JP2733352B2 - Taste modifier - Google Patents

Description

The present invention relates to a high-purity taste-modifying substance obtained by further purifying a taste-modifying substance curculin isolated from the fruit of Curcurgo latifolia.

[Conventional technology]

As a substance (taste modifying substance) that acts on the receptor membrane of the tongue and changes the taste of food, it is conventionally included in the mouth, and when eaten with a sweet substance or when eaten with a sweet substance, it gives a sweet taste. It is known that gymnema sylvestre (Gymnema sylvestre) leaves and Gymnema sylvestre leaves and jujube (Ziziphus jujuba) leaves have digiffins, which are similar to those described above. Miraculin, which is contained in the fruit of miracle fruit (Synsepulm dulcifiaum), is known as one that gives a feeling.

The above miraculin has the function as described above, but has a problem in stability and has not been put to practical use as a taste modifying substance.

In addition, Curculigo latifolia (Curculigo latif
olia ) is a plant belonging to the family Echinacea which grows naturally in West Malaysia and southern Thailand. Its fruits are known to be edible and have an appetite-enhancing effect, but other properties are not known. Not been.

[Problems to be solved by the invention]

The present inventors have previously proposed curculigo latifolia ( Cu
rculigo latifolia ) from fruit or its dried product 0.01
Protein obtained by extracting with an aqueous solution of salt having a concentration of M or more (named curculin), after eating it,
It has been found that the protein has a taste-modifying effect that gives a sweet taste when eaten or consumed with water or sour substances.
No. 63-153143 and Japanese Patent Application No. 63-277717).

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

[Means for solving the problem]

The taste modifying substance of the present invention is represented by the following amino acid sequence.

Asp Asn Val Leu Leu Ser Gly Gln Thr Leu ¹⁰ His Ala Asp His Ser Leu Gln Ala Gly Ala ²⁰ Tyr Thr Leu Thr Ile Gln Asn Asn Cys Asn ³⁰ Leu Val Lys Tyr Gln Asn Gly Arg Gln Ile ⁴⁰ Trp Ala Ser Asn Thr Asp Arg Arg Gly Ser ⁵⁰ Gly Cys Arg Leu Thr Leu Leu Ser Asp Gly ⁶⁰ Asn Leu Val Ile Tyr Asp His Asn Asn Asn ⁷⁰ Asp Val Asn Gly Ser Ala Cys Cys Gly Asp ⁸⁰ Ala Gly Lys Tyr Ala Leu Val Leu Gln Lys ⁹⁰ Asp Gly Arg Phe Val Ile Tyr Gly Pro Val ¹⁰⁰ Leu Trp Ser Leu Gly Pro Asn Gly Cys Arg ¹¹⁰ Arg Val Asn Gly ¹¹⁴ Hereinafter, the taste modifying substance of the present invention will be described in detail.

The taste modifying substance of the present invention is obtained, for example, as follows.

First, water is added to the fruit of Curculigo latifolia or its pulp, homogenized, and centrifuged. At this time, the supernatant shows a dark brown color. Further, the sediment is homogenized by adding an equal amount of water to the original fruit or its pulp, and centrifuged. This washing operation is repeated until the supernatant becomes colorless to obtain a sediment. None of the supernatants has taste modifying activity.

Subsequently, an aqueous solution of a salt having a concentration of 0.01 M or more is extracted from the obtained sediment to obtain a crude extract containing curculin.

Examples of the salt include sodium, potassium, calcium, magnesium or ammonium hydrochloride, sodium, potassium, calcium, magnesium or ammonium phosphate, sodium, potassium, calcium, magnesium or ammonium carbonate,
Sodium, potassium, calcium, magnesium or ammonium sulfate or sulfite, sodium or potassium nitrate or nitrite, sodium or calcium lactate, alum, baked alum,
Sodium acetate, sodium or potassium pyrophosphate, sodium or calcium propionate, sodium benzoate, sodium fumarate, sodium polyacrylate and the like are used.

An example of an extraction means using an aqueous solution of the salt is as follows. After the above-mentioned washing operation, an aqueous sodium chloride solution is added to the obtained sediment, homogenized, and centrifuged or filtered to obtain a crude extract containing curculin.

Next, the above crude extract containing curculin is purified as follows, and high-purity curculin (taste modifying substance of the present invention) is obtained.
Get.

The crude extract can be purified by salting out with ammonium sulfate, sodium sulfate, potassium phosphate, magnesium sulfate, sodium citrate, sodium chloride, and the like, and then performing ordinary chromatography. As an example, the precipitate obtained by salting out with ammonium sulfate is converted to CM-
High purity curculin can be obtained by subjecting it to Sepharose ion exchange chromatography followed by molecular sieve chromatography.

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

Further, since high-purity curculin has the amino acid sequence described above, it may be synthesized according to this amino acid sequence by an appropriate synthesis method, for example, solid phase synthesis, partial solid phase synthesis, fragment condensation or solution synthesis, It can also be obtained by selecting an appropriate host and using recombinant DNA technology.

〔Example〕

Example 1 (Washing and extraction with aqueous sodium chloride solution) 30 g of the flesh of Curculigo latifolia was taken, homogenized by adding 40 ml of water, and centrifuged (12,500 rpm, 60 minutes). The supernatant was brown and had no taste-modifying activity. Further, 40 ml of water was added to the obtained sediment, homogenized, and centrifuged (12,500 rpm, 20 minutes). The supernatant was colorless and had no taste modifying activity.

Next, a 0.5 M aqueous solution of sodium chloride is added to the obtained sediment, homogenized, and centrifuged (30,000 rpm, 60 minutes).
did. The resulting supernatant was colorless and showed taste-modifying activity. Further, the extraction operation with 40 ml of 0.5 M sodium chloride aqueous solution was repeated three times, and the supernatants of these three times were combined.
A crude extract containing curculin was obtained.

Example 2 (salting out with ammonium sulfate) Ammonium sulfate was added to the crude extract obtained in Example 1 so as to be 80% saturated to precipitate an active substance.
This was centrifuged (32,000 rpm, 60 minutes), and the resulting precipitate was dissolved in 100 ml of 0.01 M phosphate buffer (PH6.8).

Example 3 (CM-Sepharose ion exchange chromatography) The solution obtained in Example 2 was applied to CM-Sepharose CL-6B.
-Flowed and adsorbed on a column (diameter 2.2 cm x 18 cm, bed volume 68 ml, manufactured by Pharmacia LKB Biotechnology). Subsequently, after passing through a 0.01 M phosphate buffer (PH6.8) to remove the fraction, the curculin was eluted by a linear concentration gradient elution method of a sodium chloride solution 0 to 1.0 M (flow rate 5 ml / 1 hour,
5 ml per fraction, total eluate volume 500 ml). 280n eluted protein
m was monitored by absorption. The results are shown in FIG. The peak (B) shown in FIG. 1 is a fraction containing the taste modifying substance curculin.

Example 4 (Molecular Sieve Chromatography) Ammonium sulfate was added to the fraction indicated by the hatched portion of the peak (B) in FIG. Was precipitated. This was centrifuged (32,000 rpm, 60 minutes), and the resulting precipitate was added to 1.5 ml of 0.01 ml.
It was dissolved in M phosphate buffer (PH6.8). This concentrated solution was separated by Sephadex (Pharmacia LKB Biotechnology) G-100 column (diameter 1.6 cm × 58 cm, bed volume 160 m).
l) Using 0.01M phosphate buffer (pH6.8) containing 0.5M NaCl
(8.4 ml for 1 hour, 2.8 ml for one fraction, 182 ml for total elution). Protein was monitored by absorbance at 280 nm. The results are shown in FIG. The peak (A) shown in FIG. 2 is a fraction containing the taste-modifying substance curculin.

Reference Example 1 (SDS-polyacrylamide gel electrophoresis) The substance in the fraction shown by the hatched portion of the peak (A) in FIG. 2 obtained in Example 4 was treated with a reducing agent, and the purity and The molecular weight was confirmed by SDS-polyacrylamide gel electrophoresis containing 8M urea.

As a result, a single band was shown at a molecular weight of 12,000 daltons, and the peak (A) in FIG.
It was confirmed that the taste-modifying substance curculin in the fractions indicated by the hatched portion was pure.

Obtained from 30g of the flesh of Curculigo Latifolia,
Protein content of each curculin fraction in Examples 1 to 4,
The activity yield and the degree of purification are as shown in Table 1 below.

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

The taste-modifying activity of each of the curculin fractions (taste-modifying substances) was determined by measuring the sweetness when each sample was contained in the mouth for 3 minutes, rinsed with water, and tasted with the 0.02 M citric acid solution at various concentrations. It was measured by determining the sucrose concentration of the same sweetness as compared with the sucrose solution. The activity yields are shown in Table 1 from the above measurement results. FIG. 3 shows the relationship between curculin concentration and sweetness (equivalent to sucrose). As can be seen from FIG. 3, the activity of high-purity curculin corresponded to the sweetness of 0.3M sucrose.

Reference Example 2 (Isoelectric focusing) First System (PhastSystem ^™ , Farsia LKB)
First gel (Ph)
When isoelectric focusing of high-purity curculin was performed using astGel IEF5-8), the isoelectric point was 7.1.

Example 5 (Amino Acid Composition) The amino acid composition was determined using Waters Picotag system. That is, 10 μg of high-purity curculin contains 1% phenol
Hydrolysis with 6N-HC1 at 110 ° C. for 22 hours, phenylthiocarbamyl (PTC) conversion of the obtained amino acid, and TSK gel ODS-80 ™ column (0.46 cm × 15 cm in diameter, manufactured by Tosoh Corporation) And analyzed by HPLC. PTC-amino acids were detected by absorbance at 254 nm.

Example 6 (Preparation of S-carboxamidomethylated curculin) High-purity curculin obtained by the operations of Examples 1 to 4
7 mg was dissolved in 5 ml of 0.4 M Tris buffer containing 6 M guanidine hydrochloride, 2 mM EDTA and 60 mM dithiothreitol.
This solution was incubated at 37 ° C. for 24 hours in nitrogen gas. 0.2 g of iodoacetamide is added to this solution,
It was allowed to stand for 10 minutes, followed by 60 minutes in an ice-water bath. The resulting S-carboxamidomethylated curculin was purified using Sephadex G-25, and containing 50 mM containing 2 M urea and 2 mM EDTA.
The solvent was exchanged for an M sodium bicarbonate buffer (pH 8.0) to obtain a sample for enzyme digestion.

Example 7 (Enzymatic digestion of S-carboxamidomethylated curculin) Lysyl endopeptidase digestion of S-carboxamidomethylated curculin was carried out using 50 m of 2 M urea and 2 mM EDTA.
This was performed at 37 ° C. for 17.5 hours in a M sodium bicarbonate buffer (pH 8.0). The protein concentration was 1 mg / ml and the enzyme to substrate ratio was 1 to 12
0 (W / W). The reaction was stopped by adding HC1 to pH 2.0.

In addition, chymotrypsin digestion of S-carboxamidomethylated curculin was performed at 37 ° C. for 30 minutes under the same buffer, protein concentration and enzyme to substrate ratio as in the above digestion. The digestion reaction was stopped in the same manner as above.

In addition, tryptic digestion of S-carboxamidomethylated curculin was performed at 37 ° C. for 3 hours under the same buffer, protein concentration and enzyme to substrate ratio as in the above digestion. The digestion reaction was stopped in the same manner as above.

Example 8 (Separation of peptides) The three peptide mixtures obtained in Example 7 were
HPLC using TSK-ODS-120T (manufactured by Tosoh Corporation) column
Separated. Each peptide was eluted by a linear gradient elution method of acetonitrile containing 0.05% trifluoroacetic acid. The peptide was detected by absorption at 210 nm and each peak was collected.

The HPLC elution patterns of the lysyl endopeptidase digest, the chymotrypsin digest and the trypsin digest are shown in FIGS. 4, 5 and 6, respectively.

However, for lysyl endopeptidase digestion and trypsin digest, a 30-minute linear concentration gradient elution pattern from acetonitrile 20% (W / W) to 40% (W / W) was used. For chymotrypsin digest, acetonitrile was used. It is a 45-minute linear concentration gradient elution pattern from 10% (W / W) to 40% (W / W). In addition, the names of the peptides described later were in accordance with the names of the peaks in these HPLC elution patterns.

Example 9 (Amino Acid Composition Analysis and Determination of Amino Acid Sequence) The amino acid composition analysis of each peptide obtained in Example 8 was carried out using a Waters Picotag sys.
The results are shown in Tables 2 and 3 below. However, the values of serine and threonine were corrected assuming that the loss due to decomposition was 10% and 5%, respectively.

The amino acid sequence was determined using the 470A Applied Biosystem Protein Sequencer (Applied Biosystem Pr.
otein Sequencer). That is, the amino acid was converted to phenylthiohydantoin (PTH), and the PTH-amino acid was analyzed by HPLC using a TSK-ODS-120T column. The results are shown in Tables 4 and 5 below.

The carboxy terminal amino acid sequence was determined using carboxypeptidase by the following method. 200 μg of high-purity curculin was added to a 0.1 M N-ethylmorpholine acetate buffer (pH
8.0) Dissolve in 0.9 ml. To this solution is added 10 μg of carboxypeptidase A and the reaction mixture is incubated at room temperature. A part of the reaction solution was used for 15 minutes, 30 minutes, 60 minutes and 120 minutes.
Collect every minute. Trichloroacetic acid was added to these reaction solutions to precipitate proteins, which were removed by centrifugation, and the free amino acids in the supernatant were analyzed using a Waters 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 as shown in FIG.

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

The solid line indicates the amino acid residues identified by Edman degradation of each peptide, and the dotted line indicates the amino acid residues not identified by Edman degradation of each peptide.

The relationship between alphabets and amino acids is as follows.

[Effect of the Invention] The "taste modifying substance" of the present invention is a substance that induces sweetness composed of high-purity curculin, and is appropriately contained in foods, beverages, feeds, pet foods, drugs, and the like as a completely new type of sweet substance. Can be used.

Further, since the amino acid sequence of the "taste modifying substance" of the present invention is determined, it can be produced in a large amount by chemical and genetic engineering techniques, and is useful.

[Brief description of the drawings]

Fig. 1 shows the fruit of Curculigo latifolia washed with water,
It is a graph which shows the elution pattern of CM-Sepharose ion exchange chromatography of the taste modifier obtained by extraction and salting-out operation. FIG. 2 is a graph showing the elution pattern of Sephadex G-100 molecular sieve chromatography of the fraction indicated by the hatched portion of the peak (B) in FIG. FIG. 3 is a graph showing the activity of a taste modifying substance composed of the high-purity curculin of the present invention. FIG. 4 is a graph showing an HPLC elution pattern of a peptide obtained by lysyl endopeptidase digestion of S-carboxamidomethylated curculin. FIG. 5 is a graph showing the HPLC elution pattern of a peptide obtained by chymotrypsin digestion of S-carboxamidomethylated curculin. FIG. 6 is a graph showing the HPLC elution pattern of a peptide obtained by tryptic digestion of S-carboxamidomethylated curculin. FIG. 7 is a schematic diagram showing the amino acid sequence of curculin.

Claims (1)

(57) [Claims] 1. A taste modifying substance represented by the following amino acid sequence. Asp Asn Val Leu Leu Ser Gly Gln Thr Leu ¹⁰ His Ala Asp His Ser Leu Gln Ala Gly Ala ²⁰ Tyr Thr Leu Thr Ile Gln Asn Asn Cys Asn ³⁰ Leu Val Lys Tyr Gln Asn Gly Arg Gln Ile ⁴⁰ Trp Ala Ser Asn Thr Asp Arg Arg Gly Ser ⁵⁰ Gly Cys Arg Leu Thr Leu Leu Ser Asp Gly ⁶⁰ Asn Leu Val Ile Tyr Asp His Asn Asn Asn ⁷⁰ Asp Val Asn Gly Ser Ala Cys Cys Gly Asp ⁸⁰ Ala Gly Lys Tyr Ala Leu Val Leu Gln Lys ⁹⁰ Asp Gly Arg Phe Val Ile Tyr Gly Pro Val ¹⁰⁰ Leu Trp Ser Leu Gly Pro Asn Gly Cys Arg ¹¹⁰ Arg Val Asn Gly ¹¹⁴