NZ715250B2 - Plant extract containing diketopiperazine and method for producing same - Google Patents

Abstract

The present invention provides plant extracts, specifically tea, soybean, barley, and sesame extracts, that comprise at least one diketopiperazine(s) at a concentration of 10 µg/100 g/Bx or more, and that have a total concentration of diketopiperazines per Bx of 900 µg/100g/Bx or more. The invention further provides methods of obtaining plant extracts that comprise cyclo-leucyl-leucine and cyclo-leucyl-phenylalanine by means of high-temperature (100°C-170°C) and high-pressure (0.101-0.79MPa) for 30 to 500 minutes. The plant extracts obtained are superior in terms of their plant-based natural-substance derived flavour. As the extracts contain a large content of diketopiperazines and maintain a low Bx, the amount added to food and/or beverages may be low, which is advantageous in that it enables an increased degree of freedom in the design of food and/or drink. further provides methods of obtaining plant extracts that comprise cyclo-leucyl-leucine and cyclo-leucyl-phenylalanine by means of high-temperature (100°C-170°C) and high-pressure (0.101-0.79MPa) for 30 to 500 minutes. The plant extracts obtained are superior in terms of their plant-based natural-substance derived flavour. As the extracts contain a large content of diketopiperazines and maintain a low Bx, the amount added to food and/or beverages may be low, which is advantageous in that it enables an increased degree of freedom in the design of food and/or drink.

Description

DESCRIPTION PLANT T CONTAINING DIKETOPIPERAZINE AND METHOD FOR PRODUCING SAME TECHNICAL FIELD The present invention s to a plant extract containing a high concentration of a diketopiperazine and a method for producing the extract.

BACKGROUND ART "Dipeptides", which are each composed of two amino acids bonded to each other, have been paid attention as functional substances. Dipeptides can be provided with physical ties or novel functions that are not possessed by simple amino acids and are expected as materials having application ranges broader than those of amino acids. In particular, diketopiperazines, which are cyclic dipeptides, are known to have various physiological activities, such as an antibacterial action or an antioxidant action (Non Patent Literatures 1 and 2) and a learning motivation—improving action (Patent Literature 1), and demands for diketopiperazines are predicted to increase in the l and pharmacological fields.

In general, a piperazine is produced by, for e, chemical synthesis (Non Patent Literature 3) or an enzymatic method (Non Patent Literatures 2 and 4). In addition, a method of synthesizing a cyclic peptide having an ary amino acid sequence by a dehydration and cyclization reactions of a linear peptide in water of high—temperature and high—pressure of a ritical or tical region (Patent Literature 2) and a method of producing a cyclic dipeptide by heat treatment of a linear dipeptide or linear tripeptide in an aqueous solvent (Patent Literatures 3 and 4) have been proposed.

CITATION LIST PATENT LITERATURE Patent Literature 1: National Publication of ational Patent Application No. 2012—517998 Patent Literature 2: Japanese Patent Laid—Open No. 2003— 252896 Patent Literature 3: Korean Patent Laid—Open No. 10—2011— 0120051 Patent Literature 4: Japanese Patent No. 5456876 Patent Literature 5: Japanese Patent Laid—Open No. 2010— 166911 Patent Literature 6: National Publication of International Patent Application No. 2012—517214 NON PATENT LITERATURE Non Patent Literature 1: Peptides, 16(1), 151—164 (1995) Non Patent Literature 2: Bioscience & Industry, 60(7), 454—457 (2002) Non Patent Literature 3: J. Comb. Chem., 3, 453—460 (2001) Non Patent Literature 4: try Biology, 8, 997—1010 (2001) Non Patent ture 5: Agr. Biol. Chem., 38(5), 927—932 (1974) SUMMARY OF INVENTION TECHNICAL PROBLEM Although piperazines are thus expected to show various physiological activities in vivo, there are almost no naturally derived diketopiperazines and foods containing high concentrations of diketopiperazines. Naturally d diketopiperazines are known to be present in fermented foods, such as sherry, Shaoxing wine, soy sauce, sweet cooking rice wine, and vinegar (Non Patent Literature 5), but the contents n are significantly low. In order to take these foods for obtaining the functionality of the piperazines, considerably large amounts of the foods must be taken. Thus, none of them is practical. Coffee drinks containing Cyclo(Pro—Phe) or Cyclo(Pro—Leu) are also known (Patent Literature 5), but these diketopiperazines are highly bitter and are therefore difficult to be applied to other drinks.

Furthermore, compositions ning relatively large amounts of diketopiperazines derived from animal protein, such as collagen and meat, are known (Patent Literatures 4 and 6). However, because of their flavor, the compositions containing these piperazines derived from animal protein cannot be ly mixed with drinks mainly composed of extracts .C or juices o_ plants, such as tea drinks, coffee drinks, soybean drinks, and fruit juice drinks, or soft drinks, such as flavored water, mineral water, and carbonated drinks.

It is an object of the present invention to provide an extract ning a naturally derived and highly safe diketopiperazine at a high concentration and having good flavor and to provide a method for producing the extract.

SOLUTION TO M The present inventors, who have diligently studied for solving the mentioned problems, have found that a plant extract containing a high concentration of a diketopiperazine can be produced by subjecting a protein- containing plant to decomposition treatment to generate plant peptides and subjecting the plant es to high—temperature and high—pressure treatment in a liquid. The inventors have confirmed that this plant extract has a good taste, and have arrived at the completion of the present invention.

The present invention relates to the ing aspects: (1) A plant extract containing at least one of cyclo— alanyl—glutamine, alanyl—alanine, cyclo—seryl-tyrosine, cyclo-glycyl—leucine, cyclo—glycyl—tryptophan, cyclo—valyl— valine, cyclo—tryptophanyl-tyrosine, cyclo—leucyl—tryptophan, and cyclo~phenylalanyl—phenylalanine at a concentration of ug/lOO g/Bx or more; (2) The plant extract according to aspect (1), wherein the total amount of diketopiperazine(s) per Ex is 900 ug/lOO g/Bx or more; (3) The plant extract according to aspect (l) or (2), being a tea t, a soybean extract, or a malt extract; (4) A plant extract prepared by subjecting a protein— ning plant to decomposition treatment to generate a plant peptide and subjecting the plant peptide to high— temperature and high-pressure treatment in a liquid; (5) A method for producing a plant extract containing a high concentration of piperazines including cycloleucyl-leucine and cyclo-leucyl-phenylalanine, the method comprising a step of subjecting a plant peptide to mperature and high-pressure treatment in a liquid; (6) The method according to aspect (5), wherein the hightemperature and ressure treatment is performed in a liquid of 100°C to 170°C for from 30 minutes to several hours as heating conditions; (7) The method according to aspect (5) or (6), wherein the plant peptide is an oligopeptide; (8) The method according to any one of aspects (5) to (7), wherein the plant peptide is prepared by subjecting a plant-derived protein or a protein-containing plant to decomposition ent; (9) The method according to aspect (8), wherein the decomposition treatment is heat ent or enzyme treatment; (10) The method according to aspect (9), n the decomposition treatment is enzyme treatment, and the enzyme is an endo-type protease.

In a particular aspect, the present invention provides a plant extract comprising at least one of cyclo-alanylglutamine , alanyl-alanine, cyclo-seryl-tyrosine, cycloglycyl-leucine , cyclo-glycyl-tryptophan, cyclo-valyl-valine, [FOLLOWED BY PAGE 5a] - 5a - cyclo-tryptophanyl-tyrosine, cyclo-leucyl-tryptophan, cyclophenylalanyl-proline , and cyclo-phenylalanyl-phenylalanine at a concentration of 10 μg/100 g/Bx or more, wherein the plant extract is a tea extract, a soybean extract, a barley extract, or a sesame extract, and the total amount of diketopiperazine(s) per Bx is 900 μg/100 g/Bx or more.

In another particular aspect, the present invention provides a method for producing a plant t containing a high concentration of diketopiperazines including cycloleucyl-leucine and cyclo-leucyl-phenylalanine, the method comprising a step of subjecting a plant peptide to a treatment at 100°C to 170°C and 0.101 to 0.79 MPa for 30 to 500 minutes in a , wherein the plant peptide is a tea peptide, a soybean peptide, a barley peptide, or a sesame peptide.

ADVANTAGEOUS EFFECTS OF INVENTION According to the present invention, a plant t containing a high tration of a naturally d and highly safe diketopiperazine can be simply produced in a mass production scale t requiring a troublesome process or complicated facilities.

[FOLLOWED BY PAGE 6] BRIEF DESCRIPTION OF DRAWINGS Fig. I shows the results of quantitative measurement of cyclo-phenylalanyl—phenylalanine concentration in a plant peptide—processed product prepared from soybean protein.

Fig. 2 shows the results of quantitative measurement of cyclo—phenylalanyl—phenylalanine concentration in a plant e—processed product prepared from rice n.

Fig. 3 shows a relationship between the number of times of pre-extraction and the l rate of the soluble component.

DESCRIPTION OF EMBODIMENTS (Plant peptide) The plant extract of the present invention can be produced by subjecting plant peptides to high—temperature and high—pressure treatment in a liquid. Herein, the term "plant peptide" refers to a peptide composed of l amino acids linked by depolymerization (oligopeptide formation) of a plant—derived protein or a protein—containing plant through known decomposition treatment (e.g., decomposition treatment with heat or pressure, osition ent with an acid or alkali, or decomposition treatment with an enzyme), unless otherwise specified. :0014] The plant peptide of the present invention can be, “or example, a n peptide, a barley peptide, a wheat e, a wheat germ peptide, a pea peptide, or a rice peptide. As described below, the plant peptide may be prepared from a plant—derived protein or a protein~containing plant or may be a commercial product. Examples of the commercially available plant peptide include soybean peptides, such as HINUTE AM, HINUTE DC, and HINUTE HK (all manufactured by Fuji Oil Co., Ltd.); rice es, such as Oryza Peptide P60 (manufactured by Oryza Oil & Fat al Co., Ltd.); wheat peptides, such as Glutamine Peptide GP—lN and Glutamine e GP-N (both manufactured by Nisshin Pharma Inc.); and sesame peptides, such as Sesame Peptide KM-ZO (manufactured by KISCO Ltd. ) .

The examinations by the present inventors demonstrate that the yield of a diketopiperazine mixture varies depending on the sizes of the peptides. The plant peptides preferably include a high proportion of peptides having a molecular weight of 5000 or less, more preferably a molecular weight of 3000 or less, and particularly preferably a molecular weight of 1000 or less. In on, since the use of soybean having a high amino acid score allows generation of multiple types of diketopiperazines at a high concentration, soybean peptides are one of preferred ments.

The plant peptide of the present invention can be a peptide mixture produced using a plant—derived protein or a protein—containing plant as a raw material. Specifically, examples of the peptide mixture e those produced by known decomposition treatment (e.g., decomposition treatment with heat or pressure, decomposition treatment with an acid or alkali, or decomposition ent with an enzyme) of a raw material: a plant*derived n, such as soybean protein, wheat protein, wheat germ protein, rice protein, or sesame protein; or an edible protein—containing plant, such as a leaf (e.g., green tea leaves), a seed (e.g., barley, wheat, malt, sesame, or rice), a bean (e.g., soybean, adzuki bean, or black soybean), a potato (e.g., sweet potato or potato). Among these protein—containing plants, n, malt, and tea leaves are preferably used in the t invention. In particular, soybean and tea leaves are preferably used, and tea leaves are more preferably used. Decomposition treatment is applied to the above—mentioned plant—derived protein or the protein- containing plant as a raw material to prepare a peptide e, which is used as the plant peptide. This osition ent is performed under conditions allowing generation of oligopeptides. Specifically, the decomposition treatment is performed so as to se the proportion of peptides having a molecular weight of 5000 or less (preferably a molecular weight of 3000 or less and more preferably a molecular weight of 1000 or less).

The decomposition treatment is preferably performed with heat and/or an enzyme because of the easiness (high reaction rate) of generation of an intended oligopeptide and easiness of mass treatment. In particular, decomposition treatment with an enzyme (hereinafter, referred to as enzyme treatment) is preferably employed.

The decomposition treatment by heating is performed in a solvent for ting the plant or n from being burned. The amount of the solvent is usually about 10 to 100 parts by mass, preferably about 15 to 80 parts by mass, more preferably about 20 to 60 parts by mass, and particularly preferably about 20 to 40 parts by mass based on 1 part by mass of the plant. The solvent is preferably, for example, water, ethanol, or a mixture thereof and particularly ably water. The heating may be performed under any ions that allow generation of peptides. Examples of the g conditions include heating at 100°C or more and further at 125°C or more for 30 minutes to l hours, preferably about 2 to 7 hours. As the heat treatment equipment, for example, a pressure cooker or an autoclave can be used depending on the heating conditions. This heat treatment can be performed simultaneously with the "step of high—temperature and high—pressure treatment in a " of the present ion.

In the production of plant peptides by the enzyme treatment, the enzyme used is a proteolytic enzyme (protease) and is preferably a protease having a high endo—type decomposition ty. The protease is roughly classified into three categories: alkaline protease, neutral protease, and acid protease, based on the difference in optimum pH for the action. In addition, the origin of a protease is a plant origin, an animal origin, or a microbial origin. The enzyme may have any origin and optimum pH that do not cause disadvantageous influences, such as low decomposition efficiency or bad flavor of the resulting decomposition extract.

Examples of the bacterial protease that can be used in the present ion include Protease N, Protease NL, Protease S, and Proleather (R) FG—F (all manufactured by Amano _lO_ Enzyme Inc.); Protin NY, Protin P, Deskin, Depirays, Protin A, and Thermoase (R) (all manufactured by Daiwa Fine Chemicals Co., Ltd.); Bioplase (R) XL—416F, Bioplase (R) SP—4FG, and Bioplase (R) SP—lBFG (all ctured by Nagase ChemteX Corporation); Orientase (R) 9ON, Nucleicin (R), Orientase (R) lONL, and Orientase (R) 22BF (all manufactured by HBI Enzymes Inc.); Aloase (R) AP-lO (manufactured by Yakult Pharmaceutical [Industry Co., Ltd.); Protamex (R), Neutrase (R), and Alcalase (R) (all manufactured by mes Japan Ltd.); COROLASE N, COROLASE 7089, VERON W, and VERON P (all manufactured by AB Enzymes); Enchiron NBS (manufactured by Rakuto Kasei Industrial Co., Ltd.); and Alkali Protease GL440, Purafect (R) 4000L, Protease 899, and Protex 6L (all ctured by Genencor Kyowa Co., Ltd.). Examples of aspergillus se that can be used in the present invention include Protease A, se M, Protease P, Umamizyme, Peptidase R, Newlase (R) A, and Newlase (R) F (all manufactured by Amano Enzyme Inc.); Sumizyme (R) AP, Sumizyme (R) LP, Sumizyme (R) MP, Sumizyme (R) PP, and Sumizyme (R) LPL (all manufactured by Shinnihon als Corporation); Protin (R) FN (manufactured by Daiwa Fine Chemicals Co., Ltd.); Denapsin 2P, Denazyme (R) AP, and XP—415 (all manufactured by Nagase ChemteX Corporation); Orientase (R) 20A, Orientase (R) CNS, and Tetrase (R) 8 (all manufactured by HBI Enzymes Inc.); Molsin (R) F, PD , IP Enzyme, and AO—Protease (all manufactured by Kikkoman Corporation); Sakanase (manufactured by Kaken Pharma Co., Ltd.); Pantidase (R) YP-SS, Pantidase (R) NP—Z, and Pantidase (R) P (all manufactured by Yakult Pharmaceutical Industry Co., -11..

Ltd.); Flavourzyme (R) (manufactured by Novozymes Japan Ltd.); se (R) SS and Kokulase (R) P (both manufactured by Mitsui Lifetech Co., Ltd.); and VERON PS and COROLASE PN-L (both manufactured by AB Enzymes). es of other proteases that can be used in the present invention include actinomycete proteases (for example, se (R) AS and Actinase (R) AF (both manufactured by Kaken Pharma Co., Ltd.); and Tasinase (R) (manufactured by or Kyowa Co., Ltd.)); plant—derived proteases (for example, Papain W-40 (manufactured by Amano Enzyme Inc.), food—grade purified Papain (manufactured by Nagase ChemteX ation)); and animal pepsin and trypsin.

Among the above-mentioned proteases, from the viewpoint of the decomposition efficiency and the flavor of the resulting peptide—containing solution, the protease is preferably a bacterial protease, more ably a neutral protease derived from Bacillus subtilis or a protease derived from Bacillus amyloliquefaciens or Bacillus stearothermophilus, and particularly preferably a neutral protease derived from Bacillus subtilis.

Such a protease is used in an amount within a range of 0.1% to 20% by weight, preferably 1% to 15% by weight, more preferably 3% to 10% by weight, based on the amount of the plant—derived protein or the protein—containing plant. An amount less than the above—mentioned range cannot provide the effect of increasing the yield of peptide generation, whereas an amount higher than the mentioned range cannot achieve a considerable increase in the yield of e generation, -12_ which is disadvantageous in the cost. In the enzyme treatment, water is added to a plant—derived protein or a plant to allow the enzyme to act on the wetted protein or plant. The amount of water to be added is y about 10 to 50 parts by mass, more preferably about 10 to 30 parts by mass, and particularly preferably about 10 to 20 parts by mass, based on 1 part by mass of the dry protein or plant.

The conditions for the enzyme treatment by a protease may be determined in view of the optimum conditions for the protease and are usually at 20°C to 70°C (preferably °C to 60°C and more preferably 40°C to 60°C) for about 30 min to 24 hours (preferably 1 to 12 hours and more ably 1 to 6 hours).

Since the sites of the actions of enzymes on protein as a substrate are ent based on the types of the s, the composition of the diketopiperazine mixture prepared by the present invention can be varied. ingly, the enzyme can be selected in View of the composition of a desired diketopiperazine mixture. Two or more enzymes may be used in combination.

When a plant is used as plant es, pretreatment for reducing the amount of water—soluble protein contained in the plant is preferably performed before the step of generating peptides by the above—described decomposition treatment. The examinations by the present inventors demonstrate that the reduction in the water—soluble protein in advance considerably increases the yield of peptides ted by decomposition treatment or the yield of diketopiperazines -13.. generated by the heat treatment of the present invention.

Examples of the pretreatment for removing water—soluble prptein include a method in which soluble protein is eluted by heating a plant in a liquid, solid—liquid separation is performed to collect the solid (plant), and the solid is decomposed and a method in which a plant is ted to extraction treatment with an aqueous solvent, such as water, and the extraction residue is then decomposed (hereinafter, these methods are tively referred to as "pre— tion"). In the pre—extraction, a plant is immersed in an extracting solvent of which the weight is ably about times or more, more preferably about 15 to 150 times, the weight of the plant, and the soluble component, such as water— soluble protein, contained in the plant are eluted. In this case, the extracting solvent may be heated in advance.

Alternatively, a plant is ed in an ting solvent and the solvent may be then heated for extraction. The extracting solvent is preferably pure water and may be pure water appropriately containing an organic solvent, such as ethanol. The ting solvent may contain minerals for appropriately adjusting the hardness thereof.

The pre—extraction may be performed at any extraction temperature and is usually performed at about 50°C to 100°C, preferably about 60°C to 95°C, and more preferably about 70°C to 90°C. The extraction time is about 1 min to 24 hours, preferably about 3 min to 20 hours. The extraction conditions such as extraction temperature and time are adjusted such that the removal rate of the soluble component -14_ in the resulting tion residue is 60% or more, ably 70% or more, more preferably 80% or more, more preferably 90% or more, and particularly preferably 95% or more. The l rate of the soluble component means the relative proportion of the solid collected with the extracting solution when the maximum amount of the removable soluble component is defined as 100%, and is the value calculated by the expression: "(the amount (total amount: g) of solution obtained by pre— extraction x its Brix [Bx])/(the maximum amount (g) of the removable soluble component X its Brix [Bx]) x 100(%)".

Throughout the specification, "the maximum amount of the removable soluble component" is conveniently denoted by "the amount of the on obtained by repeating, ten times, extraction with boiling water of an amount of 30 times the weight of the plant for 10 min". Throughout the specification, the term "Bx" can be measured with a commercially available Bx scale.

The pre—extraction of a plant may be med once or several times. The extract obtained by pre~extraction may be discarded or may be used by being added to a food or drink.

For example, the extract mixed with a plant extract containing a diketopiperazine prepared by the present invention can be added to a food or drink.

(Heat treatment) In the production method of the present invention, diketopiperazines are generated by subjecting such plant es to high—temperature and ressure treatment in a liquid. The liquid for the high-temperature and high~pressure _15._ treatment is preferably pure water and may be pure water appropriately containing an organic solvent, such as ethanol.

The extracting solvent may contain minerals for riately adjusting the hardness thereof. The liquid for the heat treatment is optionally concentrated or diluted to have a Brix (Bx) of about 0.1 to 50.

Throughout the specification, the term "high~ temperature and high~pressure" refers to a temperature of 100°C or more and a pressure exceeding the atmospheric pressure. As a high—temperature and high—pressure extracting apparatus, for e, a pressure—resistant extracting apparatus, a pressure cooker, or an autoclave can be used ing on the conditions.

The ature of the high—temperature and high— pressure is preferably 100°C to 170°C, more preferably 110°C to 150°C, and particularly preferably 120°C to 140%L In the case of using a pressure—resistant ting apparatus as the g apparatus, this temperature is the measured outlet temperature of the extraction column. In the case of using an autoclave as the heating apparatus, this temperature is the measured temperature at the center of the pressure vessel.

The pressure is preferably 0.101 to 0.79 MPa and more preferably 0.101 to 0.48 MPa. The g time is preferably about 30 to 500 minutes and more preferably about 60 to 300 minutes. r optimum conditions for heat treatment are within a range of time and temperature surrounded by the following coordinate systems (i) to (vi), in which time (min) -16— is plotted on the horizontal axis and temperature (°C) on the vertical axis. (i) (170°C, 30 min), (ii) (150°C, 30 min), (iii) (115°C, 180 min), (iv) (105°C, 480 min), (v) (135°C, 480 min), and (vi) (150°C, 180 min).

After the high—temperature and high~pressure treatment in a liquid, the liquid fraction is collected by optionally performing solid—liquid separation to obtain a plant extract ning a high concentration of the diketopiperazines of the present invention. The solid—liquid separation is achieved by filtration and/or centrifugation.

Although the composition of the diketopiperazines in the resulting plant extract containing a high concentration of the diketopiperazines varies depending on the origin (the type of the plant as the raw material) of the plant peptides and the type of the enzyme, the high—temperature and ressure treatment of the plant peptides of the present invention in a liquid can increase the amount of at least one diketopiperazine selected from the group consisting of cyclo— alanyl—glutamine (CAS Registry Number: 268221—76~7; Cyclo(Ala— Gln)), histidyl—proline (CAS Registry Number: 53109—32~ 3; His—Pro)), cyclo—alanyl—alanine (CAS Registry Number: 5845—61—4; Cyclo(Ala—Ala)), glycyl—proline (CAS ry Number: 3705—27—9; Cyclo(Gly—Pro)), cyclo—seryl—tyrosine (CAS Registry Number: 31—4; Cyclo(Ser—Tyr)), cyclo—prolyl— threonine (CAS Registry Number: 227777—31—3; Cyclo(Pro—Thr)), histidyl~phenylalanine (CAS Registry Number: 56586—95-9; Cyclo(His—Phe)), cyclo—alanyl—proline (CAS ry Number: -l7_ 65556—33—4; Cyclo(Ala—Pro)), cyclo—phenylalanyl-serine (CAS ry Number: 35591—00—5; Cyclo(Phe-Ser)), cyclo—glycyl- leucine (CAS Registry Number: 7—0; Cyclo(Gly—Leu)), cyclo—glycyl—phenylalanine (CA8 Registry Number: 10125—07—2; Gly—Phe)), cyclo—propyl—proline (Cyclo(Pro-Pro)), cyclo- glycyl—tryptophan (Cyclo(Gly-Trp)), cyclo—aspartyl— phenylalanine (CAS Registry Number: 5262-10—2; Cyclo(Asp—Phe)), valyl—proline (Val—Pro)), cyclo-prolyl—tyrosine (Cyclo(Pro—Tyr)), methionyl—proline (Cyclo(Met—Pro)), methionyl—methionine (Cyclo(Met—Met)), cyclo—valyl— valine (Cyclo(Val-Val)), cyclo—leucyl—proline (CAS Registry Number: 2873—36—1; Cyclo<Leu—Pro)), cyclo—tryptophanyl— tyrosine (Cyclo(Trp-Tyr)), cyclo—phenylalanyl—proline (CAS Registry Number: 3705—26—8; Cyclo(Phe-Pro)), cyclo—leucyl— tryptophan (CA8 Registry Number: 15136—34—2; Cyclo(Leu—Trp)), cyclo-phenylalanyl—tryptophan (CAS Registry Number: 82597—82v 8; Cyclo(Phe—Trp)), cyclo—leucyl-phenylalanine (CAS Registry Number: 7280—77~5; Cyclo(Leu—Phe)), cyclo—leucyl—leucine (CA8 Registry Number: 952—45—4; Cyclo(Leu—Leu)), and cyclO* phenylalanyl—phenylalanine (CAS Registry Number: 2862~51—3; Cyclo(Phe~Phe)).

In particular, the present invention is advantageous for production of a plant extract containing a high concentration of diketopiperazines including Cyclo(Leu—Leu) and Cyclo(Leu—Phe) in relatively high concentrations. The t invention is also advantageous for production of a plant t containing a high concentration of Cyclo(Phe— Phe). ...18_.

A natural plant—derived diketopiperazine containing a high concentration of a specific diketopiperazine can be selectively produced from a plant extract containing a high concentration of the diketopiperazine of the present invention by known purification treatment. Accordingly, from one viewpoint, the present invention relates to a method for producing a plant t containing a high concentration of diketopiperazines including Cyclo(Leu—Leu) and Phe-Phe), and from another int, the present invention relates to a method for producing a specific diketopiperazine (for example, Cyclo(Ala—Ala), Cyclo(Leu—Phe), Cyclo(Leu—Leu), or Cyclo(Phe— Phe)).

(Plant extract) Throughout the specification, the term "extract" refers to a liquid extract, and a "plant extract" of the present invention refers to a liquid extract prepared by extraction treatment of a plant or its processed product.

The t invention can provide a plant extract containing at least one of Cyclo<Ala—Gln), Ala—Ala), Ser—Tyr), Gly—Trp), Cyclo(Val~Val), Cyclo(Trp—Tyr), Cyclo(Leu—Trp), and Cyclo(Phe*Phe) in an amount per Bx of pg/lOO g/Bx or more.

In addition, the present invention can provide a plant extract containing diketopiperazines in a total amount of 900 pg/lOO g or more, preferably 1000 pg/lOO g or more, more preferably 2000 ug/lOO g or more, and particularly preferably 5000 ug/lOO g or more. Throughout the specification, unless otherwise specified, the total amount of _19_ diketopiperazines refers to the sum of the amounts of Cyclo (Ala—Gin), Cyclo (His—Pro), Cyclo (Ala—Ala), Cyclo (Gly—Pro), Cyclo (Ser—Tyr), Cyclo (Pro—Thr), Cyclo (His—Phe), Cyclo (Ala- Pro), Cyclo (Phe—Ser), Cyclo (Gly—Leu), Cyclo (Gly—Phe), Cyclo (Pro—Pro), Cyclo (Gly-Trp), Cyclo (Asp—Phe), Cyclo (Val—Pro), Cyclo (Pro-Tyr), Cyclo (Met—Pro), Cyclo (Met~Met), Cyclo (Val— Val), Cyclo (Leu—Pro), Cyclo (Trp~Tyr), Cyclo ro), Cyclo (Leu—Trp), Cyclo (Phe—Trp), Cyclo (Leu—Phe), Cyclo (LeU*Leu), and Cyclo (Phe—Phe).

In general, since an extract having a high Bx contains s substances (e.g., bitter substances) d from the raw material at a high concentration, the extract itself is improper as a drink, and addition of the extract to a drink is also er due to influence on the flavor or the feeling on the tongue. Accordingly, regarding the addition to drink, a lower Bx is preferred. The present invention can provide a plant extract containing a large amount of diketopiperazines of physiologically active substances and having a low Bx, i.e., a plant t having a high ratio of the content of diketopiperazines to the Ex. Specifically, provided is a plant extract having a ratio of the total amount (unit: ug/lOO g) of the above—mentioned diketopiperazines to Brix (Bx) of 900 (pg/100 g/Bx) or more, preferably 1000 (ug/lOO g/Bx) or more, more ably 2000 (pg/100 g/Bx), and further preferably 5000 (pg/100 g/Bx). The upper limit of the amount of diketopiperazines in an extract is not particularly limited and may be appropriately ined in the light of the solubility of the diketopiperazines and is usually about _20__ 1000 mg/100 g or less, preferably about 500 mg/100 g or less, and more preferably about 200 mg/100 g or less.

In the case of a plant extract prepared by applying the tion method of the present invention to a plant as a raw material, the generation of by—products is low, because no fermentation is performed. In addition, the pre—extraction reduces the amount of the soluble component to give a plant extract having a characteristic flavor of icantly low bitterness.

Such a plant t has a good flavor and also an excellent appearance without, for example, precipitation and turbidity and can be ore used directly as an t or for seasonings, drinks, and other foodstuffs without performing specific pro—treatment. The plant extract of the present invention contains a large content of diketopiperazines, but has a relative low Bx. Accordingly, the amount to be added to a food or drink (in particular, drink) may be low, which is an advantage of increasing the degree of freedom in design of a food or drink. In particular, the plant tion can be ly mixed with a drink mainly composed of an extract or juice of a plant, such as a tea drink, a coffee drink, a soybean drink, or a fruit juice drink or a soft drink, such as flavored water, mineral water, or a carbonated drink. For example, a drink mixed with a plant extract of the present invention such that the total amount of diketopiperazines is 10 ug/lOO g or more, preferably ug/lOO g or more, more preferably 40 ug/lOO g or more, and further preferably 60 ug/lOO g or more can have good taste without having bitterness.

The plant extract prepared by the t invention may be ted to, for example, clarification treatment depending on the form of the food or drink to which the plant extract is added. In such a case, the plant extract has an advantage that the ication can be easily performed, e, for example, that the extract does not n oil and includes fibers.

Examples of preferred form of the plant extract of the present invention include tea extracts, soybean extracts, and malt ts. These extracts will now be described in detail.

(Tea extract) Throughout the specification, the term "tea extract" refers to a tea extract prepared by extraction treatment of tea leaves. The tea leaves of the extraction raw material are drinkable parts by extraction of a tea plant (scientific name: Camellia is), such as leaves and stems of tea leaves.

In addition, the tea leaves may be in any form, such as a macrophyll or powder form“ The harvest time of tea leaves may be any time and is appropriately selected to obtain a desired flavor.

The plant extract (tea extract) containing a high concentration of diketopiperazines prepared by the present invention is characterized by the production process without performing fermentation to inhibit the generation of by- products and to obtain good flavor. From the viewpoint of this flavor, the tea leaves are preferably of steamed -22_ ented tea (green tea), such as sencha, bancha, houjicha, gyokuro, kabusecha, and sweet tea, or unfermented kamairi tea, such as ureshinocha, aoyagicha, or a variety of Chinese tea.

The present inventors measured the concentrations of diketopiperazines in tea extracts prepared by extracting cially available tea leaves. The results demonstrate that fermented tea contains a significantly low amount (about 0 to 200 ug/lOO g/Bx) of the diketopiperazines and that green tea does not substantially contain the diketopiperazines (see Table 1, the ement method is the same as that shown in Example 1). _23_ [Table 1] Dl- k - etOplperaZlne' Green Pu—erh Pu-erh concentration GOishicha (min) tea tea 1 tea 2 (ppm/Bx) U) Ala—Gin Q torotoooooooooqqowowo'lmma-w mfiNkOONONI—‘OOQQU‘IOOOOOWQN His—Pro Ala—Ala Ser-Tyr Pro—Thr His—Phe .1.0000000 Ala—Pro Gly—Leu Gly—Phe Pro-Pro Gly—Trp Asp—Phe Val—Pro Pro—Tyr Met—Pro ll 1—4 O‘xthJOONOU'INJNN Phe—Pro Leu—Trp Phe—Trp Leu—Phe Leu—Leu/Ile—Ile Phe—Phe OOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOQOOOOOOOO OOOOOHONOOOHl—JOOHOOONOQOHONO OOOOOOOOOOOOOOOOOOOO OOOOOI—‘OHOOOI—‘NOOI—‘OOOWONOI—‘OOO OOOOOOOOOOOOOOOOOOOQ OOOOOI—‘Of—‘OOOHNOOF—‘OOONOl—‘Ol—‘OOO Total concentration 0.0 1.9 1.2 0.8 (ppm/BX) Total concentration per O 193 119 82 unit BX (ug/lOOg/Bx) In contrast, the tea extract of the present ion contains at least one of Cyclo<A1a—Gln), Cyclo(Ala— Ala), Ser—Tyr), Cyclo(G1y—Trp), Cyclo(Val—Val), _24_.

Cyclo(Trp—Tyr), Cyclo(Leu—Trp), and Cyclo(Phe-Phe) of diketopiperazines, which are not contained in conventional teas, at a concentration of 10 ug/lOO g/Bx or more.

Alternatively, the tea extract of the present invention contains each of Cyclo (Ala—Gln), Cyclo (His-Pro), Cyclo la), Cyclo (Gly—Pro), Cyclo yr), Cyclo (Pro— Thr), Cyclo (His—Phe), Cyclo (Ala—Pro), Cyclo (Phe~Ser), Cyclo (Gly—Leu), Cyclo (Gly—Phe), Cyclo (Pro—Pro), Cyclo (Asp—Phe), Cyclo ro), Cyclo (Pro—Tyr), Cyclo (Met—Pro), Cyclo (Leu— Pro), Cyclo (Phe—Pro), Cyclo (Leu—Phe), and Cyclo (Leu-Leu) at a concentration of 0.1 ppm/Bx (10 ug/lOO g/Bx) or more. The tea extract preferably contains each of the above—mentioned piperazines at a concentration of 0.2 ppm/Bx or more, more preferably 0.3 ppm/Bx or more, further preferably 0.4 ppm/Bx or more, and particularly preferably 0.5 ppm/Bx or more. Furthermore, the tea extract can contain each of Cyclo(Gly—Trp), Cyclo<Val—Val), Cyclo(Trp—Tyr), Cyclo(Leu—Trp), Cyclo(Phe—Trp), and Cyclo(Phe—Phe) at a concentration of 0.1 ppm/Bx (10 ug/lOO g/Bx) or more, preferably 0.2 ppm/Bx or more, and more preferably 0.3 ppm/Bx or more. piperazines known to have strong bitterness are Cyclo(Leu—Pro) and Phe—Pro) of the diketopiperazines contained in coffee drinks (see Japanese Patent Laid—Open No. 2010—166911) and Cyclo(Leu—Trp) of a decomposition treatment product of casein in Research Foundation, Peptide Institute, Inc., No. 2, 1974). The tea extract of the present invention contains these diketopiperazines having strong bitterness, but the extract itself does not substantially have bitterness. An aqueous solution containing Leu—Pro), Cyclo(Phe—Pro), and Cyclo(Leu—Trp) at the same concentrations as those of the tea t have strong bitterness. It is therefore suggested that other diketopiperazines and tea—derived component present in the tea extract additively or synergistically reduce the bitterness of Cyclo(Leu—Pro), Phe—Pro), and Cyclo(Leu-Trp). In ular, a tea extract having a ratio [(B)/(A)] of the total amount (B) of the diketopiperazines having bitterness, Cyclo(Leu~Pro), Cyclo(Phe~Pro), and Cyclo(Leu—Trp), to the total amount (A) of Cyclo(Leu—Leu) and Cyclo(Leu—Phe) of 1.0 or less (preferably 0.8 or less, more preferably 0.6 or less, and particularly preferably 0.4 or less) is a diketopiperazine—containing extract not having any taste such as bitterness and can be directly added to foods and drinks (in particular, drinks).

The total amount of the diketopiperazines per Bx in the tea extract is 900 ug/lOO g/Bx or more, preferably 900 to 30000 ug/lOO g/Bx, more ably 2000 to 25000 ug/lOO g/Bx, and particularly preferably 5000 to 20000 ug/lOO g/Bx. Such a concentration range is advantageous for ing a food or drink provided with the functions (such as physiological activity) of the diketopiperazines.

Such a tea extract can be conveniently produced by decomposing protein in tea leaves to prepare tea peptides and subjecting the tea es to high—temperature and high— pressure treatment. Tea leaves abundantly contain protein at about 25% (Food Composition Table, 5th ed.). Accordingly, it _26_ can be expected to obtain tea peptides by decomposition treatment of the protein of tea leaves with an enzyme such as a protease, but the action of proteases on tea leaves cannot give a large amount of tea es. Since 80% or more of the whole protein in tea leaves are insoluble n, it is preferable to prepare tea peptides by efficiently acting a proteolytic enzyme on the protein contained in tea leaves.

Specifically, water—soluble protein is removed from tea leaves by eatment, and a proteolytic enzyme, such as a protease, is allowed to act on the resulting extraction residue to prepare tea peptides. That is, the tea extract of the present invention containing a high concentration of diketopiperazines can be conveniently produced by efficiently osing water~ insoluble protein by sequentially performing the following steps: (a) extracting tea leaves with water and collecting the extraction residue; (b) acting an endo—type se on the extraction e in the presence of water to decompose the tea leaf protein to prepare a solution containing tea peptides; (c) subjecting the on containing tea peptides to high—temperature and high~pressure treatment to prepare a reaction solution; and (d) subjecting the reaction solution to solid—liquid separation treatment to collect a solution containing diketopiperazines, or (a) extracting tea leaves with water and collecting the extraction residue; -27_ (b) acting an endo—type protease on the extraction residue in the presence of water to decompose the tea leaf protein to prepare a on containing tea peptides; (d') subjecting the solution containing tea peptides to solid—liquid separation treatment to collect a solution containing tea peptides; and (C‘) subjecting the solution containing tea peptides to emperature and high—pressure treatment to prepare a on solution containing diketopiperazines.

The conditions for each step are as described above. In the pre—extraction in the step (a), an extraction residue, such as used tea leaves obtained by extraction ent in, for example, production of tea drinks, can also be used.

Conventionally, the water—insoluble tea protein in tea leaves has not been used as a nutrient source. For example, most of more than 22000 tons of extraction residue generated in production of green tea drinks in Japan have been discarded as unused ces, but the above—described method for producing a tea t is also useful for ive utilization of such used tea leaves that have been conventionally discarded.

This method can produce tea extract containing a high concentration of Cyclo(Leu-Leu), Cyclo(Leu—Phe), and Cyclo(Ala—Ala). Specifically, the extract contains 10% (weight basis) or more of Cyclo(Leu—Leu), 10% or more of Leu—Phe), and 7% or more of Cyclo(Ala—Ala), based on the total amount of the diketopiperazines in the tea extract.

When these weight basis contents are expressed by amounts, the tea extract contains each of these diketopiperazines at a concentration of 5.0 ppm/Bx (500 ug/lOO g/Bx) or more, ably 8.0 ppm/Bx or more, and more preferably 10.0 ppm/Bx or more. The upper limit thereof is about 50.0 ppm/Bx or less, preferably about 40.0 ppm/Bx or less, more preferably about .0 ppm/Bx or less, and further ably about 30.0 ppm/Bx or less.

In addition, it was found that the trations of Cyclo(Leu-Leu), Cyclo<Leu—Phe), and Cyclo(Phe—Phe) are notably increased by repeating the water extraction (pre—extraction) of tea leaves in the step (a) more than once. Accordingly, this method is also advantageous for production of Cyclo(Phe— Phe). The present inventors confirmed that a tea extract containing 3.0 ppm/Bx or more of Phe-Phe) prepared by this method has a learning motivation-improving action.

Incidentally, a diketopiperazine having a hydrophobic functional group is known to enhance the hydrophobicity, by being circularized, to a level higher than that of the linear peptide. The results of an accelerated preservation test (55°C, 2 weeks) of the above—described tea extract demonstrate that Cyclo(Phe—Phe), which is a component having the highest hydrophobicity, is stably retained. ingly, the tea extract of the present ion is also useful as a Cyclo(Phe-Phe)—containing extract. The content of Cyclo(Phe—Phe) in the tea extract is preferably adjusted to ug/lOO g/Bx or more, 20 ug/lOO g/Bx or more, or ug/lOO g/BX or more.

(Soybean extract) Throughout the specification, the term "soybean extract" -29_ refers to a solution prepared by adding water to soybean and performing extraction treatment or milling treatment. The soybean (scientific name: Glycine max) as the raw material may be any species and may be produced in any area. Soybean in a stage of sing, such as crushed soybean, can also be used.

The soybean extract in this specification encompasses a solution prepared by adding water to a soybean protein decomposition product, as a matter of convenience.

It is said that n account for about 30% of soybean. Since the n protein does not include a large amount of water~insoluble protein, unlike the tea protein, the pre-treatment for removing water—soluble protein is not essential and may be optionally med. When the pre— treatment for removing water~soluble protein is not performed, a plant extract (soybean extract) containing a high tration of diketopiperazines can be more conveniently produced by a one~pot reaction.

The present inventors measured the concentrations of piperazines in n peptides in View of that commercially available soybean peptides (powder) have been applied with heat about 180°C to 220°C during spray drying.

The results demonstrate that a significantly low amount (about 650 ug/lOO g/Bx) of diketopiperazines are present in the commercially available soybean peptides (see Table 2).

In contrast, the soybean extract of the present ion contains at least one of Cyclo(Ala—Gln), Cyclo(Ala— Ala), Cyclo(Ser—Tyr), Cyclo(Gly—Trp), Cyclo(Val—Val), Cyclo(Trp—Tyr), Cyclo(Leu—Trp), and Cyclo(Phe—Phe) of ~30... diketopiperazines, which are not contained in conventional soybean protein decomposition products (soybean peptides), in an amount per Bx of 10 ug/lOO g/Bx or more.

In addition, the soybean extract of the present invention contains each of Cyclo (Ala—Gln), Cyclo (His—Pro), Cyclo (Ala—Ala), Cyclo ro), Cyclo (Ser-Tyr), Cyclo (Pro— Thr), Cyclo he), Cyclo (Ala-Pro), Cyclo (Phe—Ser), Cyclo (Gly—Leu), Cyclo (Gly—Phe), Cyclo (Gly-Trp), Cyclo (Asp-Phe), Cyclo (Val-Pro), Cyclo (Pro—Tyr), Cyclo (Met~Pro), Cyclo (Val— Val), Cyclo (Leu—Pro), Cyclo (Trp—Tyr), Cyclo (Phe—Pro), Cyclo (Leu—Trp), Cyclo (Leu—Phe), Cyclo (Leu—Leu) and Cyclo (Phe— Phe) at a concentration of 0.1 ppm/Bx (lO ug/lOO g/Bx) or more.

The soybean extract preferably contains each of the above— ned diketopiperazines at a concentration of 0.5 ppm/Bx or more, more preferably 0.7 ppm/Bx or more, further preferably 0.9 ppm/Bx or more, ularly preferably 1.0 ppm/Bx or more, and particularly preferably 1.2 ppm/Bx or more. rmore, the soybean extract can contain each of Cyclo(Pro—Pro) and Cyclo(Phe—Trp) at a concentration of 0.1 ppm/Bx (10 ug/lOO g/Bx) or more, preferably 0.2 ppm/Bx or more, and more preferably 0.3 ppm/Bx or more.

This soybean extract (in ular, an extract prepared using soybean or its ground product as a raw al) contains Cyclo(Leu—Pro), Cyclo(Phe—Pro), and Cyclo(Leu—Trp), which are known as diketopiperazines having strong bitterness, but the t has reduced bitterness. An aqueous solution containing Cyclo(Leu—Pro) and Cyclo(Phe—Pro) at the same concentrations as those of the soybean extract _31_ have strong bitterness. It is therefore suggested that other diketopiperazines and soybean—derived component present in the soybean extract additively or synergistically reduce the ness of Cyclo(Leu—Pro), Cyclo(Phe—Pro), and Cyclo(Leu— Trp). In particular, a soybean t having a ratio [(B)/(A)] of the total amount (B) of the piperazines having bitterness, Cyclo(Leu—Pro), Cyclo(Phe—Pro), and Cyclo(Leu—Trp), to the total amount (A) of Cyclo(Leu—Leu) and Cyclo(Leu-Phe) of 1.0 or less (preferably 0.8 or less, more preferably 0.6 or less, and particularly preferably 0.5 or less) is a diketopiperazineecontaining extract having significantly reduced bitterness and can be advantageously mixed with foods and drinks (in ular, drinks).

The total amount of the diketopiperazines per Bx in the soybean extract is 900 ug/lOO g/Bx or more, preferably 900 to 30000 ug/lOO g/Bx, more preferably 2000 to 25000 ug/lOO g/Bx, and particularly preferably 5000 to 20000 ug/lOO g/Bx. Such a concentration range is advantageous for producing a food or drink provided with the functions (such as physiological activity) of the diketopiperazines.

The soybean extract of the present invention containing a high concentration of diketopiperazines can be produced by sequentially performing the ing steps: (x) acting an endo—type protease on soybean or a soybean protein decomposition t in the presence of water to prepare a solution containing n peptides; (y) subjecting the solution ning soybean peptides to highetemperature and high—pressure treatment to prepare a -32_ on solution; and (z) subjecting the reaction solution to solid—liquid separation treatment to collect a solution containing diketopiperazines.

As in production of the tea extract, the order of the steps (y) and (2) may be exchanged. In addition, before the step (x), a step (w) of removing water~soluble protein may be performed. In the case of using soybean peptides including a large amount of di— or tripeptides as a raw al, the step (x) is performed by: (x') adding water to soybean peptides including a large amount of di- or tripeptides to prepare a solution containing the soybean peptides.

The conditions of other steps are the same as those described above.

This method can produce a soybean extract containing a high concentration of Cyclo(Leu—Leu), Cyclo(Leu—Phe), Cyclo(Ser*Tyr), and Cyclo (Pro—Thr). ically, the extract contains 8% (weight basis) or more of Cyclo(Leu-Leu), 8% or more of Cyclo(Leu—Phe), and 6% or more of Cyclo(Ser—Tyr), based on the total amount of the diketopiperazines in the soybean extract. The n extract contains each of these piperazines at a concentration of 5.0 ppm/Bx (500 pg/lOO g/Bx) or more, preferably 6.0 ppm/Bx or more, and more preferably 7.0 ppm/Bx or more. In particular, a soybean extract containing each of Leu—Leu) and Cyclo(Leu—Phe) at 10.0 ppm/Bx or more, preferably 12.0 ppm/Bx or more, can be prepared. The upper limit thereof is about 50.0 ppm/Bx or _33... less, preferably about 40.0 ppm/Bx or less, more preferably about 35.0 ppm/Bx or less, and further preferably about .0 ppm/Bx or less.

In addition, this method can provide a soybean extract containing 3.0 ppm/Bx or more, preferably 4.0 ppm/Bx or more, of Cyclo(Phe-Phe), which is not included in soybean peptides, and is therefore also advantageous for producing Cyclo(Phe-Phe) (see Examples described below). Incidentally, it has been confirmed that Cyclo(Phe—Phe), which is a highly hydrophobic component, is stably retained in this soybean extract.

(Malt extract) Throughout the specification, the term "malt extract" refers to an extract prepared by extraction ent of malt or its ground product. The soybean malt (malt) as the raw material may be any species and may be ed in any area.

In particular, barley malt, which is germinated seeds of barley, is preferably used. It is practical and efficient to use a fraction containing a large amount of protein ted from barley malt by ng the skin. The fraction containing a large amount of protein can be obtained by, for example, gradually scraping the surface of malt to remove the husk and then collecting a fraction containing a large amount of protein, such as the aleurone layer and endosperm, by scraping. Alternatively, as performed for the tea extract, an extraction residue after pre—extraction can be used. es of the extraction e include the malt pomace ted in the production of beer. -34_ A plant extract (malt extract) containing a high tration of diketopiperazines can be more conveniently produced by a one—pot reaction by using a on containing a large amount of protein as a raw material.

The malt extract of the present invention contains at least one of Cyclo(Ala—Gln), Cyclo(Ala—Ala), Cyclo(Ser—Tyr), Cyclo(Gly—Trp), Cyclo<Val—Val), Cyclo(Trp—Tyr), Cyclo(Leu-Trp), and Phe~Phe), which are diketopiperazines that have been conventionally hard to be extracted, at a concentration of ug/lOO g/BX or more.

In addition, the malt extract of the present invention contains each of Cyclo (Ala—Gln), Cyclo (His—Pro), Cyclo (Ala—Ala), Cyclo (Gly—Pro), Cyclo (Ser—Tyr), Cyclo (Pro— Thr), Cyclo (His—Phe), Cyclo ro), Cyclo (Phe-Ser), Cyclo (Gly—Leu), Cyclo (Gly—Phe), Cyclo rp), Cyclo (Asp—Phe), Cyclo (Val—Pro), Cyclo (Pro—Tyr), Cyclo (Met—Pro), Cyclo (Val— Val), Cyclo (Leu—Pro), Cyclo (Trp—Tyr), Cyclo (Phe—Pro), Cyclo (Leu~Trp), Cyclo (Leu-Phe), Cyclo (Leu—Leu) and Cyclo (Phe— Phe) at a concentration of 0.1 ppm/Bx (50 Hg/lOO g/Bx) or more.

The malt extract preferably contains each of the above— mentioned diketopiperazines at a concentration of 0.3 ppm/Bx or more, more preferably 0.4 ppm/Bx or more, further preferably 0.5 ppm/Bx or more, and particularly preferably 0.6 ppm/Bx or more.

This malt extract contains Cyclo(Leu—Pro), Cyclo(Phe—Pro), and Leu—Trp), which are known as diketopiperazines having strong bitterness, but the extract has reduced bitterness. In particular, a malt extract having -35_ a ratio [(B)/(A)] of the total amount (B) of the diketopiperazines having bitterness, Cyclo(Leu—Pro), Cyclo(Phe—Pro), and Cyclo(Leu—Trp), to the total amount (A) of Leu—Leu) and Cyclo(Leu—Phe) of 1.0 or less (preferably 0.8 or less) is a diketopiperazine~containing extract having significantly reduced bitterness and can be advantageously mixed with foods and drinks (in particular, drinks).

The total amount of the diketopiperazines per Bx in the malt extract is 900 ug/lOO g/Bx or more, preferably 900 to 30000 ug/lOO g/Bx, more ably 2000 to 25000 pg/lOO g/Bx, and particularly preferably 5000 to 20000 ug/lOO g/Bx. Such a concentration range is advantageous for producing a food or drink provided with the functions (such as physiological activity) of the diketopiperazines.

The malt extract of the present ion containing a high concentration of diketopiperazines can be produced by sequentially performing the following steps: (x) acting an endo—type protease on malt or a malt protein decomposition product in the presence of water to prepare a solution ning malt peptides; (y) subjecting the solution ning malt peptides to high—temperature and high—pressure treatment to prepare a on solution; and (z) subjecting the reaction solution to solid—liquid separation treatment to collect a solution containing diketopiperazines.

As in production of the tea extract, the order of the steps (y) and (2) may be exchanged. In addition, before the -36_ step (x), a step (w) of removing water—soluble protein may be performed. The conditions of other steps are the same as those described above.

This method can produce a malt extract containing a high concentration of Cyclo(Leu—Leu), Cyclo(Leu—Phe), and Cyclo(Ala—Ala). Specifically, the malt extract ns each of these diketopiperazines at a concentration of 5.0 ppm/Bx (500 ug/lOO g/Bx) or more, preferably 6.0 ppm/Bx or more, and more preferably 7.0 ppm/Bx or more. The upper limit thereof is about 50.0 ppm/Bx or less, preferably about 40.0 ppm/Bx or less, more preferably about 30.0 ppm/Bx or less, and further preferably about 20.0 ppm/Bx or less.

In on, this method can provide a malt extract containing 1.0 ppm/Bx or more, preferably 2.0 ppm/Bx or more, and further preferably 3.0 ppm/Bx or more of Phe—Phe) and is therefore also advantageous for ing Cyclo(Phe— Phe).

EXAMPLES The present invention will now be described based on Examples, but is not limited to the following Examples.

Throughout the specification, unless otherwise specified, the concentrations are weight basis, and the numerical value ranges each include their endpoints.

(Example 1) Production of diketopiperazine from plant peptide Soybean peptides and sesame peptides were used as plant peptides and were ted to emperature and high— pressure treatment in liquids to produce plant extracts 137_ containing high concentrations of diketopiperazines.

Specifically, 15 mL of distilled water was added to 3 g of soybean peptides (HINUTE AM, manufactured by Fuji Oil Co., Ltd.) or sesame peptides (KM—20, manufactured by KISCO Ltd.), and the mixture was put in an autoclave (manufactured by Tomy Seiko Co., Ltd.) and was subjected to high~temperature and ressure treatment at 135°C and 0.31 MPa for 3 hours. In addition, as a Comparative Example, the same peptides were used to prepare an t without being subjected to the high—temperature and ressure treatment. After the treatment, 10 mL of each solution was diluted 50—fold, subjected to membrane treatment, and then applied to LC—MS/MS to determine the concentration of each diketopiperazine. The details of the analysis conditions were as shown below. In addition, the Brix (Bx) of each plant extract ning a high concentration of diketopiperazines was measured with a digital tometer RX—SOOOa (manufactured by ATAGO Co., Ltd.), and the ratio of the total amount (unit: ug/lOO g) of diketopiperazines to Brix (Bx) was calculated.

[Formula 1] (LC—MS/MS analysis conditions) LC tus: SHIMADZU UFLC XR Column: Agilent technologies Zorbax SB—AQ 1.8 um 2.1 x 150 mm Column temperature: 40°C Mobile phase: A: 0.1% formic acid, B: methanol nt analysis Flow rate: feed ratio 0: 0.2 mL/min _38_ Injection amount: 2 uL Detector: AB Sciex 4000 Q TRAP (R) — Turbo Spray (ESI) — Scheduled multiple reaction ring (MRM) Nozzle position: top: 4 mm, side: 7 mm MRM detection window: 40 sec, Target Scan Time: 0.5 sec [Positive mode] analysis at Scheduled MRM Ion source condition: CUR 20.0, CAD 6, IS 5500, TEM 700, G81 70, G82 70 Table 2 shows the results (throughout the specification, Cyclo(Leu—Leu) denotes the sum of Cyclo(Leu— Leu) and Cyclo(Ile—Ile)). It was demonstrated that plant ts containing a high concentration of diketopiperazines can be conveniently produced by the high—temperature and high— pressure treatment in a liquid according to the present invention. In addition, it was suggested that it is possible to increase the amount of at least one diketopiperazine selected from the group consisting of Cyclo (Ala-Gln), Cyclo (His-Pro), Cyclo la), Cyclo (Gly—Pro), Cyclo yr), Cyclo hr), Cyclo (His-Phe), Cyclo (Ala~Pro), Cyclo (Phe— Ser), Cyclo (Gly—Leu), Cyclo (Gly—Phe), Cyclo (Pro—Pro), Cyclo rp), Cyclo (Asp—Phe), Cyclo (Val—Pro), Cyclo (Pro—Tyr), Cyclo (Met-Pro), Cyclo (Met~Met), Cyclo (Val-Val), Cyclo (Leu— Pro), Cyclo (Trp—Tyr), Cyclo (Phe—Pro), Cyclo (Leu~Trp), Cyclo (Phe—Trp), Cyclo (Leu-Phe), Cyclo (Leu~Leu), and Cyclo (Phe— Phe). In particular, Cyclo(Leu—Leu) and Leu-Phe) were contained at high concentrations. The content of these diketopiperazines was 21.5% in the plant extract containing them at a high concentration. _39_ [Table 2] RT piperaZine The present Comparative The present concentration (min) invention 1 t invention 2 (ppm/Bx) Cyclo(Ala—Gln) 6.8 0.0 3.0 3.7 Cyclo(His—Pro 6.7 0.9 1.8 4.4 Cyclo(Ala—Ala) 6.1 0.0 2.8 .6 Cyclo(Gly~Pro 5.3 0.0 0.3 .8 Cyclo(Ser~Tyr 11.7 0.0 1.3 .8 Cyclo(Pro—Thr 8.1 0.5 2.6 6.5 Cyclo(His—Phe 6.1 0.1 3.3 6.7 Cyclo(Ala~Pro 6.9 0.8 2.1 7.4 Cyclo(Phe—Ser) 4.3 0.1 1.3 7.8 Cyclo(Gly—Leu 4.5 0.0 4.4 t:§:68.1 Cyclo(Gly—Phe) 6.1 0.1 6.7 Cyclo(Pro—Pro 0.4 0.0 0.1 8.6 Cyclo(Gly—Trp) 1.5 0.0 0.0 8.9 6.8 9-2 0.4 9.4 0.3 9.6 Cyclo(Met—Pro) 2.7 0.5 0.1 .2 Cyclo(Met—Met)t 0.2 0.1 2.0 .2 Cyclo(Val—Val) 1.7 0.0 0.4 .7 Leu—Pro) 6.9 1.1 1.5 .5 Cyclo(Trp-Tyr) 1.0 0.0 0.3 L_11.0 Cyclo(Phe—Pro) 8.1 0.4 0.0 11.2 Cyclo(Leu—Trp) 2.7 0.0 4.2 11.8 Cyclo(Phe—Trp) 0.3 0.1 3.9 _~_' 1 .3 Cyclo(Leu—Phe) 14.5 0.3 l 5.5 12.4 Cyclo(Leu—Leu) 17.9 0.4 6.8 _4___ 12.6 Cyclo(Phe—Phe) 4.3 0.0 2.8 tration 150.7 6.5 64.6 (ppm/BX) ' Total concentration per 15067 652 6460 unit Bx (ug/lOOg/Bx) (Example 2) Production of Cyclo<Phe—Phe) from plant peptide _40_ The plant peptides used were as follows: 1) soybean peptides "HINUTE AM" (manufactured by Fuji Oil Co., Ltd.): di and tripeptides: 67%, average molecular weight: 2) soybean peptides "HINUTE DC" (manufactured by Fuji Oil Co., Ltd.): chain length: 3 to 7, average molecular weight: 1000 3) soybean peptides "HINUTE HK" (manufactured by Fuji Oil Co., Ltd.) 4) rice es "Oryza Peptide" actured by Oryza Oil & Fat Chemical Co., Ltd.): tripeptides: 40% to 50% ) wheat peptides "Glutamine e GP—lN" (manufactured by Nisshin Pharma Inc.): molecular weight: 5000 to 10000 6) wheat peptides "Glutamine Peptide GP—N" (manufactured by Nisshin Pharma Inc.): molecular : 5000 to 10000 To 3 g of the peptides of each plant was added 15 mL of distilled water, and the e was put in an autoclave (manufactured by Tomy Seiko Co., Ltd.) and was subjected to high-temperature and high-pressure treatment at 132°C and 0.29 MPa for 2 hours. After the ent, 10 mL of each solution was subjected to solid—phase extraction with OASIS MAX (manufactured by Waters Corporation). The resulting solid—phase extract was concentrated under reduced pressure and was then dissolved in 100 uL of DMSO. Using 10 uL of the solution, the concentration of cyclo—phenylalanyl— phenylalanine was determined by high—performance liquid tography (HPLC).

Table 3 shows the results. The degree of generation -41... of cyclo—phenylalanyl—phenylalanine varied depending on the type of the peptides. Soybean peptides generated a high concentration of cyclo—phenylalanyl—phenylalanine compared to the cases of using rice peptides and wheat peptides. This suggested that it is preferable to use soybean peptides including peptides having a molecular weight of 5000 or less (in particular, a molecular weight of 1000 or less) at a high proportion. Comparison of different soybean peptides suggested that it is preferable to use oligopeptides having a lower molecular weight and containing a large amount of di- and tripeptides as a raw material.

[Table 3] Soybean peptide Diketopiperazine HINUTE HINUTE HINUTE concentration Peptide AM DC HK (Hg/ml) GP-N Phe—Phe 38.4 25.8 15.1_4 3.2 ‘ 0.59 0.33 Bx 20.66 20.10 19.42 Total concentration 186 128 78 per unit Bx L(Mg/100g/Bx) (Example 3) Production of diketopiperazine from plant—derived protein A plant~derived protein subjected to osition treatment with an enzyme was used as the raw al. The plant—derived protein used was soybean protein na 900 (manufactured by Fuji Oil Co., Ltd.)) and rice protein (Oryza Protin P70 (manufactured by Oryza Oil & Fat Chemical Co., Ltd.)), and 300 mg of each n was added to 15 mL of led water. To each mixture, was added 15 mg of any of _42_ enzyme A (ProteAX), enzyme B (Newlase F3G: acid protease (endopeptidase) derived from us niveus), enzyme C (Papain W—40: se derived from Carica papaya), enzyme D (protease A "Amano" SD: protease derived from Aspergillus sp.), enzyme E (protease M "Amano" SD: protease derived from Aspergillus sp.), enzyme F (protease P "Amano" 3SD: derived from illus sp.), enzyme G (Promelain F: protease from Ananas comosus), enzyme H (Peptidase R), enzyme I (Thermoase PClOF: protease (endopeptidase) derived from Bacillus stearothermophilus), enzyme J (Protin SD—NYlO: protease derived from Bacillus sp.), and enzyme K (Protin SD—AYlO: protease derived from Bacillus sp.) (all manufactured by Amano Enzyme Inc.), and the resulting mixture was shaken and mixed at 37°C for 2 hours. This enzyme—treated solution was then subjected to heat ent t ming solid—liquid separation. The heat treatment was high—temperature and high— pressure treatment at 132°C for 2 hours in an ave (manufactured by Tomy Seiko Co., Ltd.). In addition, the soybean protein and the rice protein not treated with any enzyme were rly treated. After the treatment, 10 mL of each solution was subjected to solid-phase extraction with OASIS MAX (manufactured by Waters Corporation). The resulting solid—phase extract was concentrated under reduced pressure and was then dissolved in 100 uL of DMSO. Using 10 uL of the solution, the tration of cyclo—phenylalanyl— phenylalanine was determined by high-performance liquid chromatography (HPLC).

Fig. 1 shows the results in the case of using the -43— soybean protein, and Fig. 2 shows the results in the case of using the rice protein. Protein not subjected to osition ent with an enzyme (Untreated) also generated the diketopiperazine by heat treatment. It was demonstrated that the degree of generation of cyclo— phenylalanyl—phenylalanine varied ing on the type of the enzyme and that there was a tendency that a larger amount of the diketopiperazine was generated by the use of the protease derived from Bacillus sp.

(Example 4) Production (1) of diketopiperazine from plant As a plant, first—grade tea leaves (species: Yabukita, total nitrogen content: 6.3%) produced in Kagoshima—ken were used. The tea leaves were first ted to pre—treatment (pre—extraction, three times) for reducing the amount of water—soluble protein. That is, 200 g of boiling water was added to 10 g of the tea leaves, and the mixture was appropriately stirred for 5 min for extraction. After the completion of the extraction, the mixture was filtered through a 140—mesh filter to collect the extraction residue (used tea leaves). To the used tea leaves was poured 200 g of boiling water, and extraction was performed for 5 min. The used tea leaves were collected and were subjected to tion ent again, and the used tea leaves were collected.

The tea leaves (used tea leaves) after the pre— extraction were subjected to decomposition treatment with an enzyme. To the used tea leaves (the whole quantity) was poured 200 g of hot water of 50°C, and l g of protease (trade -44_ name: Protin NYlOO, manufactured by Daiwa Fine Chemicals Co., Ltd.) was added thereto. The mixture was reacted in a water bath of 55°C for 3 hours with stirring with a stirring bar (300 rpm) and was then maintained at 95°C for 30 min to inactivate the enzyme.

This enzyme—treated solution was subjected to heat ent in the form of a tea leaf—liquid mixture without performing solid—liquid separation. The heat treatment was performed by a high—temperature and high—pressure fluid at 135°C for 3 hours in an autoclave (manufactured by Tomy Seiko Co., Ltd.). The solution after the treatment was filtered through a l40emesh filter to obtain a tea extract (extract A).

This tea extract (extract A) (Bx: 0.99) was ed for the diketopiperazines as in e 1.

Table 4 shows the results. It was demonstrated that a tea extract including a high concentration of a plant extract containing a high concentration of diketopiperazines can be conveniently produced by subjecting tea leaves (used tea leaves) to high—temperature and highepressure ent in a liquid. In addition, it was suggested that it is possible to increase the amount of at least one diketopiperazine ed from the group consisting of Cyclo (Ala—Gln), Cyclo (His—Pro), Cyclo (Ala—Ala), Cyclo (Gly-Pro), Cyclo (Ser~Tyr), Cyclo hr), Cyclo (His—Phe), Cyclo ro), Cyclo (Phe— Ser), Cyclo (Gly-Leu), Cyclo (Gly—Phe), Cyclo (Pro-Pro), Cyclo (Gly—Trp), Cyclo (Asp—Phe), Cyclo (Val—Pro), Cyclo (Pro—Tyr), Cyclo (Met—Pro), Cyclo (Met—Met), Cyclo (Val—Val), Cyclo (Leu— Pro), Cyclo (Trp—Tyr), Cyclo (Phe—Pro), Cyclo (Leu—Trp), Cyclo _45..

(Phe—Trp), Cyclo he), Cyclo (Leu—Leu), and Cyclo (Phe— Phe. In particular, cyclo-leucyl—leucine and cyclo—leucyl— phenylalanine were contained at high concentrations. The content of these piperazines was 27.2% in the plant extract containing them at a high concentration. In the sensory evaluation of taste, this tea extract was substantially tasteless and odorless. -46..

[Table 4] Diketopipera21ne Extract A concentration (ppm/Bx) Cyclo(Ala—Gln) 6.7 Cyclo(His—Pro) 3.5 Ala—Ala) 11.3 Cyclo(Gly—Pro) 3.

Cyclo(Ser—Tyr) 8.

Cyclo(Pro—Thr) 6.

Cyclo(His—Phe) 4.

Cyclo(Ala—Pro) 3.

Phe—Ser) 7.

Cyclo(Gly—Leu) 9.

Cyclo(Gly—Phe) 5. ___, Pro—Pro) 1.

Cyclo(Gly—Trp) 2.

Cyclo(Asp-Phe) 7 Cyclo(Val—Pro) 2 Cyclo ( Pro—Tyr) 1 Cyclo(Met—Pro) 0.

Cyclo(Met—Met)t 0.

Cyclo(Val—Val 1.

Cyclo{Leu-Pro 6.

Cyclo(Trp—Tyr 1.

Cyclo(Phe—Pro) 1.

Cyclo(Leu—Trp 2.

Cyclo(Phe—Trp 0. JCDF-‘UWKOCDKOP—‘hflwOI-JNOQKOW Cyclo(Leu—Phe 17.5 Cyclo(Leu—Leu 21.5 Cyclo(Phe—Phe 4.2 Total concentration 143.3 (ppm/Bx) Total concentration per unit Bx 14326 (ug/lOOg/Bx) (Example 5) Production (2) of diketopiperazine from plant Commercially available soybean boiled in water and malt were used as plants. The soybean boiled in water and malt -47_ were each subjected to pre—extraction, three times, with boiling water in an amount of 20 times the dry weight of the plant an), as in Example 4, and were then subjected to enzyme treatment and high—temperature and high—pressure treatment in a liquid, as in Example 3, to prepare a soybean extract (extract B) and a malt extract ct C). The dry weight of the n was assumed as 36.5% of the total amount of the soybean boiled in water, based on the data of the Food Composition Table, 5th ed. The Bx of each of the extract B and the extract C was adjusted to l, and each extract was then analyzed for the diketopiperazines as in Example 1. Table 5 shows the results. It was demonstrated that plant extracts containing high concentrations of diketopiperazines can also be conveniently produced from soybean and malt. — 48 [Table 5] Diketopiperazine Extract B Extract C concentration (ppm/BX) Cyclo(Ala—Gln) (I! O M Cyclo(His—Pro) NU) Cyclo(Ala—Ala Gly—Pro Cyclo(Ser—Tyr Cyclo(Pro-Thr His-Phe Cyclo(Ala—Pro Cyclo(Phe—Ser Cyclo(Gly—Leu) Cyclo(Gly—Phe Cyclo(Pro—Pro) Cyclo Gly—Trp( ) Cyclo Asp—Phe( ) Cyclo(Val— Pro) Cyclo ( Pro-Tyr) Cyclo Met— Pro){ Cyclo(Met—Met)t ONwONHOOHHmHomrb-Ni—‘LUO‘XKOWOWW mflwmmfiwmeQE—‘w‘btfiCDKOQOOfiOl-‘N “Wm-1’7 19.. 6 l\) as. l\) .2 [\DKOQOHWOONOOOl—‘l—‘bOOMANNN‘bUJNOO wmqwkowm¢QJ>wasqqmwQI—lmwwommmo ‘Total concentration 125.5 79.4 (ppm/BX) Total concentration per unit Bx 12553 7936 (ug/lOOg/Bx) (Example 6) Production (3) of piperazine from plant The same tea leaves as those in Example 4 were used as the plant, and the influence of pre—extraction, enzyme _49_ treatment, and heat treatment thereon were ed. The s are shown in Table 6. The samples of sample Nos. 5 and 6 show that the step of generating oligopeptides from a plant and the step of generating dipeptides through cyclization of oligopeptides by high—temperature and high— pressure treatment in a liquid were simultaneously performed by heat treatment. The pre-extraction was performed as in Example 4 except that the number of times was two. The enzyme treatment was performed as in Example 4 except that the reaction temperature was 50°C. The heat treatment was also performed as in e 4 except that the heating time was changed to 8 hours. The ing tea extracts (sample Nos. 1 to 8) were ed by LC~MS/MS as in Example 1.

[Table 6] Pre— Step (a) Step (b) extraction Not done Not done Done Without. heat Not done With enzyme treatment Done treatment Not done Without enzyme treatment, with heat treatment Done (135°C, 8hr) Not done With heat W'th treatment D trea:;:zie one (135°C, 8hr) Table 7 shows the results. It was revealed that diketopiperazines are not generated if the high—temperature and ressure treatment in a liquid is not performed (sample Nos. 1 to 4). In addition, comparison of the samples of sample Nos. 5 to 8 gave the following findings: The pre—treatment (extraction treatment) increases the _50_ diketopiperazine concentration in the resulting tea extract; Although oligopeptides can be prepared by any of the heat ent and the enzyme treatment, the enzyme treatment was more effective and ent.

A plant extract (tea extract) containing a considerably large total amount of diketopiperazines per Bx, such as 900 ug/lOO g/Bx or more, was prepared by appropriately performing pre-extraction, heat treatment, and enzyme treatment. This suggests that the present invention is advantageous for a plant extract containing a high tration of diketopiperazines and production thereof. In addition, it was suggested that a plant extract (tea extract) containing each of Cyclo ln), Cyclo (His—Pro), Cyclo (Ala—Ala), Cyclo (Gly—Pro), Cyclo (Ser—Tyr), Cyclo (Pro—Thr), Cyclo (His—Phe), Cyclo (Ala—Pro), Cyclo (Phe~Ser), Cyclo (Gly— Leu), Cyclo (Gly—Phe), Cyclo (Pro—Pro), Cyclo (Asp—Phe), Cyclo (Val—Pro), Cyclo (Pro—Tyr), Cyclo (Met—Pro), Cyclo (Len—Pro), Cyclo (Phe—Pro), Cyclo (Leu—Phe), and Cyclo (Leu~Leu) at a concentration of 10 ug/lOO g/Bx or more can be prepared. It was suggested that the present invention is also useful for the production of one or more of these diketopiperazines.

Furthermore, Cyclo(Phe~Phe) was generated by performing eatment or enzyme treatment. This suggested that the present invention can provide a plant extract (tea t) containing Cyclo(Phe—Phe) at a content per Bx of ug/lOO g/BX or more. Highly hydrophobic Cyclo(Phe—Phe) was stably retained in the extract (in an aqueous solution). _51_ Evaluation of the samples of sample Nos. 5 to 8 for flavor trated that the extracts themselves do not have any taste such as bitterness. Aqueous solutions containing one of or all three of Leu-Pro), Cyclo<Phe—Pro), and Cyclo(Leu—Trp) at the same concentrations as those of the samples of sample No. 5 were prepared and were evaluated for flavor. In these solutions, since bitterness was significantly sensed, it was suggested that the presence of the diketopiperazines in a tea extract reduces bitterness.

[Table 7] Diketopiperazine No.1 No.2 No.3 No.4 No.5 concentration x) i.Ado.)No.6 No.7 Cyclo<Ala—Gln) O O Cyclo(His—Pro) O O Cyclo(Ala—Ala > Cyclo(Gly—Pro ) l Cyclo(Ser—Tyr ) Cyclo(Pro—Thr) Cyclo(His—Phe) Cyclo(Ala—Pro ) Cyclo(Phe—Ser) Cyclo(Gly*Leu ) Cyclo(Gly—Phe) Cyclo(Pro—Pro) Cyclo(Gly—Trp ) Cyclo Asp-Phe ) O1000OOOOOOOOOO OONOOONOLOOi—‘i—J ..........

Cyclo(Val-Pro) OOOOOOOOOOOOOOQOO OOOOOOOOOOOOOOOOOO }_\ ' meNmmqomu—Imw Cyclo(Pro—Tyr) o . n .

Cyclo<Met—Pro) . . . . o Cyclo(Met—Met)t i Cyclo Val—Val( Cyclo ( Cyclo(Trp Tyr .

Cyclo(Phe~Pro Cyclo ( OW‘l'mLUfiWHWOOLUQmmCOKOLbQWNUWUTCOWKO Cyclo(Phe—Trp ONHHOWF—‘OOF—‘WONNHU‘IKDQWWONQJW WKOmObeLbGth-NKOKDQN Cyclo(Leu—Phe OOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOGOO 000000000 000000000 OOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOi—‘OOOl—‘O ONHOOHOKOOONwUWF—‘OMNNN\lNWWOOLD-P—‘N 'EEEOOOONO I—AusaoomOe-O Omsk-COOOWOOOOHHOOHWWHD—‘(fltfll—Jtfii—‘I—J i GNNr—‘N 16.5 r Cyclo(LeunLeu 20.9 Cyclo(Phe—Phe 3.9 Total concentration (ppm/BX) Total concentration per unit Bx (pg/:OOg/Bx) (Example 7) Production (4) of piperazine from plant Since the ness of pre-extraction was confirmed in Example 6, the number of times of the pre—extraction was examined. First—grade tea leaves (species: Yabukita, total -53_ nitrogen content: 6.3%) produced in Kagoshima—ken were used as the plant. In order to achieve a higher concentration of diketopiperazines, the optimum number of times of pre— extraction was investigated. The pre~extraction was performed by the following ure. That is, 200 g of boiling water was added to 10 g of tea , and the mixture was appropriately stirred for 5 min for tion. After the completion of the extraction, the mixture was filtered through a l40—mesh filter, and the extract was discarded. In the level of performing the pre-extraction twice or more, 200 g of boiling water was added again to the used tea leaves collected by filtration, and the same procedure was repeated. To the used tea leaves al amount: 10 g) thus subjected to the pre-treatment from zero to three times, 200 g of hot water of 50°C was poured, and l g of an enzyme protease (Amano Enzyme Inc., Protin NYlOO) was added o. The mixture was reacted in a water bath of 50°C for 3 hours with stirring with a stirring bar (300 rpm) and was then maintained at 95°C for min to inactivate the enzyme. The resulting tea leaf~ liquid mixture was placed in an ave (Tomy Seiko Co., Ltd.) and was subjected to high—temperature and high-pressure treatment at 135°C for 8 hours, and the resulting solution was filtered through a l40—mesh filter to prepare a tea extract.

The Bx of each of the resulting extracts was measured, and the concentration of diketopiperazines was then quantitatively measured by MS as in Example 1.

Table 8 shows the results. The amount of generated diketopiperazines increased with the number of times of pre— _54_ extraction. Fig. 3 shows a onship between the number of times of pre—extraction and the removal rate of the soluble component. The removal rate of the soluble component was calculated by the expression: "(the amount (total amount: g) of solution obtained by pre-extraction x its Brix [Bx])/(the amount (g) of solution obtained by repeating ten times the tion of a plant with boiling water in an amount of 30 times the weight of the plant for 10 min x its Brix [Bx]) x lOO(%)". It was revealed that 95% or more of the soluble component can be removed by repeating pre—extraction three times. -55_ [Table 8] . . . The number of times of pre— Diketopipera21ne extraction. concentration (ppm/Bx) zero once twice tnree times Ala—Gln) 1.9 5.0 6.6 6.7 { Cyclo(His—Pro) 1.3 2.8 3.4 3.5 Cyclo(Ala—Ala) 5.8 9.6 11.9 11.3 Cyclo(Gly—Pro 1.5 2.7 3.2 3.3 Cyclo(Ser~Tyr 3.5 7.0 8.7 8.9 Cyclo(Pro—Thr 3.2 5.8 6.9 6.9 r_ His~Phe 1.3 3.0 3.9 L 4.0 Ala—Pro 1.7 2.7 3.2 L___3.2 Cyclo(Phe—Ser) 3.4 6.7 7.4 7.1 Cyclo(Gly—Leu 3.9 8.6 9.6 9.0 Gly—Phe) 1.8 4.5 5.4 5.3 Cyclo(Pro—Pro 0.8 1.5 .L_l'7 1.7 Cyclo(Gly—Trp> 0.6 1.8 2.4 2.4 Cyclo(Asp—Phe) 1.7 5.2 6.4 7.1 cyclowal—Prm 1-3 Cyclowro-Tym m— Cyclo(Met—Pro) 0.3 0.7 0.9 0.9 Cyclo(Met-Met)t 0.1 0.0 0.5 Cyclo(Val~Val) 0.3 0.8 1.1 1.1 Cyclo(Leu-Pro) 3.4 6.0 6.7 Cyclo(Trp—Tyr) 0.3 0.9 1.3 1.3 Cyclo(Phe—Pro) 0.6 1.1 1.3 Cyclo(Leu—Trp) 0.6 1.7 2.2 2.2 Cyclo(Phe—Trp) 0.2 0.6 0.8 Cyclo(Leu-Phe) 4.1 12.2 16.5 Cyclo(Leu—Leu) 6.2 16.9 20.9 21.5 Cyclo(Phe—Phe) 0.6 2.6 3.9 y 4.2 TOtal concentration 51.1 114.4 141.6 (ppm/BX) ' 143.3 Total concentration per unit BX 5114 11437 14157 (Hg/lOOg/Bx) (Example 8) Production (5) of diketopiperazine from plant Since the usefulness of enzyme treatment was confirmed in ...56_ Example 6, the type of the enzyme was examined. The enzymes examined were the following nine types: <Sample No. 9> Protin NYlOO: protease (endopeptidase) derived from us amyloliquefaciens, <Sample No. 10> Thermoase 160: heat—resistant protease (endopeptidase) derived from Bacillus stearothermophilus, <Sample No. 11> Thermoase PC1OF: protease (endopeptidase) derived from Bacillus stearothermophilus, <Sample No. 12> ProteAX: neutral protease derived from‘ Aspergillus oryzae, <Sample No. 13> protease M: neutral protease derived from Ananas comosus, e No. 14> se P: ne protease derived from Aspergillus melleus, <Sample No. 15> protease A: neutral protease derived from Aspergillus oryzae, <Sample No. 16> Peptidase R: neutral protease derived from Rhizopus oryzae, and <Sample No. 17> Newlase F3G: acid protease (endopeptidase) derived from Rhizopus niveus.

As a plant, first-grade tea leaves (species: Yabukita, total nitrogen content: 6.3%) produced in Kagoshima—ken were used. Each of used tea leaves was prepared from 10 g of the tea leaves by ming pre—extraction three times as in Example 6, 200 g of hot water of 55°C (70°C for Thermoase 160 and Thermoase PClOF) was poured thereinto, and 1 g of an enzyme was then added thereto. The e was reacted in a water bath of 55°C (70°C for ase 160 and Thermoase PClOF) _57_ for 3 hours with stirring with a stirring bar (300 rpm) and was then maintained at 95°C for 30 min to inactivate the enzyme. The resulting tea leaf—liquid mixture was placed in an autoclave (Tomy Seiko Co., Ltd.) and was ted to high— temperature and high—pressure ent at 135°C for 8 hours, and the resulting solution was filtered through a 140*mesh filter to prepare a tea extract. The Bx of each of the 'resulting extracts was measured, and the concentration of diketopiperazines was then quantitatively measured by LC—MS/MS as in Example 1.

Table 9 shows the results. It was demonstrated that the concentration of diketopiperazines icantly increases by using a bacterial enzyme having a high ptidase activity. .Among the bacterial enzymes, when neutral protease derived from Bacillus subtilis and protease derived from Bacillus stearothermophilus were used, the amount of ted diketopiperazines particularly increased.

[Table 9] Diketopiperazine tration No.9 No.10 No 11 No.12 No.13 No.14 No.15 No.16 No.17 (ppm/Bx) Cyc;o(Ala—Gln 6.74— 7.0 3.8 2.1 1.3 1.6 l. l. 0.8 Cyc;o(His—Pro 3.5 2.4 2.6 3.2 5.4 4.8 2. 4.2 1.6 .___._—J____ Cyc;o(Ala-Ala) 11.3 9.8 7.5 3.8 2.3 2.7 4. 1.4 Gly..prO 3.3 2.2 1.9 1.2 3.5 2.6 2. 1.6 Cyclo(Ser—Tyr 8.9 5.7 3.5 1.6 0.7 1.3 1. .8 Cyc:_o(Pro—Thr 6.9 5.3 3.8 4.0 7.4 6.8 5. 6.]. 3.7 Cyc:_o(His—Phe 4.0 4.5 3.9 0.5 0.1 0.1 O. 0.3 Cyc;o(Ala-—Pro 3.2 3.2 1.9—1 2.0 3.4 2.7 2. 2.9 1.6 cyc;o(phe_3er 7.1 5.3 4.9 1.5 0.8 1.2 m 0.7 Cyc;o(G1y—Leu 9.0 19.0 10.5 1.3 1.0 1.0 1.3 0.9 0.7 CyclO(Gly_phe 5.3 5.7 5.8 0.8 0.8 1.0 0.6 0.7 0.5 Cyclo(pro_Pro 1.7 1.4 0.7 1.8 1.9 0.6 1.7 .8 ———-——————-—————J——.-—————-—-—- cyaomlyqrp 2.4 2. 0.8 0.4 0.5 0.2 Cyclo<Asp~Phe 7.1 6.4 0.8 1.0 0.7 0.7 Cyc;o(Val—Pro 2.9 1.4 2.0 3.7 3.3 2.6 3.0 1.6 Cyc;o(pro_q;yr 1.8 1.2 1.2 1.3 3.5 2.8 2.7 1.1 Cyc_o(Met—pro) 0.9 0.5 0.7 1.4 0.8 1.0 0.0 cyclo(Met_Met)t 0.5 0.5 0.3 0.0 0.0 0.0 m 0.0 .0 Cyc;o(Va1—Va1) 1.1 1.4 0.7 0.1 0.0 0.1 0. 0.1 cyc;o(Leu_pro) 6.8 4.5 2.5 3.1 6.9 5.5 5.4 5.1 1.9 __._.._—_l Cyc;o(Trp—Tyr) 1.3 1.5 0.6 0.1 0.0 0.1 0.1 0.0 Cyc; (phe_pro) 1.3 1.0 0.9 1.2 3.2 2.0 2.4 2.4 0.9 CyC;O(Leu-Trp) 2.2 3 4 2.2 0.2 0.1 0.1 0.1 0.1 Cyclo(Phe_Trp) 0.8 0.5 0.0 0.0 0.0 0.0 0.0 Cyc;o(Leu-Phe) 17.5 4.1 10.2 1.1 0.1 0.1 0.1 0.2 0.3 cyc;_o(Leu_Leu) 21.5 8. 10.8 2.6 0.2 0.4 m- 1. __+___ cyclo(Phe_phe) 4.2 0.8 2.5 0.0 0.0 0.0 mm 0.1 Total concentration 143.3 112.4 92.9 36.1 49.1 44.7 40.0 40.9 21.9 Total trat.ion per 14326 11242 9286 3607 4909 4471 4001 4086 2193 unlt Bx (HQ/loog/BX) (Exanple 9) Production (6) of diketopiperazine from plant Tea extracts were produced as in Example 4 except that the concentrations of the enzyme (Protin NY100) were changed -59_ to 0% to 20% based on the amount of the tea leaves. The resulting tea extracts were subjected to sensory evaluation, and the contents of 17 types of diketopiperazines shown in Table 10 were measured by LC-MS/MS as in Example 1 and the total amount thereof was determined.

Table 10 shows the results. It was suggested that the enzyme tration should be within a range of 1% to 20% by weight based on the amount of plant raw material, preferably 3% to 15% by weight and more preferably 4% to 10% by weight. In addition, in all of the tea extracts, since the tea extract itself does not substantially have any taste, these extracts were judged to be extracts that can be used by being added to foods and drinks. In particular, the tea extracts subjected to ations of pre—extraction, enzyme treatment, and heat treatment had ent flavor.

[Table 10] Enzyme concentration Diketooiperazine RT ‘ . concentration 20% (min) ( ppm) .1 4 - 4 Cyclo (A1a~Gln . 28 .2 -6 Cyclo(His—Pro 2.4 2.9 4_2 6 - 7 Cyclo(A1a—A1a) 2.2 2.6 ——J3_7 7'4 Gly~Pro 5~9 7-5 10,5 7-8 Cyclo(Ser~Tyr 13.7 13.1 8-1 Cyclo(Pro—”hr 7.5 9.7 9.9 10.7 8-6 Cyc10(His—Phe 0.7 0.9 0.9 1,1 9-2 Cyclo(Ala-—Pro) 0.7 1.0 1.0 1.9 9-6 Cyclo(Phe—Ser 0.4 0.5 0.6 0.8 .2 Cyclo(Gly—Leu 0.2 0.3 0.2 0.3 -2 1.7 1.7 2,2 -7 Cyclo(Pro—Pro 1.6 2.1 2.2 4.0 11 Cyclo(G1y-Trp) 1.4 1.7 1.6 2.2 12. 3 Cyclo(AspPhe) 12. 4 cyclo((Val-_Pro) 11 7 12 9 15. 2 14 8 16. 2 13.3 12.2 12 6 Cyclo((Pro-Tyr) 2 2 2. 8 4.1 4. 6 Total concentration 79.2 89.9 112.4 113.2 128.8 138.6 130.9 130.5 (ppm) 1....

BX 0.56 0.63 0.79 0.82 0.99 Total 1 concentration 14146 14267 14231 13803 13010 11088 per unit Bx (ug/lOOg/Bx) (Example 10) Production (7) of diketopiperazine from plant Tea extracts were produced as in Example 4 except that the conditions for the high—temperature and high—pressure treatment were changed. Specifically, the same tea leaves as those in Example 4 were used as the plant at the same amount.

Tea 1eaves (used tea leaves) were ed by repeating pre— tion with water three times in an amount of 30 times, d of 20 times, the amount of the plant and were subjected to enzyme treatment as in Example 4 and to heat _61_ treatment with the same heat ent ent as that in Example 4 under the various heating conditions shown in Table 11. The resulting tea extracts were analyzed for the diketopiperazines in the extracts as in Example 1.

Table 11 shows the results. It was suggested that generation of diketopiperazines needs heating at 100°C or more (preferably 115°C or more and more preferably 125°C or more) for a heating time of about 30 min to 10 hours and preferably about 2 to 8 hours. - 62 _ [Table 11] Heat treatment condition Diketoconcepntprationi erazine 95°C 105°C 115°C 125°C 135°C m1nfi 3h 3h 3h 3h (ppm/Bx) Cyclo(A1a—G1n)_#_ 0.0 0.8 1.3 2.4 7.6 Cyclo(His— Pro) 0.0 0.5 1.0 1.6 2.2 Cyclo(A1a—A1a) 0.0 3.2 5.8 9.7 19.8 Cyclo(G1y— Pro) 0.0 0.3 0911.6 3.5 } Cyclo(Ser—Tyr) 0.0 0.6 1.3 3.0 5.9 Cyclo(Pro—Thr) 0.2 0.9 1.8 3.2 5.5 Cyclo(His— Phe) 0.0 0.9 1.7 2.5 2.1 Cyclo(A1a— Pro) 0.0 0.8 1.2 2.0 1 4.9 Cyclo(Phe— Ser)_J' 0.1 1.4 1.8 _i_ 2.7 6.9 Cyclo(G1y—Leu) __Jl.0 1.8 4.0 6.3 10.7 Cyclo(G1y— Phe) 0 1 1.7 3.1 5_5.7 9.2 Cyclo(Pro— Pro 0.0 0.1 0.3 0.6 1.6 Cyclo(Gly—Trp) 0.0 0.5 0.8 1.5 3.0 Cyclo<Asp— Phe 0.2 3.1 5.3 9.4 15.5 Cyclo(Val— Pro 0.1 2.1 ProTyr) 0.0 1.4 Cyclo(Ala-Gln 0.0 0.0 0.1 0.3 0.7 Cyclo(His~ Pro 0.0 0.2 0.3 0.4 0.8 Cyclo(Ala—Ala) 0.0 0.1 0.2 0.4 1.6 Cyclo(Gly— Pro 0.3 1.9 3.0 4.2 6.3 Ser—Tyr) 0.0 0.3 0.6 1.1 2.2 Cyclo(Pro—Thr) 0.0 0.0 0.3 0.7 1.4 Cyclo(His— Phe) 0.0 0.5 1.2 2.4 2.7 Ala— Pro) 0.0 0.2 0.1 0.7 1.7 Cyclo(Phe~ Ser) 0.1 1.9 4.7 9.7 14.4 Cyclo(Gly—Leu) 0.2 2.4 5.7 12.0 17.7 Cyclo(G1y— Phe) 0.0 0.7 1.5 2.4 6.0 Total 1.3 25.5 48.9 87.8 tration (ppm/BX) Total Eggcifiiagion 130 2555 4890 8781 15748 (ug/lOOg/Bx) (Example 11) Production of diketopiperazine~~ containing food or drink _63__ Tea t A produced in Example 4 and/or water in a total amount of 50 g as shown in Table 12 was added to 450 g of a commercially available PET green tea drink to prepare diketopiperazine—containing tea drinks each in the total amount of 500 g. These tea drinks were subjected to sensory evaluation of flavor. The evaluation was performed mainly for bitterness and judged by overall preference based on five criteria: very good flavor ((3), good flavor (C3), drinkable flavor (ll), flavor slightly difficult to drink (X), and flavor very difficult to drink (XIX).

Table 12 shows the results. It was confirmed that all of the tea drinks each in an amount of 500 g including 0 to 50 g of the tea extract A containing the diketopiperazine mixture of Example 4 had good flavor. This suggests that the tea t prepared by the present invention is a highly versatile material to be mixed in ing the flavor of drinks.

[Table 12] Blending quantity Total amount of Result of Water diketopiperazines tea gree:rin sensory ex:ea ract amounTotai' (g) (”g /500 ) evaluation. g (g) (g) (g) 1 450 L 0 50 500 O 2 450 5 45 500 57 © 3 450 10 40 500 115 © 4 450 20 30 500 229 © 450 30 20 500 1* 344 @ 6 450 50 O C)

Claims (9)

1. A plant extract comprising at least one of alanylglutamine , cyclo-alanyl-alanine, cyclo-seryl-tyrosine, cycloglycyl-leucine , cyclo-glycyl-tryptophan, cyclo-valyl-valine, cyclo-tryptophanyl-tyrosine, cyclo-leucyl-tryptophan, cyclophenylalanyl-proline , and cyclo-phenylalanyl-phenylalanine at a concentration of 10 µg/100 g/Bx or more, wherein the plant extract is a tea t, a soybean extract, a barley t, or a sesame extract, and the total amount of diketopiperazine(s) per Bx is 900 µg/100 g/Bx or more.

2. The plant extract according to claim 1, being a tea extract, a soybean extract, or a barley extract.

3. A method for producing a plant extract containing a high concentration of diketopiperazines including cyclo-leucylleucine and cyclo-leucyl-phenylalanine, the method sing a step of subjecting a plant peptide to a treatment at 100°C to 170°C and 0.101 to 0.79 MPa for 30 to 500 minutes in a liquid, wherein the plant peptide is a tea peptide, a soybean peptide, a barley peptide, or a sesame peptide.

4. The method according to claim 3, n the plant peptide is an oligopeptide.

5. The method according to claim 3 or 4, wherein the plant peptide is prepared by subjecting a plant-derived protein or a protein-containing plant to decomposition treatment, wherein the plant is a tea, a soybean, a barley, or a .

6. The method according to claim 5, wherein the decomposition treatment is heat treatment or enzyme treatment.

7. The method according to claim 6, wherein the decomposition treatment is enzyme treatment, and the enzyme is endo-type protease.

8. The plant t of claim 1, substantially as herein described with reference to any one of the Examples and/or Figures f.

9. The method of claim 3, substantially as herein described with reference to any one of the Examples and/or Figures thereof. seas zoEEzwozoo 5:; Amiga g mzmémmaomxa 6554433221109fififififififlfifififififl ,,,,, A B C D E F G H I J Ii UNTREATED ig. 2 9553 2 5m on 2fl 11 5w aimi lnu” mzmémmaomxa AB CDEF (3H E J K UNTREATED /'g. 3 99% 100% 1 0o0/0 mz.<m 90% 29.5.68me 5% 80% ri|._I!L....(LI:Li 75% 2L5 7o% .L: ._<._.O.