# Patent

## Title
Kokumi-imparting agent

## Abstract

The present invention encompasses a method for screening for a kokumi-imparting substance by using the calcium receptor activity as an index, a composition containing a kokumi-imparting substance obtained by the screening method, a method for producing food or drink imparted with kokumi, and food or drink imparted with kokumi.

## Background
This application is a divisional under 35 U.S.C. §120 of U.S. patent application Ser. No. 12/117,027, filed May 8, 2008, which claims priority under 35 U.S.C. §119 to Japanese Patent Application No. 2005-325300, filed on Nov. 9, 2005, U.S. Provisional Patent Application No. 60/738,562, filed on Nov. 22, 2005, Japanese Patent Application No. 2006-188458, filed on Jul. 7, 2006, and U.S. Provisional Patent Application No. 60/807,831, filed on Jul. 20, 2006, and is a continuation application under 35 U.S.C. §120 to PCT Patent Application No. PCT/JP2006/322694, filed on Nov. 8, 2006, the contents of which are incorporated by reference in their entireties. The Sequence Listing filed electronically herewith is also hereby incorporated by reference in its entirety (File Name: 2010-11-23T_US-259D_Seq_List; File Size: 1 KB; Date Created: Dec. 7, 2010).


BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method of screening for a substance which imparts kokumi, a composition containing a substance which imparts kokumi obtained by the screening method, a method for producing a food, seasoning, or drink imparted with kokumi, and a food or drink imparted with kokumi.
2. Brief Description of the Related Art
The calcium receptor (also called the calcium sensing receptor, CaSR) contains 1078 amino acids, and is classified as in class C of the seven-transmembrane receptors (G protein-coupled receptor; GPCR). The cloning of the gene for the calcium receptor was reported in 1993 (Nature, 1993 Dec. 9; 366(6455):575-80). The calcium receptor is known to cause various cellular responses through elevation of the intracellular calcium levels, etc., when activated with calcium, etc. The sequence of the human calcium receptor gene is registered with GenBank (Accession No. NM_000388), and is well conserved among many animal species.
The calcium receptor may promote or suppress various biological functions. Therefore, therapeutic agents which act as activators or inhibitors of the calcium receptor are appropriately used in the treatment of neurological diseases, hepatic diseases, cardiovascular diseases, digestive system diseases, and other diseases, depending on the pathological conditions. For example, the calcium receptor is able to detect increased levels of blood calcium in the parathyroid, and suppress secretion of the parathyroid hormone (PTH) to correct the blood calcium level. Therefore, reduction of the blood calcium level is an expected effect of administration of a calcium receptor activator. It has been reported that when a calcium receptor activator is used to treat secondary hyperparathyroidism in a hemodialysis patient, the PTH level is reduced without the calcium and phosphorus levels increasing.
Since functional studies of the calcium receptor have been conducted primarily during calcium homeostasis, applications so far typically concern bone metabolic diseases in which calcium regulation is involved. However, through analysis of genetic expression, it is now known that the calcium receptor is widely distributed in living bodies in addition to the parathyroid and kidney tissues (J. Endocrinol., 2000 May, 165(2):173-7 and Eur. J. Pharmacol., 2002 Jul. 5, 447(2-3):271-8), and the possibility that the calcium receptor is involved in many various biological functions and the etiology of many diseases has been proposed. For example, the calcium receptor is thought to be involved in the function of the liver, heart, lung, alimentary canal, lymphocyte, and pancreas. It has been confirmed that the calcium receptor is expressed in a wide range of tissues by analyses based on RT-PCR using RNAs extracted from rat tissues. Therefore, the increased importance of activators and inhibitors of the calcium receptor in various applications is becoming recognized.
Moreover, cations such as gadolinium, basic peptides such as polyarginine, polyamines such as spermine, amino acids such as phenylalanine, and so forth have been reported to be calcium receptor activators (Cell Calcium., 2004 March, 35(3):209-16).
Although many specific calcium receptor activators have been developed as described above, few of these compounds are native to living bodies, and those that are native have very low activities. Therefore, therapeutic agents containing these activators pose serious problems including side effects, permeability, and sufficient activity. For example, although it is known that amino acids act on calcium receptors, their use as calcium receptor activators is difficult due their very weak activity. Moreover, although macromolecules such as polyarginine have been reported to be an activator as described above, the activator function is based on their actions as polyvalent cations, which have irregular structures. That is, peptides having a specific structure are not known to be useful as a calcium receptor activator.
In the field of foodstuffs, substances having specific tastes have been used for many years. In particular, substances having the five basic tastes, namely, sweet, salty, sour, bitter, and umami (a delicious taste) have been widely used as seasonings. Substances which enhance these basic tastes have also been widely used. One taste that does not fall within these five basic tastes is “kokumi”. Kokumi means a taste that is not one of the five basic tastes. Kokumi is a taste that not only enhances the five basic tastes but also enhances the marginal tastes of the basic tastes, such as thickness, growth (mouthfulness), continuity, and harmony. Several methods for imparting kokumi have been reported so far. Substances that have been reported to impart kokumi include glutathione (Japanese Patent No. 1464928), heated products of gelatin and tropomyosin (Japanese Patent Laid-open Publication (KOKAI) No. 10-276709), sulfone group-containing compounds (Japanese Patent Laid-open Publication (KOKAI) No. 8-289760), a peptide containing the Asn-His sequence (WO2004/096836), and so forth.
Although the development of various kokumi-imparting substances has been attempted as described above, and those that have been commercialized have been mainly extracts of natural products, there are presently very few examples of isolation of a pure kokumi component from an extract of natural product, such as glutathione and N-(4-methyl-5-oxo-1-imidazolin-2-yl)sarcosine.
Therefore, the development of highly effective, safe, and inexpensive kokumi-imparting substances is desired, and a convenient and highly sensitive method of screening for a kokumi-imparting substance is needed for that purpose.
SUMMARY OF THE INVENTION
An aspect of the present invention is to provide a convenient and highly sensitive method for screening for a kokumi-imparting substance, a highly effective, safe and inexpensive kokumi-imparting agent, compositions containing these substances or agents, a method for producing food, seasoning, or drink imparted with kokumi, and food, seasoning, or drink imparted with kokumi.
It has been found that low molecular weight peptides, including glutathione, are able to activate the calcium receptor. Moreover, since glutathione is known to be a kokumi-imparting substance, other low molecular weight peptides which are known to be activators of the calcium receptor were evaluated for their ability to impart kokumi, and it was found that the low molecular weight peptides imparted kokumi. The present invention was accomplished on the basis of these findings.
It is an aspect of the present invention to provide a method for screening for a kokumi-imparting substance comprising utilizing calcium receptor activity as an index.
It is a further aspect of the present invention to provide the method as described above, wherein the kokumi-imparting substance enhances a taste selected from the group consisting of salty, umami, sweet, and sour.
It is a further aspect of the present invention to provide the method as described above comprising a) reacting a calcium receptor and a test substance, b) detecting calcium receptor activity of the test substance, and c) measuring the kokumi-imparting effect of the test substance having calcium receptor activity.
It is a further aspect of the present invention to provide a composition comprising the kokumi-imparting substance obtained by the method as described above.
It is a further aspect of the present invention to provide the composition as described above comprising a substance selected from the group consisting of γ-Glu-X-Gly, wherein X is an amino acid or an amino acid derivative except for Cys, γ-Glu-Val-Y, wherein Y is an amino acid or an amino acid derivative, γ-Glu-Ala, γ-Glu-Gly, γ-Glu-Cys, γ-Glu-Met, γ-Glu-Thr, γ-Glu-Val, γ-Glu-Orn, Asp-Gly, Cys-Gly, Cys-Met, Glu-Cys, Gly-Cys, Leu-Asp, D-Cys, γ-Glu-Met(O), γ-Glu-γ-Glu-Val, γ-Glu-Val-NH2, γ-Glu-Val-ol, γ-Glu-Ser, γ-Glu-Tau, γ-Glu-Cys(S-Me)(O), γ-Glu-Leu, γ-Glu-Ile, γ-Glu-t-Leu, γ-Glu-Cys(S-Me), and combinations thereof.
It is a further aspect of the present invention to provide the composition as described above, wherein X is selected from the group consisting of Cys(SNO), Cys(S-allyl), Gly, Cys(S-Me), Abu, and Ser, and Y is selected from the group consisting of Gly, Val, Glu, Lys, Phe, Ser, Pro, Arg, Asp, Met, Thr, His, Orn, Asn, Cys, and Gln.
It is a further aspect of the present invention to provide the composition as described above, which has the property of enhancing a taste selected from the group consisting of salty, umami, sweet, and sour.
It is a further aspect of the present invention to provide a food composition comprising the composition as described above, and a composition comprising at least one compound having calcium receptor activation activity.
It is a further aspect of the present invention to provide the food composition as described above, wherein the compound having calcium receptor activation activity is selected from the group consisting of calcium, protamine, polyarginine, spermine, polylysine, glutathione, cinacalcet, and combinations thereof.
It is a further aspect of the present invention to provide a method for producing a food or drink imparted with kokumi comprising adding one or more of the compositions as described above to a food or drink to a concentration of 1 mass ppb to 99.9 mass %.
It is a further aspect of the present invention to provide a method for producing food or drink imparted with kokumi comprising adding a seasoning comprising the composition as described above at a concentration of 1 mass ppb to 99.9 mass % to food or drink to a concentration of 0.01 to 10 mass %.
It is a further aspect of the present invention to provide a food or drink imparted with kokumi obtained by the method as described above.
It is a further aspect of the present invention to provide a composition containing 1 mass ppb to 99.9 mass % of γ-Glu-Val-Gly and 1 mass ppb to 99.9 mass % of a substance selected from the group consisting of calcium, protamine, polyarginine, spermine, polylysine, glutathione, and cinacalcet.
It is a further aspect of the present invention to provide a composition comprising 1 mass ppb to 99.9 mass % of a substance selected from the group consisting of glutathione, protamine, polylysine, GABA, salt forms thereof, and combinations thereof, and 1 mass ppb to 99.9 mass % of calcium or a salt form thereof.
It is a further aspect of the present invention to provide a compound selected from the group consisting of:
a) γ-Glu-X-Gly, wherein X is selected from the group consisting of Asn, Gln, His, Lys, Orn, and Arg, and
b) γ-Glu-Val-Y, wherein Y is selected from the group consisting of Leu, Ile, Ser, Thr, Met, Cys, Asp, Asn, Gln, Lys, Orn, Arg, Phe, Tyr, Pro, Hyp, Trp, His, and Abu.
It is a further aspect of the present invention to provide a method for imparting kokumi to a food or drink comprising adding a composition obtained by the method as described above to the food or drink.
It is a further aspect of the present invention to provide a method of imparting kokumi to a composition comprising adding the kokumi-imparting substance obtained by the method described above to a composition.
It is a further aspect of the present invention to provide the method as described above, wherein the kokumi-imparing substance is selected from the group consisting of γ-Glu-X-Gly, wherein X is an amino acid or an amino acid derivative except for Cys, γ-Glu-Val-Y, wherein Y is an amino acid or amino acid derivative, γ-Glu-Ala, γ-Glu-Gly, γ-Glu-Cys, γ-Glu-Met, γ-Glu-Thr, γ-Glu-Val, γ-Glu-Orn, Asp-Gly, Cys-Gly, Cys-Met, Glu-Cys, Gly-Cys, Leu-Asp, D-Cys, γ-Glu-Met(O), γ-Glu-γ-Glu-Val, γ-Glu-Val-NH2, γ-Glu-Val-ol, γ-Glu-Ser, γ-Glu-Tau, γ-Glu-Cys(S-Me)(O), γ-Glu-Leu, γ-Glu-Ile, γ-Glu-t-Leu, γ-Glu-Cys(S-Me), and combinations thereof.
It is a further aspect of the present invention to provide the method as described above, wherein X is selected from the group consisting of Cys(SNO), Cys(S-allyl), Gly, Cys(S-Me), Abu, or Ser, and Y is selected from the group consisting of Gly, Val, Glu, Lys, Phe, Ser, Pro, Arg, Asp, Met, Thr, His, Orn, Asn, Cys, and Gln.
It is a further aspect of the present invention to provide the method as described above, wherein the kokumi-imparing substance is γ-Glu-Val-Gly.
It is a further aspect of the present invention to provide the method as described above, wherein the composition is a food or drink.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the action of calcium on the calcium receptor by introducing the human calcium receptor cRNA into Xenopus laevis oocytes by microinjection. The intracellular response currents were recorded when a calcium chloride solution was added at an arbitrary concentration. The maximum intracellular currents were considered to be the response current values. No response was observed in control oocytes microinjected only with distilled water.
FIG. 2 shows the action of L-amino acids on the calcium receptor by introducing the human calcium receptor cRNA into Xenopus laevis oocytes by microinjection. The intracellular response currents were recorded when a 10 mM L-amino acid solution was added. The maximum intracellular currents were considered to be the response current values. No response was observed in control oocytes microinjected with only distilled water.
FIG. 3 shows the action of D-amino acids on the calcium receptor by introducing the human calcium receptor cRNA into Xenopus laevis oocytes by microinjection. The intracellular response currents were recorded when a 10 mM D-amino acid solution was added. The maximum intracellular currents were considered to be the response current values. No response was observed in control oocytes microinjected with only distilled water.
FIG. 4 shows the action of peptides on the calcium receptor by introducing the human calcium receptor cRNA into Xenopus laevis oocytes by microinjection. The intracellular response currents were recorded when a peptide solution was added at an arbitrary concentration. The maximum intracellular currents were considered to be the response current values. No response was observed in control oocytes microinjected with only distilled water.



DESCRIPTION OF THE PREFERRED EMBODIMENTS
Provided is a convenient and highly sensitive method of screening for a kokumi-imparting substance, a highly effective, safe, and inexpensive composition containing the kokumi-imparting agent obtained by the screening method, a method for producing food, seasoning, or drink imparted with kokumi, and food, seasoning, or drink imparted with kokumi.
The calcium receptor can also be referred to as the calcium sensing receptor (CaSR), and belongs to class C of the seven-transmembrane receptors. The “calcium receptor activity” is when binding of a substrate to the calcium receptor activates the guanine nucleotide binding protein and, as a result, transmits one or more signals. Furthermore, the “calcium receptor activator” is a substance that acts on the calcium receptor to activate the calcium receptor and, as a result, controls the functions of cells expressing the calcium receptor.
“Kokumi” means a taste that is not one of the five basic tastes: sweet, salty, sour, bitter, and umami. Kokumi also means when a substance functions to enhance other properties of the five basic tastes, such as thickness, growth (mouthfulness), continuity, and harmony. Furthermore, a “kokumi-imparting agent” or “kokumi-imparting substance” refers to an agent or substance that can enhance one or more of the five basic tastes, and also enhance other properties of the basic tastes, such as thickness, growth (mouthfulness), continuity, and harmony which accompany the basic tastes. Therefore, the kokumi-imparting agent can also be used as a sweet taste-enhancing agent, salty taste-enhancing agent, sour taste-enhancing agent, bitter taste-enhancing agent, or umami-enhancing agent. As for the intensity of kokumi, the “first and middle taste” means the taste that is experienced from 0 to 4 seconds after eating, and the “aftertaste” means the taste that is experienced 5 seconds after eating.
All the amino acids and amino acid residues which make up the peptides are L-isomers, unless otherwise specified.
Method for Screening for Kokumi-Imparting Substance
The method for screening for a kokumi-imparting substance is characterized by using calcium receptor activity as an index. Specifically, this screening method includes steps of reacting a calcium receptor and a test substance, detecting calcium receptor activity of the test substance, and measuring the kokumi-imparting effect of the test substances having calcium receptor activation activity.
The specific process steps of the screening method are exemplified below. However, the steps of the screening method are not limited to these steps. The steps are as follows:
1) A test substance is added to a system which is set up to measure the calcium receptor activity of the test substance, and the calcium receptor activity is measured.
2) The calcium receptor activity of the test substance is compared to the same system in which no test substance is added (control), and these two values are compared.
3) The test substance having a higher calcium receptor-stimulating activity when the test substance is added is chosen.
4) The kokumi-imparting effect of the chosen test substance is measured, and a test substance having a kokumi-imparting effect is chosen.
The calcium receptor activity is measured by using, for example, a measurement system using cells expressing the calcium receptor. The cells may be cells endogenously expressing the calcium receptor, or recombinant cells which have been transformed with a foreign calcium receptor gene. As the aforementioned calcium receptor activity measurement system, any system may be used without particular limitation so long as the chosen system is able to detect when an extracellular ligand (activator) specific to the calcium receptor is added to the cells expressing the calcium receptor, and the activator binds (reacts with) to the calcium receptor, or a detectable signal is transmitted into the cells in response to binding (reaction) of the activator and the calcium receptor. When the reaction of the tested compound results in calcium receptor activity, this tested compound is determined to have calcium receptor activation activity and be a kokumi-imparting compound.
The kokumi-imparting effect can be confirmed by a taste test by a human, or the like. Furthermore, the test substance used in the screening method is not particularly limited, and may include low molecular weight compounds, saccharides, peptides, proteins, and so forth.
The human calcium receptor encoded by the human calcium receptor gene (GenBank Accession No. NM_000388) is preferred. However, the chosen calcium receptor is not limited to the protein encoded by the gene of the aforementioned reported GenBank sequence, but it may also be a protein encoded by a gene having a homology of 60% or more, preferably 80% or more, more preferably 90% or more, to the aforementioned sequence, so long as the protein functions as a calcium receptor. The GPRC6A receptor and the 5.24 receptor are also known to be subtypes of the calcium receptor, and they can also be used in the screening method described herein. The calcium receptor function can be examined by expressing a gene of interest in a cell and measuring changes in the electric current, or intracellular calcium ion concentration at the time of the addition of calcium.
The origin of the calcium receptor is not particularly limited, and examples include, besides the aforementioned human calcium receptor, calcium receptors derived from animals such as mouse, rat, and dog.
As described above, the calcium receptor activity can be confirmed by using live cells which express a calcium receptor or a fragment thereof, cell membranes which express a calcium receptor or a fragment thereof, an in vitro system containing a calcium receptor or a fragment thereof, or the like.
An example using live cells is shown below. However, confirmation of the calcium receptor activity is not limited to this example.
The calcium receptor can be expressed in cultured cells such as Xenopus laevis oocytes, hamster ovarian cells, and human fetal kidney cells. The calcium receptor can be expressed by cloning a calcium receptor gene in a plasmid that contains a foreign gene and introducing the plasmid or cRNA obtained by using the plasmid as a template. To detect the reaction, electrophysiological techniques, fluorescent indicator reagents that indicate elevation of intracellular calcium level, and so forth, can be used.
Expression of the calcium receptor is first confirmed based on the response to calcium or a specific activator. Oocytes can be used that have an intracellular current with calcium at a concentration of about 5 mM, or cultured cells can be used that showed fluorescence of the fluorescent indicator reagent with calcium at a concentration of about 5 mM. Calcium concentration dependency is determined by changing the calcium concentration. Then, a test substance such as a peptide is prepared to a concentration of about 1 μM to 1 mM, and added to the oocytes or cultured cells, and the calcium receptor activity of the test substance such as the aforementioned peptide is measured.
Kokumi-Imparting Agent
The compositions described herein include those containing a kokumi-imparting substance which is obtained by the screening method. The kokumi-imparting substance is typically the active ingredient in the composition. The composition may contain, for example, one or more of the following substances: γ-Glu-X-Gly, wherein X represents an amino acid or amino acid derivative except for Cys, γ-Glu-Val-Y, wherein Y represents an amino acid or amino acid derivative, γ-Glu-Ala, γ-Glu-Gly, γ-Glu-Cys, γ-Glu-Met, γ-Glu-Thr, γ-Glu-Val, γ-Glu-Orn, Asp-Gly, Cys-Gly, Cys-Met, Glu-Cys, Gly-Cys, Leu-Asp, D-Cys, γ-Glu-Met(O), γ-Glu-γ-Glu-Val, γ-Glu-Val-NH2, γ-Glu-Val-ol, γ-Glu-Ser, γ-Glu-Tau, γ-Glu-Cys(S-Me)(O), γ-Glu-Leu, γ-Glu-Ile, γ-Glu-t-Leu, and γ-Glu-Cys(S-Me). These substances may also be referred to as “peptides and amino acids used for the present invention”. These peptides and amino acids can also be obtained by the screening method described above. Here, “amino acid” means, but is not limited to, neutral amino acids such as Gly, Ala, Val, Leu, Ile, Ser, Thr, Cys, Met, Asn, Gln, Pro, and Hyp, acidic amino acids such as Asp and Glu, basic amino acids such as Lys, Arg, and His, aromatic amino acids such as Phe, Tyr, Trp, and other amino acids such as Homoserine, Citrulline, Ornithine, alpha-Aminobutylic acid, Norvaline, Norleucine and Taurine.
The abbreviations for the amino acid residues are as follows:
(1) Gly: Glycine
(2) Ala: Alanine
(3) Val: Valine
(4) Leu: Leucine
(5) Ile: Isoleucine
(6) Met: Methionine
(7) Phe: Phenylalanine
(8) Tyr: Tyrosine
(9) Trp: Trptophan
(10) His: Histidine
(11) Lys: Lysine
(12) Arg: Arginine
(13) Ser: Serine
(14) Thr: Threonine
(15) Asp: Aspartic acid
(16) Glu: Glutamic acid
(17) Asn: Aspargine
(18) Gln: Glutamine
(19) Cys: Cysteine
(20) Pro: Proline
(21) Orn: Ornithine
(22) Sar: Sarcosine
(23) Cit: Citruline
(24) N-Val: Norvaline
(25) N-Leu: Norleucine
(26) Abu: alpha-Aminobutylic acid
(27) Tau: Taurine
(28) Hyp: Hydroxyproline
(29) t-Leu: tert-leucine
Furthermore, “amino acid derivative” means various types of derivatives of the above-mentioned amino acids, and may include, but are not limited to, unusual amino acids, non-natural amino acids, amino alcohols, substituted amino acids wherein an amino acid side chain, such as carbonyl group, amino group, and/or thiol group, is substituted with various substituents. Such substituents include an alkyl group, acyl group, hydroxyl group, amino group, alkylamino group, nitro group, sulfonyl group, and various protection groups. Such substituted amino acids include, for example, Arg (NO2): N-γ-nitro arginine, Cys (SNO): S-nitrocysteine, Cys (S-Me): S-methyl cysteine, Cys (S-allyl): S-allyl cysteine, Val-NH2: valinamide, Val-ol: valinol (2-amino-3-methyl-1-butanol).
The “O” in the formulas γ-Glu-Met(O) and γ-Glu-Cys(S-Me)(O) indicates a sulfoxide structure. The “γ” in the formula γ-Glu indicates that glutamic acid bonds to another amino acid via the γ position of the carboxy group in the glutamic acid. γ-Glu-Cys(SNO)-Gly has the following structural formula:




The following peptides have been found to impart kokumi and can be used in the compositions described herein: γ-Glu-X-Gly, wherein the X represents an amino acid or amino acid derivative except for Cys, γ-Glu-Val-Y, wherein the Y represents an amino acid or amino acid derivative, γ-Glu-Ala, γ-Glu-Gly, γ-Glu-Cys, γ-Glu-Met, γ-Glu-Thr, γ-Glu-Val, γ-Glu-Orn, Asp-Gly, Cys-Gly, Cys-Met, Glu-Cys, Gly-Cys, Leu-Asp, D-Cys, γ-Glu-Met(O), γ-Glu-γ-Glu-Val, γ-Glu-Val-NH2, γ-Glu-Val-ol, γ-Glu-Ser, γ-Glu-Tau, γ-Glu-Cys(S-Me)(O), γ-Glu-Leu, γ-Glu-Ile, γ-Glu-t-Leu, and γ-Glu-Cys(S-Me). These peptides and amino acids may be used alone, or in various combinations of two or more. Among these, compounds having a structural formula: γ-Glu-X-Gly, wherein X represents Cys(SNO), Cys(S-allyl), Gly, Cys(S-Me), Abu, or Ser), or γ-Glu-Val-Y, wherein Y represents Gly, Val, Glu, Lys, Phe, Ser, Pro, Arg, Asp, Met, Thr, His, Orn, Asn, Cys, or Gln are preferred.
Among these, the following compounds are novel substances newly synthesized: γ-Glu-X-Gly, wherein X represents Asn, Gln, His, Lys, Orn or Arg, and γ-Glu-Val-Y, wherein Y represents Leu, Ile, Ser, Thr, Met, Cys, Asp, Asn, Gln, Lys, Orn, Arg, Phe, Tyr, Pro, Hyp, Trp, H is, or Abu. Furthermore, among these novel substances, γ-Glu-X-Gly, wherein X represents Asn, Gln, His, Lys, Orn or Arg and γ-Glu-Val-Y, wherein Y represents Ser, Thr, Met, Cys, Asp, Asn, Gln, Lys, Orn, Arg, Pro or His are preferred.
Although threshold concentrations, or the minimum concentrations which allow the sensing of taste, of known taste-imparting peptides are about 0.2% ( 1/10 of the threshold concentration of MSG), and thus their practicality is poor (J. Agr. Food Chem., vol. 23, No. 1, 49-53 (1975)), the compounds of the present invention show kokumi enhancing activity at an extremely low concentration of about 0.0001 to 0.1%, and thus they are extremely useful compounds due to their extremely high activity.
Commercially available peptides and amino acids, if available, can be used in the methods and compositions described herein. Furthermore, the peptides can be obtained by using a known technique such as chemical synthesis, or synthesis via an enzymatic reaction. Chemical synthesis is convenient since the number of amino acid residues of the peptides is small, for example, 2 or 3 residues. A peptide synthesizer can be used when chemically synthesizing the peptides, either entirely or partially. Examples of such methods include, for example, a peptide solid phase synthetic method. Peptides synthesized as described above can be purified by usual means, for example, ion exchange chromatography, reversed phase high performance liquid chromatography, affinity chromatography, and so forth. These peptide solid phase synthetic methods and the following peptide purification are well known in this technical field.
Furthermore, the peptides can also be prepared by an enzymatic reaction. For example, the method described in International Patent Publication WO2004/011653 can be used. That is, one amino acid or dipeptide with an esterified or amidated carboxyl terminus can be reacted with an amino acid having a free amino group, for example, an amino acid with a protected carboxyl group, in the presence of a peptide producing enzyme, and purifying the produced dipeptide or tripeptide. Examples of the peptide producing enzyme include a culture of microorganisms having an ability to produce peptides, microbial cells separated from such culture, processed products of these cells, peptide producing enzymes derived from such microorganisms, and so forth.
Salt forms of the peptides and amino acids are also included. Pharmacologically acceptable salts may be used for the salt forms of the peptides and amino acids. Examples of salts containing an acidic group such as a carboxyl group include ammonium salts, salts with alkali metals such as sodium and potassium, salt with alkaline earth metals such as calcium and magnesium, aluminum salts, zinc salts, salts with organic amines such as triethylamine, ethanolamine, morpholine, pyrrolidine, piperidine, piperazine, and dicyclohexylamine, and salts with basic amino acids such as arginine and lysine. Examples of salts with a basic group include salts with inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, and hydrobromic acid, salts with organic carboxylic acids such as acetic acid, citric acid, benzoic acid, maleic acid, fumaric acid, tartaric acid, succinic acid, tannic acid, butyric acid, hibenzoic acid, pamoic acid, enanthoic acid, decanoic acid, teoclic acid, salicylic acid, lactic acid, oxalic acid, mandelic acid, and malic acid, and salts with organic sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.
The method for using the kokumi-imparting substances obtained by the screening method described herein, and the compositions containing one or more of these substances is not particularly limited. For example, they can be added to food, seasonings, or drinks, either alone or in combination with other various additives, etc.
Furthermore, the kokumi-imparting agent may be employed alone, or in combination with other known compounds having kokumi-imparting activity, such as glutathione and alliin, or other various additives etc., which may be arbitrarily added. Moreover, the kokumi-imparting agent may contain one or more known compounds having calcium receptor activation activity.
Examples of these known compounds having calcium receptor activation activity include cations such as calcium and gadolinium cations, basic peptides such as polyarginine and polylysine, polyamines such as putrescine, spermine, and spermidine, proteins such as protamine, amino acids such as phenylalanine and glutathione, cinacalcet, and so forth. Salt forms of these compounds are also included. It has been found that glutathione has calcium receptor activation activity.
Also, it has been found that the kokumi-imparting activities of compounds having known kokumi-imparting activity, such as glutathione, as well as the kokumi-imparting agents described herein are also improved when formulated into a composition with compounds having calcium receptor activation activity.
Any known additives typically mixed with food, seasonings, or drink can be used without particular limitation. Examples of such additives include, for example, perfumes, saccharides, sweetners, dietary fibers, vitamins, amino acids such as sodium glutamate (MSG), nucleic acids such as inosine monophosphate (IMP), inorganic salts such as sodium chloride, water, and so forth.
The amount of the kokumi-imparting substance, agent, or composition which is obtained by the screening method described herein, may be employed in an amount effective for imparting kokumi, and can be suitably adjusted depending on the purpose. However, for seasoning, food, or drink, for example, it may be 1 mass ppb to 99.9 mass %, preferably 10 mass ppb to 99.9 mass %, more preferably 10 mass ppm to 10 mass % with respect to the seasoning, foodstuff, or drink, in terms of the total amount of the kokumi-imparting substance, agent, or composition.
Therefore, by adding one or more of the kokumi-imparting substances, agents, or compositions obtained by the screening method described herein, to food or drink so that the food or drink contains approximately 1 mass ppb to 99.9 mass %, preferably 10 mass ppb to 99.9 mass %, more preferably 10 mass ppm to 10 mass % of the substances or agents, food or drink imparted with kokumi can be produced.
Furthermore, food or drink imparted with kokumi can also be prepared by adding a seasoning containing 1 mass ppb to 99.9 mass % of one or more of the kokumi-imparting substances obtained by the screening method described herein. The kokumi-imparting agents, or compositions can be added to food or drink so that the food or drink contains 0.01 to 10 mass %, preferably 0.1 to 10 mass %, of the seasoning.
The kokumi-imparting substance obtained by the screening method described herein, or the compositions described herein may be in the form of a dry powder, paste, solution, or the like, and the physical properties thereof are not particularly limited.
EXAMPLES
Hereinafter, the present invention will be more specifically explained with reference to the following non-limiting examples.
Example 1
Preparation of Gene (cRNA)
The gene encoding the calcium receptor was prepared as follows. On the basis of the DNA sequence registered at NCBI (calcium receptor: NM_000388), synthetic oligo DNAs (forward primer (N) and reverse primer (C)) were prepared based on the DNA sequence registered at NCBI for the calcium receptor (NM_000388), and used for PCR (Table 1, SEQ ID NOS: 1 and 2).







TABLE 1







Synthetic oligo DNAs (forward primer (N)


and reverse primer (C), h: human)








Code
Sequence (5′-3′)





hCASR_N
ACTAATACGACTCACTATAGGGACCATGGCATTTTAT


 



AGCTGCTGCTGG


 


hCASR_C
TTATGAATTCACTACGTTTTCTGTAACAG









The primers shown in Table 1 (hCASR_N (SEQ ID NO: 1) and hCASR_C (SEQ ID NO: 2)) were synthesized from human kidney cDNA (Clontech), and PCR was performed with Pfu ultra DNA Polymerase (Stratagene) under the following conditions: after a reaction at 94° C. for 3 minutes, a cycle of reactions at 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 2 minutes was repeated 35 times, and then a reaction was performed at 72° C. for 7 minutes. Whether amplification was attained by PCR was detected by agarose electrophoresis, staining with a DNA staining reagent, and ultraviolet irradiation. The length of the PCR products were confirmed by comparison with DNA markers of known sizes simultaneously subjected to electrophoresis. The plasmid vector pBR322 (Takara) was digested with the restriction enzyme EcoRV. The gene fragment amplified by PCR was ligated to the cleavage site of the plasmid by using Ligation Kit (Promega). The Escherichia coli DH5α strain was transformed with each ligation reaction solution, and the transformants containing the plasmid with the PCR amplification product were cloned was selected. The PCR amplification product was confirmed by DNA sequence analysis. By using this recombinant plasmid as a template together with a cRNA preparation kit (Ambion), the cRNA of the calcium receptor gene was prepared.
Example 2
Preparation of Various Samples
23 special grade amino acids were employed, including alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, ornithine, taurine (all of these from Ajinomoto), and hydroxyproline (Nakarai Tesque). Special grade amino acids were also used as D-Cys and D-Trp (Nakarai Tesque) and calcium chloride. Furthermore, the following peptide samples were used: γ-Glu-Cys-Gly (Sigma Aldrich Japan), γ-Glu-Cys(SNO)-Gly (Dojin Chemical Laboratory), γ-Glu-Ala (Bachem Feinchemikalien AG), γ-Glu-Gly (Bachem Feinchemikalien AG), γ-Glu-Cys (Sigma Aldrich Japan), γ-Glu-Met (Bachem Feinchemikalien AG), γ-Glu-Abu-Gly (Abu: α-aminobutyric acid, Bachem Feinchemikalien AG), γ-Glu-Thr (Kokusan Chemical), γ-Glu-Val (Kokusan Chemical), γ-Glu-Leu (contract manufactured product), γ-Glu-Ile (contract manufactured product), γ-Glu-Orn (Kokusan Chemical), Asp-Gly (contract manufactured product), Cys-Gly (contract manufactured product), Cys-Met (contract manufactured product), Glu-Cys (contract manufactured product), Gly-Cys (contract manufactured product), Leu-Asp (contract manufactured product), γ-Glu-Val-Val (contract manufactured product), γ-Glu-Val-Glu (contract manufactured product), γ-Glu-Val-Lys (contract manufactured product), γ-Glu-γ-Glu-Val (contract manufactured product), γ-Glu-Gly-Gly (contract manufactured product), γ-Glu-Val-Phe (contract manufactured product), γ-Glu-Val-Ser (contract manufactured product), γ-Glu-Val-Pro (contract manufactured product) γ-Glu-Val-Arg (contract manufactured product), γ-Glu-Val-Asp (contract manufactured product), γ-Glu-Val-Met(contract manufactured product), γ-Glu-Val-Thr (contract manufactured product), γ-Glu-Val-His(contract manufactured product), γ-Glu-Val-Asn (contract manufactured product), γ-Glu-Val-Gln (contract manufactured product), γ-Glu-Val-Cys(contract manufactured product), γ-Glu-Val-Orn (contract manufactured product) and γ-Glu-Ser-Gly (contract manufactured product). Glutamine and cysteine were prepared upon use, and the other samples were stored at 20° C. after preparation. Peptides having a purity of 90% or higher were used, except for γ-Glu-Cys, which was at a purity of 80% or higher. The pH was adjusted, as needed, to an approximately neutral pH with NaOH or HCl. The solution used for dissolution of amino acids and peptides, preparation of Xenopus laevis oocytes, and culture of the oocytes had the following composition: 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM Hepes, pH 7.2.
Example 3
Synthesis of γ-Glu-Val-Gly
Boc-Val-OH (8.69 g, 40.0 mmol) and Gly-OBzl.HCl (8.07 g, 40.0 mmol) were dissolved in methylene chloride (100 ml), and the solution was maintained at 0° C. Triethylamine (6.13 ml, 44.0 mmol), HOBt (1-hydroxybenzotriazole, 6.74 g, 44.0 mmol), and WSC.HCl (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, 8.44 g, 44.0 mmol) were added to the solution, and the mixture was stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate (200 ml). The solution was washed with water (50 ml), 5% citric acid aqueous solution (50 ml× twice), saturated brine (50 ml), 5% sodium hydrogencarbonate aqueous solution (50 ml× twice), and saturated brine (50 ml). The organic layer was dried over anhydrous magnesium sulfate, then the magnesium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was recrystallized from ethyl acetate/n-hexane to obtain white crystals of Boc-Val-Gly-OBzl (13.2 g, 36.2 mmol).
Boc-Val-Gly-OBzl (5.47 g, 15.0 mmol) was added to 4 N HCl/dioxane solution (40 ml), and the mixture was stirred at room temperature for 50 minutes. Dioxane was removed by concentration under reduced pressure, n-hexane (30 ml) was added to the residue, and the mixture was concentrated under reduced pressure. This procedure was repeated 3 times to quantitatively obtain H-Val-Gly-OBzl.HCl.
Then, H-Val-Gly-OBzl.HCl and Z-Glu-OBzl (5.57 g, 15.0 mmol) were dissolved in methylene chloride (50 ml), and the solution was kept at 0° C. Triethylamine (2.30 ml, 16.5 mmol), HOBt (1-hydroxybenzotriazole, 2.53 g, 16.5 mmol), and WSC.HCl (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, 3.16 g, 16.5 mmol) were added to the solution, and the mixture was stirred at room temperature for 2 days. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in heated ethyl acetate (1500 ml). The solution was washed with water (200 ml), 5% citric acid aqueous solution (200 ml× twice), saturated brine (150 ml), 5% sodium hydrogencarbonate aqueous solution (200 ml× twice), and saturated brine (150 ml). The organic layer was dried over anhydrous magnesium sulfate, then the magnesium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure. The deposited crystals were collected by filtration, and dried under reduced pressure to obtain white crystals of Z-Glu(Val-Gly-OBzl)-OBzl (6.51 g, 10.5 mmol).
Then, Z-Glu(Val-Gly-OBzl)-OBzl (6.20 g, 10.03 mmol) was suspended in ethanol (200 ml), and 10% palladium/carbon (1.50 g) was added. A reduction reaction was performed at 55° C. for 5 hours under a hydrogen atmosphere. During the reaction, a total amount of 100 ml of water was gradually added. The catalyst was removed by filtration using a Kiriyama funnel, and the filtrate was concentrated under reduced pressure to a half volume. The reaction mixture was further filtered through a membrane filter, and the filtrate was concentrated under reduced pressure. The residue was dissolved in a small volume of water, ethanol was added to deposit crystals, and the crystals were collected by filtration and dried under reduced pressure to obtain a white powder of γ-Glu-Val-Gly (2.85 g, 9.40 mmol).
ESI-MS: (M+H)+=304.1
1H-NMR (400 MHz, D2O) δ (ppm): 0.87 (3H, d, J=6.8 Hz), 0.88 (3H, d, J=6.8 Hz), 1.99-2.09 (3H, m), 2.38-2.51 (2H, m) 3.72 (1H, t, J=6.35 Hz), 3.86 (1H, d, J=17.8 Hz), 3.80 (1H, d, J=17.8 Hz), 4.07 (1H, d, J=6.8 Hz)
Example 4
Synthesis of γ-Glu-Cys(S-Me)-Gly [Cys(S-Me): S-methylcysteine]
Reduced glutathione (15.0 g, 48.8 mmol) was added to water (45 ml), and then sodium hydroxide (4.52 g, 2.2 equivalents, 107 mmol) was added little by little to the mixture while bubbled with nitrogen. Then, methyl iodide (4.56 ml, 1.5 equivalents, 73 mmol) was added to the mixture, and the mixture was sealed and stirred at room temperature for 2 hours. The reaction mixture was adjusted to pH 2 to 3 with concentrated hydrochloric acid, added with ethanol (150 ml), and stored overnight in a refrigerator. Since oily matter separated, the supernatant was removed. When the remaining oily matter was dissolved in water and ethanol was gradually added, partially crystallized oily matter was deposited. Therefore, the supernatant liquid was removed again. The residue was dissolved in water (300 ml), adsorbed onto an ion exchange resin (Dowex 1-acetate, 400 ml) applied to a column, and after washing with water, eluted with 1 N acetic acid aqueous solution. The eluate was concentrated under reduced pressure, and precipitated with water/ethanol to obtain a white powder of γ-Glu-Cys(S-Me)-Gly (5.08 g, 15.8 mmol).
FAB-MS: (M+H)+=322
1H-NMR (400 MHz, D2O) δ (ppm): 2.14 (3H, s), 2.15-2.22 (2H, m), 2.50-2.58 (2H, m), 2.86 (1H, dd, J=9.0 Hz, J=14.0 Hz), 3.03 (1H, dd, J=5.0 Hz, J=14.0 Hz), 3.84 (1H, t, J=6.5 Hz), 3.99 (2H, S), 4.59 (1H, dd, J=5.0 Hz, J=9.0 Hz)
Example 5
Synthesis of Other Peptides
γ-Glu-Met(O), γ-Glu-Val-NH2, γ-Glu-Val-ol, γ-Glu-Ser, γ-Glu-Tau, γ-Glu-Cys(S-Me)(O), γ-Glu-t-Leu, γ-Glu-Cys(S-allyl)-Gly, and γ-Glu-Cys(S-Me) were synthesized in a manner similar to that described in Examples 3 and 4.
Example 6
Evaluation of Calcium Receptor Activation Activity
To evaluate calcium receptor activation activity, a Ca ion concentration-dependent Cl ionic current measuring method using a Xenopus laevis oocyte expression system was used. If each activator is added to Xenopus laevis oocytes expressing the calcium receptor, intracellular Ca ions increase. Then, the Ca ion concentration-dependent Cl channel opens, and the intracellular current value changes as an ionic current. By measuring the change in this intracellular current value, the presence or absence of calcium receptor activation activity can be determined.
Specifically, the abdomen of Xenopus laevis was opened, and an egg batch was taken out and treated with a 1% collagenase solution at 20° C. for 2 hours to obtain individual oocytes. Into the oocytes, 50 nl of 1 μg/μl receptor cRNA or 50 nl of sterilized water per oocyte was introduced by using a micro glass capillary, and the oocytes were cultured at 18° C. for 2 or 3 days. For the culture, a solution obtained by adding 2 mM pyruvic acid, 10 U/ml of penicillin, and 10 μg/ml of streptomycin to the solution in Example 2 was used. After the culture, a test solution was added to the oocytes containing either the cRNA or sterilized water. Electrophysiological measurement was performed by using an amplifier, Geneclamp500 (Axon), and recording software, AxoScope 9.0 (Axon). The oocytes were voltage-clamped at −70 mV by the double electrode voltage clamp method, and the intracellular current was measured via the Ca ion concentration-dependent Cl ion channel. The maximum value of the intracellular current was considered as the response current value.
Example 7
Evaluation of Calcium Receptor Activation Activity of Calcium
The calcium receptor activation activity of calcium was evaluated by using the method described in Example 6. That is, oocytes containing either cRNA of the calcium receptor or sterilized water were prepared, and voltage-clamped at −70 mV by the double electrode voltage clamp method. To the voltage-clamped oocytes, calcium was added (2 mM, 5 mM, 10 mM, 20 mM), and Ca ion concentration-dependent Cl response current was measured. The results are shown in FIG. 1. From these results, it was confirmed that the cRNA of the calcium receptor was functionally expressed in the oocytes. Furthermore, since the oocytes containing water did not respond to even high concentration calcium, it was confirmed that the calcium receptor is not expressed in the oocytes.
Example 8
Evaluation of Calcium Receptor Activation Activity of L-Amino Acids
Calcium receptor activation activity of L-amino acids was evaluated by using the method described in Example 6. That is, oocytes containing either cRNA of the calcium receptor or sterilized water were prepared, and voltage-clamped at −70 mV by the double electrode voltage clamp method. To the voltage-clamped oocytes, alanine (10 mM), arginine (10 mM), asparagine (10 mM), aspartic acid (10 mM), cysteine (10 mM), glutamine (10 mM), glutamic acid (10 mM), glycine (10 mM), histidine (10 mM), isoleucine (10 mM), leucine (10 mM), lysine (10 mM), methionine (10 mM), phenylalanine (10 mM), proline (10 mM), serine (10 mM), threonine (10 mM), tryptophan (10 mM), tyrosine (10 mM), valine (10 mM), ornithine (10 mM), taurine (10 mM), or hydroxyproline (10 mM) was added, and Ca ion concentration-dependent Cl response current was measured. The results are shown in FIG. 2. By these results, it was demonstrated that cysteine, histidine, phenylalanine, tryptophan, and tyrosine had definite calcium receptor activation activity. As for the aforementioned amino acids, the activation activity was reported in Proc. Natl. Acad. Sci. USA, 2000 Apr. 25, 97(9):4814-9.
Example 9
Evaluation of Calcium Receptor Activation Activity of D-Cysteine
Calcium receptor activation activity of D-cysteine was evaluated by using the method described in Example 6. That is, oocytes containing either cRNA of the calcium receptor or sterilized water were prepared, and voltage-clamped at −70 mV by the double electrode voltage clamp method. To the voltage-clamped oocytes, D-cysteine (10 mM), L-cysteine (10 mM), D-tryptophan (10 mM), or L-tryptophan (10 mM) was added, and Ca ion concentration-dependent Cl response current was measured. The results are shown in FIG. 3. By these results, it was demonstrated that D-cysteine had definitive calcium receptor activation activity.
Example 10
Evaluation of Calcium Receptor Activation Activity of Peptides
Calcium receptor activation activity of peptides was evaluated by using the method described in Example 6. That is, oocytes containing either cRNA of the calcium receptor or sterilized water were prepared, and voltage-clamped at −70 mV by the double electrode voltage clamp method. To the voltage-clamped oocytes, γ-Glu-Cys-Gly (50 μM), γ-Glu-Cys(SNO)-Gly (50 μM), γ-Glu-Ala (50 μM), γ-Glu-Gly (500 μM), γ-Glu-Cys (50 μM), γ-Glu-Met (500 μM), γ-Glu-Thr (50 μM), γ-Glu-Val (50 μM), γ-Glu-Orn (500 μM), Asp-Gly (1 mM), Cys-Gly (1 mM), Cys-Met (1 mM), Glu-Cys (50 μM), Gly-Cys (500 μM), or Leu-Asp (1 mM) was added, and Ca ion concentration-dependent Cl response current was measured. The results are shown in FIG. 4. By these results, it was demonstrated that the aforementioned peptides had definitive calcium receptor activation activity.
Example 11
Evaluation of Calcium Receptor Activation Activity of Peptides
Calcium receptor activation activity of peptides was evaluated in the same manner as that of Example 10. Each of the peptides shown in Table 2 was added to voltage-clamped oocytes at 1000 μM, 300 μM, 100 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.3 μM, and 0.1 μM, and Ca ion concentration-dependent Cl response current was measured. The lowest concentration for which current was detected was shown in Table 2 as the activity. From these results, it became clear that these 32 peptides had calcium receptor activation activity.









TABLE 2





No.
Peptide
Activity


















1
γ-Glu-Met(O)
1000
μM


2
γ-Glu-Val-Val
1000
μM


3
γ-Glu-Val-Glu
1000
μM


4
γ-Glu-Val-Lys
1000
μM


5
γ-Glu-Val-Arg
1000
μM


6
γ-Glu-Val-Asp
1000
μM


7
γ-Glu-Val-Met
1000
μM


8
γ-Glu-Val-Thr
1000
μM


9
γ-Glu-γ-Glu-Val
1000
μM


10
γ-Glu-Val-NH2
1000
μM


11
γ-Glu-Val-ol
1000
μM


12
γ-Glu-Ser
300
μM


13
γ-Glu-Tau
300
μM


14
γ-Glu-Cys(S—Me)(O)
300
μM


15
γ-Glu-Val-His
100
μM


16
γ-Glu-Val-Orn
100
μM


17
γ-Glu-Leu
100
μM


18
γ-Glu-Ile
100
μM


19
γ-Glu-t-Leu
100
μM


20
γ-Glu-Cys(S-allyl)-Gly
100
μM


21
γ-Glu-Val-Asn
30
μM


22
γ-Glu-Gly-Gly
30
μM


23
γ-Glu-Val-Phe
30
μM


24
γ-Glu-Val-Ser
30
μM


25
γ-Glu-Val-Pro
30
μM


26
γ-Glu-Ser-Gly
30
μM


27
γ-Glu-Cys(S—Me)
30
μM


28
γ-Glu-Val-Cys
10
μM


29
γ-Glu-Val-Gln
10
μM


30
γ-Glu-Abu-Gly
3
μM


31
γ-Glu-Cys(S—Me)-Gly
3
μM


32
γ-Glu-Val-Gly
0.1
μM









Example 12
Kokumi-Imparting Activity of Peptides and Amino Acids
Calcium receptor activation activity was confirmed for the following peptides and amino acids: γ-Glu-X-Gly (X is Cys(SNO), Cys(S-allyl), Gly, Cys(S-Me), Abu, or Ser), γ-Glu-Val-Y (Y is Gly, Val, Glu, Lys, Phe, Ser, Pro, Arg, Asp, Met, Thr, His, Orn, Asn, Cys, or Gln), γ-Glu-Ala, γ-Glu-Gly, γ-Glu-Cys, γ-Glu-Met, γ-Glu-Thr, γ-Glu-Val, γ-Glu-Orn, Asp-Gly, Cys-Gly, Cys-Met, Glu-Cys, Gly-Cys, Leu-Asp, D-Cys, γ-Glu-Met(O), γ-Glu-γ-Glu-Val, γ-Glu-Val-NH2, γ-Glu-Val-ol, γ-Glu-Ser, γ-Glu-Tau, γ-Glu-Cys(S-Me)(O), γ-Glu-Leu, γ-Glu-Ile, γ-Glu-t-Leu, and γ-Glu-Cys(S-Me). Whether or not these peptides and amino acids have kokumi-imparting activity or not was determined by a sensory evaluation test, performed as follows. Samples of either alliin (S-allyl-cysteine sulfoxide: control for Kokumi-imparting activity), γ-Glu-Cys-Gly, γ-Glu-Cys, γ-Glu-Ala, or γ-Glu-Val were added to a final concentration of 0.2 g/dl to distilled water containing sodium glutamate (0.05 g/dl), inosine monophosphate (0.05 g/dl), and calcium chloride (1 mM), and kokumi-imparting activity was determined for each mixture. Sample solutions that became acidic after dissolution of the samples were adjusted to pH 6.8 to 7.2 with NaOH before use. The results are shown in Table 3.







TABLE 3







Kokumi-imparting activity of calcium receptor activators










Calcium receptor activator
Kokumi-imparting activity







γ Glu-Cys-Gly
+



γ Glu-Cys
+



γ Glu-Ala
+



γ Glu-Val
+










Example 13
Kokumi-Imparting Activity of Peptides
The intensity of the kokumi-imparting activity of each peptide having a confirmed calcium receptor activation activity was measured by a quantitative sensory evaluation test, performed as follows: Samples of either γ-Glu-Cys-Gly (glutathione), γ-Glu-Ala, γ-Glu-Met, or γ-Glu-Val were added to distilled water (0.1 g/dl) containing sodium glutamate (0.05 g/dl), inosine monophosphate (0.05 g/dl), and sodium chloride (0.5 g/dl), and the intensity of the kokumi-imparting activity was measured. Sample solutions that became acidic after dissolution of the samples were adjusted to pH 6.8 to 7.2 with NaOH before use. Sensory evaluation scores were used for evaluation based on the control sample (0 points) and the glutathione sample (3 points), and the test was performed with n=3. The results are shown in Table 4.










TABLE 4









Intensity of kokumi














First and





Concentration
middle


Sample
(g/dl)
taste
Aftertaste
Evaluation remarks














Control
—
0
0
—


γ-Glu-Cys-
0.1
3.0
3.0
Thickness, growth


Gly



(mouthfulness), and continuity






were enhanced.


γ-Glu-Ala
0.1
0.5
0.2
Although the effect was weak,






thickness was slightly enhanced.


γ-Glu-Met
0.1
1.5
0.4
Thickness, and growth






(mouthfulness) were slightly






enhanced.


γ-Glu-Val
0.1
3.0
1.0
Thickness, and growth






(mouthfulness) were enhanced






mainly for first and middle tastes









Example 14
Kokumi-Imparting Activity of Peptides
The intensity of the kokumi-imparting activity of each peptide having confirmed calcium receptor activation activity was measured by a quantitative sensory evaluation test, performed as follows: Samples of either γ-Glu-Cys-Gly (glutathione), γ-Glu-Cys, γ-Glu-Val, or γ-Glu-Val-Gly were added to distilled water (0.1 g/dl, or 0.01 g/dl as required) containing sodium glutamate (0.05 g/dl), inosine monophosphate (0.05 g/dl), and sodium chloride (0.5 g/dl), was, and the intensity of the kokumi-imparting activity was measured. The sample solutions that became acidic after dissolution of the samples were adjusted to pH 6.8 to 7.2 with NaOH before use. Sensory evaluation scores were used for evaluation based on the control sample (0 points) and the glutathione sample (3 points), and the test was performed with n=5. The results are shown in Table 5.










TABLE 5









Intensity of kokumi














First and





Concentration
middle


Sample
(g/dl)
taste
Aftertaste
Evaluation remarks














Control
—
0
0
—


γ-Glu-Cys-
0.1
3.0
3.0
Thickness, growth


Gly



(mouthfulness), and continuity






were enhanced.


γ-Glu-Cys
0.1
2.0
2.0
The effect was slightly weaker,






but substantially equivalent






compared with γGlu-Cys-Gly


γ-Glu-Val
0.1
3.0
1.0
Thickness, and growth






(mouthfulness) were enhanced






mainly for first and middle






tastes


γ-Glu-Val-
0.1
*
*
*


Gly


γ-Glu-Val-
0.01
3.0
3.0
Thickness, and continuity were


Gly



mainly enhanced. Total taste






was enhanced





* Unmeasurable: Kokumi-imparting activity was too strong and could not be measured by the sensory evaluation.






Example 15
Kokumi-Imparting Activity of Peptides
The intensity of the kokumi-imparting activity of each peptide having confirmed calcium receptor activation activity was measured by a quantitative sensory evaluation test, performed as follows: Samples of either γ-Glu-Cys-Gly (glutathione), γ-Glu-Abu-Gly, or γ-Glu-Val-Gly were added to distilled water (0.1 g/dl or 0.01 g/dl) containing sodium glutamate (0.05 g/dl), inosine monophosphate (0.05 g/dl), and sodium chloride (0.5 g/dl), and the intensity of the kokumi-imparting activity was measured. Sample solutions that became acidic after dissolution of the samples were adjusted to pH 6.8 to 7.2 with NaOH before use. Sensory evaluation scores were used for evaluation based on the control sample (0 points) and the glutathione sample (3 points), and the test was performed with n=12. The results are shown in Table 6.










TABLE 6









Intensity of kokumi













Concentration
First and




Sample
(g/dl)
middle taste
Aftertaste
Evaluation remarks














Control
—
0
0
—


γGlu-Cys-Gly
0.1
3.0
3.0
Thickness, growth






(mouthfulness), and






continuity were enhanced.


γGlu-Abu-Gly
0.01
3.0
2.0
Thickness, and growth






(mouthfulness) were






enhanced mainly for first and






middle tastes.


γGlu-Val-Gly
0.01
3.0
3.0
Thickness, and continuity






were mainly enhanced.






Total taste was enhanced.









Example 16
Activity of Peptides on Basic Tastes
The intensity of the activity on basic tastes of each peptide having confirmed calcium receptor activation activity was measured by a quantitative sensory evaluation test, performed as follows: Samples of either γ-Glu-Cys-Gly (glutathione) or γ-Glu-Val-Gly were added to distilled water (0.0001 to 1 g/dl) containing sodium glutamate (0.2 g/dl) for the umami standard, sucrose (5 g/dl) for the sweet taste standard, sodium chloride (0.7 g/dl) or the salty taste standard, or citric acid (0.05 g/dl) for the sour taste standard, and the intensity of the activity on basic tastes was measured for each sample.
Sample solutions that became acidic after dissolution of the samples with respect to the standard solutions without the samples were adjusted with NaOH to pH not lower or higher by 0.2 than pH of the standard solutions before use. Sensory evaluation scores were used to evaluate the intensity as follows: 0 points for the control sample, 1 point for fairly intense activity as compared to the control, and 2 points for intense activity as compared to the control, and the test was performed with n=12. The samples showed that the basic tastes were enhanced at concentrations within the aforementioned broad concentration range. The results for typical concentrations are shown in Table 7.











TABLE 7







γGlu-




Evaluation
Distilled
Cys-Gly
γGlu-Val-Gly
γGlu-Val-Gly


system
water
0.10 g/dl
0.005 g/dl
0.01 g/dl







Umami
0
0.7
0.7
1.5


Sweet taste
0
1.5
0.5
1.0


Salty taste
0
0.2
0.5
1.0


Sour taste
0
1.5
0.5
1.0









Example 17
Activity of Peptides for Imparting Kokumi to Consomme Soup
The intensity of activity for imparting kokumi to consomme soup of each peptide having confirmed calcium receptor activation activity was measured by a quantitative sensory evaluation test, performed as follows: Consomme soup was prepared by dissolving consomme soup powder (35% of sodium chloride, 18% of sodium glutamate, 0.2% of inosine monophosphate, 0.3% of white pepper powder, 0.5% of black pepper powder, 8.0% of beef extract powder, 3.0% of white wine powder, 2.0% of celery powder, 8.0% of Chinese cabbage extract powder, 2.5% of onion extract powder, 25.5% of lactose) at a concentration of 5 g/dl. To this consomme soup, samples of either γ-Glu-Cys-Gly (glutathione) or γ-Glu-Val-Gly was added to a concentration of 0.0001 to 1 g/dl, and the intensity of the kokumi-imparting activity was measured for each sample. The consomme soup with the samples that became acidic after dissolution of the samples with respect to the consomme soup without the samples was adjusted with NaOH to pH not lower or higher by 0.2 than pH of the consomme soup without the samples before use. Sensory evaluation scores were used for evaluation as follows: 0 points for the control sample, 3 points for intense activity as compared to the control, and 5 points for extremely intense activity as compared to the control, and the test was performed with n=12. The samples showed kokumi-imparting activity at concentrations within the aforementioned broad concentration range. The results for typical concentrations are shown in Table 8.










TABLE 8









Intensity of kokumi













Concentration
First and




Sample
(g/dl)
middle taste
Aftertaste
Evaluation remarks














Control
—
0
0
—


γGlu-Cys-Gly
0.01
3.0
3.0
Thickness, growth






(mouthfulness), and






continuity were enhanced.


γGlu-Val-Gly
0.0005
2.5
3.0
Thickness and growth






(mouthfulness) were mainly






enhanced from first and






middle taste.


γGlu-Val-Gly
0.001
3.0
3.5
Thickness and growth






(mouthfulness) were mainly






enhanced from first and






middle taste.


γGlu-Val-Gly
0.01
5.0
5.0
Thickness and continuity






were mainly enhanced.






Total taste was enhanced.


γGlu-Val-Gly
0.1
5.0
5.0
Thickness and continuity






were mainly enhanced.






Total taste was enhanced.









Example 18
Activity of Peptides for Imparting Kokumi to Japanese Clear Soup
The intensity of activity for imparting kokumi to clear soup of each peptide having confirmed calcium receptor activation activity was measured by a quantitative sensory evaluation test, performed as follows: Japanese clear soup was prepared by adding 0.5 g/dl of soy sauce and 0.6 g/dl of sodium chloride to bonito kelp stock, obtained by adding 5 g of dried kelp to 3 L of water, heating the water, adding 25 g of dried bonito flakes just before boiling, and then filtering the water containing kelp and bonito flakes. To this clear soup, samples of either γ-Glu-Cys-Gly (glutathione) or γ-Glu-Val-Gly was added to a concentration of 0.0001 to 1 g/dl, and the intensity of the kokumi-imparting activity was measured for each sample. The clear soup with the samples that became acidic after dissolution of the samples with respect to the clear soup without the samples were adjusted with NaOH to pH not lower or higher by 0.2 than pH of the clear soup without the samples before use. Sensory evaluation scores were used to evaluate as follows: 0 points for the control sample, 3 points for intense activity as compared to the control, and 5 points for extremely intense activity as compared to the control, and the test was performed with n=12. The samples showed kokumi-imparting activity at concentrations within the aforementioned broad concentration range. The results for typical concentrations are shown in Table 9.










TABLE 9









Intensity of kokumi













Concentration
First and




Sample
(g/dl)
middle taste
Aftertaste
Evaluation remarks














Control
—
0
0
—


γGlu-Cys-Gly
0.01
2.0
2.0
Thickness and growth






(mouthfulness) were






enhanced.


γGlu-Val-Gly
0.0005
2.5
3.0
Thickness, growth






(mouthfulness) and






continuity were mainly






enhanced from first and






middle taste.


γGlu-Val-Gly
0.001
3.5
4.0
Thickness, growth






(mouthfulness) and






continuity were mainly






enhanced from first and






middle taste.


γGlu-Val-Gly
0.01
5.0
5.0
Thickness, and






continuity were mainly






enhanced. Total taste






was enhanced.


γGlu-Val-Gly
0.1
5.0
5.0
Thickness, and






continuity were mainly






enhanced. Total taste






was enhanced.









Example 19
Activity of Peptides for Imparting Kokumi to Corn Soup
The intensity of the activity for imparting kokumi to corn soup of each peptide having confirmed calcium receptor activation activity was measured by a quantitative sensory evaluation test, performed as follows: to commercially available corn soup, samples of either γ-Glu-Cys-Gly (glutathione) or γ-Glu-Val-Gly were added to a concentration of 0.0001 to 1 g/dl, and the intensity of the kokumi-imparting activity was measured for each sample. The corn soup with the samples that became acidic after dissolution of the samples with respect to the corn soup without the samples was adjusted with NaOH to pH not lower or higher by 0.2 than pH of the corn soup without the samples before use. Sensory evaluation scores were used to evaluate as follows: 0 points for the control sample, 3 points for intense activity as compared to the control, and 5 points for extremely intense activity as compared to the control, and the test was performed with n=12. The samples showed kokumi-imparting activity at concentrations within the aforementioned broad concentration range. The results for typical concentrations are shown in Table 10.










TABLE 10









Intensity of kokumi














First





Concentration
and middle


Sample
(g/dl)
taste
Aftertaste
Evaluation remarks














Control
—
0
0
—


γGlu-Cys-Gly
0.01
3.0
3.0
Richness, thickness and






growth (mouthfulness) were






enhanced.


γGlu-Val-Gly
0.0005
2.5
3.0
Sweet taste, growth






(mouthfulness) and






continuity were mainly






enhanced from first and






middle taste.


γGlu-Val-Gly
0.001
3.5
4.0
Sweet taste, growth






(mouthfulness) and






continuity were mainly






enhanced from first and






middle taste.


γGlu-Val-Gly
0.01
4.5
5.0
Growth (mouthfulness) and






continuity were mainly






enhanced. Total taste was






enhanced.


γGlu-Val-Gly
0.1
5.0
5.0
Growth (mouthfulness) and






continuity were mainly






enhanced. Total taste was






enhanced.









Example 20
Activity of Peptides for Imparting Kokumi to Curry Sauce
The intensity of the activity for imparting kokumi to curry sauce of each peptide having confirmed calcium receptor activation activity was measured by a quantitative sensory evaluation test, performed as follows: to curry sauce prepared in a conventional manner by using commercially available curry roux, samples of either γ-Glu-Cys-Gly (glutathione) or γ-Glu-Val-Gly were added to a concentration of 0.0001 to 1 g/dl, and the intensity of kokumi-imparting activity was measured for each sample. The curry sauces with the samples that became acidic after dissolution of the samples with respect to the curry soup without the samples were adjusted with NaOH to pH not lower or higher by 0.2 than pH of the curry soup without the samples before use. Sensory evaluation scores were used to evaluate as follows: 0 points for the control sample, 3 points for intense activity as compared to the control, and 5 points for extremely intense activity as compared to the control, and the test was performed with n=12. The samples showed kokumi-imparting activity at concentrations within the aforementioned broad concentration range. The results for typical concentrations are shown in Table 11.










TABLE 11









Intensity of kokumi














First





Concentration
and middle


Sample
(g/dl)
taste
Aftertaste
Evaluation remarks














Control
—
0
0
—


γGlu-Cys-Gly
0.01
3.0
3.0
Richness, thickness and






continuity were enhanced.


γGlu-Val-Gly
0.001
2.5
3.0
Mildness, richness and






growth (mouthfulness)






were mainly enhanced.


γGlu-Val-Gly
0.005
3.5
4.0
Mildness, richness and






growth (mouthfulness)






were mainly enhanced.


γGlu-Val-Gly
0.01
5.0
5.0
Richness and continuity






were mainly enhanced.






Total taste was enhanced.


γGlu-Val-Gly
0.1
5.0
5.0
Richness and continuity






were mainly enhanced.






Total taste was enhanced.









Example 21
Kokumi-Imparting Activity Observed when Peptides and Additives Such as Known Calcium Receptor Activators were Used in Combination
The intensity of the kokumi-imparting activity of each peptide having confirmed calcium receptor activation activity and a known calcium receptor activator used in combination was measured by a quantitative sensory evaluation test, performed as follows: Samples of γ-Glu-Cys-Gly (glutathione) or γ-Glu-Val-Gly (0.0001 to 1 g/dl), or these samples combined with a calcium receptor activator such as calcium lactate, protamine or polylysine, or GABA (addition concentration: 0.0001 to 1 g/dl) were added to distilled water containing sodium glutamate (0.05 g/dl), inosine monophosphate (0.05 g/dl), and sodium chloride (0.5 g/dl), and the intensity of the kokumi-imparting activity was measured. Sample solutions that became acidic after dissolution of the samples were adjusted to pH 6.8 to 7.2 with NaOH before use. Sensory evaluation scores were used to evaluate as follows: 0 points for the control sample, 3 points for intense activity (as intensity of 0.05 g/dl γ-Glu-Cys-Gly and 0.005 g/dl γ-Glu-Val-Gly), and 6 points for extremely intense activity (as twice intensity of 0.05 g/dl γ-Glu-Cys-Gly and 0.005 g/dl γ-Glu-Val-Gly), and the test was performed with n=12. The samples showed kokumi-imparting activity at concentrations within the aforementioned broad concentration range. The results for typical concentrations are shown in Table 12. As a result, a compound known to have kokumi-imparting activity, glutathione, can have improved kokumi-imparting activity when used with a known calcium receptor activator or the like such as calcium.









TABLE 12








Intensity of




kokumi


















First








and




Peptide
Concentration

Concentration
middle




sample
(g/dl)
Additive
(g/dl)
taste
Aftertaste
Evaluation remarks
















—
—
—
—
0
0



γGlu-Cys-
0.05
—
—
3.0
3.0
Thickness, growth


Gly





(mouthfulness) and








continuity


γGlu-Val-
0.005
—
—
3.0
3.0
Thickness and


Gly





continuity


—
—
Calcium
0.25
0.5
0.5
Thickness




lactate






—
—
Protamine
0.005
1.5
1.0
Growth








(mouthfulness)


—
—
Polylysine
0.001
0.5
0.5
Thickness


—
—
GABA
0.025
0.5
0.5
Thickness


γGlu-Cys-
0.05
Calcium
0.25
3.5
4.0
Thickness, growth


Gly

lactate



(mouthfulness) and








continuity


γGlu-Cys-
0.05
Protamine
0.005
4.5
4.5
Thickness, growth


Gly





(mouthfulness) and








continuity


γGlu-Cys-
0.05
Polylysine
0.001
3.5
4.0
Thickness, growth


Gly





(mouthfulness) and








continuity


γGlu-Cys-
0.05
GABA
0.025
4.5
4.5
Thickness, growth


Gly





(mouthfulness) and








continuity


γGlu-Val-
0.005
Calcium
0.25
5.0
5.0
Thickness and


Gly

lactate



continuity


γGlu-Val-
0.005
Protamine
0.005
4.5
4.5
Thickness, growth


Gly





(mouthfulness) and








continuity


γGlu-Val-
0.005
Polylysine
0.001
4.5
4.5
Thickness and


Gly





continuity


γGlu-Val-
0.005
GABA
0.025
4.5
4.5
Richness and


Gly





thickness









INDUSTRIAL APPLICABILITY
The specific amino acids and peptides having calcium receptor activation activity are also useful as kokumi-imparting substances. In particular, as shown in Examples 12 to 21, several kinds of dipeptides and tripeptides were newly discovered to be kokumi-imparting substances, and since they are peptides, they can be used in the field of foodstuffs in which high safety is demanded. In addition, since a method for screening for a kokumi-imparting substance utilizing calcium receptor activation as an index has been developed, so-called high throughput screening can be used, and thus development of a still more highly efficient kokumi substances is possible.
While the invention has been described in detail with reference to preferred embodiments thereof, it will be apparent to one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention. All the cited references herein are incorporated as a part of this application by reference.

## Claims

1. A method for preparing a food or beverage that contains a kokumi-imparting substance, the method comprising:
A) a first step of detecting the calcium receptor activating ability of a test substance, which is an indication of the ability to impart kokumi, by using recombinant cells which have been transformed with a foreign calcium receptor gene, wherein the test substance enhances a taste selected from the group consisting of salty taste, umami taste, sweet taste, sour taste, and combinations thereof;
B) a second step of measuring the kokumi-imparting effect of the test substance for which calcium receptor activating ability has been detected in the first step;
C) selecting a substance which is effective to both activate the calcium receptor and impart kokumi, and
D) combining the substance selected in step C) with a food/beverage, thereby producing said food or beverage that contains a kokumi-imparting substance.

2. The method of claim 1, wherein the kokumi-imparting effect of the test substance is measured by a quantitative sensory evaluation test.