenwit aconstantp 1.Tesmetho

UsigW~aapotinatrilcntinngheTW seueceaG WY

contle]Trainno poictionapoducedhGT ontilndertteodionhatet enduring

enzymatic reaction was kept constant on the alkaline side. A commercially available WPC was dissolved in ion-exchanged water to a concentration of 16% (w/w). The resultant solution was heated to 50'Cand then adjusted to pH 7.5 with NaOH to obtain a WPC solution. Protease P "Amano" 3SD was added to the WPC solution to a concentration of 0.1% (w/v) to start the reaction. The reaction was carried out at 50°C for 5 hours using a jar fermenter (microbial culture system BMJ-02NC with a total volume of 2L, manufactured by ABLE Corporation) with the addition of a neutralizing agent (NaOH) to maintain the pH at 7.5. Samples were collected at 1 hour and

5 hours after the start of the reaction to measure the amount of GTWY in the reaction

solution.

[0029]

2. Test results

The amount of GTWY in the reaction solution was 701.6 g/ml after 1 hour and 851.6

ptg/ml after 5 hours. When these samples were freeze-dried, their respective GTWY

contents were 4.4 mg/g and 5.3 mg/g. Thus, a composition containing a high amount of

GTWY was obtained.

[0030]

[Example 5] Trial production of a high GTWY content material

1. Test method

(1) Test method of the present invention

Using WPC as a protein material containing the GTWY sequence, a high GTWY

content material was produced on trial under the condition that the pH at the start of an

enzymatic reaction was raised to the alkaline side. The trial production was conducted at

two levels (Table 3). A commercially available WPC was dissolved in ion-exchanged

water to a concentration of 16% (w/w). The resultant solution was heated to 50°C, and

then adjusted to pH 8.5 with NaOH. Protease P "Amano" 3SD was added thereto at 0.06%

(w/v) for level 1 and 0.1% (w/v) for level 2 to prepare reaction solutions for levels 1 and

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. The reaction solutions were heated at 90°C for 20 minutes to

inactivate the enzyme and then freeze-dried to obtain a high GTWY content material 1 and

a high GTWY content material 2.

[0031]

(2) Comparative example

For comparison, a GTWY material was produced by an existing production method

with reference to the method of Patent Literature 1. A commercially available WPC material was dissolved in ion-exchanged water to a concentration of 10% (w/w). The resultant solution was heated to 50°C and then adjusted to pH 7.0 with NaOH to obtain a WPC solution. Protease P "Amano" 3SD was added to the WPC solution to a concentration of 0.2% (w/v) to carry out an enzymatic reaction at 50°C for 5 hours. Subsequently, 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 production method (a 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 assuming the molecular weight of GTWY to be 525.54 and the molecular weight of WY to be 367.41. The high GTWY content material of the present invention had a very high GTWY content of 5.1 mg/g for level 1 and 3.4 mg/g for level 2, and a low WY content of 0.2 mg/g for level 1 and 0.1 mg/g for level 2. Accordingly, the GTWY/WY molar ratio was 17.0 for level 1 and 23.5 for level 2, both of which were high values. Furthermore, the high GTWY content material had an average molecular weight of 786 Da for level 1 and 1024 Da for level 2. The GTWY material of the comparative example had a GTWY content of 0.8 mg/g and a WY content of 0.3 mg/g, and its GTWY/WY molar ratio was 1.9. This material had an average molecular weight of 309 Da. The pH at 30 minutes after the start of the enzymatic reaction was 6.2 and the pH at 5 hours after the start was 6.0, indicating that the degradation of GTWY occurred during the enzymatic reaction. The above results have demonstrated that using the technology of the present invention can provide a material with a high GTWY content, a high GTWY/WY molar ratio, and a high average molecular weight in a shorter time.

[0033]

[Table 3]

46-J

4--j

N t-4 Cr

0) 4-J

to CZI 0

o

00 CI

0

4 (U N CD

a) C

> U 000

to to 0

-= N

0) 0)

[0034

[Example~~S 6] Sesr vlaints ftehg TYcnetmtra

1. Test method

The high GTWY content material 2 prepared in Example 5 and the GTWY material

produced by an existing production method (the comparative example) were compared in

terms of bitterness. Nine panelists were asked to place 20 mg of the powdered material on

their tongues, taste it, and rate its bitterness on a four-point scale based on the following

criteria:

3 points: Very bitter

2 points: Bitter

1 point: Slightly bitter

0 points: Not bitter

[0035]

2. Test results

As a result of comparison of the average scores by the ten panelists, it was confirmed

that the GTWY material of the comparative example, whose average score was 2.4 points,

had a strong bitterness, whereas the high GTWY content material of the present invention,

whose average score was 1.4 points, had less bitterness.

[0036]

[Table 4] Sensory evaluation results High GTWY content GTWYmaterialofthe Raw material WPC material 2 of the GTWYamatereale present invention comparativeexample Bitterness score o 1.4 (average) 2.4

[0037]

[Example 7] Sensory evaluation test of milk to which the high GTWY content material is

added 1. Test method

A sensory evaluation was conducted on milk to which the high GTWY content

material prepared in Example 5 was added. The milk was prepared by adding 0.67% of the

high GTWY content material 2 to commercially available milk (GTWY content: 2.3

mg/100 g). For comparison, a milk sample to which 1.45% of the GTWY material prepared in the comparative example of Example 5 was added (GTWY content: 1.1 mg/100 g), and milk to which no GTWY material was added were prepared. Ten panelists were asked to drink the three samples, and rate its bitterness and tastiness on a four-point scale as below:

3 points: Very bitter

2 points: Bitter

1 point: Slightly bitter

0 points: Not bitter

3 points: Tasty

2 points: Slightly less tasty

1 point: Less tasty

0 points: Not tasty

[0038] 2. Test results

As a result of comparison of the average scores by the ten panelists, the sample to

which the GTWY material prepared in the comparative example was added had bitterness

and had a lower score for tastiness. On the other hand, the sample to which the high GTWY

content material 2 was added had almost no bitterness and had only a slight difference in

tastiness rating as compared with the sample to which no GTWY material was added.

These results have confirmed that using the high GTWY content material makes it possible

to ingest GTWY at a high concentration without impairing the flavor of a product.

[0039]

[Table 5] jeKI C%]

> (

4

0

4.4

0

[0040]

[Example 8] Influence of the initial pHof enzymatic reaction on the production of GTWY from WPC when an alternative enzyme derived from Aspergillus melleus is used 1. Test method In order to investigate enzymes that can be used in the technology ofthe present invention, the influence of the initial pHofan enzymatic reaction on the production of GTWY was examined by using aprotease derived from Aspergillus melleus that is not Protease P"Amano" 3SD used in Example 1,i.e., Proteinasefrom Aspergillus melleus

(Sigma). A commercially available WPC was dissolved in ion-exchanged water heated to

50°C to a concentration of 13% (w/w), and the pH was adjusted to 6.5 to 9.0 with NaOH.

Proteinase from Aspergillus melleus (Sigma) was added thereto to a concentration of 0.1%

(w/v) to start the reaction. Samples were collected at 2 hours after the start of the reaction

(after the reaction) to measure the pH and GTWY content of the reaction solution.

[0041]

2. Test results

Table 6 shows the initial pHs, and the amounts of GTWY and pHs after the reaction.

As in the case of using Protease P "Amano" 3SD, it was confirmed that the more alkaline

the initial pH, the greater the amount of GTWY produced. Therefore, it was confirmed

that a variety of proteases derived from Aspergillus melleus can be used in the present

invention without any type restriction.

[0042]

[Table 6]

Level Initial pH pH after reaction GTWY amount after reaction (pg/mL)

( 6.5 6.20 429 (2 7.5 6.75 652 @ 9.0 7.01 945

[0043]

[Example 9] Preparation of GTWY-containing capsule

10 g of the high GTWY content material 2 prepared in Example 5, 33.5 g of lactose,

56.0 g of soluble starch, and 0.5 g of magnesium stearate were mixed together, and the

mixture was granulated by a conventional method and encapsulated to produce GTWY

containing capsules of the present invention.

[0044]

[Example 10] Preparation of GTWY-containing beverage

10 g of the high GTWY content material 2 prepared in Example 5 was dissolved in

690 g of deionized water. The resultant solution was heated to 400 C, and then mixed by

stirring for 20 minutes at 9,500 rpm using an ultra disperser (ULTRA-TURRAX T-25;

manufactured by IKA Japan K.K.). To this were added 100 g of maltitol, 2 g of acidulant,

20 g of reduced starch syrup, 2 g of flavoring, and 176 g of deionized water. This mixture was poured into 100 ml glass bottles, sterilized at 95°C for 15 seconds, and then sealed tightly to prepare 10 bottles (100 ml each) of a GTWY-containing beverage of the present invention.

[0045]

[Example 11] Preparation of GTWY-containing milk beverage 100 g of the high GTWY content material 2 prepared in Example 5 and 9.9 kg of milk were mixed, heated to 40°C, and then mixed by stirring for 10 minutes at 6,000 rpm using a TK homomixer (TK ROBO MICS; manufactured by Tokushu Kika Kogyo Co., Ltd.). This mixture was sterilized by heating at 130°C for 2 seconds, and then cooled to 10°C or lower to produce 10 kg of a GTWY-containing milk beverage of the present invention.

[0046]

[Example 12] Preparation of GTWY-containing fermented milk 100 g of the high GTWY content material 2 prepared in Example 5, 1600 g of skim milk powder, 300 g of glucose, and 7700 g of deionized water were mixed together, and held at 95°C for 2 hours to be heat-sterilized. The resultant solution was cooled to 37°C, and 300g of a lactic acid bacteria starter (Lb. casei) was inoculated therein. After mixing by stirring, the mixture was fermented to pH 4.0 in an incubator kept at 37°C. After the pH reached 4.0, the mixture was cooled to 10°C or lower to produce 10 kg of 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 high GTWY content material 2 prepared in Example 5, 200 g of sodium citrate, and 700 g of deionized water were mixed together and emulsified at 85°C. After the emulsification, the cheese was packed into a carton and cooled at 5°C for two days and nights to produce 20 kg of GTWY containing cheese of the present invention.

[0048]

[Example 14] Preparation of GTWY-containing cream 4.5 kg of rapeseed hydrogenated oil, 40 g of lecithin, 10 g of monoglycerin fatty acid ester, and 10 g of sorbitan fatty acid ester were mixed together to prepare an oil phase. Next, 100 g of the high GTWY 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 were mixed together to prepare an aqueous phase. The oil phase heated to 70°C was added to the aqueous phase heated to 65°C little by little with stirring, and mixed by stirring for 10 minutes at 6,000 rpm 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 GTWY-containing cream of the present invention.

[0049]

[Example 15] Preparation of GTWY-containing pudding

A pudding mix was prepared by mixing 2000 g of honey, 100 g of the high GTWY

content material 2 prepared in Example 5, 770 g of skim milk powder, 300 g of

mascarpone, 700 g of liquid starch syrup, 500g of granulated sugar, 250 g of fresh cream,

200 g of butter, 400 g of sweetened egg yolk, 40 g of gelatin, 15 g of agar, 120 g of locust

bean gum, and 4605 g of deionized water. The pudding mix was mixed by stirring for 10

minutes at 6,000 rpm using a TK homomixer (TK ROBO MICS; manufactured by Tokushu

Kika Kogyo Co., Ltd.), and heated to 60°C for dissolution. Then, 100 g-portions of the

pudding mix were poured into cups and cooled to produce 100 cups of GTWY-containing

pudding of the present invention.

[0050]

[Example 16] Preparation of GTWY-containing milk powder

1 kg of the high GTWY content material 2 prepared in Example 5, 8.3 kg of skim milk

powder, and 90 kg of deionized water were mixed together, heated to 40°C, and mixed by

stirring for 10 minutes at 6,000 rpm using a TK homomixer (TK ROBO MICS;

manufactured by Tokushu Kika Kogyo Co., Ltd.). This mixture was spray-dried to produce

about 10 kg of GTWY-containing powdered milk of the present invention.

[0051]

[Example 17] Trial production of GTWY-containing soft drink

A GTWY-containing soft drink was produced on trial by performing enzyme

treatment in the production process of a soft drink. 16 g of commercially available WPC

was dissolved in 84 g of distilled water. This WPC solution was heated to 50°C, and then

adjusted to pH 8.5 with NaOH. Protease P "Amano" 3SD was added thereto to a

concentration of 0.1% (w/v) to carry out an enzymatic reaction at 50°C for 30 minutes. In

a separate container, 10 g of skim milk powder, 100 g of sugar, 5 g of citric acid, 2.5 g of trisodium citrate, and 5 g of pectin were dissolved in 778 ml of distilled water in advance. The resultant solution and the enzyme-treated WPC solution were mixed together, sterilized by heating at 90°C for 20 minutes, and poured into 100 ml plastic bottles.

Industrial Applicability

[0052] According to the method of the present invention, it is possible to increase the content of a specific peptide simply by adjusting the pH when a whey protein-containing material is hydrolyzed with a protease, and peptides can be selectively concentrated. In addition, since the method does not require expensive equipment, it can be easily implemented in factories. Further, the short enzymatic reaction time allows high production efficiency and continuous production in the production line. Furthermore, since GTWY is efficiently produced while suppressing excessive degradation of protein, the GTWY-containing composition of the present invention has an average molecular weight of 500 or more and hence has the advantage of good flavor. When added to a food or drink, the GTWY-containing composition of the present invention can increase the peptide content without adversely affecting the original flavor of the food or drink, and therefore has a high utility value.

Claims

1. A method for producing a peptide composition, wherein

the peptide composition is produced by reaction of a protease with a whey protein

containing material, and

a reaction solution has a pH of more than 7.0 at a start of the reaction of the protease.

2. The method according to claim 1, wherein the reaction is carried out for 5 minutes or

more and 5 hours or less.

3. The method according to claim 1 or 2, wherein the whey protein is P-lactoglobulin. 4. The method according to any one of claims 1 to 3, wherein the protease is derived

from Aspergillus melleus.

5. The method according to any one of claims 1 to 4, wherein the peptide composition

is a GTWY-containing composition.

6. The method according to any one of claims 1 to 5, wherein the peptide composition

and a molar ratio of GTWY to WY of 2.0 or more.

7. A method for producing a peptide composition, wherein

containing material, and

a pH of 6.6 or more is maintained in a reaction solution in which the reaction of the

protease takes place.

8. The method according to claim 7, wherein the reaction is carried out for 5 minutes or

more and 5 hours or less.

9. The method according to claim 7 or 8, wherein the whey protein is P-lactoglobulin. 10. The method according to any one of claims 7 to 9, wherein the protease is derived

from Aspergillus melleus.

11. The method according to any one of claims 7 to 10, wherein the peptide composition

is a GTWY-containing composition.

12. The method according to any one of claims 7 to 11, wherein the peptide composition

and a molar ratio of GTWY to WY of 2.0 or more.

13. A method for suppressing degradation of GTWY in a method for producing a GTWY- containing composition, wherein the GTWY-containing composition is produced by reaction of a protease derived from

Aspergillus melleus with a whey protein-containing material, and

the degradation of GTWY is suppressed by setting a pH of a reaction solution at a

start of the reaction of the protease to be more 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 claim 14, wherein the peptide composition is a

GTWY-containing composition having a molar ratio of GTWY to WY of 2.0 or more.

16. A food or drink product, comprising the peptide composition according to claim 15,