JPH07118142A - Taste-related protein extractant - Google Patents

Abstract

PURPOSE:To obtain a taste-related protein extractant capable of extracting a taste-related protein which exists on the tongue and plans an important role in manifestation of sweetness, flavor, etc., and of changing taste sensitivity of the tongue. CONSTITUTION:This taste-related protein extractant comprises a suspension of liposome containing an amide bond-containing phosphatidylcholine of formula I (R<1> and R<2> are 8-24C alkyl or 8-24C alkenyl), preferably 1,2- dimyristoylamido-1,2-deoxyphosphatidylcholine(DDPC). The suspension of liposome comprises the compound of formula I and a phospholipid for forming an ordinary liposome, preferably a phosphatidylcholine of formula II (R<3> and R<4> are R<1> and R<2>) such as egg yolk lecithin or dimyristoylphosphatidylcholine(DMPC) and a mixture of DDPC and egg yolk lecithin or DMPC in the molar ratio of 4:6 may be cited as the suspension of liposome. The extractant is brought into contact with the surface of epithelium of the tongue to extract a taste-related protein existing on the surface of epithelium of the tongue.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention has a pronounced sweetness on the tongue,
The present invention relates to a taste-related protein extractant capable of extracting a taste-related protein that plays an important role in the expression of umami and the like, and capable of changing the taste sensitivity of the tongue, and an extraction method using the same.

[0002]

2. Description of the Related Art As substances that stimulate taste cells on the tongue,
Acid, salt, bitter substance, sweet substance, umami substance (amino acid)
Etc. are known. Of these, salty taste, sour taste, and bitterness are said to be the tastes caused by these causative substances acting on the lipid membrane on the tongue. For example, regarding the relationship between bitter substances and lingual lipid membranes, the response threshold for various bitter substances of azolectin liposomes and the human taste threshold show a good correlation. It has been found that it is a hydrophobic site similar to that of a bilayer membrane [membrane (MEMBRANE), 13 (3). 144-151,
(1988)].

On the basis of this finding, studies on bitter substances, acids, and salts have been carried out using general lipid bilayer membranes.

On the other hand, regarding sweetness and umami, when the tongue is treated with a proteolytic enzyme, the nerve response disappears.
It is considered that the response is expressed via a receptor protein that is specific to the sweet and umami substances.

However, this on-lingual protein is present on the surface of the tongue and, since it is a membrane protein, it is difficult to extract and research has not progressed so much.

[0006]

Therefore, it has been difficult to change the taste sensitivity of the tongue for sweet and umami substances except when a protease is used. Therefore, an object of the present invention is to provide a method for extracting a protein involved in taste, to change the taste sensitivity of the tongue, and to serve for regulation of intake of sweet substances and the like.

[0007]

In view of such circumstances, the present inventors have conducted diligent research and as a result, if a liposome suspension containing a phosphatidylcholine represented by the following general formula (1) was brought into contact with the tongue, The present inventors have completed the present invention by finding that proteins involved in taste can be extracted, and that tastes related to sweetness and umami decrease after extraction.

That is, the present invention provides the following general formula (1)

[0009]

[Chemical 3]

A taste-related protein extract comprising a liposome suspension containing a phosphatidylcholine having an amide bond represented by the formula (wherein R ¹ and R ² represent an alkyl or alkenyl group having 8 to 24 carbon atoms). It is a thing. The present invention also provides a method for extracting a taste-related protein that is apparent on the surface of the tongue epithelium, which comprises contacting the taste-related protein extract with the surface of the tongue epithelium. Furthermore, the present invention provides a method for measuring a change in taste sensitivity, which comprises measuring a change in the electrical potential of the glossopharyngeal nerve before and after contacting the taste-related protein extract with the surface of the tongue epithelium.

R ¹ and R of phosphatidylcholine having an amide bond represented by the general formula (1) used in the present invention
^The alkyl or alkenyl group represented by 2 has 8 to 8 carbon atoms.
It is 24, but particularly preferably 10 to 20. Specifically, lauroyl group, myristoyl group, palmitoyl group and the like are preferable. A particularly preferred phosphatidylcholine having an amide bond is DDPC (1,2-dimyristoylamide-1,2-deoxyphosphatidylcholine).
Is mentioned. The phosphatidylcholine (1) having an amide bond can be produced by the method described in JP-A-61-267509.

The liposome suspension in the present invention is prepared by a known method, for example, a vortexing method (Vortexing method or Hyd), from the phosphatidylcholine (1) and an ordinary phospholipid for forming a liposome.
(Ration method) [Bangham, A .;
D. , Standish, H .; H. & Hatkin
S.J. C. J. Hol. Biol. , 13 , 238
(1965)]. As the liposome-forming phospholipid, more specifically, for example, soybean, extracted from egg yolk and the like, purified lecithin, lipids such as sphingomyelin, and synthetic lipids having a dipeptide bond near the polar head, The following general formula (2)

[0013]

[Chemical 4]

Phosphatidylcholine represented by the formula (wherein R ³ and R ⁴ represent an alkyl or alkenyl group having 8 to 24 carbon atoms), for example, egg yolk lecithin, dimyristoylphosphatidylcholine (DMPC) and the like are preferable.

A preferred combination of phosphatidylcholine (1) having an amide bond and ordinary phospholipids for forming liposomes is one in which DDPC and egg yolk lecithin or DDPC and DMPC are mixed in a ratio of about 4: 6 (molar ratio). The liposome suspension obtained as described above can be used as a taste-related protein extractant.

This extraction can be carried out by bringing the above liposome suspension into contact with the surface of the tongue epithelium.
Specifically, the liposome suspension may be repeatedly poured on the tongue. The tongue applied here is a tongue of animals and a taste sensation in which proteins are involved in taste expression, but in the present invention, tongues of mammals, amphibians, reptiles, birds and fish are particularly preferable. The amount of the liposome suspension used for the tongue is very important with respect to the amount of extracted protein, and for example, DDPC: DMPC = 1 ml of the liposome suspension for one tongue of a bullfrog.
It is preferable to add 10 ml of a liquid containing 5 mg of 4: 6, and for human, DDPC: DMPC = 4: 6 is 15 to 45.
It is preferable to add 30 ml of a solution containing mg.

The taste-involved protein extracted as described above is reconstituted on the liposome while retaining its activity, and it can be used as a study of taste and taste-involved protein, for example, artificial membrane taste sensor.

The change in taste sensitivity can be measured by measuring the change in the potential of the glossopharyngeal nerve before and after the contact of the liposome suspension of the present invention with the surface of the tongue epithelium. When the liposome suspension of the present invention is used as a mouthwash before meals, for example, salt and bitterness are strongly felt, so that salt in foods can be reduced and further sweetness is not felt. For this reason, the sweet food becomes unfavorable, and it is considered that the application of the sweet food can be inhibited.

[0019]

INDUSTRIAL APPLICABILITY The taste-related protein extract of the present invention can easily extract a taste-related protein involved in the expression of sweetness, umami, etc. on the tongue, and the obtained extract is a taste-related protein. Can be used for research. Furthermore, the change in taste sensitivity can be known by measuring the potential change of the glossopharyngeal nerve before and after the taste-related protein extract of the present invention is brought into contact with the surface of the tongue epithelium. On the other hand, the taste-related protein extract of the present invention can change the taste sensitivity of the tongue by being mixed with a mouthwash and the like, and can also be expected to have an effect of suppressing feeding of sweet substances.

[0020]

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

Example 1 From egg yolk, the method described in the literature [W. S. Singleto
n, H. S. Gray, H.M. L. Brown, J.M. L.
White, J. et al. Am. All Chem. Soc. ，
42 , 53 (1965)], and egg yolk phosphatidylcholine (EggPC) was extracted and purified using an alumina column. Its purity was confirmed by TLC. Also,
2,3-diaminopropionic acid from JP-A-61-267
No. 509 [Junzo Sunamoto et al., The Chemical Society of Japan, 1987]
(3), p569-574].
C was synthesized. 10.6 mg of the above EggPC and DDPC
Was mixed with 7.0 mg and dissolved in 2 ml of chloroform in an eggplant-shaped flask. Chloroform was distilled off under reduced pressure using a rotary evaporator, and the mixture was left overnight in a reduced pressure desiccator. Then, the obtained solution was swollen with 1 ml of PBS (pH 7.38) on a vortex mixer and stirred. Then, using a UR200P probe type ultrasonic crusher (manufactured by Tommy), ultrasonic irradiation was performed at 0 ° C. and 25 W for 5 minutes under a nitrogen gas stream to obtain a desired liposome suspension.

Example 2 Extraction of frog tongue protein: Bullfrog is anesthetized (urethane anesthesia) and the tongue is pulled out from the oral cavity and fixed with the taste-sensing surface facing up. Then DDP in 1 ml
C: DMPC = 4: 6 Lipid aqueous solution (10 ml) containing 5 mg of lipid was added to the tongue with a spid, the solution was received by a petri dish on the back of the tongue, and the solution was sucked with a spoid. A liposome suspension in which reconstituted proteins related to taste on the tongue were reconstituted was obtained.

Example 3 Effect of liposome for extracting protein on tongue: A silver / silver chloride electrode was connected to the glossopharyngeal nerve of bullfrog to measure a change in taste response as a nerve potential change, and the liposome treatment shown in Example 2 was performed for 1 hour. The measured values were compared before and after the application. The results are shown in FIGS. From FIGS. 1 and 2, for sucrose and 1-alanine, which are sweet components, the amount of change in nerve potential when taste was stimulated was clearly decreased immediately after the treatment as compared with that before the treatment. Subsequently, the liposomes in the obtained liposome suspension were separated, and the amount of extracted protein in the liposomes was quantified using a fluorescent indicator (flurane). As a result, it was confirmed that 133 μg / ml of protein was extracted.

Example 4 Analysis of Extracted Protein: Example 2
The liposome extract described in 1) and the protein obtained by washing the tongue with ion-exchanged water before liposome extraction were analyzed by SDS-PAGE and then compared and examined. The results are shown in FIGS. 5 and 6. From the figure, it is clear that a difference in high molecular weight (60 KD or more) was observed, and it was found that the protein was extracted by the liposome suspension.

Example 5 Selective masking of taste by optimizing extraction conditions: Generally, salt, acid and bitterness are said to be expressed by acting on the lipid membrane on the tongue, while sweet and umami are present on the tongue. It is considered to be a taste expressed by acting on a possible receptor protein. Therefore, a liposome extraction method for mainly extracting the receptor protein on the tongue without damaging the lipid on the tongue was examined using the liposomes described in Example 2. The results are shown in Fig. 7. As a result, it was found that only the sweetness can be selectively masked immediately after extraction if the extraction time is 30 minutes.

[Brief description of drawings]

FIG. 1 is a graph showing changes in nerve potential when L-alanine was stimulated before and after treatment with the extractant of the present invention in Example 3.

FIG. 2 is a graph showing changes in nerve potential when sucrose stimulation was performed before and after treatment with the extractant of the present invention in Example 3.

FIG. 3 is a graph showing changes in nerve potential when quinine hydrochloride was stimulated before and after treatment with the extractant of the present invention in Example 3.

FIG. 4 is a graph showing changes in nerve potential when L-leucine was stimulated before and after treatment with the extractant of the present invention in Example 3.

FIG. 5 is a diagram showing the results of protein analysis obtained by washing the tongue with ion-exchanged water in Example 4.

FIG. 6 is a diagram showing the analysis results of proteins extracted by the extractant of the present invention in Example 4.

7 is a diagram showing the results of selective masking of taste with the extractant of the present invention in Example 5. FIG.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication G01N 27/327

Claims (4)

[Claims] 1. The following general formula (1): (In the formula, R ¹ and R ² represent an alkyl or alkenyl group having 8 to 24 carbon atoms) A taste-related protein extractant comprising a liposome suspension containing a phosphatidylcholine having an amide bond. 2. A liposome suspension having the following general formula (2): The taste-related protein extract according to claim 1, which contains a phosphatidylcholine represented by the formula (wherein R ³ and R ⁴ represent an alkyl or alkenyl group having 8 to 24 carbon atoms). 3. A method for extracting a taste-related protein manifested on the surface of the tongue epithelium, which comprises contacting the surface of the tongue epithelium with the extract for taste-related protein according to claim 1 or 2. 4. A method for measuring a change in taste sensitivity, which comprises measuring a change in the potential of the glossopharyngeal nerve before and after contacting the taste-related protein extract according to claim 1 or 2 with the surface of the tongue epithelium.