JP2017217005A - Saltiness enhancement peptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a saltiness enhancer and a saltiness enhancing method suitable for food and drink with high safety, and provide a tryptophan-containing peptide having the capability to activate hENaC.SOLUTION: A saltiness enhancer contains a dipeptide of at least one of Trp-X and X-Trp, where X is an amino acid selected from Phe, Leu, Trp, Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Lys, Met, Pro, Ser, Thr, Tyr, and Val.SELECTED DRAWING: Figure 1

Description

Sodium chloride (NaCl) is an essential nutrient for humans (Crit
. Rev. Food Sci. Nutr. , 2009, 49, 841-851: Non-patent document 1), which is a typical component constituting salty taste. On the other hand, excessive intake of salt contributes to an increase in blood pressure and is thought to cause stroke and heart disease. From the viewpoint of preventing an increase in the risk of lifestyle-related diseases due to excessive intake of salt, the Ministry of Health, Labor and Welfare stated that the target intake of salt in adults is male in “Japanese dietary intake standards (2010 edition): Non-patent document 2”. It is set at less than 9 g / day and less than 7.5 g / day for women. WHO recommends a salt intake of less than 5 g / day (Report of a WHO Forum and Technical
Meeting, 5-7 October 2006, Paris, France, W
HO; Geneva, Switzerland, 2007. 7: Non-patent document 3).

However, according to the 2009 National Nutrition and Health Survey (Non-patent Document 4), the intake of salt by adults is 11.1 g / day for men and 9.6 g / day for women, which is far from the target value. is there.
From this point of view, there is a strong demand to reduce the intake of salt, but the role of food is not only to give a salty taste, but also to interact with taste such as suppressing bitterness and enhancing sweetness and umami. (Food Qual. Pref., 2003, 14, 1
11-124: Non-patent document 5), salt-reduced foods in which the amount of salt added is reduced and the taste is significantly reduced, so that the suppression of salt intake has not progressed.

Therefore, search for salt substitutes and salty taste enhancing materials has been widely carried out. As a salt substitute,
A method of replacing a part of sodium chloride (NaCl) with potassium salt (KCl), magnesium salt (MgCl2), or ammonium salt (NH4Cl) is known. However, these have a drawback that the taste quality of the food is remarkably deteriorated because they have bitterness, astringency, astringency, and the like. Ornithyl taurine, ornithyl-β-alanine as a salty peptide,
Peptides composed of basic amino acids such as glycyrrhizin (J. Agric. Food C
hem. , 32, no. 5, 1984: Non-Patent Document 6) has been reported, but the effect is slight.

As a salty taste enhancing substance, for example, an equimolar mixture of L-arginine and L-aspartic acid (US Pat. No. 5,145,707: Patent Document 1), a peptide obtained by hydrolyzing collagen having a molecular weight of 50,000 daltons or less ( JP-A-63-3766: Patent Document 2), thaumatin which is a sweet protein (JP-A-63-137658: Patent Document 3), egg white protein,
Protein hydrolysates such as gelatin, soybean protein, wheat protein, corn protein, fish protein, milk protein or meat protein (Japanese Patent Laid-Open No. 7-289197: Patent Document 4), trehalose (Japanese Patent Laid-Open No. 10-66540: Patent Document 5) ), A decomposition solution obtained by digesting and decomposing a mixture of black koji made with black koji mold having citric acid-producing ability and koji made with koji mold (JP-A-2-5345)
No. 6: Patent Document 6), a cetylpyridium salt as a cationic surfactant alone or a mixture of a cetylpyridium salt and a basic amino acid such as arginine or lysine (Japanese Patent Publication No. 3-5025)
17: Patent Document 7), saturated fatty acid monocarboxylic acid having 3 to 8 carbon atoms (JP-A-5-1843)
26: Patent Document 8), yeast extract (Japanese Patent Laid-Open No. 2000-37170: Patent Document 9), peptide obtained by hydrolyzing and deamidating protein (International Publication No. 01/03961)
No. 3: Patent Document 10), a taste improver mainly composed of a neutralized salt produced by reacting a basic amino acid and citric acid (Japanese Patent Application Laid-Open No. 2003-144088: Patent Document 11), and many other methods. Has been proposed.

However, from the viewpoint of salt reduction effect, flavor, economy, safety, etc., it has not yet reached an effective technology or technology that meets the needs of consumers. There is a strong demand for safe and effective salt reduction technology that does not impair the flavor.
Taste substances taken into the oral cavity are received by organs called miso that are distributed on the surface of the tongue, pharynx, and larynx. There are different types of cells in the taste buds: basal cells, support cells, and taste cells. Among them, taste cells can be tasted, and when further expanded, taste cells on the cell membrane surface of the taste cells. Taste receptors, which are the entrance to recognition, are expressed. When a tastant is sensed by a taste receptor, cell membrane depolarization occurs via intracellular signaling,
Neurotransmitters are released toward the taste nerve projecting on the taste cells. The signal transmitted from the taste cell finally reaches the brain, which is the end point of taste recognition, and is recognized as a taste. In order to make the salty taste strong, it is only necessary to amplify the signal somewhere in this series of flows, but the material acting on the taste receptor that is the entrance to taste recognition is an essential salty taste enhancing material. is expected.

Taste receptors have been discovered one after another due to recent advances in molecular biology and genomics (Natur)
e 2006, 444, 288-294: Non-Patent Document 7), an evaluation method using cultured cells in which these taste receptors are expressed has been developed. Furthermore, by using this method, a sweetness-enhancing material (Proc. Natl. Acad. Sci. USA, 20
10, 107, 4746-4751: Non-patent document 8) and bitterness-suppressing materials that suppress bitterness (Curr. Biol., 2010, 20, 1104-1109: Non-patent document 9) have been found. From the above reports, it is reasonable to consider that a material that directly acts on the salty taste receptor and enhances the signal can also be a salty taste enhancing material.

Although there are still many unclear points regarding the mechanism of salty taste, the salty taste receptor was developed in 2010 by amiloride-sensitive epithelial channel (ENaC: Epithelial Na Channel).
Is a complex composed of α, β, and γ subunits and has been reported to be involved in the sensation of low concentrations of salt in rodents such as mice and rats, ie, preference salt (Natur).
e 2010, 464, 297-301: Non-Patent Document 10). The coding DNA (cDNA) for the α, β, and γ subunits of the human epithelial sodium channel (hENaC) has also been isolated and these genes have been cloned (Proc. Natl. Acad).
. Sci. U. S. A. , 1994, 91, 247-251, and Genomics,
1995, 28, 560-565: Non-Patent Documents 11 and 12), each subunit is considered to be necessary for the formation of a functional sodium channel even in humans (Nat).
ure, 1994, 367, 463-467: Non-patent document 13).

Therefore, if a material that activates hENaC is searched using a cultured cell evaluation system in which the α, β, and γ subunits of hENaC are expressed, it is considered that the material is likely to enhance salty taste. Methods for searching for compounds having ENaC activation ability using a cultured cell evaluation system have been constructed (Japanese Patent Application Nos. 2006-518896 and 2009-519912: Patent Document 1).
2 and 13), a compound S3969 that activates hENaC has been found (J
. Biol. Chem. 2008, 283 (18), 11981-11994: Non-patent document 14). Furthermore, it has been discovered that an analogous compound of S3969 also has the ability to activate hENaC and is excellent in the effect of enhancing the salty taste of sodium chloride to humans by coexisting with sodium chloride (WO / 2011/010748: Patent Document 14). ). However, the above hE
Any material that activates NaC is a synthetic compound that does not exist in nature, and the safety of eating has not been verified.

US Pat. No. 5,145,707 JP 63-3766 JP 63-137658 A JP-A-7-289197 JP-A-10-66540 JP-A-2-53456 Special table 3-550217 JP-A-5-184326 JP 2000-37170 A International Publication No. 01/039613 JP2003-1444088 Japanese Patent Application No. 2006-518896 Japanese Patent Application No. 2009-519912 WO / 2011/010748 Crit. Rev. Food Sci. Nutr. , 2009, 4 9, 841-851 Japanese food intake standards (2010 edition) Report of a WHO Forum and Technical Meeting, 5-7 October 2006, Paris, France, WHO; Geneva, Switzerland, 2007. 7 2009 National Nutrition and Health Survey Food Qual. Pref. , 2003, 14, 111-1 24 J. et al. Agric. Food Chem. , 32, no. 5, 198 4 Nature 2006, 444, 288-294 Proc. Natl. Acad. Sci. USA, 2010, 1 07, 4746-4751. Curr. Biol. , 2010, 20, 1104-1109 Nature 2010, 464, 297-301 Proc. Natl. Acad. Sci. U. S. A. , 199 4,91,247-251 Genomics, 1995, 28, 560-565. Nature, 1994, 367, 463-467. J. et al. Biol. Chem. 2008, 283 (18), 119 81-11994.

An object of this invention is to provide the salty taste enhancer and salty taste enhancing method with high safety | security suitable for food-drinks. Another object of the present invention is to provide a tryptophan-containing peptide having hENaC activation ability.

Inventors have diligently studied and have a dietary experience, and a dipeptide containing at least one tryptophan (Trp) as a highly safe constituent amino acid has an activating action of hENaC and coexists with sodium chloride. Thus, the inventors have found that the salty taste of the salt is excellent and the present invention has been completed.
That is, the present invention includes the following inventions:
Item 1, a salty taste enhancer containing at least one dipeptide of Trp-X or X-Trp, wherein X is Phe, Leu, Trp, Ala, Arg, Asn, As
p, Cys, Gln, Gly, His, Ile, Lys, Met, Pro, Ser, Th
An amino acid selected from r, Tyr, and Val is shown.
Claim | item 2, The method to adjust the salty taste of food-drinks including the process of mixing the dipeptide of Claim 1, and food-drinks.
Claim | item 3 and the food / beverage products containing the dipeptide of Claim 1, and food / beverage products.
Item 4. An activator of hENaC containing the dipeptide according to claim 1 or 2.
It is.

The Trp-containing dipeptide (Trp-X or X-Trp) of the present invention has the ability to activate hENaC and has the effect of enhancing salty taste by coexisting with salt. Therefore, by using the salty taste enhancer of the present invention, the salty taste in salt-reduced foods is improved, and the amount of salt used can be reduced. In addition, the Trp-containing dipeptide of the present invention is a dipeptide present in natural food materials and is considered to be extremely safe because it is a dipeptide contained in materials having a long dietary experience.
Therefore, for hypertensive patients, cardiovascular disease patients, kidney disease patients, etc. who are restricted in intake of sodium, and healthy people who are taking a low-salt diet from the viewpoint of lifestyle-related disease prevention Therefore, it is considered useful for providing a low-salt diet.

The measurement example of hENaC activation ability using the membrane potential dye method in Example 1 is shown. The measurement example of hENaC activation ability by the electrophysiological method in Example 2 is shown.

The present invention relates to a salty taste enhancer containing, as an active ingredient, a dipeptide having at least one tryptophan as a constituent amino acid.
In the present invention, the dipeptide containing at least one tryptophan as a constituent amino acid is tryptophan-amino acid (Trp-X) or amino acid-tryptophan (XT).
rp). Among these dipeptides, Trp-Ala
Trp-Arg, Trp-Asn, Trp-Asp, Trp-Cys, Trp-Gln
Trp-Gly, Trp-His, Trp-Ile, Trp-Leu, Trp-Lys
, Trp-Met, Trp-Phe, Trp-Pro, Trp-Ser, Trp-Thr
, Trp-Trp, Trp-Tyr, Trp-Val, Ala-Trp, Ile-Trp
, Leu-Trp, Lys-Trp, Phe-Trp, Tyr-Trp, an effect is recognized, more preferably Trp-Leu, Trp-Trp, Trp-
Particularly strong effects are observed in Phe, Trp-Cys, Trp-Met, and Ile-Trp.

The dipeptide may be a synthetic product or a product extracted and purified from a natural product. For example, Trp-
Regarding Leu, it is thought that it can be isolated and purified from “tofu-yo”, which is a food fermented with tofu from red yeast rice (Biosci. Biotechnol. Biochem).
. 67 (6), 1278-1283, 2003). Further, the protein can be used after being degraded with a proteolytic enzyme and purified. For example, it is considered that Trp-Trp can be obtained from egg white lysozyme, and Trp-Phe can be obtained by enzymatic treatment and purification of the α subunit of β-conglycinin. These may be a highly purified one kind of dipeptide, a mixture of a plurality of dipeptides, or may be used in a state containing amino acids or tripeptides. If it is necessary to reduce the amount added depending on the food to be added, it may be highly purified, and when used for food that does not affect the taste or flavor of ingredients other than dipeptide, it may be of low purity. .

The salty taste enhancer containing the dipeptide of the present invention exhibits a salty taste enhancing action when added alone to a salt-containing food or drink, but the effect is further improved by using a guanidinyl compound or a salt thereof in combination. In this case, arginine is particularly preferable as the guanidinyl compound to be used. Arginine and its salts can be either commercially available or synthesized and purified by conventional methods. The amount to be added is 0.005 with respect to 1 part by weight of dipeptide.
It is preferable to add at ˜400 parts by weight, particularly 0.05 to 100 parts by weight.

In addition, when these compounds are added, the pH tends to be alkaline, so it is preferable to adjust the pH. The pH may be adjusted using a suitable acid, preferably citric acid, acetic acid, lactic acid, succinic acid, fumaric acid, phosphoric acid, malic acid, phosphoric acid, gluconic acid, sulfuric acid, particularly preferably hydrochloric acid.

Further, inorganic salts such as potassium chloride and magnesium chloride may be combined. These may be commercially available, and the amount added is 0.005 to 10 per 1 part by weight of dipeptide.
It is preferably added at 00 parts by weight, particularly 0.1 to 200 parts by weight.

The present invention also contemplates a salty taste enhancing method using the salty taste enhancer of the present invention. The salty taste of the food can be enhanced by adding the salty taste enhancer of the present invention obtained by the above method to a food or drink containing salt. As a guideline for addition, although depending on the food to be added, 0.001 to 0.5% by weight of the dipeptide of the present invention in the food, and 0.
About 25% of salt can be reduced by adding about 0.1 to 1.0% by weight of potassium chloride or magnesium chloride. It becomes. By adjusting the amount of the salty taste enhancer of the present invention according to the desired degree of salt reduction using this as a guide, a salt-reduced food or drink can be obtained. In the salty taste enhancing method of the present invention, it is possible to achieve good quality salty taste enhancement with little bitterness or astringency imparting, which was a problem of conventional techniques.

The use in food can be widely applied, such as ramen, udon, soba, potage, consomme, bouillon, miso soup, soups such as soup, noodles such as ramen, udon, soba, yakisoba, pasta Examples include seasonings for foods such as breads, soy sauce, miso and dressing, or seasoning powders for snacks.
Examples of the present invention will be described below, but the present invention is not limited thereto.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to these Examples. In the examples, commercially available dipeptides (TRP-X or X-Trp)
However, dipeptides synthesized by themselves or obtained by enzymatic degradation may be used.

─Evaluation of hENaC activation using membrane potential dye method─
HEK293T cells were inactivated 10% fetal bovine serum (Inv
dulbecco's modified Eagle's me
It was cultured in a dium (DMEM, Invitrogen) at 37 ° C. in the presence of 5% CO 2. Each subunit of hENaCα, hENaCβ, and hENaCγ is mixed at a ratio of 1: 1: 1 Lipofectamine LTX + Plus reagent (Invitro
gen).

The amount and method of plasmid introduction were in accordance with the product manual. Six hours after gene introduction, the above-described cells were treated with 96-well black, clear-bottomed Cell
The cells were diluted with BIND surface plate (Corning Inc.) on the next day or the next day so that the cells were in a confluent state, re-plated in 100 μl aliquots, and cultured for 12-40 hours at 37 ° C. in the presence of 5% CO 2. Remove DMEM medium, assay buffer (130 mM NaCl, 10 mM glucose, 5 mM KCl, 2
mM CaCl2, and 1.2 mM MgCl2 in 10 mM HEPES, p
After rinsing with H7.4; hereinafter referred to as HEPES buffer), the membrane potential sensitive dye (Molecul) dissolved in HEPES buffer was filled with 50 μl of HEPES buffer.
ar Devices membrane potential kit Blue, M
Olecular Devices Inc. ) Was loaded at 50 μl. The concentration of the membrane potential sensitive dye was in accordance with the product manual. After standing at 27 ° C. for 10-60 minutes in the dark,
At Flex Station 3 (Molecular Devices Inc.), fluorescence intensity (excitation: 530 nm, detection: 565 nm, cut-off: 550 nm) was observed at 27 ° C. every 2 seconds, and after 20 seconds from the start of observation, a double concentration HEPES buffer was used. 100 μl of the dissolved sample solution was added, and the fluorescence was monitored for another 100 seconds. The difference between the fluorescence intensity 80 seconds after addition of the sample solution and the fluorescence intensity before addition of the sample solution is expressed by ΔRFU (relative fluo).
response units).

The expression plasmids for hENaCα, hENaCβ, and hENaCγ are Open Bi.
The cDNA shown in the table below was purchased from ossystems, and the coding region was amplified by PCR and then ligated to the insertion site shown in Table 1 of the pEAK10 expression vector. Table 1 shows the sequence information of each subunit of hENaC.

A measurement example in which the activation effect of hENaC using the membrane potential dye method was evaluated for Trp-X and X-Trp type dipeptides is shown in FIG. HEPES as a negative target
Buffers, S3969 as J.P. Biol. Chem. , 2008, 2
83 (18), 11981-11994, and synthesized and used. The final concentration of the sample was 1 μM for S3969 and 1 mM for dipeptide. Therefore, S396
9 was dissolved in HEPES buffer so that 2 μM and dipeptide was 2 mM,
Samples were prepared. 100 mM for compounds that are difficult to dissolve in HEPES buffer
First, a DMSO solution was prepared and diluted with HEPES buffer. It can be said that the hENaC activation effect is higher as the evaluation sample has a larger change in the RFU value. FIG. 1 shows a measurement example of the hENaC activation effect using the membrane potential dye method. Table 2 shows the hENaC activation effect using the membrane potential dye method.

Significance test was performed by one-way analysis of variance (ANOVA) and then by multiple analysis using Dunnett's method. Trp-Cys, Trp-Leu, Trp-Phe, Trp-Trp
, Ile-Trp, Phe-Trp, Tyr-Trp showed a statistically significant difference. Although no statistically significant difference was observed in other dipeptides, hENa
A tendency to activate C was observed.

─Evaluation of hENaC activation by electrophysiological methods─
The electrophysiological approach is to apply hENaCα, hENaCβ,
A method of observing a change in current value due to addition of a dipeptide by a two-electrode membrane potential fixation method using a microinjected oENaCαβγ-expressing oocyte mixed with equal amounts of hENaCγ subunit cRNA. Collection of Xenopus oocytes, microinjection of cRNA into the oocytes, and measurement of current values by the two-electrode membrane potential fixation method (Motonao Nakamura, Takao Shimizu: Xenopus oocyte experiments, experimental medicine Vol. .1
1, no. 3, 224-232 (1993)). hENaCα, h
The gene sequences of the subunits of ENaCβ and hENaCγ are as shown in Table 1 of Example 1.

Xenopus oocytes are microinjected with a mixture of equal amounts of cRNA of hENaCα, hENaCβ, and hENaCγ subunits, and MBS buffer (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO 3, 0.3 mM).
Ca (NO3) 2 · 4H2O, 0.41 mM CaCl2 · 2H2O, 0.82 mM
The cells were cultured at 16 ° C. for 12 to 72 hours in the presence of 10 μM amiloride in MgSO 4 .7H 2 O, in 15 mM HEPES, pH 7.6). Current measuring device (Warner In
The oocyte was set in Ocycle Clamp OC-725C (manufactured by Instruments), the electrode was inserted, the voltage was clamped to -60 mV, and the current value was measured. The buffer for the current value measurement was ND96 (96 mM NaCl, 2.5 mM KCl, 1 mM CaC.
l2, 1 mM MgCl2 in 5 mM HEPES, pH 7.5) was used. Set the oocyte of the amperometric device and the volume of the perfusion tank for measuring the current value is 100 μl, and the activity of ENaC is measured by measuring the current change when ND96 solution in which 25 μl of the evaluation compound is dissolved is added. Chemical ability was evaluated. Therefore, the evaluation compound was prepared by dissolving in ND96 so as to have a concentration 5 times the final concentration. When the solubility was low, it was first dissolved in DMSO to 100 mM and then diluted with ND96 to a concentration 5 times the final concentration.
The perfusion tank of the measuring device was perfused with ND96 (2 ml / min.) So that the added sample solution could be washed away. Examples of the two-electrode potential fixing method are shown in Table 3. Moreover, the measurement example of the hENaC activation effect by the two-electrode potential fixing method is shown in FIG.

Effect of amiloride: Effect of A and evaluation sample: ENaC determined from the ratio of B
After quantification by activation and test by one-way analysis of variance (ANOVA), significant difference test was performed by multiple analysis by Dunnett's method. Table 4 shows hENa by electrophysiological methods.
C activation effect is shown.

As shown in Table 4, in particular, Trp-Leu, Trp-Trp, Trp-Phe, Trp-
It was confirmed that Cys, Trp-Met, and Ile-Trp have significant ENaC activation ability. In addition, it was confirmed that these compound groups had a salty taste enhancing effect according to the methods described in Examples 3 and 4 below.

─Evaluation of salty taste enhancement effect by sensory evaluation─
1. Trp-Leu, Trp-Tr that showed high hENaC activation ability in Evaluation Example 2 using chicken soup
For p, Trp-Phe, and in addition to this, arginine hydrochloride + Trp-Phe was examined for the presence or absence of a salty taste enhancing effect by a sensory test. After diluting commercially available chicken bouillon soup concentrate (chicken bouillon EA, Ariake Japan Co., Ltd.) 5 times with boiling water,
Purified salt was added to adjust the salt concentration to 0.5%. Is this 0.5% salt-containing chicken soup used as a control, whereas 0.02% of dipeptide and 0.1% of arginine hydrochloride are dissolved, and the saltiness is enhanced by comparison with the control solution Verified. For the control, the salty taste was greatly enhanced by ++, the enhanced salt by ++, the slightly enhanced salt by +, and the unchanged salt by-.
Table 5 shows the salty taste enhancement effect using chicken soup.

From the results of Table 5, it can be seen that the product of the present invention has not only the ability to activate hENaC but also the salty taste enhancing effect. It was also observed that the effect was increased by mixing with arginine hydrochloride.

2. From the result of Evaluation Example 3 using instant noodle udon soup, the arginine hydrochloride + Trp-Phe addition group had the most excellent salty taste enhancing effect, so how much salt can be reduced by using this formulation. It was evaluated using udon soup for instant noodles. The noodle soup for instant noodles is prepared as shown in Table 6 and 1
Evaluation was performed by dissolving in 000 ml of hot water. Table 6 shows the powdered udon soup blending.

The salt concentration of the soup prepared by dissolving the control formulation and the reduced salt formulation with 1000 ml of hot water was determined to be 1.04% (W / V) and 0.79% (W / V), respectively. From this, it can be said that the low-salt blended soup is 25% salt-reduced from the control soup.
Arginine hydrochloride and Trp-Phe were added to the soup containing reduced salt so as to have the amounts shown in Table 7, and an Arg-HCl + Trp-Phe addition test group was prepared. ++ for those with strong titers
Somewhat strong, +, and some weak,-. The results are shown in Table 7.

As shown in Table 7, 25% of salt can be reduced by adding Trp-Phe and arginine hydrochloride to the salt-reduced formulation, and the accompanying taste and odor were not observed in the conventional salt reduction method. .

Claims (2)

A method for producing a salty food or drink, comprising a step of adding a salty taste enhancer comprising one or more dipeptides selected from the group consisting of Trp-Leu, Trp-Trp and Trp-Phe. Food and drink with enhanced salty taste containing 0.01 to 0.5% by weight of one or more dipeptides selected from the group consisting of Trp-Leu, Trp-Trp and Trp-Phe.

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