JPH03190899A - Substance for improvement of taste - Google Patents

Abstract

NEW MATERIAL:A substance for improvement of taste having an amino acid sequence represented by the formula. USE:A sweetening substance for foods, beverages, feeds, pet foods, medicines, etc. PREPARATION:For example, water is added to sarcocarp of gluculigo latifolia and the resultant mixture is homogenized. The homogenized mixture is centrifuged and the resultant precipitate is collected. To the collected precipitate, 0.5N sodium chloride is added followed by homogenization. The homogenized material is centrifuged and a supernatant is collected. To the collected crude extract, ammonium sulfate is added till the concentration is 80% saturation and the generated precipitate is separated by centrifugation. The separated precipitate is then dissolved in 0.01M phosphate buffer solution and the obtained solution is subjected to an ion-exchange chromatography. The adsorbed substances are eluted by the linear-concentration gradientelusion method using 0-1.0 M sodium chloride solution and the eluted active fraction is treated using the molecular sieve chromatography method and purified, thus obtaining the objective substance for improvement of taste having an amino acid sequence of the formula.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a highly purified taste modifier obtained by further purifying tartarin, a taste modifier separated from the fruit of Talcribo latifolia.

[Conventional technology]

Substances that act on the receptor membrane of the tongue to change the taste of food (taste modifiers) have traditionally been used during the day to modify sweetness when a sweet substance is eaten or when eaten together with a sweet substance. Gymnema Sylvester (G
Gymnemic acid contained in the leaves of ymnema 5ylvestre) and jujube (Ziziphllsjuju).
It is known that didyfin is contained in the leaves of Miracle Fruit (Synsepul), which gives a sweet taste when eating sour substances in the same manner as above.
Miraculin contained in the fruit of M. dulcifium is known.

Although the miraculin described above has the above-mentioned functions, it has stability problems and has not been put to practical use as a taste modifier.

Also, Tarkuribo Latifolia (Kondorifuji).

Order 1atifo (a) is a plant of the genus Prunus genus in the family Prunus elegans that grows naturally in western Malaysia and southern Thailand.The fruit is edible and is known to have an appetite-stimulating effect. No other properties are known.

[Problem to be solved by the invention]

The present inventors previously discovered a protein (named tartarin) obtained by extracting the fruit of Curculigo latifolia or its dried product with an aqueous solution of salt at a concentration of 0.01 M or more. discovered that it is a protein that has a taste-modifying effect that makes you feel sweet when you eat or drink water or a sour substance after eating it (Japanese Patent Application No. 631531, No. 43 and Patent Application No. 63-
27771.7).

The present invention provides a taste modifier consisting of highly purified tartarin (hereinafter referred to as high purity tartarin) obtained by highly purifying the above-mentioned curculin, and its amino acid sequence has also been determined.

[Means to solve the problem]

The taste modifier of the present invention has the following amino acid sequence.

Asp Asn Val Leu Leu
Ser Gly Gin Thr Leu” Olds Aha Asp His Ser Le
u Gin Ala Gly Ala”Tyr
Thr Leu Thr lie Gin
Asn Asn Cys Asn30Leu
Val l, ys Tyr Gin As
n Gly Arg Gin l1e40Tr
p Aha Ser Asn Thr As
p Arg Arg Gly 5er50Gl
y Cys Arg Leu Thr Leu Leu
Ser Asp Gly"Asn Leu Va
l Tie Tyr Asp His As
n Asn Asn"Asp Val Asn G
ly Ser Ala Cys Cys Gly As
p"Δla Gly Lys Tyr Aha
Leu Val Leu Gin Lys
90Asp Gly Arg Phe Val
Ile Tyr Gly Pro Val
IO'Leu Trp Ser Leu Gly Pr
o Asn Gly Cys Arg”.

Arg Val Asn Gly”’ Hereinafter, the taste modifier of the present invention will be explained in detail.

The taste modifier of the present invention can be obtained, for example, as follows.

First, water is added to Curculigo latifolia fruit or its pulp, homogenized, and centrifuged. At this time, the supernatant exhibits a deep brown color. Further, water in an amount equal to that of the original fruit or its pulp is added to this sediment, homogenized, and centrifuged. Repeat this water washing operation until the supernatant becomes colorless to obtain a precipitate.

None of the supernatants has taste-modifying activity.

Subsequently, the obtained precipitate was mixed with 0.01. A crude extract containing tartarin is obtained by extraction with an aqueous salt solution having a concentration of M or higher.

The above salts include sodium, potassium, calcium, magnesium or ammonium hydrochlorides, sodium, potassium, calcium, magnesium or ammonium phosphates, sodium, potassium, calcium, magnesium or ammonium carbonates, sodium, potassium, Calcium, magnesium or ammonium sulphates or sulphites, sodium or potassium nitrates or nitrites, sodium or calcium lactates, alum, calcined alum, sodium acetate, sodium or potassium birophosphates, sodium or calcium propionates. Salt, sodium benzoate, sodium fumarate, sodium polyacrylate, etc. are used.

An example of the extraction method using an aqueous solution of the above salt is as follows. After the above water washing operation, an aqueous sodium chloride solution is added to the resulting precipitate, homogenized, and centrifuged or filtered to obtain a crude extract containing tartarin.

Next, the crude extract containing curculin is purified as follows to obtain highly purified curculin (the taste modifier of the present invention).

The crude extract can be purified by salting out with ammonium sulfate, sodium sulfate, potassium phosphate, magnesium sulfate, sodium citrate, sulfur/lium chloride, etc., and then by ordinary chromatography. - For example, the precipitate obtained by salting out with ammonium sulfate, CM
- subjected to sepharose ion exchange chromatography;
By further subjecting it to molecular sieve chromatography, high purity curtaline can be obtained.

The amino acid sequence of high-purity curculin is determined by hydrolyzing the high-purity curculin with an enzyme such as trypsin, chymotrypsin, or lysyl endopeptidase, and then purifying each peptide fragment by HPLC using an aqueous reversed-phase column. This is done by determining the structure of this peptide fragment.

Furthermore, since high-purity curculin has the above-mentioned amino acid sequence, it may be synthesized according to this amino acid sequence by an appropriate synthesis method, such as solid-phase synthesis, partial solid-phase synthesis, fragment condensation, or solution synthesis. Choose a suitable host
It can also be obtained using recombinant DNA technology.

(Example) Example 1 (Washing with water and extraction with aqueous sodium chloride solution) Take 30 g of Curculigo latifolia pulp and add 40 ml
of water, homogenize, and centrifuge (12,50O
rpm, 60 minutes). This supernatant exhibited a brown color and had no taste-modifying activity. Furthermore, 40 d of water was added to the obtained sediment, homogenized, and centrifuged (1, 2,500
rpm for 20 minutes). This supernatant was colorless and had no taste-modifying activity.

Next, a 0.5M aqueous sodium chloride solution was added to the resulting precipitate, homogenized, and centrifuged (30,000 rpm,
60 minutes). The obtained supernatant was colorless and exhibited taste-modifying activity. Furthermore, the extraction operation using 40 m2 of 0.5 M sodium chloride aqueous solution was repeated three times, and the supernatants from these three times were combined to obtain a crude extract containing tartarin.

Example 2 (Salting out with ammonium sulfate) Ammonium sulfate was added to the crude extract obtained in Example 1 to achieve 80% saturation to precipitate the active substance. The precipitate obtained by centrifuging this (32,00 Orpm, 60 minutes) was
100 ml of 0°01M phosphate buffer (PH6,8)
dissolved in

Example 3 (CM-Sepharose ion exchange chromatography) The solution obtained in Example 2 was purified by CM-Sepharose CL-
6B-column (2.2 cm diameter x 1 Bcm.

The mixture was poured into a 68 mL bed (manufactured by Pharmacia LKB Biotechnology) for adsorption. Subsequently, after removing the pass-through fraction with 0.01M phosphate buffer (pr16.8),
Tartarin was eluted using a linear concentration gradient elution method with a sodium chloride solution of 0-1°0M (flow rate 5 ml/1 hour,
1 fraction 5 ml, total eluate volume 500 mj. Eluted proteins were monitored by absorption at 280 nm. The results are shown in Figure 1.

The peak (B) shown in FIG. 1 is the peak containing the taste modifier tartarin.

Example 4 (Molecular sieve chromatography) Example 3
The active substance was precipitated by adding ammonium sulfate to both the shaded areas of peak (B) in FIG. This is centrifuged (32
, 00 Orpm for 60 minutes) was added to 1.5 liters of 0.01M phosphate buffer (+1). 8). Sephadex (Pharmacia L)
Using a G-100 column (diameter 1.6 cm x 58 cm, bed volume 160 mR) (manufactured by K B Biotechnology), O, Ol, M phosphate buffer containing 0.5 M NaCl (
(pH 6.8) (flow rate 8.4 mf/1 hour, 1 fraction 2.8 ml, total elution volume 182 mR).

Protein was monitored by absorption at 280 nm. The results are shown in Figure 2. The peak (A) shown in FIG. 2 is the peak containing the taste modifier tartarin.

Reference Example 1 (SDS-polyacrylamide gel electrophoresis) The purity and molecular weight of both substances obtained in Example 4 and shown in the shaded area of peak (A) in FIG. Confirmed by 5DS-polyacrylamide gel electrophoresis.

As a result, the molecular weight was 12,000 Daltons.
), it was confirmed that the taste modification substance tartarin shown in the shaded area of peak (A) in FIG. 2 was a pure product.

Obtained from 30g of pulp of Curculigo latifolia,
The protein content, activity yield, and degree of purification of each curculin fraction are shown in Table 1 below.

The protein content was measured by the method of Lowry et al.

The activity was determined by holding the sample in the mouth for 3 minutes, rinsing the mouth with water, and comparing the sweetness of a 0.02M citric acid solution with sucrose solutions of various concentrations. It was measured by determining the sugar concentration.

The results are shown in FIG. As can be seen from Figure 3, the activity of highly purified talcrine was equivalent to the sweetness of 0.3M sucrose.

Reference Example 2 (Isoelectric Focusing) Phast I Gel (PhastGelIEF5-
When high purity talcrine was subjected to isofocus electrophoresis using 8), the isoelectric point was 7.1.

Example 5 (Amino acid composition) Amino acid composition was determined using Waters Picotag system.
It was carried out by In other words, 107 tB of pure rutalin
110 by 611-HCI containing 1% phenol.
Hydrolysis was carried out for 22 hours at °C, and the resulting amino acid was converted into phenylthiocarbamylate (PTC).
It was analyzed by H P I-C using TIl (manufactured by Tosoh Corporation). PTC-amino acids were detected by absorbance at 254 nm.

Example 6 (Preparation of S-carboxyamidomethylated talcrine) Highly purified talcrine 7 obtained by the operations of Examples 1 to 4
+ng to 0°4M containing 6M guanidine hydrochloride, 2mM ED2 TA and 60mM dithiothreitol.
) Dissolved in Squirrel buffer 5G. This solution was incubated in nitrogen gas at 37°C for 124 hours.

0.2 g of iodoacetamide was added to this solution, and the mixture was allowed to stand at room temperature for 10 minutes, and then in an ice water bath for 60 minutes. The resulting S-carboxyamidomethylated talcrine was mixed with 2M urea and 2mME using Sephadex G-25.
50mM bicarbonate containing DTA) IJum buffer (p
The solvent was exchanged with oe, o) and used as a sample for enzymatic digestion.

Example 7 (Enzymatic digestion of S-carboxyamidomethylated talcrine) S-carboxyamidomethylated talcrine was subjected to sylendopeptidase digestion at 37°C in 50mM sodium bicarbonate buffer (pH 8,0) containing 2M urea and 2mM EDTA. C. 17. I went for 5 hours.

The protein concentration was 1 mg/ml and the enzyme-to-substrate ratio was 1.
The ratio is 120 (W/W). For the reaction, add MCI and adjust the pH
It stopped by setting it to 2,0.

In addition, chymotryptic digestion of S-carboxyamidomethylated talcrine was performed at 37°C for 130 minutes under the same buffer, protein concentration, and enzyme-to-substrate ratio as in the second digestion.

The digestive reaction was stopped in the same manner as described in Section 2.

Additionally, tryptic digestion of S-carboxyamidomethylated talcrine was performed at 37°C for 13 hours under the same buffer, protein concentration, and enzyme-to-substrate ratio as in the above digestion. Digestion reactions were stopped in the same manner as above.

Example 8 (Separation of peptides) The three types of peptide mixture obtained in Example 7 were T
Separation was performed by HPLC using a SK-ODS-1,20T (manufactured by Tosoh Corporation) column. Each peptide is 0.05
Elution was performed using a linear gradient elution method of acetonitrile containing % trifluoroacetic acid. Peptides were detected by absorption at: 210 nm and each peak was collected.

The HPLC elution patterns of the lysyl endopeptidase digest, chymotryptic digest, and tryptic digest are shown in Figures 4, 5, and 6, respectively.

However, for lysyl endopeptidase digests and topsin digests, acetonitrile 20% (W/W)
The elution pattern is a 30-minute linear gradient elution pattern from 40% (W/W) to 40% (W/W), and the 45-minute linear gradient elution pattern from Kimo1-Ripmo% (W/W) to 40% (W/W). be. Furthermore, the names of the peptides described below are based on the names of the peaks in these HPLC elution patterns.

Example 9 (Amino acid composition analysis and determination of amino acid sequence) Amino acid composition analysis of each peptide obtained in Example 8 was carried out using the Waters Pico tag system (Waters Pico).
tag system), and the results are shown in Table 2 and Table 3 below. However, for serine and threonine, the loss due to decomposition is 10% and 5%, respectively.
The corrected value is shown as .

The amino acid sequence was determined using a 47OA Abride Hyosystem Blotin Sequencer (Applfed
Biosystem Protein 5equenc
er) was performed. That is, amino acids are converted into phenylthiohydantoin (PT
H) and convert this PTH-amino acid into TSK-O
Analysis was performed by HPLC using a DS-120T column. The results are shown in Table 4 and Table 5 below.

The carboxy-terminal amino acid sequence was determined by the following method using carboxybebutitase. 200 μg of high-purity tartaline was added to O, IMN-ethylmorpholine acetate buffer (pH 8,0). Melt to 9mA. To this solution, 10 μg of carboxybeptidase A is added and the reaction mixture is incubated at room temperature. A part of the reaction solution was
Samples are taken every 15, 30, 60 and 120 minutes. To these reaction solutions, 1~lychloroacetic acid was added to precipitate the protein, which was removed by centrifugation.
ters Picotag system). As a result, it was revealed that the carboxy-terminal amino acid residue was glycine.

The amino acid sequence determined by the above method is 6 As shown in FIG.

In FIG. 7, LEP, CH and T represent peptides from lysyl endopeptidase, chymotrypsin and tryptic digestion, respectively, and N represents the amino acid sequence determined from the N-terminus of high purity tarculin by Edman degradation.

Further, solid lines indicate amino acid residues identified by Edman degradation of each peptide, and dotted lines indicate amino acid residues not identified by Edman degradation of each peptide.

Furthermore, the relationship between alphahet and amino acids is as follows.

Aspartic acid Aspartic acid Glutamic acid Glutamine Methionine Cysteine Serine Three letter code One letter code Asp D 8sn N Glu E Ginmouth Met M Cys C 3er S Glycine Histidine Threonine Alanine Proline Arginine Tyrosine Valine Isoleucine Leucine Phenylalanine Lysine Amino acid CH-H3 l-4B CH- 5 C1+-6 CI-1,0 l-13 Amino acid EP-2 EP-3 LEP~5 EP-6 -11 Talculin 85x (B) Glx (Z) Cys (C) Ser (S) 2.0 (2 )"],,0(1) 2.0(2) 0,7(1,) 1.0(1,) 3,2(3) 11(1) 0.6(1) 3.0(3 ) 1.9 (2) 2.0 (2) 9.9 (11) 1.4 (1) 0.8 (1) 2.3 (3) 4.2 (4) Thr (T) 3.0 (3) 2,0 (2) 1.2(1) (5) 8Ia (A) Pro (P) ], 3(1) 3.1(3) 2.4(3) 0.7( 1) 1.2(1) Tyr(Y) 1.0(1) 1.0(1)
1. ．． 9(2) 1.1(1) 1.9(
2) (5)Vat(V) 1.1(1)
1.7(2) 1. ．． 7(2) 2.2(3
) 2.5(3) (8)11e(D
1.0 (1) 1.9 (2) 0.7
(1) 0.7 (1) (4) Lys (K) Trp (gull 0.8 (1) 0.9 (1) 0.9 (1) ND (1) ND (1) 2.3 (2 ) 11e(1) Leu (L) Phe (F) Lys (K) Trp (W) 1.3(1) 0.8(1) ND(1)" 0.8(1,) 1,6(2 ) 0.8 (1) 4.2 (4) 1.1. (1) 4.5 (4) 0.9 (1) 1.0 (1) 2.7 (3) 1.0 (1) 0.7 (1) ND (1)” Total (12) (7) (13) (21) (43) (18) Total (7) (33) (50) (24) (37) (114) Yield Values corrected as ratio (χ) 6.1 18.9 17.5 38.1 15.0 19.3 yield (χ) 55.2 72.4 83.0 68.5 31.3 are shown.

9 0 1 2 [Effects of the Invention] The "taste modifier" of the present invention is a sweet taste-inducing substance made of highly purified talcrine, and is a completely new type of sweet substance that can be used in foods, beverages, feed, vet food, drugs, etc. It can be used by appropriately containing it.

Moreover, since the amino acid sequence of the "taste modifier" of the present invention has been determined, it can be produced in large quantities by chemical and genetic engineering techniques, and is thus useful.

[Brief explanation of drawings]

Figure 1 shows the fruit of Curculigo radiofolia, washed with water,
1 is a graph showing the elution pattern of a taste modifier obtained by extraction and salting-out operations in CM-Sepharose ion exchange chromatography. Figure 2 shows the results of Sephadex G-100 molecular sieve chromatography of both of the shaded areas of peak (B) in Figure 1. It is a graph showing a discharge pattern. FIG. 3 is a graph showing the activity of the taste modifier made of highly purified talcrine of the present invention. FIG. 4 is a graph showing the HPLCi extraction pattern of peptides obtained by S-carboxyamidomethylated talcrine glue endopeptidase digestion. Figure 5 shows HPLC analysis of peptides obtained by chymotryptic digestion of S-carboxyamidomethylated talcrine.
It is a graph showing an elution pattern. FIG. 6 is a graph showing the HPI and C elution patterns of peptides obtained by trypsin digestion of S-carboxyamidomethylated talcrine. FIG. 7 is a schematic diagram showing the amino acid sequence of tarculin. Patent application business Yoshie Hara 3 4 JP-A-3 190899 (9) JP-A-3 190899 (10)

Claims (1)

[Claims] A taste modifier having the following amino acid sequence. [There is a gene sequence]